INDUCTION OF APOPTOSIS BY NOVEL AMIDOXIMES INVOLVES
DECREASED HISTONE ACETYLATION AND p300 INHIBITION
A DISSERTATION
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
IN THE GRADUATE SCHOOL OF THE
TEXAS WOMAN’S UNIVERSITY
COLLEGE OF ARTS AND SCIENCES
BY
SUDHEER DHANIREDDY, B. Sc., M. Sc.
DENTON, TEXAS
DECEMBER 2012
ACKNOWLEDGMENTS
Firstly, I would like to thank my mentor and supervisor, Dr. Michael
Bergel, for giving me the opportunity to work in his lab and also for his
motivation, guidance, and a positive outlook throughout my PhD
without whom it would have been impossible to reach this goal. I would
also like to thank my committee Drs. Heather-Conrad Webb,
Nathaniel Mills, Camelia Maier, and Huanbiao Mo for their guidance
and advice towards my PhD.
A huge thank you to all my colleagues in the lab who have been very
friendly and helped me with all I needed throughout the course of my
PhD.
I would also like to thank Dr. McIntire for giving me an opportunity to
teach at TWU, which helped me gain experience in teaching.
Finally, many thanks to my parents, wife, sisters and uncle who have
encouraged me to pursue my higher studies at TWU, and I also
thank all my friends for their support at every juncture throughout my
time at university.
2
ABSTRACT
SUDHEER DHANIREDDY
INDUCTION OF APOPTOSIS BY NOVEL AMIDOXIMES INVOLVES
DECREASED HISTONE ACETYLATION AND p300 INHIBITION
DECEMBER 2012
The eukaryotic nucleus contains chromatin which consists of repeated units of
DNA wrapped 1.6 times around an octamer of histone proteins called core
particle. The folding and unfolding of chromatin can be regulated by the
deacetylation and acetylation levels of lysine residues on the histones. Histone
acetyltransferases (HATs) and histone deacetylases (HDACs) are enzymes that
modulate the degree of acetylation of histones, therefore regulating the levels of
gene expression. This project tested the growth inhibition effect of nine novel
amidoximes (JJMB 1-9) on six malignant cell lines. Four out of nine amidoximes
screened induced cell death specifically in various malignant cell lines as
determined by MTS cell viability assay. By DNA fragmentation assay, detection of
caspase-3 activation and FACS analysis, it was shown that the amidoximes
induced apoptosis by through the activation of caspase-3 in colon carcinoma
(HCT-116) cells. By FACS analysis it was shown that the amidoximes induced
cell cycle arrest mainly at G1 phase. In vivo acetylation studies (in cultured cells)
revealed that JJMB 5, 6, 7 and 9 inhibited the acetylation of histones H3 and H4.
JJMB 5, 6 and 9 but not JJMB 7 reversed the effect of the HDAC inhibitor, TSA in
3
colon carcinoma suggesting that they may inhibit HATs in the cell. In vitro assays
revealed that amidoxime, JJMB 9, but not the other amidoximes is indeed an
inhibitor of HAT p300. None of the amidoximes inhibited the HAT-GCN5.
Exploring the sequence and causal relationship between the reduced acetylation
and apoptosis in HCT-116 cells (colon carcinoma) indicated that JJMB 9 may
induce apoptosis by inhibiting HATs in the cell. In contrast JJMB 7 may induce
apoptosis by an unknown mechanism and the apoptosis in turn may inhibit the
acetylation of the histones H3K9 and H4K5 in HCT-116 cells. To the best of our
knowledge this is the first report of HAT inhibition activity by novel amidoximes
that was associated with an anti-proliferative effect. These amodoximes can be
potential therapeutic agents since overexpression of p300 and the HAT activity is
seen in diseases such as cancer, polyglutamine expansion diseases and
respiratory diseases.
4
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS​
ABSTRACT​
TABLE OF CONTENTS​
LIST OF TABLES​
LIST OF FIGURES​
LIST OF ABBREVIATIONS​
ii
iii
v
viii
ix
xi
Chapters
I. INTRODUCTION​
1
STRUCTURE OF CHROMATIN​
1
The 10 nm chromatin fiber​
2
The 30 nm higher chromatin fiber​
3
The 300 nm -1440 nm higher order chromatin​
4
POST-TRANSLATIONAL MODIFICATIONS​
6
Histone acetylation and its effects​
6
Acetylation of non-histone proteins​
8
Histone acetylation in chromatin assembly​
9
Regulation of transcription by PTMs of histone tails​
11
Interplay between post-translational modifications of histones​ 13
HAT families​
13
GNAT family​
14
MYST family​
15
p300/CBP family​
17
TAFII250​
18
Histone Deacetylases (HDACs)​
20
HATs and DISEASES​
21
Cancer​
22
Polyglutamine expansion diseases​
23
Asthma and COPD​
25
HAT INHIBITORS​
26
Bisubstrate analogs​
26
Anacardic Acid​
28
Garcinol​
29
Curcumin​
31
5
CELL CYCLE REGULATION AND APOPTOSIS​
33
II. MATERIALS AND METHODS​
Chemicals​
Cell culture​
Treatment of cells with amidoximes​
MTS assay​
Cell cycle analysis​
DNA fragmentation assay​
In vitro HAT assay​
Western blot analysis​
Statistical Analysis​
37
37
37
38
38
39
39
40
41
43
III. RESULTS​
Amidoximes induced death in human cancer cell lines​
Amidoximes induced apoptosis in HCT-116 cells as determined
by DNA fragmentation assay​
Amidoximes induced apoptosis is caspase-3 dependent​
Flow cytometric analysis of sub-G1 apoptotic population in
HCT-116 cells treated with amidoximes​
Flow cytometric analysis of sub-G1 apoptotic population in DU-145
cells treated with amidoxime JJMB 9​
Amidoximes induce cell cycle arrest in HCT-116 cells​
Amidoximes with a demonstrated anti-proliferative effect reduced
core histone acetylation in colon carcinoma cells (HCT-116)​
Amidoximes reverse the TSA induced hyperacetylation​
The effect of concomitant treatment with TSA and amidoximes on
cell survival​
JJMB 9 is a p300 inhibitor​
Amidoximes did not inhibit the GCN5 in vitro​
Amidoximes did not induce mitotic arrest in HCT-116 cells​
Exploring the sequence and causal relationship between the
reduced acetylation and apoptosis in HCT-116 cells treated with
amidoximes​
44
44
IV. DISCUSSION​
Structure based induction of cell death in malignant cell lines
by amidoximes JJMB 1-9​
Amidoximes causes histone hypoacetylation in HCT-116 cells​
Three out of four amidoximes reversed the TSA induced
hyperacetylation​
Novel amidoximes-induced apoptosis in HCT-116 cells is
85
6
54
57
59
61
63
66
70
73
75
79
81
83
86
90
91
caspase dependent​
94
Inhibition of acetylation precedes the caspase activation upon JJMB 9
treatment but not with JJMB 7​
95
Possible future applications of amidoximes​
98
V. REFERENCES​
7
102
LIST OF TABLES
Table
Page
1. List of acetylation sites​
10
2. HAT families, complexes and their function​
19
3. HATs and their in vitro substrates​
20
4. HDAC families, complexes and their function​
21
5. Induction of death in malignant cells by amidoximes JJMB 1-9 and the HAT
inhibitor garcinol​
47
6. The effect of amidoximes on the cell cycle distribution of HCT-116 cells
following a 24 hour treatment​
65
7. Structure based induction of cell death in malignant cell lines by
amidoximes JJMB 1-9​
89
8
LIST OF FIGURES
Figure
Page
1. Structure of nucleosome​
2
2. Levels of chromatin organization​
5
3. Regulation of acetylation on lysine residue by HATs and HDACs​
7
4. Structure of GCN5/PCAF​
14
5. Structure of Esa1​
16
6. Structure of CBP/p300​
17
7. Structure of TAFII250​
18
8. Chemical structures of Lys-CoA and H3-CoA-20​
27
9. Structure of anacardic acid and CTPB​
29
10. Chemical structures of garcinol and its derivatives LTK-14
and LTK-15​
31
11. Chemical structure of curcumin​
32
12. Chemical structures of amidoximes JJMB 1-9​
36
13. Survival rate of malignant cell lines treated with JJMB 4​
48
14. Survival rate of malignant cell lines treated with JJMB 5​
49
15. Survival rate of malignant cell lines treated with JJMB 6​
50
16. Survival rate of malignant cell lines treated with JJMB 7​
51
17. Survival rate of malignant cell lines treated with JJMB 9​
52
18. Survival rate of malignant cell lines treated with garcinol​
53
19. Model showing the pathways leading to DNA fragmentation through
caspase dependent and caspase independent pathways​
55
20. Amidoximes induced apoptosis in HCT-116 cells as determined
by DNA fragmentation assay​
56
21. Amidoximes-induced apoptosis is caspase-3 dependent​
58
22. The effect of amidoximes on the sub-G1 population of HCT-116 cells​ 60
23. The effect of amidoximes on the sub-G1 population of HCT-116 cells​ 61
24. The effect of amidoxime, JJMB 9 on the sub-G1 population of
DU-145 cells​
62
25. The effect of amidoximes on the cell cycle distribution of HCT-116
9
cells​
64
26. Amidoximes JJMB 5, 6, 7 & 9 induced cell cycle arrest in HCT-116
cells​
65
27. Effect of JJMB 5, 6, 7, 9 and garcinol on cellular core histone
acetylation​
68
28. Percentage of standardized acetylation on H3K9 (28.i) and H4K5
(28.ii)​
69
29. Inhibition of HDACs by TSA induces hyperacetylation of
core histones​
71
30. Amidoximes reverse the HDAC inhibitor, TSA induced
hyperacetylation​
72
31. Reversing the TSA effect by amidoximes doesn’t block cell death​
74
32. Diagram showing the experimental design of in vitro HAT assay in the
presence and absence of inhibitor​
76
33. In vitro P300 inhibition assay​
77
34. Percentage of standardized acetylation of H4K5​
78
35. In vitro GCN5 inhibition assay​
80
36. JJMB 7, 9 and garcinol did not arrest the cells in mitosis​
82
37. The kinetics of caspase-3 activation relatively to the inhibition of
acetylation in HCT- 116 after treatment with JJMB 7, JJMB 9 and
garcinol​
84
38. Model showing the mode of action of JJMB 7 in inhibiting
HCT-116 cells​
100
39. Model showing the mode of action of JJMB 9 in inhibiting
HCT-116 cells​
101
10
LIST OF ABBREVIATIONS
AA— anacardic acid
ADA— HAT complex includes Gcn5, Ada2 and Ada3
AD— alzheimer’s disease
AIF— apoptosis inducing factor
AML— acute myeloid leukemia
AP-1— activated protein 1
Apaf— apoptotic protease activating factor 1
AR— androgen receptor
ATR— ataxia telangiectasia and Rad3 related
ATM— ataxia telangiectasia mutated
ATP— adenosine tri-phosphate
CAF1— chromatin assembly factor 1
CARM1— cofactor associated arginine methyltransferase 1
CBP— creb binding protein
CGN— cerebellar granule neurons
11
CK2— casein kinase 2
COPD— chronic obstructive pulmonary disease
CRX: cone-rod homeobox-containing gene
CTPB — carboxyl-terminated polybutadiene
DFF40— DNA fragmentation factor 40
DNMT3— DNA methyl transferase 3
DMSO— dimethyl sulfoxide
DTT— dithiothreitol
EDTA— ethylenediaminetetraacetic acid
ELP— elongate protein 3
Endo G— endonuclease G
EZH2— enhancer of zeste homologue 2
FACS— fluorescent activated cell sorter
GCN5— HAT Gcn5
GNAT— Gcn5-related N-acetyltransferase
HAT— histone acetyltransferase
HDAC— histone deacetylase
HMT— histone methyltransferase
HMKT— histone methyl lysinetransferase
HMRT— histone methyl argininetransferase
HP1— heterochromatin protein 1
12
HIV— human immunodeficiency virus
IKKα— conserved helix-loop-helix ubiquitous kinase
Jmjc— Jumonji domain containing histone methylase
LSD1— lysine specific demethylase 1
LSA— lysophosphatidic acid
MLL— mixed lineage leukemia
MSK— mitogen-and stress-activated protein kinase
Mst-1— mammalian sterile twenty kinase 1
MOZ— monocytic leukaemia zinc finger protein
MORF— MOZ-related factor
MEF-2—myocyte enhancer factor-2
MTS— 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium, inner salt
NCP— nucleosomal core particle
NHK1— nucleosomal histone kinase-1
NIMAK— never In mitosis gene A kinase
NuA— nucleosome acetyltransferase of histone H4
NLS— nuclear localization signal
NURF— nucleosome remodeling factor
NuRD— nucleosome remodeling histone deacetylase
ODC— ornithine decarboxylase
13
PARP— poly (ADP-ribose) polymerase
PCAF— p300/CREB associated factor
PRMT— protein arginine methyl transferase
PADI4— peptidyl arginine deIminase 4
PHD— plant homeobox domain
PI— propidium iodide
PVDF— polyvinylidene difluoride
RSK2— ribosomal protein S6 kinase
RTS— rubinstein-taybi syndrome
ROS— reactive oxygen species
Sas— something about silencing
SAGA— Spt-Ada_Gcn5-acetyltransferase
SBMA— spinobulbar muscular atrophy
SCA 7— spinocerebellar ataxia type 7
SET— SET domain of protein (named after Su(var)3-9, E(z), and Trx drosophila
protein)
SMC— structural maintenance of chromosome
SWI/SNF— switching/surcose non-fermenting
SP1— Stimulating Protein 1
Ste20— Sterile 20 kinase
S(uv)3-9— Supressor of Variegation 3-9
14
TIP60— TAT-Interactive Protein 60 KDa
TAF1— TATA box binding protein (TBP)- associated factor of RNA Pol II
TAZ— Zn binding domain
TAFII250— TATA box binding protein (TBP)- associated factor of RNA Pol II 250
TFIID— transcription factor IID
TNF-α— tumor necrosis factor α
TRAIL— TNF-related apoptosis-inducing ligand
TSA— trichostatin A
USP22— ubiquitin-specific protease 22
UAS— upstream activating sequences
URS1— upstream repressing sequences 1
XCAP— xenopus chromosome-associated Protein
15
16
CHAPTER I
INTRODUCTION
Epigenetics is the study of heritable changes in gene expression without any
alteration to the primary DNA sequence. The epigenetic modifications that cause
the alterations in the gene expression are DNA methylation and post-translational
modifications (PTMs) of the N-terminal tails of core histones. These epigenetic
modifications modulate the structure of chromatin thereby regulating its
accessibility to various transcriptional factors and the proper functioning of the
cell. Disruption or alterations in epigenetic machinery play an important role in
causing cancer, neurodegenerative and respiratory diseases (Sharma et al.,
2010). Nucleosomes, the structural unit of chromatin takes the center stage of
epigenetic gene regulation. The structural and functional aspects of chromatin
are discussed in the chapter below.
THE STRUCTURE OF CHROMATIN
The human genome comprises of 3 x 109 base pairs of DNA measuring
approximately 2 meters in length. The compaction of the chromosomal DNA to fit
into the nucleus is carried out by a nucleoprotein complex called chromatin. The
basic unit of chromatin is a nucleosome (≈ 11 nm in diameter) which is composed
of DNA wrapped around histone proteins (Watson and Losick, 2011). The
nucleosome core particle (NCP) is a structure composed of an octamer of core
1
histones which contain two copies of H2A, H2B, H3 and H4 wrapped around by
147 bp of DNA. The histones H3 and H4 form a dimer and then the two dimmers
form a heterotetramer of H3 and H4. H2A and H2B form a heterodimer two of
which constitute each core particle. (Watson and Losick, 2011). The
histone-histone interactions and histone-DNA interactions in the NCP play an
important role in the stabilization of the nucleosome (Fig. 1)
Fig 1: The structure of nucleosome (Watson and Losick, 2011)
The 10 nm chromatin fiber
The
nucleosomes
are
arranged
in
a
continuous
DNA
helix
as
a
“bead-on-a-string” structure called a nucleosomal array which is about 11 nm in
diameter (Lodish and Matthew, 2008). This structure forms the lowest functional
and less compacted unit of chromatin (Christopher L Woodcock, 2001). Histones
are highly basic due to the presence of high proportion (≈ 28%) of lysine and
arginine residues. In every nucleosome, histones interact with DNA at 14 binding
sites with 142 hydrogen bonds making it a very stable structure. These
2
interactions are irrespective of the DNA sequence which is different for each
NCPs (Karolin Luger, 1998b). The histone tails are parts of histones at their
N-terminals that protrude out from the nucleosomal disk and make contacts with
DNA to further stabilize the structure (Fig. 1) (Karolin Luger, 1998a). The
negatively charged phosphate groups of the DNA backbone interact with the
positive charges of the histone tails. The tails of the histones undergoes various
post-translational modifications (Watson and Losick, 2011). These modifications
are important for regulating the folding and unfolding of chromatin thereby giving
accessibility to the DNA for various functions such as gene expression,
replication, DNA repair and apoptosis.
