altered gene and protein expressions in colo 320 cell

Biomedicine: 2020; 40(2): 134- 142 April - June 2020

www.biomedicineonline.org Biomedicine- Vol. 40 No. 2: 2020

Altered gene and protein expressions in COLO 320 cell lines by Bauhinia variegata Linn.

Gayathri Gunalan1 and K. Vijayalakshmi2

1Research Officer (Biochemistry), Siddha Regional Research Institute (CCRS), Kuyavarpalayam, Puducherry, India-605013. 2Associate Professor, Department of Biochemistry, Bharathi Women’s College, Chennai, Tamil Nadu, India-600108.

(Received: December 2019 Revised: April 2020 Accepted: May 2020)

Corresponding author: Gayathri Gunalan. Email: [email protected]

ABSTRACT

Introduction and aim: Bauhinia variegata is a medium-sized deciduous tree belonging to the family

Caeselpinaceae. Various parts of this medicinal plant have been used in different systems of medicine for the

treatment of many ailments. The present study aims to unravel the role of active fractions on various pro and anti-

apoptotic gene expression in COLO 320 cell lines by RT-PCR and to study the effect of active fractions on the

expression of various pro and anti-apoptotic proteins in COLO 320 cell line by Western blotting.

Materials and Methods: The active fractions of ethanol extract of Bauhinia variegata (EBV) was obtained by

silica gel chromatography followed by MTT assay. The gene expression of TGF-β, COX – 2, iNOS, c-Myc, k-Ras,

β- Catenin, Bax, and Caspase 9 were studied by using reverse transcriptase PCR. The expression of the above gene

products was also studied using Western blotting.

Results: Upon treatment with active fractions, the expression of anti-apoptotic genes and proteins like TGF-β,

COX – 2, iNOS, c-Myc, k-Ras, and β- Catenin were decreased whereas the expression of pro-apoptotic genes and

proteins like Bax and Caspase 9 were increased.

Conclusion: From the above results, a tentative mechanism of the EBV's active fraction can be elucidated. Thus,

the present study may throw light on the mechanism of action of EBV's active fractions. Further identification of

the exact lead of EBV might help in the discovery of new anti-colon cancer drugs.

Keywords: Bioactivity; anticancer; flow cytometry; apoptosis; medicinal plants; colon cancer.

INTRODUCTION

atural products remain a prolific source for

the discovery of new drugs and drug leads

even from the Vedic period. Recent data

suggests that 80% of drug molecules are natural

products or natural compounds inspired (1).

Recently, it has been suggested that drug discovery

should not always be limited to single-molecule

discovery. The current belief ‘one drug – one

disease’ approach would become unsustainable in

future. Hence, poly herbal formulations or active

fractions of plant extracts could be analysed as an

alternative for the treatment for many diseases.

Medicinal and aromatic plants have

pharmacologically active molecules such as

flavonoids, saponins, tannins, alkaloids, essential oils

and other chemical compounds of diverse therapeutic

value. These plants have demonstrated their

contribution to the treatment of diseases such as

diabetes, malaria, sickle-cell anaemia, mental

disorders, and microbial infections. According to the

World Health Organisation, 80% of the world

population uses medicinal plants in the treatment of

diseases. Medicinal plants are rich in many

phytonutrients, which has proven cytotoxic, and

apoptogenic potential towards various types of

cancers including colon cancer. These molecules

belong to various phytochemical families and trigger

different signalling pathways (2).

Bauhinia variegata Linn. is a medium-sized

deciduous tree belonging to the family

Caeselpiniaceae. It is commonly known as Kachnar

in Hindi, Segappu Mandarai in Tamil, and Mountain

Ebony in English. Various parts of these plant-like

flowers, leaves, stem, bark, root, and seeds are

popular in various systems of medicine like

Ayurveda, Siddha, Unani, and Homeopathy in India

for the cure of many diseases. Following a large

number of claims on the curable properties of B.

variegata, considerable efforts have been made by

various researchers to justify its efficacy through

pharmacological investigations.

