FLAVONOIDS
• Flavonoids comprise of a huge group of polyphenolic compounds
encompass a benzo-γ-pyrone core moiety.
• Chemically flavonoids rely upon a fifteen-carbon skeleton
possessing of two benzene rings.
• Flavonoids occur in nature as aglycones, glycosides, and methylated
derivatives.
• Their basic structure is a skeleton of diphenylpropane, namely, two
benzene rings (ring A and B) linked by a three carbon chain that
forms a closed pyran ring with benzenic A ring. Therefore, their
structure is also referred to as C6-C3-C6.
HEPATOCELLULAR
CARCINOMA
• Hepatocellular carcinoma (HCC) is a crucial growth
of malignant tumor cell of the liver and takes place
mainly in patients with fundamental chronic liver
disease and cirrhosis.
• The cells of origin are supposed to be the hepatic
stem cells, although this remainder the question of
examination.
• Tumors development progresses with local increase
in size, intra-hepatic spread, and outlying metastases.
https://emedicine.medscape.com/article/197319-overview
SCHEMATIC UNDERSTANDING OF
HEPATO CELLULAR CARCINOMA
POLYAMINES
• Natural polyamines play a essential role in eukaryotic cell growth,
division, and proliferation.
• Here in the context polyamine stands for the attachment of more
than one amine group to the flavonoids nucleus with varying groups
attached with the C ring of flavonoids nucleus.
• Both indigenous and synthetic polyamines have been attached to
varied pharmacophore to provide various biological functions, such
as cancer inhibition, neuroprotection, and anti-parasitic properties.
• Recently, there is growing interest in the relationship between the
polyamines and tumor migration.
Q. Li et al. / European Journal of Medicinal Chemistry 121 (2016) 110-119
MAJOR RESEARCH PROBLEM
The major research problem depicted is that
the authors were likely to assess whether the
synthesized polyamine modified flavonoids
were able to inhibit the hepatocellular
carcinoma.
DESCRIPTION
• Authors synthesized the series of polyamine conjugates of flavonoids with
a naphthalene moiety.
• After synthesizing the analogs they evaluated them for their anti-hepatocellular carcinoma attributes with the help of in vitro as well as in vivo
assays.
• Authors performed the study of the apoptosis on the cell lines at different
concentrations assessed by HCS using AO/EB staining on HepG2 cells.
• Biological materials and methods
– Immuno-fluorescence staining for caspases and p-p38.
– In vitro migration assay.
– Cellular apoptotic evaluation.
– ROS, MMP and LYS/pH staining experiments.
– Immuno-fluorescence staining for p53, p-p38, p-JNK, Bcl-2.
– Subcutaneous xenograft of H22 cells in Kunming mice.
– H22 cells lung metastasis models.
SYNTHETIC SCHEME
In vitro structure-activity relation study
• The ability of the flavonoids to differentiate between cancerous and noncancerous cells was evaluated with an MTT assay.
• The two positive controls, Mito (MITOXANTRONE) and
Que (QUERCETIN), had contradictory affects in this assay. Mito killed
more QSG7701 (noncancerous) cells than the analogous cancerous cells at
the equivalent concentration, and Que showed more encouraging cell
selectivity.
• The cancer cell selectivity of synthesized flavonoids 8a-8d was better to
than that of Mito.
• Nevertheless, only compound 8a displayed cell type selectivity that
resembled to that of Que.