The linker histone H1 binds on one end to the linker DNA and the other end
to the center of 146 bp DNA associated with the nucleosomes. This binding
facilitates the tightening of the DNA association to the nucleosome. (Watson and
Losick, 2011).
The 30 nm higher order chromatin fiber
The 30 nm fibers are the first level of higher order chromatin structure and they
repress the transcription due to the inaccessibility of DNA to transcription factors.
Two models have been proposed to explain the formation of 30 nm chromatin
fiber – Solenoid model and zigzag model. In the Solenoid model the array of
nucleosomes is folded next to each other in a simple one start helix with six
nucleosomes per turn forming a super helical structure (Watson and Losick,
3
2011, Tremethick, 2007). In the zigzag model nucleosomes are arranged in a
random zigzag fashion when the linker criss-cross between the nucleosomes not
leaving a space for a central axis as in the solenoid. This model is also called the
two start helix (Tremethick, 2007, Watson and.Losick, 2011). The formation of 30
nm fiber, whether the solenoid or zigzag, is mediated by the interaction between
the N-terminal tail of histone H4 and the acidic patch of H2A.Z (Tremethick,
2007). The amino acids 14-19 in the N-terminal tail of histone H4 are essential
for the nucleosome-nucleosome interaction in the 30 nm chromatin fiber
(Benedetta Dorigo and Timothy, 2003). Controversy still exists in regard to the
relevance of solenoid versus zigzag structures in the nucleus.
The 300 nm-1400 nm higher order chromatin
Further condensation of the 30 nm fiber into higher-order chromatin fibers of 300
nm, 700 nm and 1400 nm takes place as well (Fig 2). The 30 nm fiber forms
large radial loops attached to a central chromosomal axis called chromosomal
scaffold or matrix. The regions of DNA in the loop that are attached to the
chromosomal scaffold are called scaffold attachment regions (SARs) or matrix
attachment regions (MARs). The chromosomal scaffold is rearranged depending
on the stage of the cell cycle (Watson and Losick, 2011, Lodish and Matthew,
2008).
A number of non-histone proteins are also involved in the condensation into
the higher-order chromatin. One of the important components of scaffold proteins
4
is the topoisomerase II (topo II), which is helpful in the condensation and
decondensation of the coiled loops in both mitotic and interphase nuclei
(Strunnikov, 1998). The promotion of chromatin compaction by Topo II is through
the interaction between Topo II and the H1 attached to the chromatin (Kohji
Hizume and Kunio, 2007). The attachment sites for Topo II are the AT-rich
sequences which are lined along the chromosomal axis (Anna-Lisa, 1999). The
interphase chromosome is around 300 nm in width whereas the highly
compacted form measures around 1400 nm (Lodish and Matthew, 2008). The
condensation of chromatin into a metaphase chromosome involves a special
class of proteins called structural maintenance of chromsome proteins (SMC).
These SMC proteins are a part of condensin complex (five subunit complex) –
SMC proteins (XCAP-C and XCAP-E), XCAP-D2, XCAP-G and XCAP-H which
function together to carry out the chromosome condensation in an ATP
dependent manner (Strunnikov, 1998, Rea et al., 2000).
5
Figure 2: Levels of chromatin organization from open naked DNA to the highly
condensed metaphase chromosome.
(http://beyondthedish.wordpress.com/tag/chromatin-fiber)
POST-TRANSLATIONAL MODIFICATIONS
The N-terminus of the histone tails has 19-39 residues that extend from the
nucleosomal disc. These tails undergo various post-translational modifications
(PTMs) such as acetylation, phosphorylation, methylation, ubiquitination and
ribosylation that help in transcriptional regulation, DNA repair, apoptosis, cell
proliferation, differentiation and cellular signaling. (Rea et al., 2000).
Histone acetylation and its effects
Acetylation of the N-terminal lysine residues of histone tails is one of the most
important
and
well
studied
post-translational
modifications.
Histone
acetyltransferases (HATs) transfer the acetyl group from acetyl Co-A donor onto
the ε-amino group (on the R-group) of specific lysine residues of histone tails.
This causes the neutralization of the positive charge on the lysine residues and
therefore weakens the contacts between DNA and histones establishing an open
chromatin state and gene expression. Histone deacetylases (HDACs) on the
other hand, removes the acetyl group from the lysine residue restoring the
positive charge on the histone tails, and causing the compaction of chromatin
and subsequently subsequently in most cases, gene repression. Core histone
6
acetylation is a reversible process (Fig. 3). Cell homeostasis is maintained by the
proper balance of HATs and HDACs activity (Watson and Losick, 2011, Lodish
and Matthew, 2008).
Figure 3: Regulation of acetylation on lysine residue by the negating activities of
HATs and HDACs (http://www.web-books.com/MoBio/Free/Ch4G.htm).
Acetylation as well as other PTMs of histones serves as a mark to histone
modifying enzymes and non-histone proteins that regulate transcription which is
referred to as a histone code (Sameez Hasan, 2002). Apart from transcription,
acetylation is also involved in a number of other processes such as DNA repair,
histone deposition and telomere silencing. For instance, Esa1 (in yeast) and its
homolog Tip60 (in humans) are involved in DNA double stranded break repair by
acetylating the H4 tails of nucleosomes near the break site. This helps in
unfolding the chromatin and therefore increasing the accessibility for the repair
factors to the damaged site (Bird et al., 2002, Murr et al., 2006). The
7
hypoacetylation of H3K9 serves as a marker for the X-chromosome inactivation
(He and Lehming, 2003). Exception to the histone acetylation is the acetylation of
H4K12 by Hat1p, which is essential for the silencing of telomere (Kelly et al.,
2000).
Acetylation of non-histone and structural proteins
In addition to histone acetylation, studies indicate that HATs can also acetylate a
range of non-histone proteins. Like histone acetylation, non-histone acetylation is
also reversible and many HDACs are reported to deacetylate the non-histone
proteins. Since the discovery of p53 as a non-histone protein target of
acetylation, many new proteins have been discovered. Tumor suppressor gene,
p53 is acetylated by CBP/p300 on various lysine residues of its C-terminal DNA
binding domain. Acetylation modifies the conformation of p53 and increases its
DNA binding affinity (Luo et al., 2004). It was reported that HDAC1 and SirT1
deacetylate p53 and decreases its DNA binding affinity (Juan et al., 2000). A
number of other transcriptional factors are regulated by acetylation such as
STAT3, the c-MYC oncogene and MyoD. In addition to transcriptional factors,
structural proteins such as α- tubulin are also regulated by acetylation. MEC-17,
an acetyltransferases related to Gcn5 is identified as a specific acetyltransferase
of α-tubulin that targets lysine 40 and increases stability of microtubules (Akella
et al., 2010). HDAC6 and SirT2 were found to deacetylate lysine 40 of α-tubulin
and cause microtubule depolymerization (Matsuyama et al., 2002, Zhang et al.,
8
2003). Protein acetylation plays an important role not only in regulation of
transcription but also in several other cellular processes as evident by various
non transcriptional protein targets such as α-tubulin.
Histone acetylation in chromatin assembly
Acetylation is also linked to chromatin assembly during replication. During this
process, the H3/H4 tetramer is first deposited to the DNA followed by assembly
of H2A/H2B dimers. H3 and H4 are first synthesized in the cytoplasm with H4
specifically diacetylated at K5 and K12. It is shown that Hat1 helps in the
acetylation of these histones. During the histone deposition, CAF1 (complex of
p150, p60 and p48) a histone chaperon, interacts with the acetylated H3/H4 in
the cytoplasm and facilitates the deposition of H3/H4 dimers to the newly
assembled chromatin (Grunstein, 1997, Brownell and Allis, 1996). Table1
summarizes the list of core histone acetylation sites and the histone modifiers.
9
Table 1: List of acetylation sites (Clarke, 1999, Kimura and Horikoshi, 1998,
Schiltz et al., 1999, Suka et al., 2001, Grant et al., 1999, Bird et al., 2002, Ikura et
al., 2000, Kelly et al., 2000, Ye et al., 2005)
Histone
H2A
H2B
H3
10
Modification
K4Ac
K5Ac
K7Ac
K5Ac
K11Ac
K12Ac
K15Ac
K16Ac
K20Ac
K4Ac
Histone modifier
Esa1
Tip60, p300/CBP
Esa1
p300
GCN5
p300/CBP
p300/CBP
GCN5, Esa1
p300
Esa1
Role or function
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
Transcriptional activation
K5Ac
GCN5
Transcriptional activation
Ac.K9
p300, GCN5
Transcriptional activation
K14Ac
K18Ac
GCN5,PCAF,
Esa1,Tip60,Sas3,
p300
Esa1, Tip60
GCN5
K23Ac
p300/CBP
GCN5
K27Ac
K56Ac
p300/CBP
GCN5
Spt10
Transcriptional activation
DNA repair
Transcriptional activation,
DNA repair
Transcriptional activation
Transcriptional activation,
DNA repair
Transcriptional activation
Transcriptional activation
Transcriptional activation
K5Ac
K8Ac
H4
K12Ac
K16Ac
Esa1, Tip60
p300
GCN5, PCAF
Esa1, Tip60
Hat1
Esa1, Tip60
p300
Transcriptional activation, DNA repair
Transcriptional activation
Transcriptional activation
DNA repair
Histone deposition, telomere silencing
Transcriptional activation, DNA repair
Transcriptional activation
GCN5,
Transcriptional activation
Esa1, Tip60
DNA repair
Sas2
Eurchromatin
(Ac- acetylation, K – lysine)
Regulation of transcription by PTMs of histone tails
Regulation of gene expression is controlled by transcription factors binding to the
promoter region, covalent modifications of histones and non-histone proteins,
and
the
activity
of
chromatin
remodelers
such
as
SWI/SNF, NURF.
Post-translational modifications (PTMs) of core histone tails regulate the
chromatin structure and therefore modulate the binding and activities of
transcription factors to the target genes. Therefore, PTMs control the expression
of target genes by acting as ON/OFF switch (Lodish and Matthew, 2008).
Transcription of genes occurs by recruiting ATP-dependent chromatin
remodelers, HATs and transcription factors to the sites of transcription. Most of
the HATs are a part of large multi-subunit complexes such as SAGA/GCN5
complex or NuA4/Tip60 complex, while CBP/P300 acts either in complexes or as
independent transcriptional coactivators. In S. cerevisiae, to enable transcription
of genes from the highly condensed mitotic chromosome, chromatin remodelers
11
such as SWI/SNF are recruited to the upstream activating sequences (UAS) and
loosen up the chromatin. SAGA/GCN5 complex is then recruited to acetylate the
nucleosomal histones and thus allowing the general transcription factors such as
TBP, TAFII250 and RNA pol II to bind to the promoter (Lodish and Matthew,
2008, Lemieux and Gaudreau, 2004). SWI/SNF complexes contain ATPase
activity that aids in the disruption of histone-DNA interactions. Gene silencing
results from binding of large multi-subunit corepressor complexes containing
HDACs. In yeast, multi-subunit complex containing Rpd3 and Sin3 binds to the
repressor UME6. UME6 when bound to URS1 DNA sequence along with Rpd3
and Sin3 forms a corepressor complex that removes the acetyl groups and
causes chromatin compaction (Kadosh and Struhl, 1997). This results in the
inaccessibility of transcriptional factors and RNA pol II to the promoter and
therefore gene repression. This mechanism is conserved in higher organisms. In
mammals, HDAC 1 and HDAC 2 are a part of mSin3, CoREST and Mi-2/NuRD
corepressors; HDAC 3 is a part of N-CoR and SMRT complexes that functions in
silencing gene expression (You et al., 2001, Denslow and Wade, 2007, Jones
and Shi, 2003, Heinzel et al., 1997).
In contrast to acetylation, histone methylation causes transcriptional
activation or repression depending on the sites modified. For instance, H3K4
methylation is a marker for repression at the rDNA locus in S.cerevisiae which is
mediated by Set1. In another example, H3K9 methylation is involved in formation
12
of heterochromatin by interaction of methyl transferase Su(var)3-9 with HP1.
Preceding to HP1 binding to tri-me H3K9, deacetylation of H3K9 by HDAC1/2
takes place which is followed by the methylation by methyltransferase
Su(var)3-9.
HDAC1/2
was
also
shown
to
associate
with
the
DNA
methyltransferase DNMT3B at heterchromatin regions along with the recruitment
of ATP chromatin remodeling complexes. Methylation of H3K9 by HP1 continues
to the neighboring nucleosomes and causes the spreading of heterochromatin
(Honda et al., 2012). Methylation of H3K79 by histone H3 methyltransferase,
dot1 causes a telomeric silencing (He and Lehming, 2003). Histone methylation
is mainly involved in gene silencing but it is also reported to cause gene
activation. For example, gene activation of pS2 involves the recruitment of
CARM1 methylase to the promoter and H3K17 methylation (Bauer et al., 2002).
Interplay between post translational modifications of histones
Recent studies showed that the covalent histone modifications such as
acetylation, methylation and phosphorylation also affect each other during the
transcription of genes. For instance, Lo et al. showed that phosphorylation of
H3S10 mediates the recruitment of SAGA/GCN5 complex and subsequent
acetylation of H3K14. This suggests that the phosphorylation of H3S10 serves as
an activating event that enhance the acetylation of H3K14 by HATs (Lo et al.,
2000). It was also reported that lysine methylation is regulated by the
phosphorylation of H3. Methylation of H3K9 by SUV39H1 is inhibited when
13
H3S10 is phosphorylated. Similarly, H3S10 phosphorylation is inhibited when
H3K9 is methylated (Rea et al., 2000). On histone H4, methylation of H4R3 by
PRMT1 promotes the acetylation of H4K8 and H4K12 by P300 (Wang et al.,
2001).
HAT families
The mechanism of acetylation and its relation with transcriptional activation was
established in 1960’s (Borrow, 1996) but the identification and role of enzymes
performing this process began in 1996 with the discovery of HAT GCN5 (Kuo et
al., 1996). As of now, many enzymes and enzyme complexes have been
discovered which possess the intrinsic HAT activity. HATs are divided into two
groups based on their origin and function – type A HATs function as
transcriptional co-activators in the nucleus acetylating the nucleosomal histones
and non-histone proteins; whereas, the type B HATs help in acetylating free
histones in cytoplasm and transport them to the nucleus for chromatin assembly
(Roth et al., 2001). Together, type A and type B HATs fall into three major families
along with few transcriptional factors that possess HAT activity.
GNAT (Gcn5-related N-acetyltransferases) family
The members of this family include the closely related Gcn5, PCAF, Tetrahymena
p55 and more distantly related proteins such as Hat1, Elp3 and Hpa2. These
proteins function not only as transcriptional co-activators but also in histone
deposition (Hat1), telomere silencing (Hat1) and transcriptional elongation (Elp3).
14
GCN5/PCAF protein family members have two functional domains – a 160
residue HAT catalytic domain that helps in the activation of target genes and a
C-terminally located bromodomain which helps in the recognition of acetylated
lysines on target substrates (Fig. 4) (Ornaghi et al., 1999).
Figure 4: Structure of GCN5/PCAF showing N-terminal HAT catalytic domain
and a C-terminal bromodomain (Marmorstein, 2001).
GCN5 was first isolated as a nuclear HAT from yeast and initially seemed to
potentially acetylate free histones but it could not acetylate nucleosomal histones
in vitro (Table 2 and 3) (Pal et al., 2004). Yeast GCN5 functions as a member of
two multi-subunit complexes - the ADA complex and the SAGA complex (Table
2). Both these complexes preferentially acetylate histone H3, and to a lesser
extent, H2B (Table 2 and 3). (Grant et al., 1997). GCN5, as a part of SAGA
complex along with Usp22 function to maintain the telomere (Atanassov et al.,
2009).
PCAF was identified as a protein interacting with p300/CBP. It can acetylate
both free histones and nucleosomal histones but preferentially acetylates H3K14
for cellular processes. (Sameez Hasan, 2002). PCAF is a part of a complex
which is similar to the yeast SAGA complex. PCAF is also involved in other
biological processes apart from transcriptional activation. Upon DNA damage,
PCAF acetylates lysine 320 of P53 increasing its binding ability to the damaged
15
DNA (Liu et al., 1999). PCAF is also involved in muscle differentiation by
acetylating MyoD (important regulator of muscle differentiation) and changing its
conformation thereby increasing its ability to bind to the target DNA (Sartorelli et
al., 1999).