The leaves of B. variegata are used in the treatment

of skin diseases and stomatitis. They are also

reported to be anti-tumour and used to treat obesity.

The leaves are rich in reducing sugar and vitamin C.

The aqueous and ethanol extracts of B. variegata

have shown significant antioxidant activity (3). The

nonwoody aerial parts of B. variegata have shown

the anti-inflammatory activity against lipo-

polysaccharides and interferon-γ induced nitric oxide

and cytokines (4). Ethanol extract of B. variegata

leaves has shown hypoglycaemic activity. The

methanol extract of B. variegata leaves exhibited

antibacterial and antifungal activity against a wide

variety of microorganisms (5). The anti-carcinogenic

and anti-mutagenic potential of B. variegata leaves

were evaluated in Swiss albino mice using the

melanoma tumour model (6). Considering the diverse

N

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Gunalan and Vijayalakshmi: Altered gene and protein…… Bauhinia variegata Linn.


medicinal properties of B. variegata, the present

study was undertaken to evaluate the effect of B.

variegata on COLO 320 (human colon cancer cell

lines) proliferation and to unravel its mechanism of

action by using in vitro techniques.

In our previous studies, the active fractions of

B.variegata leaves showed appreciable cytotoxicity

towards COLO 320 cell lines (7). Hence, the present

study was aimed to determine the effect of active

fractions on the expression of various genes and their

products (proteins) which are involved in colon

carcinogenesis.

MATERIALS AND METHODS

Cell lines and reagents

The COLO 320 DM human colon cancer cell line

(human colon adenocarcinoma established, ATCC

catalog: CCL 220) was obtained from the American

Type Culture Collection (ATCC), USA. Rosewell

Parker Memorial Institute (RPMI) medium, Fetal calf

serum (FCS), DMSO, penicillin-streptomycin

solution, and all other reagents used in the present

study were of analytical grade and purchased from

Sigma (St Louis, MO). ONE STEP-RNA reagent

was purchased from Biobasic Inc. Reverse

Transcription system kit was purchased from

Promega, France. Integrated DNA Technologies and

Ocimum Biosolutions synthesized primer sequences.

Maintenance and storage of cell culture

COLO 320 DM was used for the study. The cell line

was grown as monolayers up to 80% confluence in

RPMI 1640 supplemented with 10% FBS and 1%

Penicillin/Streptomycin at 37°C, 5% CO2 and

humidified air.

Plant material and extraction

B.variegata leaves were collected from Chennai and

it was authenticated by Director, Plant Anatomy

Research Centre (Authentication reference no.

PARC/2010/670 dated 22/12/2010).

The leaves were washed with water, shade dried, and

powdered coarsely. The crude extract was obtained

after maceration with 95% ethanol at room

temperature for 72 h and repeated till exhaustion of

the material. Thereafter, the ethanol crude extract

was distilled, evaporated, and dried under reduced

pressure to yield ethanol extract of B.variegata

leaves, EBV (yield 8%).

Preparation of active fractions

The ethanol extract of B. variegata leaves was

separated through silica gel G (60-120) column

chromatography with various solvents of increasing

polarity (n-hexane, chloroform, ethyl acetate, and

methanol) in gradient steps and final elution was

performed with 100% methanol. All the fractions

were applied to the pre-coated silica gel TLC plates

and chromatographed using the appropriate solvent

system. Plates were examined under UV and visible

light to combine similar fractions, thus resulting in

11 different fractions, which were designated from

Fraction F1 to Fraction F11. Finally, fractions were

concentrated under vacuum. All the 11 fractions were

subjected to cytotoxicity assays like MTT assay

using human colon cancer cell lines, COLO 320.

Among the 11 fractions, two (Fraction I and Fraction

II) were found to have good cytotoxic activity and

hence they were assigned as active fraction I and II

respectively (7).