In vitro inhibitory rates of novel conjugates
NO
R, n
SMMC 7721(%)
HepG2 (%)
QSG 7701 (%)
30μM
100μM
30μM
100μM
30μM
100μM
8a
(CH2)3NH2 , 2
5.1
87.1
7.9
84.2
<5
41.8
8b
(CH2)4 NH2 , 2
<5
39.8
<5
16.1
<5
10.6
8c
(CH2)3NH(CH2)4
(CH2)3NH2 , 3
7.6
32.5
<5
28.9
<5
22.4
8d
(CH2)4NH(CH2)4
(CH2)3NH2 , 3
70.2
85.5
62.0
75.9
46.7
79.4
8e
(CH2)2 N(CH3)3, 2
<5
16.6
<5
7.5
<5
17.9
8f
(CH2)2 CH3, 1
14.6
28.4
6.0
21.6
19.6
41.7
Que
29.5
62.9
33.2
56.3
<5
36.0
Mito
20.5 d
62.2e
41.2d
78.6d
75.81d
82.5e
The inhibitory rates were determined at two different concentrations. Each sample is the mean of three
independent experiments.
(B )Que. Quercetin; (c)Mito. Mitoxantrone. (D) The inhibitory rates were determined at the conc of 1 mM.
(e )The inhibitory rates were determined at the concentration of 5 mM.
In vitro structure-activity relation study
• Two HCC cell lines (HepG2, SMMC7721) were chosen for the
in vitro screening of newly synthesized flavonoid-polyamine
conjugates.
• Polyamines and/or flavonoids were evaluated for selectivity
between tumor and normal cells using one normal hepatocyte
cell line (QSG7701).
• The inhibitory activity of target compounds against tumor cells
was measured by traditional MTT tests.
(3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide)
formazan
RESULTS
• The concentrations of new compounds i.e 8a-8f were chosen as 30μM and
100 μM.
• Compound 8a displayed favorable specificity among hepatocellular
carcinoma cells and usual hepatocytes , and the amalgamation of 8a with
aspirin resulted in synergistic inhibition of in vitro tumor cell in both
growth as well as migration.
• The 8a-aspirin combination also retarded H22 liver tumor growth and
metastasis in tumor in lungs and enhanced body weight index in animal
models.
• 8a augmented the expression of apoptosis-related proteins such as p-p38, pJNK, p53 and Bcl-2, an effect that was further improved by aspirin.
CONCLUSION / RESULTS
• The respective IC50 values of 8a for HepG2, H22 (Murine
carcinoma cells used in mouse models) and QSG7701 cells
were 65.0 μM, 32.1 μM and over 100 μM, respectively.
• Compound 8a exhibited a more advantageous dose-dependent
inhibition against HepG2 and H22 while retaining the
favorable cell selectivity with respect to QSG7701 cells.
• Authors also tried the combination of the the effect of novel
flavonoids-polyamine conjugates with 800 μM aspirin, and
found that the aspirin was able to increase the potency of the
compound 8a against HepG2 (from 65.0 μMto 59.2 μM), and
H22 (from 32.1 μM to 21.1 μM)
In vitro migration assay.
• Metastases are a serious trouble in cancer therapy. Number of
reports discovered that some natural and synthetic flavonoids
are successful metastasis inhibitors.
• The conventional Transwell Tests and Woundhealing Assays
to scrutinize the capacity of 8a alone or with aspirin to inhibit
tumor cell attack to evaluate the ability of the either 8a alone
or in combination with aspirin.
IN VITRO MIGRATION ASSAY.
RESULTS
Cellular Apoptotic Evaluation
• To investigate whether novel flavonoid-polyamine conjugates induce cell
apoptosis, HepG2 cells treated with compound 8a were doubly stained by
AO (Acridine Orange) with a green fluorescence and EB (Ethidium
bromide) with an orange-red fluorescence, respectively.
• AO penetrates the intact cell membranes and intercalates into the nuclear
DNA, which is suitable for the detection of apoptosis in the early stages.
EB is excluded from viable cells with integral membranes and is used to
discern cell necrosis and late apoptosis by intercalating into the nuclear
DNA after passing through the damaged cell membrane. As shown in Fig.
5A, 8a induced apoptosis in a dose-responsive manner.
• Ethidium bromide)
(Acridine Orange)
(Ethidium bromide)
RESULT
Cellular Apoptotic Evaluation
• The mitochondrial membrane potential (MMP) is a decisive
parameter related to mitochondrial function in cells.
