Current Research Journal of Biological Sciences 2(6): 396-401, 2010
ISSN: 2041-0778
© M axwell Scientific Organization, 2010
Submitted date: September 10, 2010
Accepted date: October 09, 2010
Published date: November 25, 2010
Azurin as Antitumor Protein and its Effect on the Cancer Cell Lines
1
Mervat S. M ohamed, 2 Said A. Fattah and 1 Howay ada M . Mostafa
1
Chemistry Department, Biochemistry Speciality,
2
Biophysics Department, Faculty of Science, Cairo University, Egypt
Abstract: As a low molecular weight redox protein was elaborated from the patho genic bacteria Pseudomonas
aeruginosa, azurin is one of the representative bacterial products used in the treatment of tumours. In this study,
Pseudomonas aeruginosa was isolated from lungs of cystic fibrosis patients in Egypt, and identified by 16S
rRNA. Azurin ge ne w as am plified using the suitab le PCR programme. The gene was cloned into pG EM -T easy
vector and sequenced. Then subcloned into the overexpression vector pET-28a (+) using E. coli BL21 as
expression bacteria. The SDS-PAGE analysis showed that the gene overexpressed in E. coli. The recombinant
protein was purified by one step purification using Ni+ + resin. The effect of azurin on proliferation and apop tosis
of human breast cancer cell line (MCF7), human hepatocellular carcinoma cell line (HEPG2) and human colon
carcinoma cell line (HCT116) and human normal melanocytes (HFB4) cell line was studied. The results showed
that azurin strongly inhibited the proliferation of both colon carcinoma cell line and breast cancer cell line.
W hereas unclear result of the effect of azurin on hepatocellular carcinoma cell line was exhibited. At the same
time, azurin didn’t show any effect on the human norma l melanocytes.
Key w ords: Antitumor, azurin, cell lines, Pseudom onas aeruginosa
INTRODUCTION
Live or attenuated patho genic bacteria or the ir
products were used in the treatment of cancer (Da
Rocha et al., 2001; Chakrab arty, 2003; Sinha, 2003). A
significant regression of subcutane ous tumours in mice
was observed by combining anaerobic bacteria w ith
variou s chemoth erape utic agents (T angri et al., 2001 ).
Azurin is a small globular metalloprotein, endowed
with redox activity, involv ed in th e bac terial
denitrification process (Edward et al., 1992; Webb and
Loppnow, 1999). It is acting as an electron transfer shuttle
in Pseudom onas aeruginosa and other bacteria. The
presence of the copper ion gives this protein a number of
features, including an intense blue color, a high reduction
potential and a small parallel hyperfine coupling in the
electron spin resona nce spectru m (A dma n, 199 1). It has
been reported to induce and trigger apoptosis in several
human cancer cells selectively. These findings in vitro
have been confirmed in nude mice bearing tumour
xenog raft in vivo. Furthermore, it is completely lack of
toxicity. At necropsy, none of the treated mice showed
any histological evidence of toxicity and all of the viscera
were within normal limits (Yamada et al., 2002a, b;
Punj et al., 2004). Azurin capability of indu cing apoptosis
in tumour cells by p53 stabilization makes this protein
suitable for being em ploye d as anticancer agent.
The p53 tumor supp ressor is involv ed in m ultiple
central cellular processes, including transcription, DNA
repair, genomic stability, cell cycle control, and apoptosis;
it is functionally inactivated in many human canc ers
(Harris, 1996). Azurin was discovered to enter
mammalian cells, such as J774 macrophage-like cells
(Yamada et al., 2002a) and cancer lines such as
melanoma UISO -Me l-2 and M CF-7 breast-cancer cells,
and cause cell death (Goto et al., 2003; Punj et al., 2003;
Yamada et al., 2004a). Upo n entry into these cells, azurin
appeared to form a complex with p53, somehow raising
its intracellular levels. The increased amount of p53 then
triggered apoptosis in the cells through enhanced Bax
formation and release of mitochondrial cytoc hrom e c in
the cytosol. Many viral and mammalian proteins can
mod ulate p53 function by physical interaction; how ever,
azurin is the first bacterial protein reported to form a
complex with p53. The support for the azurin-p53
complex was only based on g lycerol-gradient
ultracentrifugation and pull-down experiments using
GST-fusion constructs (Yamada et al., 2004a, b;
Punj et al., 2004). However, as a potent inducer of
apoptosis, the precise mechanism of azurin-induced
apoptosis is still unclear. In this study, the effect of the
recombinant azurin was studied on the different cancer
cell lines and human normal melanocytes.