Treatment with the active fractions

Active fractions (25μg/ml) of extract were dissolved

in dimethyl sulfoxide (DMSO) (Sigma, USA) and

were used for further treatment. 5 × 105 COLO 320

cells were seeded in (3mL total volume) 96-well

multi dishes and were incubated with the active

fractions at the IC50 concentration (7.8, 15.62, 31.25

µM/ml for an active fraction I and 15.62, 31.25, 62.5

µM/ml for active fraction II) for 48 hrs at 37°C in

complete growth medium and used for both gene and

protein expression studies.

Gene expression studies

The expression of genes was studied by using reverse

transcriptase-PCR (RT-PCR). The housekeeping

gene β- Actin was used as a control. At the end of

incubation, the cells were rinsed twice with PBS and

trypsinized in trypsine- 0.02% EDTA mixture. After

centrifugation for 5 min at 500 ×g at 4°C, the

supernatant was removed, and the pellet was used for

RT-PCR studies.

RT-PCR

Total RNA from cell lines was isolated using ONE

STEP-RNA Reagent (Biobasic Inc.). RNA quality

and integrity were confirmed through the A260/A280

ratio and agarose gel electrophoresis respectively.

After RNA isolation, RNA was immediately reverse

transcribed with Reverse transcription system of

Promega, France. For RT-PCR reaction, 1-2µg of

RNA was used which corresponds to 1-10 µl of total

RNA isolate. The cDNA was prepared and Gene

Specific PCR was used to amplify TGF-β, COX-2,

iNOS, c-myc, k-ras, β-catenin, Bax, and Caspase 9

separately. The housekeeping gene, namely β-actin,

was also done in order to assess the quality of PCR.

Primer sequences (synthesized by Integrated DNA

Technologies and Ocimum Biosolutions) used to

analyze different genes are given in Table 1. PCR

products were separated on a 1.5% agarose gel and

stained with ethidium bromide. The gel was run at 50

V for 90 min and the intensity of individual bands

was semi-quantitatively assessed using NIH Image.

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Table 1: Primer sequences used for RT-PCR analysis

Gene

Name

Forward Primer Reverse Primer

TGF-β 5′-CGCCATCTATGAGAAAACC-3′ 5′-GTAACGCCAGGAATTGT-3’

Cox-2 5′-CAAAAGCTGGGAAGCCTTCT-3’ 5′-CCATCCTTGAAAAGGCGCAG-3′

iNOS 5’-CGAGGAGGCTGCCTGCAGACTGG-3’ 5’-CTGGGAGGAGCTGATGGAGTAGTA-3’

c-myc 5’-AATGAAAAGGCCCCCAAGGTAGTTATCC-3’ 5’-GTCGTTTCCGCAACAAGTCCTCTTC-3’

k-ras 5′-GAGTTTGTATTAAAAGGTACTGGTGGA-3′ 5′-TGTATCAAAGAATGGTCCTGCAC-3′

β -catenin 5’-AAATGGTCCGATTAGTTTCCT-3’ 5’-TGAATGAATTAAAAGTTTAATTCTG-3’

Bax 5′- ACCAAGAAGCTGAGCGAGTGTC- 3′ 5′- GGCAGACCGTGACCATCTTTGT- 3′

Caspase-9 5’-ATGGACGAAGCGGATCGG-3’ 5’-CCCTGGCCTTATGATGTT-3′

β-Actin 5′-ACTGGCTTGTTCAAAGG-3′ 5′-CAGCGTGTAAACGGAG-3’

Protein Expression Studies

Preparation of the protein extracts

Briefly, COLO-320 cells (1.5×106) were seeded onto

100mm culture dishes in the presence or absence of

extract and were treated for 24h with indicated

concentrations. The medium was removed and the

cells were washed with PBS (0.01M, pH 7.2) for

several times and lysed on ice in lysis buffer

containing 100 μg/ml phenylmethylsulfonyl fluoride

(PMSF), 50mM Tris base at pH 8.0, 150mM NaCl,

1% NP-40, and 1g/ml aprotinine. The supernatants

were collected by centrifugation at 10,000×g for

5min at 4°C and were used as the cell protein

extracts. The harvested protein concentration was

measured using a protein assay kit (Bio-Rad).