MYST family
MYST is another major family of HATs named after its founding members – MOZ,
Ybf2, Sas2, Sas3 and Tip60. Tip60 was the first member of MYST family
identified with HAT activity. Members of MYST family participate in two HAT
complexes – the NuA3 complex (sas3) and NuA4 complex (Esa1) (Allard et al.,
1999,
Roth
et
al.,
2001). MYST family members contain N-terminal
chromodomain (a domain that binds to methylated lysines on histones) and Zinc
binding HAT catalytic region located C-terminal to the chromodomain. Some of
the members in MYST family lack the chromodomain (example: Tip60, Sas2-3)
and some contains a PHD domain (a domain that binds to methylated lysines on
histones) instead of chromodomain (example: MOZ, MORF) (Fig. 5) (Roth et al.,
2001).
​
Figure 5: Structure of Esa1 showing Zinc binding domain within HAT catalytic
C- terminal region and an N -terminal chromodomain (Marmorstein, 2001).
16
Esa1p, a member of MYST family is highly homologous to MOF and Tip60. It
can acetylate free histones but not nucleosomal histones in vitro. Esa1p as a part
of NuA4 complex specifically acetylates histone H4 and positively regulate
transcription (Table 2 and 3) (Allard et al., 1999). Also Esa1p is required for cell
cycle progression, providing a link between H4 acetylation, transcriptional
activation and cell cycle control (Clarke, 1999) (Table 2). Members of MYST
family such as MOZ and MORF form fusion proteins like MOZ-CBP and
MORF-CBP, leading to acute myeloid leukemia (AML) (Katsumoto et al., 2008).
CBP/p300 family
CBP/p300 family includes the most studied and well characterized HAT
proteins-p300 and CBP. They act as transcriptional co-activators and are
involved in other biological processes as well. CBP and p300 and are highly
homologous proteins that share a lot of similarties in their structure and function.
They were identified as proteins that were found to interact with viral oncoprotein
E1A and CREB binding protein respectively (Roth et al., 2001).
p300/CBP
contains three major domains which include cysteine-histidine rich domains (TAZ,
PHD and ZZ) which aid in DNA binding, bromodomain that recognizes the
acetylated lysine residues and C-terminal HAT catalytic domain. The HAT domain
17
in p300/CBP which is 500 residues longer than in GCN5/PCAF helps in its
extensive transcriptional regulation (Fig. 6) (Marmorstein, 2001).
Figure 6: Structure of p300/CBP showing the N-terminal bromodomain, three
cystein-histidine rich domains (TAZ, ZZ and PHD) and HAT catalytic domain
(Marmorstein, 2001).
p300/CBP function as transcriptional co-activators where they are recruited to
the promoter region by specific transcription factors to acetylate core histones
(Marmorstein, 2001). CBP and p300 have shown to acetylate core histones both
in vitro and in vivo (Table 2 and 3). They preferentially acetylate H3 and H4; and
H2A & H2B to a lesser extent. The HAT activity of p300 is regulated by several
factors such as viral oncoprotein E1A. The N-terminal region of E1A binds to the
p300/CBP-PCAF complex and displaces PCAF. The c-terminal region of E1A
binds to HAT catalytic domain of p300 and inhibit its activity providing a
mechanism for p300 regulated transcription (Chakravarti et al., 1999)
TAFII250
TAFII250, is a transcriptional factor and a part of TFIID complex along with TBP
and many other TAFs. In this complex, TBP recognizes the TATA element along
with other general TAFs and forms the preinitiation complex to which RNA pol II
is recruited (Watson and Losick, 2011). TAFII250 possess an intrinsic HAT
activity capable of acetylating both free and nucleosomal histones (Mizzen et al.,
1996) (Table 2 and 3). TAFII250 also has an intrinsic kinase activity. Thus,
18
TAFII250 is composed of N-terminal kinase domain, HAT catalytic domain and
c-terminal
bromodomain
(Fig.
7)
(Marmorstein,
2001).
TAFII250
autophosphorylates several of its own serine residues and also phosphorylates
Family
Type of HAT
Complex
Function
Gcn5
PCAF
Elp3
Esa1
SAGA, Ada
PCAF
None
NuA4
Sas3
NuA3
Transcription activation
Transcription activation
Transcription elongation
Transcriptional activation,
Cell cycle progression
Gene silencing
Tip60
NuA4
DNA repair
MOZ
None
Leukemogenesis
CBP/p300 CBP
None
Transcription activation
P300
None
Transcription activation
TAFII250
None
Transcription elongation
GNAT
MYST
TAFII250
RAP74 (subunit of TFIIH) underlying a possible mechanism in transcriptional
initiation (Dikstein et al., 1996).
Figure 7: Structure of TAFII250 with N-terminal kinase domain, HAT catalytic
domain and c-terminal bromodomain (Marmorstein, 2001).
Table 2: HAT families, several HATs, HAT complexes and their function (Grant et
al., 1999, Grant and Berger, 1999, Kimura and Horikoshi, 1998, Mizzen et al.,
1996, Clarke, 1999, Ogryzko et al., 1996)
19
20
Table 3: HATs and their in vitro substrates (Grant et al., 1999, Liu et al., 1999,
Kimura and Horikoshi, 1998, Clarke, 1999, Takechi and Nakayama, 1999,
Mizzen et al., 1996, Schiltz et al., 1999, Kelly et al., 2000)
Type of HAT
Gcn5
Acetylation of free
histones
H3 and H4
Acetylation of nucleosomal
histones
H3 only (SAGA, Ada)
PCAF
H3 and H4
H3 and H4
Tip60
H3, H4 and H2A
Poorly acetylates H3, H4 and H2A
Esa1
H3, H4 and H2A
Sas3
H3, H4 and H2A
Prefers H3, H4 and H2A (NuA4
complex)
H3 and H4 (NuA3 complex)
Hat 1
H4
H4
TAFII250
H3 and H4
None
CBP/p300
H3, H4, H2A and H2B
H3, H4, H2A and H2B
Histone Deacetylases (HDACs)
HDACs are the enzymes that catalyze the removal of acetyl groups from lysine
residues of core histone causing in most cases to the compaction of chromatin.
Mammalian HDACs are divided into four classes: Class I HDACs include
HDAC1, 2, 3 and 8, and are homologous to yeast Rpd3. Class II HDACs are
divided into two major subgroups based on domains and are homologous to
yeast Hda1. Class IIa HDACs include HDAC 4, 5, 7 and 9 and class IIb HDACs
include HDAC 6 and10 (Gregoretti, 2004, Gregoire, 2007). Class III HDACs,
called sirtuins are NAD+ dependent HDACs. They include SirT 1-7, and are
21
homologous to the yeast Sir2 (Gray, 2001). Recently class IV HDACs was
classified, which includes HDAC11. HDAC11 has conserved residues in its
catalytic center that are shared both by class I and class II HDACs. (Gregoretti,
2004) (Table 4). Most of the HDACs work in complexes to repress or silence the
expression the genes.
Table 4: HDAC families, complexes and their function (Gregoretti et al., 2004,
De ruijter et al., 2003).
Family
Class I
Type of
HDAC
HDAC1,
HDAC2
HDAC3
Complex
Function
Sin3, NuRD,
Co-REST
SMRT, N-CoR
Transcription silencing
HDAC8
None
Transcription silencing
HDAC4, 5 , 7
and 9
Transcription silencing and
blocking muscle differentiation.
Transcription silencing
Class IIb
HDAC6
HDAC10
SMRT,
N-CoR;
interacts with
MEF2
HDAC 11
N-CoR
Class III
SirT1-7
None
Trancription silencing
Class iv
HDAC11
HDAC6
Not known
Class IIa
Deacetylation of tubulin
Trancription modulator
HATs AND DISEASES
Epigenetic modulators such as HATs, HDACs and HMTs play a role not only in
transcriptional regulation but also in DNA repair, apoptosis, cell proliferation,
differentiation and cellular signaling. A proper function of these modulators is
22
important for cellular homeostasis. Any aberration or malfunctioning can lead to
diseases such as cancer, heart diseases, neurological diseases and respiratory
diseases. The following section summarizes various diseases that are caused by
dysregulation of histone acetyltransferases (HATs).
Cancer
Mutations in HATs can lead to various types of cancers and other diseases. For
example acute myeloid leukemia (AML), a disorder of hematopoietic progenitor
cells is caused by fusion of transcriptional co-activators by chromosomal
translocations. Some of the fused genes that cause AML are MOZ-p300,
MOZ-CBP, MOZ-TIF2, MORF-CBP, MORF-p300, MLL-CBP and MLL-p300
(Katsumoto et al., 2008). MOZ-CBP was the first fusion gene discovered that
was involved in AML. The fusion is due to the translocation of MOZ from
chromosome 8 to chromosome 16 adjacently to p300 {t(8;16) (p11;q13)}. This
translocation causes a breakpoint in CBP at amino acid 266 deleting CBP’s NLS
while the breakpoint in MOZ at 1547 leaves MOZ with DNA binding Zn finger
domain and HAT domain but not the M-rich domain. MOZ-CBP, the resultant
fusion with two HAT domains activates Nf-kβ dependent transcription and
promotes leukemogenesis. The transcriptional regulation of target genes differs
from one fusion protein to another fusion protein (Katsumoto et al., 2008, Borrow
et al., 1996). It was also reported that in many of microsatellite instability (MSI+)
colon cancer cell lines such as RKO and HCT15 have high frequency of
23
mutations in p300 and CBP genes probably causing a reduced DNA repair and
accumulation of mutations in these cells (Ionov et al., 2004).
.
It was also reported that Tip60, (a MYST family HAT), and an androgen
receptor activator is accumulated in the nucleus of prostate cancer cells (LnCap)
and CWR22 (xenograft mice model) possibly causing the progression of prostate
cancer (Halkidou et al., 2003). Another study, reported a significant down
regulation of Tip60 mRNA levels in human colon and lung carcinomas,
suggesting a prominent role of Tip60 in these cancers (Lleonart et al., 2006).
Tip60 is also involved in skin cancer and could be detected by the increased
levels of ODC (Ornithine decarboxylase) and polyamine metabolism in the cell.
Interestingly, elevated HAT activity was observed in K6/ODC mice with skin
cancer. These elevated HAT activity was correlated with an abnormal increase in
the protein levels of Tip60 and its variant Tip53 but not with elevated mRNA
levels (Hobbs et al., 2006). Due to the diverse functions of Tip60 in transcriptional
regulation, apoptosis, repair and cellular signaling, its role in different cancers
also varies.
Polyglutamine expansion diseases
HATs are also involved in polyglutamine expansion diseases (CAG expansion)
such as HD (Huntington’s disease), SCA-7 (Spinocerebellar ataxia type 7) and
SBMA (Spinal-bulbar muscular atrophy). Proteins with expanded polyglutamine
such as ataxin-7 and AR receptors interact with HATs/ transcriptional factors such
24
as CBP, TAFII250, PCAF, SP1 and p53, and greatly affect the gene expression
pattern and causes neurodegeneration. Expanded polyglutamine proteins
change their conformation from a monomer to toxic inclusion bodies (Shao and
Diamond, 2007). SBMA is a type of polyglutamine disease with a CAG expansion
in the exon 1 of AR receptor. CBP interacts with androgen receptors (AR) in
normal cells and its interaction is further enhanced in mutant AR. CBP is
sequestered to nuclear inclusions formed in CAG expanded cells (as opposed to
its diffused pattern in the nucleus of normal cells) causing a disruption in the
regulation of targeted genes. The same is observed in transgenic mice
expressing AR112∆ and also in patient’s tissue. Overexpression of CBP reduced
the toxicity induced by polyglutamine expansion in SBMA (McCampbell et al.,
2000). A similar phenomenon is also observed in Huntington’s disease (HD)
where CBP is sequestered to nuclear inclusions, which leads to the alteration of
CBP regulated gene expression and cellular toxicity (Nucifora et al., 2001).
SCA-7 (Spinocerebellar ataxia type 7) is another polyglutamine disease
characterized by cerebellar and retinal degeneration dystrophy caused by the
mutant ataxin-7 (CAG expansion). Interestingly, ataxin-7 is also a part of human
STAGA transcriptional co-activator complex. Polyglutamine expanded ataxin-7
inhibits the HAT activity of STAGA and thereby inhibiting the acetylation of
CRX-photoreceptor (Cone-rod homeobox protein) target genes resulting in the
retinal degeneration in SCA-7 (Palhan et al., 2005).
25
Another example of involvement of HATs in neurodegenerative diseases is
observed in PCAF knockout mice that have a phenotype of short term memory
loss whereas the CBP knockout mice resulted in long term memory loss. The
phenotypes of the mice suggested that the embryonic inactivation of PCAF
resulted in the alteration of gene expression, impairment of neuronal activation
and signaling involved in short term memory loss (Maurice et al., 2008).
RTS (Rubinstein-Taybi Syndrome) is another neuro related genetic disease
that is characterized by facial abnormalities, broad thumbs and mental
retardation.
These
symptoms
are
caused
due
to
the
chromosomal
rearrangements in chromosome 16 and mutations in the CBP gene. RTS
patients have one functional copy of the CBP allele and one copy of mutated or
truncated CBP allele. This leads to defects in the thought processes, neural
differentiation and reduced CBP HAT activity (Wang et al., 2010, Petrij et al.,
1995).
Asthma and COPD
Respiratory diseases like asthma and COPD (Chronic obstructive pulmonary
disorder) are caused by the increased expression of pro-inflammatory genes.
Core histone modifications such as acetylation, methylation and phosphorylation
play an important role in the activation of these genes. For example, biopsies
from asthma patients showed a significant increase in HAT activity and reduced
levels of HDAC activity especially HDAC1 and HDAC 2 (Ito et al., 2002). In
26
another study, when rats were exposed to cigarette smoke, it resulted in much
reduced HDAC activity that correlated with elevated H4 acetylation. This in turn
led to the activation of proinflammatory genes such as NF-Kβ and Ap-1. The
possible reason for the activation of these genes could be due to the interaction
with transcriptional factors (Marwick et al., 2004). Use of corticosteroids and
theophylline in COPD and asthma drastically reduced the HAT activity and
increased the HDAC activity (Marwick et al., 2004, Cosio et al., 2004)
HAT INHIBITORS
The multiple functions HATs and HDACs in gene expression and DNA repair also
means that their malfunctioning results in disease or lethality. Focus has been
recently given to designing drugs that can activate or inhibit HATs for therapeutic
purposes. Inhibitors of HDACs are well studied and several of them are already
in clinical trials and in clinical use. However, as of now, only few synthetic and
natural inhibitors of HATs have been discovered and some of which have shown
promising results for therapeutic purposes. Nonetheless, there is a limited
knowledge of their mechanism of action inside the cell. Some of the known HAT
inhibitors and their potential as therapeutic agents will be discussed below.
Bisubstrate analogs
Bisubstrate analogs (Lys-CoA and H3-CoA-20) are the first HAT inhibitors
designed specifically to inhibit p300 and PCAF. The first synthetic HAT inhibitors
designed were Lys-CoA and H3-CoA-20 (Fig. 8). Lys-CoA specifically inhibited
27
p300 with an IC50 of 500 nM whereas H3-CoA-20 inhibited PCAF and GCN5 with
an IC50 of 300 nM (Ontario D. Lau, 2000). When methyl group and phenyl group
were substituted in Lys-CoA, effective Lys-CoA analogs were obtained with a 4
folds lower IC50 than the parent structure (Sagar et al., 2004) . p300 HAT
inhibition studies reveal that bisubstrate analogs potentially inhibit the enzyme by
ping-pong mechanism (Sagar et al., 2004). Ping-pong mechanism is a
bisubstrate reaction in which the enzyme reacts with first substrate to form a
product and a modified enzyme. The modified enzyme will then react with a
second substrate to form a second product and regaining original enzyme form at
the end of the reaction. The idea of synthesizing H3-CoA-20 and Lys-CoA came
from a prior study on serotonin-N-acetyltransferase bisubstrate analogs that
effectively inhibited the HAT activity by placing an acetyl linker between amine
substrate and CoA (Sagar et al., 2004).
Figure 8: Chemical structures of Lys-CoA and H3-CoA-20 (Sagar et al., 2004).
28
The major drawback of these compounds is their poor cell permeability due to
the presence of negatively charged phosphate groups (Fig. 8). Linking of Tat
peptide sequence to H3-CoA-20 generated a potent cell permeable PCAF/GCN5
inhibitor. Cole and his group synthesized a series of Lys-CoA derivatives linked
with amino acid backbone. They found that these compounds were cell
permeable but the efficacy was reduced by 8 fold in comparison to the parent
compound. These compounds can be further used to test the function of p300,
PCAF and GCN5 in transcriptional regulation and also for therapeutic purposes
against neurodegenerative diseases, COPD, asthma and cancer (Zheng et al.,
2005). These compounds were the first synthesized and the most potent HAT
inhibitors designed so far.