MATERIALS AND METHODS
All products inc luding (kits, vectors, and enzymes)
were purchased from Prom ega, and a ll procedures w ere
carried out at 4ºC. Unless noted otherwise.
Isolation and identification of bacterial strain: The
work was conducted in the Cairo University during the
Corresponding Author: Mervat S. Mohamed, Chemistry Department, Biochemistry Speciality , Cairo University, Egypt
396
Curr. Res. J. Biol. Sci., 2(6): 396-401, 2010
period of 200 7-2009. B acterial strain was characterised
and identified on the basis of physiological
micro biological and b ioche mica l chara cterisatio n
(Duguid, 1996) also it was subjected to further molecular
characterization by PCR amplification for partial
sequence of the 16S rR NA gene. U niversal primers pairs
9F and 1512R were used to amplify ~1500 bp. These
primers are used to amplify the 16S rRNA gene of most
eubacteria (William et al., 1991). PC R prod ucts were
purified by Wizard® SV Gel and PCR cleaning up system
kit following the protocol provided by the supplier and
then resolved by electrophoresis on 1% agarose gel. The
purified product was sequenced. The sequence of the
16SrRNA of the strain was compared with other
published 16S rRNA sequences using the BLAST
program of the N CB I datab ase (h ttp://w w w .
ncbi.nlm.nih.gov). That was to determine the nearest
phylogenetic neighbours.
The culture was incubated at 37ºC with shaking at 250
rpm to an OD600 of approximately 0.5. The entire 3 mL
culture was added to 100 mL LB medium containing
kanamycin at final concentration of 30 :g/mL. The
culture was sh aken at 37 ºC until the OD 600 of 0.5-1. Just
prior to induction, the 100 ml culture was split into 2×50
mL cultures. IPTG was added to final concen tration of 1
mM to one of the 50 mL cu ltures and the other culture
was used as an uninduced control. Bo th cultures we re
incubated with shaking at 37ºC for 2, 4 and 6 h. Bacteria
were then pelleted by centrifugation at 6,000 rpm at 4ºC
in each time and resuspended in a phosp hate buffer (20
mM Sodium phosphate pH 8.0, 500 mM Sodium
chloride) the cells were then lysed by boiling for 5 min
and debris was removed by centrifugation at about 5000
rpm at 4ºC. The cell crude cell lysate was analyzed using
SDS P AG E gel electrophoresis.
Recombinant protein purification: Protein purification
was performed according to manufacturer’s protocol
using column chromatography. The fractions of the target
protein was collect and assayed by a modified SDSPAG E gel electroph oresis (Laem mli, 1970).
PCR amplification of azurin gene: Azurin gene was
amp lified from genome of isolated bacterial strain by
using previously designed primers; AZU-f (5'GAATT CCGCCCACCTG CCT-3') and A ZU -r (5'A A G C T TTCATG C A G C G G A TC G -3 ') to am plify
~522bp. The amplification reaction was carried out using
the following (PC R) reaction conditions: an initial
denaturation step at 94ºC (1 min), followed by (30 cycle)
of denaturation step at 95ºC (1 min), with the following
prime r-specific anne aling tem peratu res at 60ºC (1 min),
primer extension at 72ºC (10 min), and cooling to 4°C
forever . The PCR product was applied to 1% agarose gel
electrophoresis in comparison with DNA molecular
weight marker 8 / HindIII. The purified product was
sequenced. The obtained sequences were analyzed for
similarities to other known sequences found in the
GenBank database u sing BL AST program of the NCBI
database. Sequences with the greatest similarity to our
products w ere extracted from the database and aligned.