Western Blotting

Equal amounts of proteins from each extract were

applied to 12% SDS–polyacrylamide gel and electro-

transferred onto PVDF membrane. Proteins were

blocked overnight with 5% non-fat dried milk in

PBS-T at 2-8°C. After washing in PBS containing

0.1% Tween 20 for 3 times, the membrane was

incubated with the specific primary antibodies

[namely anti-Bax (1:500), anti-Caspase 9 (1:1000),

anti-β-actin (1:5000), and anti-iNOS (1:500)

antibodies in 5% (w/v) skim milk in PBST. After

overnight incubation at 4°C, the membrane was then

washed three times with TBST, incubated further

with alkaline phosphatase-conjugated goat anti-

mouse antibody or anti-rabbit antibody at room

temperature for 2 hours, and then washed three times

with TBST. After reaction with horseradish

peroxidase conjugated goat anti-mouse antibody,

chemiluminescence ECL PLUS detection reagents

following the manufacturer’s procedure (Amersham

Bioscience) visualized the immune complexes (8).

Statistical Analysis

The statistical evaluation involved a two-way

analysis of variance (ANOVA) followed by Duncan's

multiple range test (DMRT). Statistical significance

was set at P<0.05.

RESULTS AND DISCUSSION

Anti-apoptotic genes and protein expression

Gene and protein expression of β – Catenin,

iNOS, and COX-2

The RT-PCR and immune blot analysis of β-catenin,

iNOS, and COX-2 expressions in COLO 320 cell

lines upon treatment with the active fractions I and II

were shown in figure 1, 2, 7 and 8. In the present

study, COLO 320 cell lines were treated with both

the active fraction I and active fraction II separately

at the concentration range of 7.8 – 62.5 μM/ml. After

incubation, the gene and protein expressions were

studied using RT-PCR and Immunoblotting

respectively. On treatment with the active fractions,

the expression of β-catenin, iNOS, and COX-2 genes

and their products (protein) were down-regulated. At

IC50 concentration (10.3μM/ml), active fraction I

decreases the expression of β-catenin, iNOS and

COX-2 proteins much significantly (P< 0.001, P<

0.001 and P< 0.001 respectively) than Active

Fraction II. β- Catenin is reported to play a critical

role in NO induction of COX-2 in colon epithelial

cells. Furthermore, Howe et al., demonstrated that β-

catenin stimulates COX-2 expression (9). More

recently, it has been reported that NO increases

PEA3 expression through β-catenin/APC pathway

and directly augments the COX-2 promoter activity

of the PEA3/p300 in YAMC cells (10). In the present

study, the decreased expression of β-catenin with

active fractions (I and II) treatment was observed.

Dashwood et al., (2002) in which they documented

that β-catenin expression was decreased by the

treatment with green tea, white tea, and EGCG (11),

reported a similar finding. Down-regulation of β-

catenin might decrease the expression of its target

genes like iNOS and COX-2 in COLO 320 cells.

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Biomedicine: 2020; 40(2): 134- 142 April - June 2020


Fig. 1: Impact of fraction I on anti-apoptotic genes: The level of genes was normalized to the level of β-actin using a Molecular

Dynamics densitometer and expressed as relative intensity with β-actin. *** - P<0.001, ** - P<0.01, *- P<0.05

COX-2, as well as iNOS enzymes, has been

recognized as a promising target for colon cancer

prevention. Consequently, many novel COX-2

inhibitors and NO inhibitors have been considered to

be chemo-preventive agents on DMH/AOM-induced

colon carcinogenesis (12). Many natural products

like curcumin, astaxanthin, and ginsenosides

decrease the expression of COX-2 in AOM induced

colon cancer model (13, 14). Morin, a plant flavonoid

supplementation to DMH administered rats down-

regulated NF-κB pathway and its downstream

inflammatory mediators like tumour necrosis factor-

alpha (TNF-α), interleukin 6 (IL-6), cyclooxygenase

2 (COX-2) and prostaglandin (PGE-2) (15). In the

present study, similar findings were observed when

COLO 320 cells were treated with active fractions of

B. variegata. The active fractions, by decreasing the

expression levels of iNOS and COX-2 might allow

the COLO 320 cells to enter the apoptotic pathway

and thereby prevents cancer progression.