Natural HAT inhibitors (and an activator)
Anacardic Acid
Anacardic acid (AA) is a small compound extracted from cashew nutshell and is
also known as 2-hydroxy-6-pentadicyclobenzoic acid (Fig. 9). AA is a potent
inhibitor of p300 with an IC50 of 8.5 µM and PCAF with an IC50 of 5 µM. Inhibition
kinetic studies reveal that AA is a non-competitive inhibitor of p300/PCAF HAT
activity (Fig. 9). AA could also inhibit the HAT Tip60 in vitro and blocked the Tip60
dependent activation of ATM which is required for cell survival upon ionizing
radiation. Thus
29
AA, by inhibiting Tip60 sensitizes the tumor cells to ionizing
radiation (Sun et al., 2006). AA is also shown to inhibit NF-Kβ which is activated
by carcinogens, and by inflammatory stimuli through the inhibition of IKK
activation, IKβα phosphorylation and p65 phosphorylation. By inhibiting NF-Kβ,
the genes that inhibit apoptosis, promote proliferation, angiogenesis and invasion
in cancer cells were also down-regulated. This study demonstrated that AA is a
potent anti-proliferative, anti-angiogenic, pro-apoptotic and anti-inflammatory
agent (Sung et al., 2008). To acquire specificity towards a particular HAT, the
acidic group on the phenolic ring in AA is modified into amide derivative. An
interesting compound CTPB was obtained by this modification of AA. In vitro
assay revealed that CTPB enhanced p300 HAT activity rather than inhibited it but
it did not enhance the PCAF HAT activity confirming its specificity towards p300.
This is the only HAT activator discovered so far (Balasubramanyam et al., 2003).
Figure 9: Structure of anacardic acid and CTPB (Balasubramanyam et al.,
2003).
Garcinol
30
Another natural compound that can inhibit HATs is a derivative from garcinia
indica commonly called as garcinol (polyisoprenylated benzophenone) (Fig.10).
Inhibition studies revealed that garcinol is a potential inhibitor of PCAF with an
IC50 of 5 µM and p300 with an IC50 of 7 µM. Kinetic studies indicated that garcinol
competes with histone substrate for the active site of p300/PCAF, thus acting as
a competitive inhibitor (Balasubramanyam et al., 2004a).
Due to the high toxicity of garcinol, efforts are made to develop specific and
non-toxic small molecule inhibitors of p300. A highly specific and non-toxic
compound, LTK-14 was derived when 14’ position of parent garcinol was
monosubstituted by methoxy group. LTK-15, another garcinol derivative, was
obtained when substituted with acetoxy group at 13 and 14 positions. These
compounds were highly specific to p300 with an IC50 of 6 µM (Mantelingu et al.,
2007). Structural studies reveal that molecular cyclization of garcinol to LTK-14
induced specificity towards p300 and reduced the PCAFs ability to interact with
LTK-14. Molecular docking studies indicate that LTK-14 was unable to make any
hydrogen bonds with PCAF’s HAT domain while garcinol made two hydrogen
bonds with PCAF HAT domain. Aromatic rings in garcinol and LTK-14 are
important for the recognition of acetyl CoA binding site while the non-aromatic
side groups are required for competitive inhibition of the histone substrate
(Fig.10) (M. Arif, 2009).
31
LTK-14 is also able to stop the formation of viral syncitia and
subsequently the multiplication of HIV. From microarray analysis of garcinol
treated HeLa cells, it is observed that 72% of the tested genes are
downregulated and 28% of genes are upregulated. Some of the downregulated
genes
include
antiapoptotic
proteins
such
as
Bcl-2,
Fas ligand and
protooncogenes. Upregulated genes include proapoptotic genes such as
caspase 4 and CED-6. The above data clearly show a therapeutic potential of
garcinol as a strong anti-cancer agent that possibly could be used as a
therapeutic agent in other diseases as well (Mantelingu et al., 2007).
Figure 10: Chemical structures of garcinol and its derivatives LTK-14 and LTK-15
(Balasubramanyam et al., 2004a).
Curcumin
Curcumin is a natural polyphenolic compound extracted from the rhizome
curcuma longa (Fig. 11). Curcumin is a specific and potent inhibitor of p300 with
an IC50 of 25 µM. It has good cell permeability and anticancer properties,
inhibiting cell proliferation and inducing apoptosis in HeLa cells. Inhibition kinetic
32
studies showed that curcumin doesn’t compete with histone substrate or Co-A
and
thus
acts
as
a
non-competitive
and
non-reversible
inhibitor
(Balasubramanyam et al., 2004b). It inhibits p300/CBP through a covalent bond
with the help of two Michael acceptor motifs in their structure (Manzo et al.,
2009). Curcumin was also shown to stop the multiplication of HIV virus by
inhibiting the acetylation of HIV-Tat protein which is required for its multiplication
(Balasubramanyam et al., 2004b). Curcumin is reported to prevent cardiac
hypertrophy in rats by inhibition of p300 in cardiomyocytes leading to the
repression of hypertrophic response genes such as MEF-2 and GATA4. The
repression of these genes happens by the inhibition of p300’s to the mediator
complex and thus disruption of the p300/GATA4 complex (Tatsuya Morimoto,
2008). Curcumin also has a potential anti-inflammatory effect. It is reported to
reduce the reactive oxygen species (ROS) which in turn inhibits the activation of
a proinflammatory gene NF-Kβ (Manzo et al., 2009).
Curcumin is a well-studied HAT inhibitor and is already tested in clinical trials
against several diseases such as AD, Leukemia, mental disorders and
hematological disorders. Out of a total of 31 clinical trials reported for curcumin,
ten of the trials have already been completed. An interesting fact is that Indians
have a low percentage of Alzheimer’s disease is probably due to their extensive
use of curcumin in their curries (Manzo et al., 2009). Since some HAT inhibitors
cause cell cycle arrest and apoptosis (Balasubramanyam et al., 2004b,
33
Balasubramanyam et al., 2004a) , in the next section, these processes will be
shortly described.
Figure 11: Chemical structure of curcumin with two phenolic rings joined by a
linker (Balasubramanyam et al., 2004b).
CELL CYCLE REGULATION AND APOPTOSIS
The regulation of cell cycle and apoptosis is vital for the cell survival and
proliferation. Abnormalities or dysregulation in these processes can result in
mutations, leading to cell death or cancer. Events such as cell differentiation,
division, proliferation and survival are all governed by the balance between a
number of regulatory proteins in the cell.
The eukaryotic cell cycle is divided into four stages – G1, S, G2 and M phase.
G1 phase, also known as gap 1 phase, is the stage in which the cell prepares for
the process of DNA replication. S-phase is the synthesis phase where the DNA is
duplicated. G2 is the gap 2 phase during which the cell prepares for the process
of cell division. M phase also known as mitotic phase is the stage during which
the two sister chromatids segregate and divide to form two daughter cells. Apart
from these stages, G0 is the phase during which the cell exits from the cell cycle
34
and become quiescent (Karp, 2005). Cell cycle is controlled by a group of
kinases called as CDKs (Cyclin dependent kinases). CDKs are activated when
associated with their regulatory subunits, called as cyclins. Cyclin-CDK
complexes phosphorylate various substrates that are involved in cell cycle
progression. Depending on the type of cyclin forming a complex with CDK, its
function varies in the cell cycle. The degradation of cyclins and other cell cycle
regulators are targeted during the cell cycle progression (Lodish and Matthew,
2008).
For decades, thousands of small molecule inhibitors have been discovered
that target the cell cycle regulators such as CDKs, aurora kinases etc, induced
cell cycle arrest either in G1, S, or G2/M, stopping the progression of cancer cell
cycle. This in turn triggers the activation of apoptotic process in the malignant cell
lines.
Cell death is divided into two distinct processes – apoptosis and necrosis.
Necrosis is a form of cell death during which the cell explodes and releases
toxins that injures or damages the neighboring cells causing inflammation.
Apoptosis is a form of programmed cell death which is initiated through several
pathways in response to various environmental, physiological and toxicological
stimuli (Lodish and Matthew, 2008).
The apoptotic process can be triggered by death signals received at the cell
membrane such as Fas ligand, TRAIL, TNFα etc, (Schulze-Osthoff et al., 1998).
35
This triggers caspase-8 to initiate the pathway inside the cell that leads to
execution of DNA fragmentation and other apoptotic events (VG, 1993).
Caspases are a family of cysteine aspartic proteases that plays a key role in cell
death. Caspase-8 can either directly activate the caspase-3 to execute the DNA
fragmentation or it can activate pro-apoptotic proteins such as bid, bax, bim and
inactivate the antiapoptotic proteins such as bcl-2 that causes the loss of
mitochondrial transmembrane potential (Micheau and Tschopp, 2003). This loss
triggers the release of cytochrome c from the mitochondria to the cytoplasm.
Cytochrome c, when released from the cytoplasm causes the oligomerization
and binding of Apaf-1 to caspase-9 resulting in the formation of apoptosome
complex (Lodish and Matthew, 2008). This complex in turn gets activated and
causes the DNA fragmentation via DFF45 (DNA fragmentation factor 45) or
PARP endonuclease (Kitazumi et al., 2010). Caspase independent mechanism of
DNA fragmentation occurs mainly through the cleavage of mitochondrial proteins,
Apoptosis Inducing Factor (AIF) and Endonuclease G. These cleaved proteins
translocate to the nucleus and cause high molecular weight DNA fragmentation
and intra-nucleosomal breaks (Otera et al., 2005, Lily Y. Li, 2001)
Since HAT inhibitors are becoming very seeked after compounds to be used
in several diseases, this research goal was to explore the potential
cell-proliferation inhibitory effect of three novel groups of amidoximes. These
compounds have not just an amidoximes group which potentially cant inhibit
36
HDACs but also one-three aromatic carbon rings that have a potential HAT
inhibitory effect. As a part of our study we also wanted to test if these
amidoximes will induce apoptosis of malignant cell lines and how they will affect
the cell cycle. The mechanism of action of these amidoximes will be explored as
well.
Figure 12: Chemical structures of amidoximes JJMB 1-9 used in this study.
37
CHAPTER II
METHODS AND MATERIALS
Chemicals
Amidoximes used in this research were synthesized by Dr. James Johnson from
the Department of Chemistry and Biochemistry, TWU, Denton, TX, USA. Garcinol
(a p300 inhibitor) and Anacardic Acid (a GCN5 inhibitor) were purchased from
Enzo Life Sciences (Farmingdale, NY).
Cell culture
The cancer cell lines HCT-116 (colorectal carcinoma), DU-145 (prostate
carcinoma), SK-OV-3 (ovarian adenocarcinoma), HLF-a (lung epidermoid
carcinoma), MCF-7 and MDA-MB-231 (breast adenocarcinoma), and NHDF
38
(normal human dermal fibroblasts) were obtained from ATCC (Manassas, VA,
USA). All the cell lines but HCT-116 and NHDF were maintained in Dulbecco’s
Modified Eagle’s Medium (Gibco/BRL, Carlsbad, CA, USA) and supplemented
with 10% fetal bovine serum (Benchmark, Waukegan, IL, USA) and 1%
penicillin-streptomycin (Gibco/BRL, Carlsbad, CA, USA). HCT-116 and NHDF
cells were maintained in McCoy’s 5A Medium and fibroblast basal medium
respectively (ATCC, Manassas, VA, USA). All cells were grown in an incubator at
370C, 100% humidity and 5% CO2. Cells in log phase were taken and plated
22x103 cells (DU-145), 25x103 cells/well (HCT-116), 20x103 cells/well (MCF-7), 16
x103 cells/well (MDA-MB-231), 31x103 cells/well (SK-OV-3), 30x103 cells/well
(HLF-a) and 16x103 cells/well (NDHF) in 96-well tissue culture plates (Corning,
Lowell, MA, USA) and were grown for 24 hours.
Treatment of malignant cells with amidoximes
Twenty four hours after plating in 96 well plates, the cells were treated with
amidoximes in DMSO at various concentrations ranging from 2 µM to 500 µM.
Control cells were treated with DMSO alone. The amount of DMSO used for
controls was 0.5%__ the highest DMSO concentration used for the treatment
groups.
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-te
trazolium) viability assay was performed after the cells were incubated with
amidoximes for 48 hours (72 hours for MCF-7).
39
MTS assay
The MTS assay is a colorimetric based cytotoxicity assay which determines the
number of viable cells based on the mitochondrial dehydrogenase activity. Cell
viability was tested after 48 hour incubation with the amidoximes. The medium in
each well was replaced with 100 µl of fresh medium and 20 µl of reagent solution
(Promega, Madision, WI, USA) containing MTS and an electron coupling reagent
PMS (phenazine methosulfate). After 4 hours of incubation, the cell viability was
determined by measuring the absorbance at 490 nm using a synergy HT 96-well
plate reader (Bio-Tek Instruments Inc, Winooski, VT, USA) (www.promega.com).
The assays were performed in triplicates and absorbance values were corrected
by subtracting the background absorbance (Medium and MTS reagent without
cells). The GI50 of the amidoximes were measured (GI50 is defined as the
concentration of the compound that causes 50% of cell death). All the
experiments were repeated three times and average as well as standard
deviation was calculated.
Cell cycle analysis
HCT-116 cells were plated in 60 mm2 tissue culture dish at 1x106 cells/dish and
let to grow for 24 hours. Medium was removed and replaced with medium
containing amidoximes for 24 hours. Cells were harvested by trypsinization,
pelleted by centrifugation and then fixed with 70% ethanol and incubated
overnight at 200C. Cells were centrifuged and washed twice with PBS (Cellgro,
40
Manassas, VA, USA) to remove residual ethanol. Washed cells were incubated
with propidium iodide (PI) (40 μg/μl) /RNase (100 μg/μl) solution (BD Biosciences
San Diego, CA, USA) for 20 minutes and passed through nylon mesh to remove
clumped cell. The cells were subjected to flow cytometric analysis (BD FACS
Calibur) to measure DNA content by PI incorporation. This analysis gives a
detailed DNA distribution curve of various cell cycle stages along with apoptotic
cells (sub-G1) located to the left of the G1 phase (Yan et al., 2012). The
distribution of cells in the sub-G1, G1, S, and G2/M phases of the cell cycle was
determined using Flowing software (University of Turku, Turku, Finland).
DNA fragmentation assay
This assay serves as an indicator for apoptosis, and was performed to probe the
extent of chromatin fragmentation after treating the colon cancer cells (HCT-116)
with amidoximes. HCT-116 cells (3x106) were plated in 100 mm2 cell culture petri
dish and allowed to grow at 370C for 24 hours. The growth medium was replaced
with fresh medium containing amidoximes at concentrations approximately equal
to the GI50 values. After 24 hours, cells were centrifuged and the pellet was lysed
by incubating in digestion buffer (100 mM NaCl, 10 mM Tris-Cl, 25 mM EDTA,
0.5% SDS and 0.2 mg/ml proteinase K) overnight at 500C. The lysate was
treated with RNase (50 µg/ml) at 370C for 1 hour. The lysate was then
centrifuged (Beckman Coulter microfuge 22R centrifuge) at 14,000 rpm for 30
minutes to separate the fragmented DNA and genomic DNA. The supernatant
41
was collected and an equal amount of solution containing phenol: chloroform:
isoamyl alcohol (25:24:1) (Sigma, MO, USA) was added to the tube. The
aqueous layer (upper layer) was transferred into a fresh tube and an equal
volume of ethanol was added to precipitate the DNA. The sample was then
centrifuged at 4000 rpm for 2 minutes and supernatent was discarded. The pellet
is washed twice with 70% ethanol by centrifuging at 4000 rpm for 2 minutes. The
supernatant was discarded and the pellet was dried by inverting the tube. The
pellet was then suspended by adding 40 µl of water. The DNA was quantified and
20 µg of DNA was loaded and run on 1.5% agarose gel electrophoresis (Jänicke
et al., 1998). The gels were photographed using Alpha Innotech Imager (Protein
Simple, Santa Clara, CA, USA).
In vitro HAT Assay
An in vitro HAT assay was performed to test if amidoximes are able to inhibit the
recombinant HAT p300 or the recombinant HAT GCN5 in vitro. Two µg of core
histones extracted from chicken erythrocytes (Millipore, Billerica, MA, USA) were
incubated in HAT assay buffer containing TBE (0.44 M Tris-base, 0.44 M Boric
acid, 0.5 M EDTA pH 8) and 72 µM Acetyl-CoA (Sigma Aldrich, MO, USA) in the
presence and absence of amidoximes at various concentrations. Fourty ng of
human recombinant full length p300 (Calbiochem, NJ, USA) or 50 ng of mouse
recombinant full length GCN5 (purified from a plasmid, gift of Dr. Sharon Dent,
M.D. Anderson) was added to each tube except for histones alone control. The
42
final reaction volume was 30 µl. Reactions was done at 370C for 45 minutes and
stopped by adding 2X SDS dye and heating to 950C for 15 minutes
(Balasubramanyam et al., 2004a). The samples were run on 15% SDS-PAGE
(sodium dodecyl-sulfate polyacrylamide gel electrophoresis) and western blots
were performed with antibodies against Ac-H4K5 and Ac-H3K9 for p300 HAT
assay and Ac-H3K14 for GCN5 HAT assay. These antibodies were directed
against the histone lysines reported to be the respective targets of p300 and
GCN5 (Grant et al., 1999).