Cloning of azurin gen e in pGEM ®-T easy vec tor:
DNA fragments were cloned into pGEM-T easy
vector system , then the (azurin/pGEM ) construct was
introduced into the Ma ch1™ - T1® E. coli competent
cells. Screening and selection of white/blue colonies was
performed. The large scale plasmid containing
(azurin /p GE M ) construct was prepared according to
manu facturer’s instructions. Digestion of the produced
plasmids by EcoR1/ Hind III were carried out at 37ºC for
2 h to relea se the azurin gene and was detected by gel
electrophoresis.
Measurment of potential cytotoxicity by SRB assay:
After determination of protein concentration using
Bradford Protein Co ncentration Assay, the potential
cytotoxicity of azurin was tested (Philip et al., 1990).
SRB assay was done in the National Cancer Institute,
Egy pt.
Normal mela nocytes (HFB4), Breast cell line
(MC F7), Liver cell line (HEPG2) and colon cell line
(HCT116) were used in this study. Cells were plated on
96-m ultiwell plate (104 cells/well) for 24 h befo re
treatment with azurin to allow attachment of cell to the
wall of the plate. Different concentration of azurin under
test (0, 1, 2.5, 5 and 10 :g/mL) were added to the ce ll
monolayer triplicate wells were prepared for each
individual dose. Monolayer cells were incubated w ith
azurin for 48 h at 37ºC and in atomosphere of 5% CO 2 .
After 48 h, cells were fixed, washed and stained with
Sulfo- Rhadoamine - B stain. Excess stain was washed
with acetic acid and attach ed stain was recovered with
Tris EDTA buffer. Color intensity was measured in an
ELISA reader. The relation between surviving fraction
and azurin concentration is plotted to get the survival
curve of each tumour cell line after the specified azurin.
RESULTS
Expression of the recombinant azurin: Azurin gene was
subcloned into the T7-inducible vector (pET-28a (+)) then
azurin / pET-28a(+) construct was transformed into BL21
(DE3)-pLysS E.coli. A single colony from BL21 having
the construct w as inoculated in 3 mL of LB broth culture
containing kanamycin at final concentration 30 :g/mL.
Identification of the bacterial isolate by 16S rRNA
gene: Partial sequence analysis of the amplified 16S
rRNA gene co nfirmed the classification of the isolate as
a member of the genus Pseudomonas where it showed the
highest degree of similarity (98%) to that of pseudomonas
aeruginosa.
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Curr. Res. J. Biol. Sci., 2(6): 396-401, 2010
amino acid sequences of that gene were analyzed for
similarities to other known sequences published
previously on data base. The similarity search of the
partial nucleotide se quence o f azurin gene form
Pseudomonas aeruginosa exhibited significant similarity
to azurin gene on other species.
Large scale plasm id preparation of Azurin: The
pGEMAzu plasmid was prepared according to
manufacturer’s protocol and then digested by
EcoR 1/HindIII to validate the plasmid as shown in Fig. 2.
Subcloning of azurin into expression vector pET28a(+): pET vectors containing a sequence encoding
6xH is at the N-terminus of the inserted gene was used to
express the azurin gene. Both pET vector and pGEM Azu
construct were digested using the same restriction
enzymes EcoRI/HindIII. pGEMA zu was digested by the
enzymes to isolate the azurin ge ne then ligated into the
pET vector. The ligated plasmid w as transformed into
competent cells of BL21(DE3) strain. Target cells
containing the construct were selected using Kanamycine.