Fig. 2: Impact of fraction II on anti-apoptotic genes: The level of genes was normalized to the level of β-actin using a Molecular


On increasing the concentration of both the active

fractions, there was an increase in the down-

regulation of all the three genes and their protein

products. The increased activity of active fraction I

may be due to its phytochemical content. By GC-MS

analysis, bioactive constituents of active fraction I

was identified, and their biological functions were

described in previously published paper (7). Active

fraction I contain many hydrophobic substances

(lipids). These lipid fractions may influence the

down-regulation of β-Catenin, iNOS, and COX-2.

Reddy et al., previously reported a similar finding.

They stated that the lipid fractions of wheat bran

have tumour-inhibiting properties and decrease the

expression of β-Catenin, iNOS, and COX-2 (16).

Thus, the present study is in agreement with the

previous findings. Thus, EBV's active fractions might

help in the prevention of colon cancer.

Gene and protein expression of k-ras

In the present study, the impact of EBV active

fractions on the expression of the k-ras gene and its

product (protein) was analysed by incubating the

COLO 320 cells with the active fractions and so

examined by RT-PCR and western blotting

procedures respectively. Both active fractions I and

active fraction II decreased the expression of k-ras in

an exceedingly dose-dependent manner (Figures 3, 4,

7, and 8) significant down-regulation was observed

for both the active fraction (P< 0.001). Many authors

have studied the overexpression of the k-ras gene in

colon carcinoma (17), pancreatic cancer, and in

follicular and undifferentiated carcinomas of the

thyroid. As the k-ras were down regulated upon

active fractions treatment, it might prevent the

promotion of colon cancer. A similar finding was

reported by Reynoso-Camacho et al., in which they

state that lutein, a carotenoid, down regulated the

expression of k-Ras gene/ protein (18). Singh et al.,

also demonstrated that D, L, α-difluoromethyl

ornithine (DFMO), an ornithine decarboxylase

inhibitor, and piroxicam, a NSAID have decreased

the expression of the k-Ras protein in AOM model of

colon cancer (19). Besides, its target genes like c-

137



myc, iNOS, and COX-2 might also get down

regulated by the inactivation of k-Ras protein and

leads to the induction of apoptosis.

Gene and Protein Expression of c-myc

In the present investigation, c- myc gene and protein

expression was studied in active fractions treated

COLO 320 cells. The expression of both the gene

and protein were decreased by active fractions

treatment (Figure 3, 4, 7, and 8). Active fraction I

and II down-regulates c-myc expression significantly

(P<0.01). The c-myc gene is amplified in various

human cancers, including lung carcinoma, breast

carcinoma, and colon carcinoma. The c-myc gene is

frequently deregulated and overexpressed in colon

cancer, and strategies designed to inhibit c-myc

expression in cancer cells may have considerable

therapeutic value. In one study, an inverse correlation

between increased APC expression level and

decreased c-myc mRNA expression was reported

(20). Tanaka et al., have reported that auraptene and

collinin might inhibit inflammation and oxidative

stress via a c-myc molecule and thus they exhibit

protective effect after treatment with AOM and DSS

(21).

Fig. 3: Impact of fraction I on anti-apoptotic genes: The level of genes was normalized to the level of β-actin using a Molecular


Xing et al., have already reported that the down-

regulation of c-myc inhibited cell growth and

induced apoptosis in COLO 320 cells (22). Vadde et

al., have reported that Triphala has suppressed the

protein levels of c-Myc and cyclin D1, key proteins

involved in proliferation, and induced apoptosis

through elevation of Bax/Bcl-2 ratio (23). Since the

active fractions decrease the c-myc expression, they

might decrease the expression of its target genes like

iNOS and COX-2. Thus, the active fractions may

induce apoptosis through down-regulation of COX-2

and thereby inhibits cell proliferation and tumour

progression.