Western blot analysis
After incubating the colon cancer cells (HCT-116) with amidoximes for 24 hours,
the whole cell lysate was collected by using 1X SDS buffer (100 mM Tris-Cl, 4%
SDS, 100 mM DTT, 20% glycerol and mini complete protease inhibitor tablet
(Roche, Branford, CT, USA). The extracted lysate was boiled at 950C for 20
minutes to denature DNA. The lysate was centrifuged at 14,000 rpm for 30 sec
and the protein concentration was measured by using a detergent and reducing
agent compatible – 660 nm Protein Assay kit (Pierce, IL, USA). The cell extracts
were resolved on 15% SDS-PAGE to separate proteins based on molecular
weight. The equal loading was confirmed after Coomassie staining by
densitometric analysis, using Alpha Innotech Imager and Fluorchem HD2
software. Once the protein loading was confirmed by SDS-PAGE, the proteins
were transferred onto PVDF membrane (Millipore, MA, USA) at 28 mA for 2
43
hours using a trans-blot semi-dry transfer machine (Bio-rad, CA, USA). The
membrane was blocked for 45 minutes in blocking buffer, PBS-Tween (1X PBS
with 0.1% tween 20) containing 5% non-fat dry milk (Nestle carnation, Solon,
OH). After rinsing with 1X PBS, the membrane was incubated overnight at 4oC
with one of the following primary antibodies directed against: acetylated H4K5 (r)
(0.08 µg/ml), acetylated H3K9 (1 µg/ml), acetylated H3K14 (r) (0.4 µg/ml),
phosphorylated H3S10 (r) (0.4 µg/ml) or caspase-3 (r) (0.1 µg/ml). The
membrane was then washed with PBS-Tween and incubated with horseradish
peroxidase conjugated secondary antibody (Goat anti-Rabbit) targeted against
the primary antibody obtained from rabbit. The membrane was washed with
PBS-Tween and exposed to ECL plus (Amersham, NJ, USA) according to
manufacturer’s instructions. The membrane was then exposed to X-ray film
(Amersham, NJ, USA) and scanned using Alpha Innotech Imager and Fluorchem
HD2 software.
Statistical Analysis
Values from triplicate experiments was presented as means ± SD/SE. One-way
analysis of variance (ANOVA) was performed to assess the differences between
groups using SPSS (IBM corporation, Armonk, NY). Statistical significance
between control groups and treated groups were analyzed by post hoc Dunnett’s
44
test. For in vivo acetylation studies, non-parametric tests such as Kruskal Wallis
and Man-Whitney U tests were also performed. Levels of significance were
designated as P < 0.001, P < 0.01 or P < 0.05 depending on the experiment.
CHAPTER III
RESULTS
Amidoximes induced death in human cancer cell lines
45
The anti-proliferative effect of nine novel amidoximes was examined on the
following malignant cell lines: HCT-116 (colorectal carcinoma), DU-145 (prostate
carcinoma), SK-OV-3 (ovarian adenocarcinoma), HLF-a (lung epidermoid
carcinoma), MCF-7 and MDA-MB-231 (breast adenocarcinoma) and NHDF
(normal human dermal fibroblasts). The cells were incubated for 48 hours (72
hours for MCF-7) with amidoximes at concentrations ranging from 0.2 µM to 500
µM and the cell viability was measured using an MTS assay. Garcinol, a
polyisoprenylated benzophenone, is a natural compound that reduces core
histone acetylation by inhibiting p300 and PCAF at micromolar concentrations. It
was used as a positive control for the experiments (Balasubramanyam et al.,
2004a). The GI50 of the amidoximes were measured (GI50 is defined as the
concentration of the compound where it causes 50% of cell death) (Table 5). As
expected, the positive control, garcinol, significantly inhibits 20-60% growth in all
the malignant cell lines tested but not in normal cells (Table 5 and Fig 18).
Amidoximes JJMB 1, 2, 3 and 8 did not induce cell death in any of the malignant
cell lines tested (Table 5). In contrast, JJMB 4 significantly killed all of the
malignant cells tested except for MDA-MB-231 (Breast), but also induced death
in normal cells (Table 5 and Fig. 13). JJMB 5 significantly induced cell death in
HCT-116 (Colon) (GI50 of 36 µM ± 5) and MCF-7 (Breast) (GI50 of 90 µM ± 4) cells
at low micromolar concentrations but HLF-A (Lung) (GI50 of 335 µM ± 4) and
MDA-MB231 (GI50 of 125 µM ± 4) cells required higher micromolar concentrations
46
for cell death (Table 5 and Fig. 14). JJMB 6 significantly and specifically induced
cell death in HCT-116 cells (Colon) (GI50 of 17 µM ± 4) but none of the other cell
lines tested were sensitive to JJMB 6 (Table 5 and Fig. 15). JJMB 7 significantly
induced death in HCT-116 cells (Colon) (GI50 of 36 µM ± 2) and MDA-MB-231
cells (Breast)(GI50 of 85 µM ± 3) at low micromolar concentrations but HLF-A
cells (Lung) (GI50 of 275 µM ± 3) were killed at higher micromolar concentration
(Table 5 and Fig. 16). JJMB 9 significantly induced death in HCT-116 (Colon)
(GI50 of 29 µM ± 5), HLF-A (Lung) (GI50 of 40 µM ± 7) and MCF-7 (Breast) (GI50 of
30 µM ± 5) cells, all of them at low micromolar concentrations (Table 5 and Fig.
17). MDA-MB-231 cells (Breast) were sensitive to JJMB 5 and JJMB 7 but
MCF-7 cells (Breast) were only sensitive to JJMB 5 and JJMB 9 (Table 5). None
of the amidoximes were toxic to the normal cells except for JJMB 4 (GI50 of 73 µM
± 2) (Table 5). HCT-116 showed sensitivity to all the amidoximes except JJMB 1,
2, 3 and 8 (Table 5). These results suggest that different amidoximes have
different specificities towards inhibiting the malignant cell lines. Since HCT-116
cells (Colon) were sensitive to the low levels of amidoximes that induced death in
one or more malignant cell lines, we used this cell line for further studies.
Cancer cells are formed due to mutations in the genome caused by carcinogens
such as environmental toxins, UV radiation etc (Steller, 1995). The mutations
47
accumulate in the cell until they result in the formation of fully fledged malignant
cell. These cells are usually resistant to cell cycle arrest and apoptosis
(Thompson, 1995). Treating the cancerous cells with amidoximes might trigger
cell cycle arrest and apoptosis.
48
49
50
Figure 13: Survival rate of malignant cell lines treated with JJMB 4. Growth
levels of six malignant cell lines and NHDF cells demonstrating a
concentration-dependent killing of cells by amidoxime JJMB 4. Cells were
cultured and incubated with JJMB 4 for 48 hours (72 hours for MCF-7) before cell
viability was measured by MTS assay as described in materials and methods.
Garcinol, a known HAT inhibitor was used as a positive control. One-way ANOVA
was performed to assess the differences between groups. Differences in means
between control and JJMB 4 treated groups were analyzed by post-hoc
Dunnett’s test,*p < 0.05. Values are mean ± SD, n = 3.
51
Figure 14: Survival rate of malignant cell lines treated with JJMB 5. Growth
levels of six malignant cell lines and NHDF cells demonstrating a
concentration-dependent killing of cells by amidoxime JJMB 5. Cells were
cultured and incubated with JJMB 5 for 48 hours (72 hours for MCF-7) before cell
viability was measured by MTS assay as described in materials and methods.
Garcinol, a known HAT inhibitor was used as a positive control. One-way ANOVA
was performed to assess the differences between groups. Differences in means
between control and JJMB 5 treated groups were analyzed by post-hoc
Dunnett’s test,*p < 0.05. Values are mean ± SD, n = 3.
52
53
Figure 15: Survival rate of malignant cell lines treated with JJMB 6. Growth
levels of six malignant cell lines and NHDF cells demonstrating a
concentration-dependent killing of cells by amidoxime JJMB 6. Cells were
cultured and incubated with JJMB 6 for 48 hours (72 hours for MCF-7) before cell
viability was measured by MTS assay as described in materials and methods.
Garcinol, a known HAT inhibitor was used as a positive control. One-way ANOVA
was performed to assess the differences between the groups. Differences in
means between DMSO treated and JJMB 6 treated groups were analyzed by
post-hoc Dunnett’s test,*p < 0.05. Values are mean ± SD, n = 3.
54
55
Figure 16: Survival rate of malignant cell lines treated with JJMB 7. Growth
levels of six malignant cell lines and NHDF cells demonstrating a
concentration-dependent killing of cells by amidoxime JJMB 7. Cells were
cultured and incubated with JJMB 7 for 48 hours (72 hours for MCF-7) before cell
viability was measured by MTS assay as described in materials and methods.
Garcinol, a known HAT inhibitor was used as a positive control. One-way ANOVA
was performed to assess the differences between groups. Differences in means
between control and JJMB 7 treated groups were analyzed by post-hoc
Dunnett’s test,*p < 0.05. Values are mean ± SD, n = 3.
56
Figure 17: Survival rate of malignant cell lines treated with JJMB 9. Growth
levels of six malignant cell lines and NHDF cells demonstrating a
concentration-dependent killing of cells by amidoxime JJMB 9. Cells were
cultured and incubated with JJMB 9 for 48 hours (72 hours for MCF-7) before cell
viability was measured by MTS assay as described in materials and methods.
Garcinol, a known HAT inhibitor was used as a positive control. One-way ANOVA
was performed to assess the differences between groups. Differences in means
between control and JJMB 9 treated groups were analyzed by post-hoc
Dunnett’s test,*p < 0.05. Values are mean ± SD, n = 3.
57
Figure 18: Survival rate of malignant cell lines treated with garcinol. Growth
levels of six malignant cell lines and NHDF cells demonstrating a
58
concentration-dependent killing of cells by garcinol. Cells were cultured and
incubated with garcinol for 48 hours (72 hours for MCF-7) before cell viability was
measured by MTS assay as described in materials and methods. Garcinol, a
known HAT inhibitor was used as a positive control. One-way ANOVA was
performed to assess the differences between groups. Differences in means
between control and garcinol treated groups were analyzed by post-hoc
Dunnett’s test,*p < 0.05. Values are mean ± SD, n = 3.
Amidoximes induced apoptosis in HCT-116 cells as determined by DNA
fragmentation assay
Apoptosis is marked by several morphological and biochemical changes inside
the cell such as blebbing, formation of apoptotic bodies, caspase signaling etc.
Inter-nucleosomal DNA fragmentation is one of the characteristic markers of the
apoptotic cell death (Fig. 19). For this study, DNA fragmentation assay was used
to determine if the amidoximes induce apoptosis. HCT-116 (colon) cell line was
selected for further experiments because of its sensitivity to all the amidoximes.
HCT-116 cells were treated with amidoximes at concentrations approximately
equal to GI50 values. DNA was extracted after 24 hours and the fragmentation of
DNA was confirmed by agarose gel electrophoresis. The fragments generated
were in a typical oligonucleosomal sizes in treated cells. The vehicle treated cells
did not show DNA fragmentation (Fig. 20). Garcinol, which is a positive control
59
also showed fragmentation of DNA (Fig. 20, lane 7). JJMB 6 revealed intense
fragmentation of DNA as compared to amidoximes JJMB 5, 7 and 9 (Fig. 20 and
lane 4). The intensity of DNA fragmentation induced by JJMB 9 is less reduced
compared to JJMB 5, 6 and 7 (Fig. 20 and lane 6). These results indicate that the
studied amidoximes induced apoptosis in HCT-116 cells.
Figure 19: Model showing the pathways leading to DNA fragmentation through
caspase dependent and caspase independent pathways. DNA fragmentation
occurs either by extrinsic pathway or intrinsic pathway. Extrinsic pathway is
60
triggered by the binding of death ligands of TRAIL (TNF-related apoptosis
inducing ligand) or Fas ligand to the death receptors (DRs) in two ways. In the
first type, caspase-8 activates the caspase-3 without the involvement of
mitochondria. In type II, caspase-8 activates bid (BH3-interacting domain)
resulting in the suppression of anti-apoptotic factors such as bax (Bcl-2
associated X-protein) and bad (Bcl-2-associated death promoter). This causes
the release of Cyt C (Cytochrome C) from mitochondria. Cyt c activates the
caspase-9 which in turn activates caspase-3. Activated caspase-3 translocates
into the nucleus and causes the DNA fragmentation either through DFF40 (DNA
fragmentation factor 40) or PARP (poly ADP ribose polymerase). Caspase
independent apoptotic pathway occurs through the release of mitochondrial
proteins AIF (Apoptosis inducing factor) or Endo G (Endonuclease G) into the
nucleus causing DNA fragmentation (Based on the review from Kitazumi and
Tsukahara, 2011).
61
Figure 20: Amidoximes induced apoptosis in HCT-116 cells as determined by
DNA fragmentation. DNA fragmentation assay was performed on colon cancer
cells (HCT-116) following treatment with JJMB 5, 6, 7 and 9 for 24 hours at
various concentrations. DNA was extracted and analyzed on 1.5% agarose gel.
The DNA appeared to be fragmented upon treatment with amidoximes but not in
DMSO (vehicle) treated cells. Garcinol was used as a positive control.
Amidoximes induced apoptosis is caspase-3 dependent
Apoptotic DNA fragmentation in cells can occur either by caspase dependent
mechanisms or caspase independent mechanisms. Caspase independent
mechanism of DNA fragmentation occurs mainly through the cleavage of
mitochondrial proteins, Apoptosis Inducing Factor (AIF) and Endonuclease G
(Fig. 19). These cleaved proteins translocate to the nucleus and cause high
molecular weight DNA fragmentation and intra-nucleosomal breaks (Lily Y. Li,
2001, Otera et al., 2005). Previous studies have shown that caspase-3 is an
essential protease in the caspase signaling cascade towards apoptosis and its
activation is required for the fragmentation of DNA during apoptosis (Jänicke et
al., 1998). Caspase-3 is the main executioner caspase, and induction of
apoptosis in the cells causes the cleavage of procaspase-3 (32 KDa) into active
62
form which is detected at 17/19 KDa (Slee et al., 2001). The active form
translocates into the nucleus and activates either DFF40 or PARP causing
fragmentation
of
DNA
(Chen
et
al.,
2000).
To
determine
if
the
amidoximes-induced apoptosis by DNA fragmentation is caspase-3 dependent,
Western blot analysis was performed against procaspase-3 after a 24 hour
treatment with amidoximes. Western blot analysis revealed an increase in the
active caspase-3 with concomitant decrease in the expression of procaspase-3 in
cells treated with higher concentrations of amidoximes (Fig. 21). Caspase-3
assay also revealed more effective activation of procaspase-3 by JJMB 6
compared to amidoximes JJMB 5, 7 and 9, which is consistent with the DNA
fragmentation assay (Fig. 20). This finding is not surprising in light of the
relatively highest potency of JJMB 6 in killing the HCT-116 cells with the lowest
GI50 of 17 µM (Table 5). Garcinol, which is used as a positive control also caused
the activation of caspase-3 at 25 µM and 50 µM. (Fig. 21.e).
63
Figure 21: Amidoximes-induced apoptosis is caspase-3 dependent. The figure
shows the effect of JJMB 5, 6, 7 and 9 on caspase-3 cleavage following
treatment for 24 hours with concentrations ranging from 8 μM to 45 μM. Colon
cancer cells (HCT-116) were incubated with the amidoximes for 24 hours and
then lysed. Coomassie staining of the SDS-PAGE was done to demonstrate
equal loading of the protein lysates. Western blot analysis was performed by
using antibody against procaspase-3. This antibody also recognizes cleaved
caspase-3. Garcinol was used as a positive control. All the amidoximes caused a
significant reduction in procaspase-3 levels seen at lanes with higher
concentrations of amidoximes and this was correlated with an increase in the
levels of cleaved caspase-3 (17/19 KDa).
Flow cytometric analysis of sub-G1 apoptotic population in HCT-116 cells
treated with amidoximes
During the process of apoptosis, following DNA fragmentation, a portion of the
DNA is lost and these cells with reduced DNA content appear as a sub-G1
population on the DNA distribution curve located to the left of the G1 phase in a
flow cytometry analysis. We evaluated the level of apoptosis induced by
amidoximes in concentrations close to the GI50 values in HCT-116 (Colon) cells
using FACS analysis of PI stained fixed cells (Fig. 22, 23). The DMSO treated
cells showed 5.95% of sub-G1 population (Fig. 22.a). Following the 24 hour
treatment with amidoximes, all amidoximes caused significant increase in the
levels of sub-G1 apoptotic populations. JJMB 6 caused the highest percentage of
64
sub-G1 population (33.65 ± 2.1%, P < 0.001) (Fig. 22.c) compared to JJMB 5
(17.32 ± 1.97%, P < 0.01) (Fig. 22.b), JJMB 7 (23.41 ± 3.42%, P < 0.001)
(Fig.22, d) and JJMB 9 (16.31 ± 1.43%, P < 0.01) (Fig. 22.e). The percentage of
apoptosis induced by JJMB 6 at 20 µM (33.65 ± 2.1%, P < 0.001) is more than
the percentage of apoptosis induced by garcinol at 40 µM (31.4 ± 5.06%, P <
0.001) (Fig. 22.c & 22.f). The increase in sub-G1 population confirming the loss
of DNA after 24 hour treatment demonstrated that the apoptosis induced by
amidoximes resulted in DNA fragmentation.