Plasm id was prepared from several clones, and to screen
for presence for the azurin construct, EcoRI/HindIII
double digestion was performed. The source vector, pET28a(+), gave a band of about 5000 bp whether digested
by EcoRI alone or with both EcoRI and HindIII. If the
AZ UR IN gene had been successfully cloned into pET28a(+), two bands (about 5000bp and 522 bp) would be
expected upon EcoRI and HindIII digestion. Figure 3
demonstrates the cutting patterns achieved for the
positively identified DN A construct. The clone produced
Fig. 1: Agarose gel electrophoresis of amplified azurin gene of
pseudomonas aeruginosa; M: DNA Molecular weight
marker 8 / HindIII, Lane 1: negative control, Lane 2:
amplified azurin gene of pseudomonas aeruginosa
Primer design and PCR am plification of azurin gene:
Specific primer pairs were designed for the PCR
amplification and sequencing of azurin gene of
Pseudomonas aeruginosa. The primers were designed
based on the known sequence of azurin in Pseudomonas
aeruginosa strain. Figure 1 represents the agarose gel
electrophoretic visualize of the PCR product of azurin
gene. Am plified azurin gene was purified and then
sequenced by using the designed primers; after omitting
unspecific bases, the obtained nucleotide sequence was
translated into the amino acid sequence using ExPASy
Translate Tool. The obtained nucleotide and deduced
Fig. 2: Restriction enzymes digeestions of pGEMAzu construct, lane 1, 4: DNA molecular weight marker 8 / HindIII, Lane 2:
Digested construct (pGEMAzu) by EcoR1/HindIII, Lan 3: Uncut construct
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Curr. Res. J. Biol. Sci., 2(6): 396-401, 2010
Fig. 3: Restriction enzymes digestions of pETAzu28 construct, Lane 1, 4: DNA molecular weight marker 8 / HindIII, Lane 2:
Digested pET-28a by EcoR1 / HindIII, Lane 3: Uncut pET-28a, Lane 5: Digested construct (pETAzu28) by EcoR1 / HindIII,
Lane 6: Uncut construct
Fig. 5: SDS-PAGE gel electrophoresis showing purification of
Azurin under nature condition
M:
Portein marker molecular weight (from top 45,
35, 25, 18, 14, 4KDa)
Lane 1: Total protein extract
Lane 2: Flow through
Lane 3: Wash
Lane 4: Elute of azurin at pH 8
Fig. 4: SDS-PAGE of total protein extracted from BL21
transformed with pETAzu construct
M:
Protein marker of BL21 sample without
induction
Lane 1: Soluble fraction of BL21 sample without
induction
Lane 2: Insoluble fraction of BL21 sample without
induction
Lane 3: Soluble fraction of BL21 sample induced with
1mMIPTG after 2 h
Lane 4: Insoluble fraction of BL21 sample after induced
with 1mMIPTG after 2 h
Lane 5: Soluble fraction of BL21 sample after induced
with 1mMIPTG after 4 h
Lane 6: Insoluble fraction of BL21 sample after induced
with 1mMIPTG after 4 h
Lane 7: Soluble fraction of BL21 sample after induced
with 1mMIPTG after 6 h
Lane 8: Insoluble fraction of BL21 sample after induced
with 1mMIPTG after 6 h
the DNA shown in lane 5 was chosen for further study on
the construct and named pETazu28.
Gene expression and protein purification: For the
expression of azurin protein in bacteria, the pETazu28
construct was transformed into competent cells of
BL21(DE3). This strain of E. coli contains a chromosomal
copy of the gene coding for the T7 RNA polymerase
under control of the lacUV5 promoter. In this strain,
lacUV5 is the only promoter known to direct transcription
by T7 RNA polymerase and is inducible by IPTG. Thus,
addition of IPTG to the growing culture of bacteria
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Curr. Res. J. Biol. Sci., 2(6): 396-401, 2010
caspases that indu ces apoptosis in cancer cells. p53
normally stops cells that are damaged from reproducing
and encourages them to commit apoptosis, but a majority
of cancer cells have damaged or missing p53. Now, they
dem onstrate that a smaller, 28 amino-acid fragment of
azurin also enters cancer cells selectively, but not in any
of the normal cells tested. This small m olecu le could
poten tially be used as a vehicle for cancer-targeted
chemotherapy (Arsenio et al., 2007a). So in this study an
attempt was done to isolate, clone and study the effects of
azurin on proliferation of human breast cancer cell line
(MC F7), hum an he patoc ellular carcinoma cell line
(HEP G2), human colon carcinoma cell line (HCT116) and
huma n normal melanocytes (HF B4).