Fig. 4: Impact of fraction II on anti-apoptotic genes: The level of genes was normalized to the level of β-actin using a Molecular


Gene and protein expression of TGF-β

In the current investigation, the impact of EBV active

fractions (I and II) on the expression of TGF - β gene

and protein were studied. From the result, it was

found that the active fractions, both I and II (Figure

3, 4, 7, and 8) could down-regulate the expression of

TGF- β significantly (P< 0.001). TGF- β can then

decrease the expression of its target genes like COX-

2 and thus it mediates apoptosis in COLO 320 cells.

Increased expression of TGF-β was observed in

animal breast cancer model (24). Han et al., has

reported elevated TGF-β expression in skin

carcinogenesis (25). Ragini et al., reported that TGF

– β was down-regulated by EGCG in human

138



bronchial epithelial 21 BES cells (26). In the present

investigation, it was found that TGF – β gene/protein

was inactivated by the active fractions. This result

was in agreement with that of previous findings.

Pro-apoptotic Genes and Protein expression

Bax

In the present investigation, total Bax RNA levels of

COLO 320 cells treated with active fraction was

analyzed using Reverse Transcriptase PCR

individually. PCR products were analysed on 1.5%

agarose gel. The effect of EBV's active fractions on

the expression of the Bax gene was represented in

Figures 5 and 6. The immunoblot of Bax protein was

shown in figure 9. The expression of Bax was

significantly (P< 0.001) increased upon treatment

with the active fractions. Both the active fraction I

and active fraction II increases Bax expression

significantly equal. Higher expression of the Bax

gene induces mitochondrial permeability transition,

which may lead to the release of cytochrome c from

the mitochondria and results in apoptotic death of the

cells. The up-regulation of Bax protein may cause the

release of cytochrome c, which again activates

caspases 3, 9, and finally, apoptosis occurs. Bax

plays a central role in regulation and commitment to

programmed cell death. Bax inhibits the action of the

Bcl2, the apoptosis preventing gene and may directly

induce apoptosis. Thus, in COLO 320 cell lines due

to an increase in Bax expression, the caspases

activity may increase leading to apoptosis

sequentially. Many natural products were found to

have an influence on Bax gene/protein expression.

Green tea polyphenols like EC, ECG, EGC, EGCG

increases the expression of Bax in mouse skin tumors

(27). Survival was also high in groups with high Bax

expression. Khan et al., has reported that oral

administration of naturally occurring chitosan-based

nano formulated green tea polyphenols, EGCG can

up-regulate Bax expression in pancreatic cancer (28).

Bahadori et al., have also reported that the chrysin

exerted its anticancer potential by upregulating

caspase 3, caspase 9, and Bax proteins (29). A

similar finding was observed in the present study

also. Upon active fractions (I and II) treatment, the

pro-apoptotic Bax level was increased and thus it

induces apoptosis in COLO 320 cells via caspase-9

up-regulation.

Fig. 5: Impact of fraction I on pro-apoptotic genes: The level of genes was normalized to the level of β-actin using a Molecular Dynamics

densitometer and expressed as relative intensity with β-actin. *** - P<0.001, ** - P<0.01, *- P<0.05

Fig. 6: Impact of fraction II on pro-apoptotic genes: The level of genes was normalized to the level of β-actin using a Molecular

Dynamics densitometer and expressed as relative intensity with β-actin *** - P<0.001, ** - P<0.01, *- P<0.05

139



Fig. 7: Impact of fraction I on anti-apoptotic proteins: The level of proteins was normalized to the level of β-actin using a Molecular


Fig. 8: Impact of fraction II on anti-apoptotic proteins: The level of proteins were normalized to the level of β-actin using a Molecular

Dynamics densitometer and expressed as relative intensity with β-actin *** - P<0.001, ** - P<0.01, *- P<0.05

Caspases- 9

Fig. 5, 6, 9, and 10 depict the impact of active

fractions on the pro-apoptotic gene and protein

expression. In the present investigation, active

fraction treated COLO 320 cell lines increases the

expression of caspase- 9 significantly. Both the

active fraction I and II increases the expression of

caspase-9 (P<0.001) significantly. This confirms that

the active fractions may induce apoptosis In vitro by

mitochondrial dysfunction and cell cycle inhibition.