65
Figure 22: The effect of amidoximes on the sub-G1 population of HCT-116 cells.
Flow cytometry distribution curves of DNA content showing the sub-G1 apoptotic
population in HCT-116 cells treated for 24 hours with a) DMSO b) JJMB 5 (30
µM) c) JJMB 6 (20 µM) d) JJMB 7 (30 µM) e) JJMB 9 (20 µM) and f) Garcinol (40
µM). FACS analysis was done on 10,000 cells per trial and the experiment was
repeated three times.
66
Figure 23: The effect of amidoximes on sub-G1 population of HCT-116 cells.
This graph represents three repetitions of the experiment shown in figure 22. The
graph demonstrates that the percentage of the cells in the sub-G1 phase was
increased significantly in response to the treatment with amidoximes when
compared to DMSO treated cells. Values are mean ± SD, n=3. FACS analysis
was done on 10,000 cells per trial and the experiment was repeated three times.
Differences between treated and control groups were analyzed by one-way
analysis of variance and significance was determined by Dunnett’s post hoc test
(***P < 0.001, **P < 0.01).
Flow cytometric analysis of sub-G1 apoptotic population in DU-145 cells
treated with amidoxime JJMB 9.
From the MTS cell viability assay, we established that JJMB 9 did not induce
death in DU-145 cells (prostate carcinoma) (see Table 5). Our hypothesis is that
the amidoximes that did not induce death in cancer cells will not induce apoptosis
in cancer cells. To determine if JJMB 9 induced apoptosis in DU-145 (prostate
carcinoma) cells 24 hours after treatment, we performed FACS analysis of PI
stained fixed cells. The results indicate that there was no significant increase in
67
the percentage of cells in sub-G1 apoptotic population in response to JJMB 9 at
20 µM in comparision to the DMSO treated cells (Fig. 24a and 24b). These
results reveal that the apoptosis is not induced by the compounds that did not
induce death in cancer cells.
Figure 24: The effect of amidoxime, JJMB 9 on the sub-G1 population of DU-145
cells. a) Flow cytometry distribution curves of DNA content showing the sub-G1
apoptotic population in HCT-116 cells treated with i) DMSO ii) JJMB 9 (20 µM).
b) No significant difference in the percentage of the cells in the sub-G1 phase
was observed in response to the treatment with JJMB 9 when compared to
DMSO treated cells. Values are mean ± SD, n=3. FACS analysis was done on
10,000 cells per trial and the experiment was repeated three times. Differences
between treated and control groups were analyzed by one-way analysis of
variance and significance was determined by Dunnett’s post hoc test (p < 0.01).
68
Amidoximes induce cell cycle arrest in HCT-116 cells (colon carcinoma)
Cancer cells usually exhibit cell cycle abnormalities and causes irregular
proliferation of cells (Preston-Martin et al., 1990). Anti-cancer drugs are known to
cause not only apoptosis but also cell cycle arrest either in the G1, S or G2/M
phases (Malumbres and Barbacid, 2009). To test whether the amidoximes induce
cell cycle arrest, HCT-116 cells were incubated with amidoximes for 24 hours at
approximately GI50 concentration and cell cycle distribution was measured by PI
staining. FACS analysis produce a typical DNA histogram with three populations,
in which the first peak represents the G1 phase, the middle trough represents the
S phase and second peak represents the G2/M phase (Fig. 25). Cell cycle
analysis revealed that the percentage of cells in G1 phase increased from 52.5 ±
1.45% (DMSO treated) to 58.2 ± 0.67% (20 µM JJMB 6, P<0.01), 59.39 ± 0.65%
(30 µM JJMB 7, P<0.01), 63.53 ± 1% (20 µM JJMB 9, P<0.01) and 61.56 ± 1.4%
(40 µM garcinol, P<0.01), respectively (Table 6, Fig. 26) . No significant increase
was observed between the DMSO treated (52.5 ± 1.45%) and 30 µM JJMB 5 (54
± 1.45%, P<0.01) in G1 phase. However, JJMB 5 caused an increase in the
percentage of G2/M cells from 26.71 ± 1.07% (DMSO treated) to 30.95 ± 0.43%
(30 µM JJMB 5, P<0.01). Conversely, the percentage of cells at the S phase
decreased from 20.58 ± 0.8% (DMSO) to 13.66 ± 0.25% (30 µM JJMB 5,
69
P<0.01), 9.89 ± 0.86% (20 µM JJMB 6, P<0.01), 14.77 ± 1.75% (30 µM JJMB 7,
P<0.01), 15.74 ± 1% (20 µM JJMB 9, P<0.01) and 16.37 ± 0.98 % (40 µM
garcinol, P<0.01), respectively (Table 6, Fig. 26). These results suggest that
JJMB 6, 7, 9 and garcinol induced cell cycle arrest at G1 phase whereas JJMB 5
induced an arrest at G2/M phase.
70
Figure 25: The effect of amidoximes on the cell cycle distribution of HCT-116
cells. Representative DNA distribution curves of HCT-116 cells following 24 hour
incubation with a) DMSO b) JJMB 5 (30 µM) c) JJMB 6 (20 µM) d) JJMB 7 (30
µM) e) JJMB 9 (20 µM) and f) Garcinol (40 µM). FACS analysis was done on
10,000 cells per trial and the experiment was repeated three times. For the cell
cycle analysis, we excluded the sub-G1 from the DNA distribution curve and the
100% population represents the cells in G1/S, S and G2/M phases.
Table 6: The effect of amidoximes on the cell cycle distribution of HCT-116 cells
following a 24 hour treatment. Values represent mean ± SD of three individual
experiments.
71
DMSO
JJMB 5 30 µM
G1%
52.5 ± 1.5
54.0 ± 0.7
S%
20.6 ± 0.8
***13.7 ± 0.3
G2/M%
26.7 ± 1.1
**30.1 ± 0.4
JJMB 6 20 µM
JJMB 7 30 µM
***58.2 ± 0.7
***59.4 ± 0.6
***10.0 ± 0.9
***14.8 ± 1.8
26.2 ± 0.6
25.2 ± 0.7
JJMB 9 20 µM
***63.5 ± 1.0
***15.7 ± 1.0
24.9 ± 0.2
Garcinol 40 µM
***61.8 ± 1.4
**16.4 ± 0.1
***20.3 ± 2.0
Figure 26: Amidoximes JJMB 5, 6, 7 & 9 induced cell cycle arrest in HCT-116
cells. The percentages of HCT-116 cells in G1, S and G2/M phases of the cell
cycle following 24 hour incubation with i) DMSO ii) JJMB 5 (30 µM) iii) JJMB 6
(20 µM) iv) JJMB 7 (30 µM) v) JJMB 9 (20 µM) and vi) Garcinol (40 µM). JJMB 5
arrested the cells at G2/M check point while JJMB 6, 7 & 9 arrested cells at G1/S
check point. Values are mean ± SD, n=3. FACS analysis was done on 10,000
cells per trial and the experiment was repeated three times. Differences between
treated and control groups were analyzed by one-way ANOVA and significance
was tested with Dunnett’s post hoc test (***P < 0.001, **P < 0.01).
Amidoximes with a demonstrated anti-proliferative effect reduced core
histone acetylation in colon carcinoma cells (HCT-116)
The amidoximes (JJMB 1-9) used in my research contain an amidoxime group,
characteristic of some HDAC inhibitors (Finnin et al., 1999). However, some of
these compounds have a six carbon ring connected by a linker, characteristic of
some HAT inhibitors (Balasubramanyam et al., 2004b, Balasubramanyam et al.,
2004a). To test if the amidoximes induced reduction of acetylation or increased
acetylation levels of core histones, we performed Western blot analysis against
acetylated H3K9 and acetylated H4K5 on the whole cell lysates after treating
HCT-116 cells with amidoximes for 24 hours at various concentrations. The
72
experiment was repeated three times. Histone H3 lysine 9 (H3K9) and histone
H4 lysine 5 (H4K5) are well studied and important sites involved mainly in
transcriptional regulation (Allard et al., 1999). Western blot analysis revealed that
amidoximes JJMB 5, 6, 7, 9 and garcinol showed a dose dependent inhibition of
core histone acetylation on H3K9 and H4K5 (Fig. 27). The levels of core histone
acetylation on H3K9 and H4K5 are completely inhibited at 40 µM by treatment
with JJMB 5 and JJMB 9 (Fig. 27, a and d). JJMB 6 inhibited the acetylation
levels on H3K9 and H4K5 seen at 30 µM and 45 µM (Fig. 27, b). The reduction in
the acetylation levels on H3K9 and H4K5 appeared at 40 µM with JJMB 7
treatment (Fig. 27, c). Garcinol showed inhibition of core histone acetylation on
both acetylation sites at 25 µM and 50 µM (Fig. 27, e). These results suggest
that, amidoximes that induced death of HCT-116 cells reduced the core histone
acetylation in vivo.
To determine if induction of death in cancer cells is correlated with reduced
core histone acetylation, this experiment was repeated with DU-145 cells
prostate carcinoma). By MTS cell viability assay, we demonstrated that DU-145
cells were not killed by JJMB 9 (see Table 5). Therefore, we treated DU-145 cells
for 24 hours with increasing doses of JJMB 9 and Western blot analysis was
performed to test the acetylation levels of core histones with antibodies against
Ac-H3K9 and Ac-H4K5. The results showed that JJMB 9 did not inhibit the levels
of acetylation on Ac-H3K9 and Ac-H4K5 in DU-145 cells (Fig. 27, f). These
73
results suggest that the inhibition of core histone acetylation occurred only in the
cells that were killed by amidoximes.
The reduced core histone acetylation in cells treated with amidoximes can be
explained mainly in three ways:
1)​ The amidoximes inhibit histone acetyltransferases (HATs) directly or indirectly.
2)​ The amidoximes activate histone deacetylases (HDACs) directly or indirectly.
3)​ The amidoximes increase the expression levels of HDAC genes or decreases
the expression levels of HAT genes in the cell.
74
Figure 27: Effect of JJMB 5, 6, 7, 9 and garcinol on cellular core histone
acetylation. The acetylation levels of core histones in HCT-116 cells were
measured following treatment for 24 hours at concentrations ranging from 0.04
μM to 80 μM. HCT-116 cells (Panels a–e) were incubated with the amidoximes
for 24 hours and then lysed. Western blot analysis was conducted by using
antibodies against Ac-H3K9 and Ac-H4K5. Coomassie staining of the SDS-PAGE
was done to demonstrate equal loading of the protein. a) Significant reduction in
acetylation levels is seen at a concentration of 20 μM and higher after JJMB 5
treatment. b) JJMB 6 treatment resulted in reduction of acetylation levels at 30
μM on H3K9 and H4K5. c) JJMB 7 treatment resulted in reduced acetylation
levels at 40 μM and higher concentrations. d) JJMB 9 treatment resulted in
complete inhibition of acetylation levels seen at 40 μM. e) Garcinol, used as a
positive control, also showed reduced core histone acetylation seen at 25 µM
and 50 µM. f) No change in acetylation levels was observed in DU-145 cells
treated with JJMB 9 Note: DMSO was used as a vehicle control and applied at
75
concentrations used at the highest amidoxime treatment, in percentage ranging
from 0.04% - 0.08%.
Figure 28: Percentage of standardized acetylation on H3K9 (28.a) and H4K5
(28.b) was calculated by normalizing the acetylation levels to the O.D of the core
histones obtained by densitometry of coomassie staining. This graph represents
the means of 3 individual experiments described in figure 27. One-way ANOVA
was performed to determine the differences between the groups. Pair-wise
comparison was done to find out the significance between control group and
treatment groups. These results were confirmed by Kruskal Wallis and
76
Mann-U-Whitney non-parametric tests. Levels of significance were designated as
*p<0.05. Data is reported as the mean ± SEM (n = 3).
Amidoximes reverse the TSA induced hyperacetylation
TSA (trichostatin A) is an HDAC inhibitor that inhibits class I and II HDACs but
not class III HDACs (Matsuyama et al., 2002). The inhibition of HDACs causes
the hyperacetylation of lysine residues on the core histone tails. In the previous
experiment, we demonstrated that amidoximes caused reduction in core histone
acetylation in HCT-116 cells (see Fig. 27, 28). The primary reason for the
reduced acetylation levels caused by amidoximes could be due to the inhibition
of HATs. Our hypothesis is that, cells treated with a combination of HDAC
inhibitor, TSA and amidoximes would reverse the TSA induced hyperacetylation.
(See
Fig.
29).
To
determine
if
amidoximes
can
reverse
the
TSA
induced-hyperacetylation, we treated cells with each amidoxime alone, TSA
alone and each amidoxime together with TSA for 24 hours and cell lysates were
extracted. Western blot analysis was performed using antibodies against
acetylated H3K9 and acetylated H4K5. DMSO treated cells (Lane 1, Fig. 30a 30e) showed basal acetylation of core histones, followed by a dramatic increase
in acetylation levels upon TSA treatment (Lane 2, Fig. 30a - 30e). The positive
controls, amidoxime alone (Lane 6, 30a -30d) and garcinol alone (Lane 5, Fig.
30e) showed inhibition of acetylation levels compared to DMSO treated cells
(Lane 1, Fig. 30a - 30e). The co-treatment of HCT-116 cells with the positive
control, garcinol at 25 µM and TSA resulted in the reduced acetylation acetylation
77
levels of core histones when compared to TSA alone treated cells (Lane 2, Fig.
30e). The co-treatment of HCT-116 cells with amidoximes JJMB 5 (20 µM and 40
µM) or JJMB 6 (30 µM and 45 µM) or JJMB 9 (20 µM and 40 µM) and TSA
showed reduced acetylation levels compared to TSA alone treated cells (Lane 2,
Fig. 30a - 30e). These results suggest that JJMB 5, 6, 9 and garcinol effectively
reversed the TSA induced hyperacetylation at higher concentrations, whereas
JJMB 7 did not reverse the TSA induced hyperacetylation (Fig. 30). Results also
support the possibility that amidoximes JJMB 5, 6 & 9 are HAT inhibitors but the
possibility of histone hypoacetylation could be an indirect effect as well. On the
other hand, JJMB 7 is most probably not a HAT inhibitor based on these results.
Figure 29: Inhibition of HDACs by TSA induces hyperacetylation of core
histones. Inhibition of HATs on the other hand causes hypoacetylation.
78
Figure 30: Amidoximes reverse the HDAC inhibitor, TSA induced
hyperacetylation. HCT-116 cells were treated with amidoximes alone (at the
indicated concentrations), TSA alone (0.3 µM) and amidoximes concomitantly
with TSA for 24 hours and cell lysates were extracted. Western blot analysis was
performed using the antibodies against Ac-H3K9 and Ac-H4K5. JJMB 5, 6 and 9
effectively reversed TSA indued hyperacetylation whereas JJMB 7 did not affect
the TSA induced hyperacetylation. Garcinol also opposed the TSA induced
hyperacetylation at 25 µM.
79
The effect of concomitant treatment with TSA and amidoximes on cell
survival
From the previous experiment, we learned that treatment of HCT-116 cells with
the combination of TSA and amidoximes reversed the TSA induced
hyperacetylation in most cases (see Fig. 30). Acetylation of core histones is
generally involved in activation of gene expression whereas deacetylation of core
histones is generally involved in gene repression. Our hypothesis was that
reversing the TSA effect by amidoximes would also block the cell death induced
by TSA or by the amidoximes. In other words, reversing the TSA effect by
amidoximes would bring the global acetylation and gene expression levels to
normal steady state levels and therefore the cells would survive. To determine if
the amidoximes can block the TSA induced cell death, we treated HCT-116 cells
for 24 hrs with amidoximes alone, TSA alone, and amidoximes in various
concentrations concurrently with TSA. MTS assay was performed to determine
cell viability. The results revealed that TSA alone and amidoximes alone
effectively induced cell death in HCT-116 cells when compared to DMSO treated
cells (Fig. 31). When we compared the cells treated with TSA alone and TSA
incubated along with amidoximes JJMB 5, 6, 7, 9 and garcinol, no significant
blocking of cell death was observed in any of the compounds tested except for
JJMB 5 at 8 µM with TSA. These results indicate that the reason for the cell
80
death is not simply a change in the global acetylation steady state of core
histones but something more complex which will be addressed in the discussion.