• As a MMP specific cationic fluorescent dye, Rh123 was used
to measure the variation of MMP, which accumulates in
healthy mitochondria with intact membrane potential and is
absent in the depolarized mitochondria
Rh123 (specific cationic fluorescent dye)
Cellular Apoptotic Evaluation
• Lysosomes participate in various cell death processes such as
apoptosis and senescence.
• Thus, the lysosomal mass/pH (LYS/pH) variation was detected by a
basic fluorescent probe Lyso-Tracker Red which selectively
accumulates in acidic lysosomes during cytotoxicity.
BASIC FLUORESCENT PROBE LYSO-TRACKER RED
Cellular Apoptotic Evaluation
RESULT
ROS, MMP and LYS/pH Etaining Experiments.
• It is known that polyamine conjugates may
produce reactive oxygen species (ROS).
• The elevated ROS content may in turn
increase MMP loss and LYS/pH destabilization,
which generally activates the cell death.
Therefore, ROS generation was measured by
the DCFH-DA assay.
(2’, 7’-dichlorodihydrofluorescein diacetate)
DCFH-DA Assay Result
• The authors observed a dose dependent increase in ROS, as
shown in Fig. 5B. In general, 60 μM 8a and 80 μM Que
showed minor differences in their effects on MMP, LYS/pH,
and ROS. Further efforts were subsequently conducted to
investigate if 8a and Quercetin possess a similar apoptotic
mechanism.
Immuno-fluorescence staining for p53, p-p38, pJNK, Bcl-2.
• The expression of p-p38 and p-JNK (phospho Jun-aminoterminal kinase) was upregulated by 8a in a dose dependent
manner.
• The upregulated expression of p-p38 and p-JNK generally
induced the activation of p53, followed by the
downregulation of Bcl-2 (B cell CLL/Lymphoma 2) family.
• The alteration of protein expression of both p53 and Bcl-2 was
observed as expected in the experiments.
Immuno-fluorescence staining for
p53, p-p38, p-JNK, Bcl-2.
Immunofluorescence staining for
caspases and p-p38
• Candidate 8a and Que activated caspase 3, indicating caspasedependent apoptosis.
• Further analyses using similar methodology were done
for caspases 8 and 9, the upstream proteins of caspase 3. Que
activated both caspase 8which is closely associated with the
extrinsic death receptor pathway, and caspase 9, which is
involved in the intrinsic mitochondrial pathway. 8a had no
impact on caspase 9, but activated caspase 8
• 8a up-regulated the expression of phospho p38 (p-p38)
potently, while Que had a moderate impact on p-p38.
H22 cells lung metastasis models
• Animals had a better tolerance for 8a (20 mg/kg) than Mito
(0.4 mg/kg) in the pre-trials for MTD (maximum tolerated
dose), which is in agreement with their respective cell
selectivity.
• compound 8a was injected into mice with a median dosage
(10 mg/kg, half of the MTD) to avoid detrimental effects.
H22 cells lung metastasis models
RESULTS
CONCLUSION
• A series of flavonoid-polyamine conjugates were designed and
synthesized. The in vitro evaluation using HCC cell lines
revealed cytotoxicity only at relatively high concentration.
• Compound 8a showed excellent cell selectivity for cancerous
cell lines, and showed significant in vitro and in vivo anticancer and anti-metastatic properties, which were enhanced by
co-administration with aspirin.
• The 8a-aspirin combination did not cause any obvious signs of
toxicity, as indicated by favorable body weight indices.
REFERENCE
Synthesis and biological properties of polyamine modified flavonoids
as hepatocellular carcinoma inhibitors
Qian Li et al; European Journal of Medicinal Chemistry 121 (2016) 110-119
Received 13 January 2016
Received in revised form
15 March 2016
Accepted 11 April 2016
Available online 19 May 2016