The recom binan t azurin was successfully expressed
in BL2 1 E. co li as a soluble protein. Azurin has several
interesting characteristics. N ot only is it released from P.
aeruginosa on co ntact w ith cancer cells, but once
released, it enters preferen tially to cancer cells and o nly
marg inally to normal cells (Yamada et al., 2005 ). Azu rin
also inhibits growth of cancer cells as by interfering
in the
signaling pathways an d angiogen esis
(Arsenio et al., 2007 b), dem onstrating the multiple
pathways through which it exerts its inhibitory action.
Azurin is not only a potential anticancer drug candidate,
but it also forms complexes with many surface proteins,
thereby interfering in their entry to the host cells and
significa ntly suppressing their grow th (Ch audhari et al.,
2006). Thus azurin could potentially be used as a drug in
the treatment of such unrelated diseases as cancer. The
cytotoxic effect of the recombinant azurin was
successfully tested against human breast cancer cell line
(MC F7), human hepatocellular carcinoma cell line
(HEPG2) and human colon carcinoma cell line (HCT116)
and hum an no rmal m elano cytes (HFB4) cell line. The
study showed that when the concentration of azurin was
increased, the proliferation of colon carcinoma cell line
was strongly inhibited. The proliferation of breast cancer
cell line decreased to reach a plateau at azurin conc. 6
ug/uL. Whereas the proliferation of hepatocellular
carcinoma cell line gave a fluctuation resu lt and data
couldn’t be explained. Finally no effect was detected on
human normal melanocytes, making it a promising
antitumour drug.
Fig. 6: The relationship between azurin concentration and the
cells surviving rate
induces T7 R NA polym erase, which in turn transcribes
the target DN A in the plasmid. Transformed cells were
grown on liquid L.B. medium at 37ºC as described
previou sly and protein was induced by the addition of
IPTG and extracted from b acterial cells . The protein was
expressed as am ino-term inal H is-tagged pro teins.
Figure 4 shows the SDS-PAGE of soluble and
insoluble proteins extracted from BL21.
Purification of soluble protein was done by Ni-NTA
Gel by column chromatography. Figure 5 shows the
recombinant protein having a theoretical molecular weight
of 14.5 KD a because of the additional 6 amino acids,
histadine at the amino-termins of the protein. The effect
of the azurin on the different cell lines was studied by
SRB assay, and the data is shown in Fig. 6.
DISCUSSION
Much effort has been spent over the years in
developing wild-type or attenuated bacterial and viral
strains for the treatment of cancer (Jain and Forbes, 2001;
Kirn, 2000). Pseudomonas aeruginosa is known to secre te
the protein azurin as a weapon against invaders as
cancers, parasites and viruses. The production of such
weapons by pathogenic bacteria could provide important
insights into ho w a patho gen responds in the postcolonization state to impede o ther intruders for its own
surviv al. Moreover, these molecules might find use in the
pharmaceutical industry as next-generation therapeutics.
Pseudomonas aeruginosa, that grows in the soil and
marshes but is often found in the lung s of cystic fibrosis
patients, may offer a new route to cancer therapy. Based
on this surprising discovery, the researchers have shown
that P. aeruginosa preferentially enters human melanoma
and breast cancer cells, triggering apoptotic cell death.
They further discovered that azurin sets off this death
sequence by forming a complex with the well-known
tumor suppressor protein p53, stabilizing it, and activating
ACKNOWLEDGMENT
The au thors are thankful to the staff members in the
chemistry departm ent, biochem istry unit, Cairo University
for their valuable help.
ABBREVIATIONS
SDS-PAGE: Sodium dode cyl sulfate polyacrylamide gel
electrophoresis
SRB:
Sulforhodamine B
400
Curr. Res. J. Biol. Sci., 2(6): 396-401, 2010
PCR:
LB:
IPTG:
Polymerase chain reaction
Luria-Bertani broth
Isopro pyl-$-D thiogalactopyranoside
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