Figure 31: Reversing the TSA effect by amidoximes doesn’t block cell death.
HCT-116 cells were treated with amidoximes alone (at the indicated
concentrations), TSA alone (0.3 µM) or TSA incubated along with JJMB 5, 6, 7
and 9 at various concentrations and MTS cell viability assay was performed after
24 hours. MTS assay revealed that incubating amidoximes JJMB 5, 6, 7, 9 and
81
garcinol along with TSA in HCT-116 cells didn’t block the cell death except in
TSA and 8 µM of JJMB 5. One-way ANOVA was performed to assess the
differences between groups. Differences in means between TSA treated and
TSA along with amidoximes groups were analyzed by post-hoc Dunnett’s test,*p
< 0.05. Values are mean ± SD, n = 3.
JJMB 9 is a p300 inhibitor
The previous experiments have shown the inhibition of core histone acetylation in
HCT-116 cells when treated with amidoximes (see Fig. 27). This was also
supported by the fact that JJMB 5, 6, and 9 reversed the TSA induced
hyperacetylation suggesting that inhibition of core histone acetylation levels could
be due to the inhibition of histone acetyltransferases (HATs) (see Fig. 30). This
prompted us to examine if these amidoximes can inhibit the HATs in vitro. To test
this hypothesis, we selected p300, a HAT from p300/CBP family which is capable
of acetylating free and nucleosomal histones both in vitro and in vivo (Ogryzko et
al., 1996). The HAT inhibitory activity was assayed using full length human
recombinant p300, purified core histones from chicken erythrocytes and acetyl
coA as the substrates (Fig. 32). Western blot analysis was performed to
determine if the amidoximes inhibited the p300-dependent acetylation on H3K9
and H4K5. Lane 1 represents the basal acetylation levels of H3K9 and H4K5.
Increase in the acetylation levels was observed in lane 2 upon p300 addition
(Fig. 33). As expected garcinol inhibited the p300-dependent acetylation on
H3K9 at 20 µM and at 5 µM on H4K5 compared to DMSO. JJMB 5, 6 and 7
didn’t inhibit p300-dependent acetylation in vitro (Fig. 34). In contrast, JJMB 9
was found to inhibit p300 activity in a dose dependent manner with an IC50 of 40
82
µM (Fig. 33 and 34). The p300 mediated acetylation was almost completely
inhibited at 1000 µM by JJMB 9 compared to DMSO (*p<0.05) (Fig. 34). Garcinol
completely inhibited the p300-dependent acetylation at 100 µM whereas JJMB 9
inhibited at 1000 µM with a ten-fold difference. This suggests that garcinol is
potent inhibitor of p300 compared to JJMB 9 (Fig. 33).
​
83
Figure 32: Diagram showing the experimental design of in vitro HAT assay in the
presence and absence of inhibitor.
84
Figure 33: In vitro P300 inhibition assay. In vitro histone acetyltransferase
inhibition (HAT) assay was performed using acetyl CoA (72 µM) and purified core
histones as substrates for the HAT p300 (Calbiochem) in the absence or
presence of increased concentrations of JJMB 5, 6, 7 and 9 (8 µM–1000 µM).
DMSO was used as a vehicle control (lane 2). Garcinol, a known HAT p300
inhibitor was used as a positive control. Reaction mixtures were run on 15%
SDS-PAGE and Western blot was performed using antibodies against Ac-H4K5
and Ac-H3K9. JJMB 5, 6 and 7 did not inhibit the acetylation activity of p300
whereas JJMB 9 and garcinol inhibited the p300 acetylation activity in a dose
dependent manner.
85
Figure 34: Percentage of standardized acetylation of H4K5. p300 dependent
acetylation activity of histone H4K5 was inhibited by JJMB 9 in a dose dependent
manner as compared to the DMSO control. One-way ANOVA was performed to
determine the significance of th4 differences between the groups. Post-hoc
Dunnett’s test was done to find out the significance between control group and
treatment groups. Levels of significance based on post-hoc Dunnett’s test were
designated as *p<0.05 & **p<0.01. Data is reported as the mean ± SEM of three
experiments.
Amidoximes did not inhibit the GCN5 in vitro
Since amidoximes JJMB 5, 6 and 7 did not inhibit the p300 HAT in vitro, we
tested the possibility that these amidoximes inhibit another type of HAT. To test
this hypothesis, we selected GCN5, GNAT family HAT, which is capable of
acetylating histones both in vitro and in vivo (Grant et al., 1997). The HAT
inhibitory activity was assayed using full length mouse recombinant GCN5
(purified in our lab) and purified core histones and acetyl coA as the substrates
(Fig. 11A). Western blot analysis was performed to determine if the amidoximes
inhibit the GCN5-dependent acetylation of Ac-H3K14. For GCN5 inhibition assay,
86
we used only anti Ac-H3K14 antibody since rGCN5 predominantly acetylates
histone H3 at lysine 14 (Grant et al., 1999). Lane 1 represents the basal
acetylation levels of H3K9 and H4K5. Increase in the acetylation levels was
observed in lane 2 upon GCN5 addition (Fig. 35). As expected anacardic acid
inhibited the GCN5-dependent acetylation at 40 µM (Fig. 35). The results
revealed that JJMB 5, 6, 7 and 9 did not inhibit GCN5-dependent acetylation in
vitro (Fig. 35).
87
Figure 35: In vitro GCN5 inhibition assay. This assay was performed using acetyl
CoA (72 µM) and purified core histones (2 µg) as substrates for the HAT GCN5
(purified) in the absence or presence of increased concentrations of JJMB 5, 6, 7
and 9 (8 µM–500 µM). Anacardic acid, a known HAT GCN5 inhibitor was used as
a positive control. Reaction mixtures were run on 15% SDS-PAGE and western
blot was performed using antibodies against Ac-H3K14. JJMB 5, 6, 7 and 9 did
not inhibit the acetylation activity of GCN5 whereas anacardic acid inhibited the
GCN5 acetylation activity in a dose dependent manner.
Amidoximes did not induce mitotic arrest in HCT-116 cells
Acetylation of lysine residues on core histone tails causes the unfolding of
chromatin which prevents the formation of higher order chromatin. Histone
deacetylation causes chromatin condensation and is involved in the formation of
higher order chromatin structure which peaks at metaphase of mitosis. Previous
studies have shown that inhibition of HDACs by HDAC inhibitors causes defects
in mitotic progression and therefore mitotic arrest (Shin et al., 2003). Also a study
reported that depleting the HDAC 3 from the HeLa S3 cells hindered the
88
chromosome alignment at the metaphase plate (Warrener et al., 2010). At the
same time, depletion of HATs such as p300, CBP and PCAF resulted in the
mitotic catastrophe such as delay in mitotic progression, formation of multipolar
spindles, multinucleation and aneuploidy etc (Ha et al., 2009). These studies
suggest that both HATs and HDACs are important for mitotic progression. To
determine if the amidoximes induced mitotic arrest in HCT-116 cells, we carried
out a time dependent treatment of HCT-116 cells with JJMB 7, 9 and garcinol.
JJMB 9 was shown as a HAT (p300) inhibitor and JJMB 7 was shown to
decrease acetylation by a different mechanism than inhibiting HAT, therefore both
of them were chosen for this experiment. Cell lysates were extracted after 3, 6,
12 and 24 hours. Cells treated with DMSO were used as a vehicle control.
Phosphorylated histone H3 serine was used a marker for mitosis. Western blot
analysis was performed against P-H3S10. The results indicated that there is a
significant decrease in the levels of P-H3S10 during the treatment, suggesting
that JJMB 7, 9 and garcinol did not induce mitotic arrest in HCT-116 cells (Fig.
36). These results were supported by our PI-based cell cycle analysis (Fig. 25
and 26).
89
Figure 36: JJMB 7, 9 and garcinol did not arrest the cells in mitosis. HCT-116
cells were treated with JJMB 7 (80 µM), JJMB 9 (40 µM), garcinol (50 µM) and
cell lysates were extracted after 3 hours, 6 hours, 12 hours and 24 hours.
P-H3S10 was chosen as the marker for mitosis. JJMB 7, 9 and garcinol reduced
the levels of P-H3S10 in a time dependent manner suggesting that the cells are
not arrested in mitosis.
Exploring the sequence and causal relationship between the reduced
acetylation and apoptosis in HCT-116 cells treated with amidoximes.
90
Previous studies have shown that apoptosis caused the inhibition of acetylation
of histone H4 (Kitazumi and Tsukahara, 2011). We therefore wanted to check the
sequence of the events of apoptosis and acetylation inhibition relative to each
other. To test this hypothesis, we selected JJMB 7, JJMB 9 and garcinol for this
experiment. From the previous experiments, we have shown that JJMB 7 did not
reverse the TSA effect and also did not inhibit p300 and GCN5 in vitro (see
Fig.30, 33 and 35). We also established that the inhibition of core histone
acetylation by JJMB 9 could be due to the inhibition of p300 (see Fig. 27, 33 and
34). To explore our question, we carried out a time dependent treatment of
HCT-116 cells with JJMB 7, 9 and garcinol. Cell lysates were extracted after 3, 6,
12 and 24 hours. Cells treated with DMSO were used as a vehicle control.
Caspase-3 activation and cleavage was used as the marker for early apoptosis
and acetylation levels were measured using the antibodies against Ac-H3K9 and
Ac-H4K5. Western blot analysis was performed against procaspase-3, Ac-H3K9
and Ac-H4K5. Treatment of HCT-116 cells with JJMB 7 revealed that the
activation of caspase-3 started at 12 hours while the inhibition of acetylation on
H3K9 and H4K5 was seen at 24 hours (Fig. 37.a). Treatment of HCT-116 cells
with JJMB 9 and garcinol revealed that activation of caspase-3 started at 12
hours while the inhibition of acetylation on H3K9 and H4K5 started at 6 hours
(Fig. 37.b, c). These results indicate that JJMB 9 may induce apoptosis by
inhibiting HATs in the cell. In contrast JJMB 7 may induce apoptosis by a different
91
mechanism and the apoptosis in turn may inhibit the acetylation of the histones
H3K9 and H4K5 in the cells. This conclusion is supported by Figure 30 that
shows that JJMB 7 cannot reverse the activity of TSA.
Figure 37: The kinetics of caspase-3 activation relatively to the inhibition of
acetylation in HCT- 116 after treatment with JJMB 7, JJMB 9 and garcinol. a)
Treatment of HCT-116 cells with JJMB 7 revealed that peak of caspase-3
activation occurred at 24 hours while the inhibition of acetylation on H3K9 and
H4K5 was also seen at 24 hours. b) Treatment of HCT-116 cells with JJMB 9
revealed that activation of caspase-3 started at 12 hours while the inhibition of
acetylation on H3K9 and H4K5 started at 6 hours. c) Treatment of HCT-116 cells
with garcinol revealed that peak of caspase-3 activation occurred at 24 hours
while the inhibition of acetylation on H3K9 and H4K5 started at 12 hours.
92
CHAPTER IV
DISCUSSION
Our major goal in this work was to determine the effect of nine novel amidoximes
on various malignant cell lines and to explore the mechanism of action of these
compounds. The amidoximes (JJMB 1-9) used in my research contain 1-3
amidoxime groups, characteristic of some HDAC inhibitors such as SAHA and
TSA (Richon et al., 1998, Finnin et al., 1999). On the other hand, some of these
compounds have 1-3 methylbenzene rings joined by a linker, characteristic of
some
HAT
inhibitors
such
as
curcumin,
garcinol
and
pentamidine.
(Balasubramanyam et al., 2004a, Balasubramanyam et al., 2004b, Kobayashi et
al., 2010). All the above discussed compounds have been shown to be promising
candidates against diseases such as cancer, bipolar diseases and COPD. We
hypothesized that the amidoximes used in this research would be effective
anti-cancer agents by inducing apoptosis and cell cycle arrest. By MTS cell
viability assay, we found that four out of nine amidoximes screened induced cell
death specifically in various malignant cell lines (Table. 5). By DNA fragmentation
assay, detection of caspase-3 proteolysis and FACS analysis, we showed that
the amidoximes induced apoptosis by fragmentation of DNA through the
activation of caspase-3 in HCT-116 (colon) cells (Fig. 20, 21 and 22). By FACS
analysis we have shown that amidoximes induced cell cycle arrest mainly at G1
phase (Fig. 26).
93
Previous studies have shown that the inhibition of HATs cause histone
hypoacetylation (Balasubramanyam et al., 2004a), whereas the inhibition of
HDACs causes the histone hyperacetylation (Richon et al., 1998). Treatment of
HCT-116 cells with amidoximes JJMB 5, 6, 7, 9 and garcinol for 24 hrs revealed a
significant decrease in the levels of core histone acetylation indicating that these
compounds may be HAT inhibitors (Fig. 27 and 28). The co-treatment of
HCT-116 cells with the HDAC inhibitor, TSA along with amidoximes revealed that
JJMB 5, 6 & 9 reversed the effect of TSA induced hyperacetylation supporting
the hypothesis that JJMB 5, 6, and 9 are HAT inhibitors (Fig. 30). By in vitro HAT
inhibition assay we have shown that one of our amidoximes, JJMB 9 is an
inhibitor of p300 but not JJMB 5, 6 and 7 (Fig. 33).
Structure-function relationship in induction of cell death in malignant cell
lines by amidoximes JJMB 1-9
Amidoximes are chemical compounds that have an amide group (-NH2) attached
to the oxime group (=N-OH) (Fylaktakidou KC, 2008). MTS cell viability assay
revealed that five out of nine novel amidoximes induced cell death in various
malignant cell lines. The nine novel amidoximes synthesized by Dr. J. Johnson,
TWU fall into 4 categories (Fig. 12). JJMB 1, 2 and 8 have similar chemical
structure with amide and oxime group on either side attached by variable lengths
of carbon linker (CH2). JJMB 3 has an amide and oxime group attached to a
methylbenzene ring with an extension of carbon linker (CH2). Amidoximes, JJMB
94
5, 6, 7 and 9 have similar chemical structures with two methylbenzene rings on
either end but vary by carbon linker (CH2) of different lengths. JJMB 4 is an
amidoxime with three methylbenzene rings joined by a carbon linker (CH2). From
table 5, which determined the growth inhibitory effect of nine novel amidoximes
on six malignant cell lines, we can interpret interesting facts about the efficacy of
the compounds based on their structures.
There is a correlation between the number of methylbenzene rings in the
series of compounds tested and their cytotoxicity. JJMB 1, 2, 3 and 8 did not
induce cell death in any of the malignant cell lines tested, indicating that no
methylbenzene ring or one methylbenzene ring in their structure did not cause an
anti-proliferative effect (Table 5 and 7). JJMB 4 significantly induced cell death in
all the malignant cell lines tested except for MDA-MB-231 but also killed normal
cells, indicating that the presence of three methylbenzene rings in its structure
was highly cytotoxic to the cells (Table 5, 7 and Fig. 13). Amidoximes JJMB 5, 6,
7 and 9 significantly induced cell death in various malignant cell lines but not in
normal cells, indicating that the presence of two methylbenzene rings is not
cytotoxic to the normal cells (Table 5 and 7).
Among the two methylbenzene ring compounds, we observed a general rule
that, the longer the carbon linker, the more efficient the compound was in killing
the cells. Amidoxime, JJMB 7 with the shortest 2-carbon linker induced
significant cell death in HCT-116 cells at low micromolar concentrations but
95
HLF-A (Lung) and MDA-MB-231 (Breast) cells were killed at higher micromolar
concentrations (Table 5, 7 and Fig. 16). JJMB 5 with a slightly longer linker
(3-carbon) also induced significant cell death in HCT-116 cells (Colon) at low
micromolar concentrations and HLF-A (Lung), MDA-MB-231 and MCF-7 cells
(Breast) were killed at higher micromolar concentrations (Table 5, 7 and Fig. 14).
In contrast, JJMB 6 with a 4-carbon linker, induced cell death in HCT-116 cells
(colon) with two times potent than JJMB 5 and 7 that have 3- or 2-carbon linker,
respectively. Surprisingly JJMB 6-induced cell death is restricted to only HCT-116
cells (Colon) (Table 5, 7 and Fig. 15). We observed that, JJMB 9 with the largest
linker (7-CH2 & N) significantly induced cell death in HCT-116 (colon), HLF-A
(Lung) and MCF-7 (Breast) at low micromolar concentrations (Table 5, 7 and Fig.
17). To the best of our knowledge, this is the first report of an amidoxime HAT
inhibitor and the first report indicating that the length of the linker between the
two aromatic rings in amidoximes correlates with the increased cytotoxicity of a
HAT inhibitor. Until now, none of the HAT inhibitors with three aromatic rings were
reported to be toxic to the normal cells unlike JJMB 4.