Many chemopreventive agents act by activation of

caspases 9 and 3. In the present, study also, the active

fractions I and II up-regulates caspase 9 and thus

induces apoptosis. A similar finding was reported by

Rashmi et al., have documented the effect of

curcumin on Caspase 9 expression in colon cancer

cell lines SW480 and SW620 (30). Khan et al., also

reported the up-regulation of caspase 9 by the oral

140



administration of chitosan-based EGCG nano-

formulation (28). The proteolytic activation of

upstream caspases, which in turn activates the

effector caspases, may ultimately involve in the

proteolysis of cellular components of the apoptotic

cell. Thus, apoptosis was stimulated in COLO 320

cell lines by the active fractions of EBV. Hence, the

active fractions could act as chemo-preventive agents

for colon cancer.

Fig. 9: Impact of fraction I on pro-apoptotic proteins: The level of proteins was normalized to the level of β-actin using a Molecular


Fig. 10: Impact of fraction II on pro-apoptotic proteins: The level of proteins were normalized to the level of β-actin using a Molecular

Dynamics densitometer and expressed as relative intensity with β-actin) *** - P<0.001, ** - P<0.01, *- P<0.05

CONCLUSION

From the above observations, a tentative mechanism

of the active fractions of EBV can be constructed as

shown in figure 11. On treatment with the active

fractions, k-ras and β-catenin were down regulated.

This shows that the active fractions have protected

these genes from mutations; thereby the downstream

targets like c-myc, iNOS, and COX-2 were not

activated. Cell proliferation was inhibited by the up-

regulation of pro-apoptotic genes like Bax and

caspase 9. Since caspase 9 is an initiator caspase, it

may form an apoptosome and induce apoptosis in

colon cancer cells. Thus, the present investigation

may throw light on the mechanism of action of

EBV's active fractions. Further identification of the

exact lead of the active fractions might help in the

discovery of new anti-colon cancer drugs.

Fig. 11: Tentative mechanism of EBV’s active fractions

ACKNOWLEDGMENT

All authors have contributed substantially to the

design, performance, analysis, and manuscript

writing. The authors sincerely acknowledge (Late)

Dr. A. Saraswathy, Ex-Director, Captain Srinivasa

Murthy Drug Research Institute, Arumbakkam,

141



Chennai, for her mentorship and valuable suggestion

during the project work.

CONFLICT OF INTEREST

The authors confirm that this article content has no

conflicts of interest.

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https://www.sciencedirect.com/science/journal/07533322

https://www.sciencedirect.com/science/journal/07533322

https://www.sciencedirect.com/science/journal/07533322/107/supp/C

http://www.ncbi.nlm.nih.gov/pubmed?term=Hongxing%20Z%5BAuthor%5D&cauthor=true&cauthor_uid=18454692

http://www.ncbi.nlm.nih.gov/pubmed?term=Nancai%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=18454692

http://www.ncbi.nlm.nih.gov/pubmed?term=Wen%20S%5BAuthor%5D&cauthor=true&cauthor_uid=18454692

http://www.ncbi.nlm.nih.gov/pubmed?term=Guofu%20H%5BAuthor%5D&cauthor=true&cauthor_uid=18454692

http://www.ncbi.nlm.nih.gov/pubmed?term=Yanxia%20W%5BAuthor%5D&cauthor=true&cauthor_uid=18454692

http://www.ncbi.nlm.nih.gov/pubmed?term=Hanju%20H%5BAuthor%5D&cauthor=true&cauthor_uid=18454692

http://www.ncbi.nlm.nih.gov/pubmed/18454692

http://www.ncbi.nlm.nih.gov/pubmed/18454692

altered gene and protein expressions in colo 320 cell

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