MCF-7 and MDA-MB-231 are breast adenocarcinoma cells; the former is ER
(estrogen receptor) positive cell line and the latter is ER negative cell line (Brooks
et al., 1973, Cailleau et al., 1974). Interestingly JJMB 9 induced cell death in ER+
MCF-7 cells but not in ER- MDA-MB-231 cells while JJMB 7 induced cell death in
MDA-MB-231 cells but not in MCF-7 cells (Table 5 and 7). This indicates that,
96
may be JJMB 9-induced cell death in MCF-7 cells could be through ER receptors
or ER receptor pathway. During the ER regulation, the estrogen receptors
interact with p300 and help in the transcription of its target genes such as BRCA1
(breast cancer type 1 susceptibility protein) and AP-1 (activator protein 1). It
might be possible that JJMB 9 might be targeting the downstream genes of ER
pathway such as p300 that could block the transcription of target genes thereby
inhibiting the growth of MCF-7cells. The fact that the various amidoximes killed
different types of malignant cell lines indicates that each compound has a
different mechanism of action in killing the malignant cell lines.
Table 7: Structure based induction of cell death in malignant cell lines by
amidoximes JJMB 1-9
97
Amidoximes caused histone hypoacetylation in HCT-116 cells
The amidoximes (JJMB 1-9) used in my research contain an amidoxime group,
characteristic of some HDAC inhibitors such as SAHA and TSA (Richon et al.,
1998, Finnin et al., 1999). Some of these compounds have a methylbenzene ring
connected by a linker, characteristic of some HAT inhibitors such as curcumin
and garcinol (Balasubramanyam et al., 2004a, Balasubramanyam et al., 2003).
When tested, the amidoximes caused histone hypoacetylation in HCT-116 cells.
98
We have observed that the treatment of HCT-116 cells with amidoximes JJMB 5,
6, 7, 9, and the positive control, garcinol caused the reduction in the acetylation
levels of histone H3 lysine 9 and histone H4 lysine 5. This finding suggested a
possibility that the amidoximes tested caused HAT inhibition and thereby
reduction in core histone acetylation (Fig. 27 and 28).
In agreement to above results, previous studies revealed natural HAT
inhibitors such as curcumin, garcinol, anacardic acid and synthetic HAT inhibitors
such as isothiazanoles reduced core histone acetylation through the inhibition of
CBP/p300, PCAF, GCN5, Tip60 and other HATs (Balasubramanyam et al., 2003,
Balasubramanyam et al., 2004a, Balasubramanyam et al., 2004b, Stimson et al.,
2005). A number of other mechanisms which are not based on direct HAT
inhibition could also explain histone hypoacetylation; this will be discussed below.
Three out of four amidoximes reversed the TSA induced hyperacetylation
but could not block the cell death
The inhibition of HDACs by TSA causes the hyperacetylation of core histone
lysine residues. We demonstrated that amidoximes caused reduction in core
histone acetylation in HCT-116 (colon) cells (Fig. 27, 28). The primary reason for
the reduced acetylation levels caused by amidoximes could be due to the
inhibition of HATs. To support this, we assessed whether the hyperacetylation
99
induced by HDAC inhibition can be reversed by the amidoximes that were
hypothesized to be HAT inhibitors. Based on the Western blot analysis, we found
that JJMB 5, 6, 9 and garcinol, effectively reversed the TSA induced
hyperacetylation at higher concentrations, whereas JJMB 7 did not reverse the
TSA induced hyperacetylation (Fig. 30). These result suggests that the histone
hypoacetylation induced by JJMB 5, 6, and 9 may be due to the inhibition of
HATs while JJMB 7-induced histone hypoacetylation could be due to an
activation of HDACs or might increase the expression levels of HDAC genes.
To test if the studied amidoximes inhibit HATs, we performed in vitro HAT
inhibition assay using full length recombinant human p300, a member of
CBP/p300 family. In vitro assay showed that JJMB 9, but not JJMB 5, 6 or 7, is
an inhibitor of p300 but not JJMB 5, 6 and 7 (Fig. 33 & 34). This led us to
investigate whether these amdoximes and particularly JJMB 5 and 6 inhibit HATs
from a different family. Therefore, we performed in vitro HAT inhibition assay with
GCN5, a member of GNAT family (Marmorstein, 2001) (Fig. 35). None of the
amidoximes inhibited GCN5-dependent acetylation (except the positive control,
anacardic acid) suggesting that JJMB 5, 6 and 7 might be inhibiting different
family of HATs which have not been tested or might have a different mechanism
of causing reduced core histone acetylation.
Based on the published structures of HAT inhibitors, our amidoximes show
some similarity to the curcumin (Balasubramanyam et al., 2004b), thus we
100
assume that our amidoximes would also be non-competetive inhibitors of HATs. If
this hypothesis is correct, it is also logical that like the curcumin that has two
aromatic rings connected by a 7-carbon linker, JJMB 9 with a 7-carbon linker is
the most potent inhibitor, thus this length provides the best interaction for the
aromatic rings with the HATs non-competetive inhibitor binding site.
Histone hypoacetylation can also be caused due to the activation of HDACs.
For example, lysophosphatidic acid (LSA) induces histone hypoacetylation of
H3K9 and H4K12 by the activation of HDAC1 but not HDAC2 (Ishdorj et al.,
2008).
The reversal of TSA-induced hyperacetylation by amidoximes JJMB 5, 6 and
9 led us to investigate the effect of concomitant treatment of TSA and
amidoximes on the cell survival. To our surprise, we found that none of the
amidoximes blocked the cell death induced by TSA except for JJMB 5 at 8µM
(Fig. 31). To explain this apparent conflict let us note that, in the Western blot
analysis, we have seen the reversal of global steady state core histone
acetylation levels, but gene-specific core histone acetylation might vary. Inhibition
of HDACs by TSA leads to only a 2% change in expression of total genes in
human genome (Kajstura et al., 2007). At the same time, inhibition of HATs
probably leads also to only a small percentage of gene expression alteration. The
genes targeted by TSA-induced HDAC inhibition may be different from the genes
that are targeted by garcinol or amidoxime-induced HAT inhibition. For example,
101
GATA4, a tumor-suppressor gene involved in many gastric cancers is
trimethylated causing the inactivation of the gene. Treatment by TSA that
induced hyperacetylation of histone H3 and H4 as well as demethylation of
H3K4, caused the activation of GATA4 (Yamamura and Kishimoto, 2012). No
studies have reported that GATA4 is the target for garcinol. This indicates that
gene targets for HDAC inhibitors such as TSA and HAT inhibitors such as
garcinol and probably amidoximes are different, even though some genes could
be commonly targeted by all these compounds. Recent study showed that using
the HAT inhibitor, curcumin in combination with the HDAC inhibitor, TSA, in ER
negative breast cancer cells showed greater anti-proliferative effects, and
increased apoptosis which was not accomplished with either agent alone (Yan et
al., 2012). These studies underscore the fact that HAT inhibitors and HDAC
inhibitors though seemingly counteract each other activity on the global
chromatin level; they don’t negate each other necessarily on the single gene
level.
Furthermore, Wang et al. showed that both HATs and HDACs are detected in
high levels in the active genes. Since decades, it was known that HATs are
recruited to the promoter region to acetylate the nucleosomal histones and unfold
the actively transcribed regions. The findings that HDACs are present at higher
concentrations in active genes suggest that HDACs recruitment to the
transcribed regions might play a role in removing the acetyl groups as the RNA
102
polymerase is moving on during elongation. These findings indicate that the
dynamic interplay between the HATs and HDACs in active genes is vital for gene
expression and that both HATs and HDACs are required for proper transcription.
Thus, blocking both HAT and HDACs would not compensate for each other in
this respect. Our results are in accord with these findings since TSA that inhibit
HDACs and amidoximes that inhibit HATs though globally compensate each
other, locally are causing the deregulation of chromatin structure and therefore
enhanced cell death.
Novel amidoximes-induced apoptosis in HCT-116 cells is caspase
dependent
Anti-cancer drugs are known to induce apoptosis and some of them could also
cause necrosis in malignant cell lines (Mailloux et al., 2001). Studies on apoptotic
pathways indicated that apoptosis can be caspase-dependent or independent
(Fig. 19) (Lodish and Matthew, 2008). Previous studies have shown that
caspase-3 is an essential protease required to cause DNA fragmentation during
apoptosis (Otera et al., 2005). To find out if the amidoximes induced apoptosis in
malignant cell lines, we used apoptotic markers such as detection of caspase-3
proteolysis, DNA fragmentation and increase in sub-G1 apoptotic cell population.
The sub-G1 peak in the DNA distribution curve represents the apoptotic cells
reduction in the DNA content due to the shedding of apoptotic bodies (Kajstura et
al.,
103
2007).
Our
data
shows
that
the
amidoximes
induced
a
concentration-dependent apoptosis in HCT-116 cells at micromolar range. JJMB
5, 6, 7, 9- and garcinol-induced apoptosis by DNA fragmentation, is executed by
capase-3 activation (Fig. 20, 21 & 22). Although caspase-dependent apoptosis is
involved in many pathological processes and in tissue development, apoptosis
can also occur without the involvement of caspases through mitochondrial
proteins, Endo G or AIF (Lily Y. Li, 2001, Otera et al., 2005). For example,
Berberin, an isoquinoline alkaloid obtained from plants, induced caspaseindependent apoptosis by inducing AIF release from cytosol and translocation
into the nucleus in mouse immorto-Min colonic epithelial (IMCE) cells where it
causes DNA fragmentation (Wang et al., 2012). In contrast, the induction of DNA
fragmentation by protein kinase C inhibitor, safingol, was through the activation of
endonuclease G but not AIF or caspases (Hamada et al., 2006).
Inhibition of acetylation precedes the caspase activation upon JJMB 9
treatment but not upon JJMB 7 treatment
Our research demonstrated that amidoximes JJMB 5, 6, 7 and 9 induced
apoptosis and were associated with reduction of acetylation levels in HCT-116
cells. But only JJMB 9 was unequivocally proven as a HAT inhibitor. JJMB 7
unlike JJMB 9 didn’t reverse the TSA’s activity, and didn’t inhibit p300 or GCN5
(Fig. 30, 33 and 35). These findings raised the question of causal relationship
between acetylation inhibition and apoptosis. Does inhibition of acetylation
causes apoptosis, or apoptosis causes inhibition of acetylation? Previous studies
104
have shown that apoptosis caused the inhibition of acetylation of histone H4
(Fullgrabe, 2010). So our question was a valid one. We therefore checked the
sequence of the events of apoptosis and acetylation inhibition relatively to each
other.
Treatment of HCT-116 cells with JJMB 7 revealed that the activation of
caspase-3 started after 12 hours while the inhibition of acetylation on H3K9 and
H4K5 was seen at 24 hours (Fig. 37). This suggests that JJMB 7 may induce
apoptosis before the histone acetylation is inhibited by an undefined mechanism,
and the apoptosis in turn causes the inhibition of acetylation of the histones
H3K9 and H4K5 in the cells. A relevant study in neurodegeneration showed that
LK (K+ deprived)-induced neuronal apoptosis in cerebellar granule neurons
(CGNs) caused the degradation of CBP and decreased HAT activity which
resulted in reduced histone H3 and H4 acetylation. In vitro experiments revealed
that caspase-6 acted as an effector caspase that cleaved CBP. When CGNs
were treated with caspase-6 inhibitor, Z-VEID-fmk, CBP degradation was
ameliorated, suggesting also an in vivo degradation of CBP by caspase-6
(Mailloux et al., 2001). A number of biochemical changes occur during apoptosis
to the chromatin and DNA such as chromatin condensation, DNA fragmentation
and release of nuclear material into the cytosol. These events suggest the
possibility of link between apoptosis and histone post-translational modifications.
Some of the modifications such as phosphorylation of H2A.X, H2B and H3 and
105
dephosphorylation
of
H1 have already been established (Ajiro, 2000,
Fylaktakidou KC, 2008). Chornet et al. analyzed the acetylation levels of histones
in apoptotic cell lysate and in nuclear pellet of jurkat cells upon etoposide
treatment. They found a global histone hypoacetylation of H3 and H4 in apoptotic
cell lysate, although no significant degradation of histones were observed. Apart
from the histone hypoacetylation, released histone H4 fraction into cytosol
showed a significant trimethylation as well (Johnson et al., 2009).
In contrast to JJMB 7 effect, treatment of HCT-116 cells with JJMB 9
revealed that the activation of caspase-3 started at 12 hours while the inhibition
of acetylation on H3K9 and H4K5 started at 6 hours (Fig. 37). This suggests that
JJMB 9 might have induced apoptosis by inhibiting HATs in the cell. Similar to
JJMB 9, studies on HAT inhibitors such as curcumin, garcinol, anarcardic acid
etc., induced apoptosis in variety of malignant cells by inhibiting CBP/p300,
PCAF, GCN5 and Tip60 (Balasubramanyam et al., 2004a, Balasubramanyam et
al., 2003, Balasubramanyam et al., 2004b). Studies also indicated that HAT
inhibitors that sensitized cancer cells to TRAIL (TNF-related apoptosis-inducing
ligand) and IR (Infra-red) induced apoptosis (Prasad et al., 2010 and Yingli sun et
al., 2006). For example, anacardic acid inhibited the HAT activity of Tip60 that
lead to the activation of ATM protein kinase, which in turn sensitized HeLa cells
to cytotoxic effects of ionizing radiation (Sun et al., 2006).
106
Exploring the sequence and causal relationship between the reduced
acetylation and apoptosis in HCT-116 cells upon treatment with JJMB 7 and
JJMB 9 suggested that histone hypoacetylation can be caused as a result of
apoptosis by JJMB 7 or due to the direct inhibition of HATs in the cell by JJMB 9.
Possible future applications of amidoximes
HATs and HDACs play an important role not only in transcriptional regulation but
also in DNA repair, apoptosis, cell proliferation, differentiation and cellular
signaling. A proper functioning of HATs and HDACs is important for cellular
homeostasis. Any aberration or malfunctioning can lead to diseases such as
cancer, heart diseases, neurological and respiratory diseases.
It is reported that the HAT Tip60 (member of MYST family), an androgen
receptor activator is accumulated in the nucleus of prostate cells (LnCaP human prostate cancer cells) and in CWR22 mice cells (xenograft mouse model)
possibly causing the progression of prostate cancer (Halkidou K, et al., 2000).
Tip60 is also involved in skin cancer and can be detected by the increased levels
of ODC (ornithine decarboxylase) and polyamine metabolism in the cell.
Interestingly, elevated HAT activity was observed in Knock out/ODC mice with
skin cancer. These elevated HAT activity is correlated with an abnormal increase
in the protein levels of Tip60 and its variant Tip53 (Hobbs et al., 2006). Similarly,
p300 was also shown to activate the androgen receptor suggesting its role in the
progression of prostate cancer cells. When biopsies of 95 patients with prostate
107
cancer were analyzed, p300 expression levels were very high. Knocking down
the p300 by siRNA decreased the cell proliferation and increased apoptosis in
LNCaP human prostate cancer cells (Debes et al., 2003).
Biopsies from lungs of asthma patients showed a significant increase in HAT
activity and reduced levels of HDAC activity especially HDAC 1 and HDAC 2 (Ito
K, et al., 2002). In another study, when rats are exposed to cigarette smoking, it
resulted in a sharply reduced HDAC activity correlating with the elevated H4
acetylation. This in turn led to the activation of pro inflammatory genes such as
NF-Kβ and Ap-1 (Marwick et al., 2004).
Also, prostate cancer studies indicated that HAT PCAF is involved in the
resistance of PC3 cells to the chemotherapy drug, cisplatin. PCAF expression
was upregulated in cisplatin resistant cells. Down regulation of PCAF by siRNA
sensitized PC3 cells to cisplatin and other chemotherapeutic drugs (Hirano et al.,
2010).
The above studies demonstrate that HATs are upregulated in various
diseases such as cancer, COPD and asthma. By using HAT inhibitors such as
garcinol, curcumin, and the amidoximes reported in this work, HAT activity can be
inhibited which might help in treating these diseases.
108
109
Figure 38: Model showing the mode of action of JJMB 9 in inhibiting HCT-116
cells (Colon): The cellular stress induced by JJMB 9 in HCT-116 cells cause the
reduced core histone acetylation by inhibiting p300 HAT. This would probably
cause the cell cycle arrest at G1 phase thereby triggering apoptosis. This causes
the activation of caspase-3 and therefore translocates into the nucleus and
trigger either DFF40 or PARP causing the DNA fragmentation.
110
Figure 39: Model showing the mode of action of JJMB 7 in inhibiting HCT-116
cells (Colon). The cellular stress induced by JJMB 7 in HCT-116 cells cause the
cell cycle arrest at G1 phase thereby triggering apoptosis. This causes the
activation of caspase-3 and therefore translocates into the nucleus and trigger
either DFF40 or PARP causing the DNA fragmentation. The apoptosis in turn
cause the reduced core histone acetylation by an unknown mechanism.
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