P53 SENSITIZES HUMAN COLON CANCER CELLS TO … · Ismail A. Ismail 1, Mohamed S. Gabry 2, Shimaa K....

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The Egyptian Journal of Biochemistry & Molecular Biology VOL 30 (N.2)185- 202 Dec. 2012

P53 SENSITIZES HUMAN COLON CANCER CELLS TO

HESPERIDIN THROUGH UPREGULATION

OF BAX AND P21

Ismail A. Ismail1, Mohamed S. Gabry

2, Shimaa K. Abdalla

2, Mona

A. Ibrahim2

1Laboratory of Molecular Cell Biology, Department of

Zoology, Faculty of Science, Assiut University,

Assiut 71516, Egypt. 2Department of Zoology, faculty of Science, Helwan University, Egypt.

Received 8/5/2012– Accepted 5/8/2012

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ABSTRACT

Hesperidin, a flavonoid found mainly in citrus, was reported to

inhibit growth and proliferation of several cancers, including colon

cancer cells. However, the question does p53 tumor suppressor protein

is required for the effect of hesperidin is not yet clarified. In the

present study, the effect of hesperidin on p53-expressing (HCT116

p53+/+

) and p53-knockout (HCT116 p53-/-

) human colon cancer cells

was investigated. Hesperidin inhibits cell growth of both HCT116

p53+/+

and HCT116 p53-/-

cells, however, it was more effective in p53-

expressing cells. Hesperidin induced G1 cell cycle arrest in only

HCT116 p53+/+

cells however induction of reactive oxygen species

(ROS) and apoptosis was induced in both cells. Furthermore,

hesperidin activates the proapoptotic (Bax) and cyclin dependent

kinase inhibitor (p21) in only HCT116 p53+/+

cells. Interestingly,

using p53 transcriptional inhibitor (pifithrin-α), hesperidin-inducing

Bax and p21 upregulation in only HCT116 p53+/+

cells was reduced

by cotreatment with pifithrin-α without inducing any changes in

HCT116 p53-/-

cells. Altogether; these results showed that hesperidin

induced apoptosis and G1 cell cycle arrest in colon cancer cells in a

p53/Bax - dependent and – independent , and p53/p21 - dependent

Ismail A. Ismail et all


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manners; respectively.

Keywords: Hesperidin; Colon Cancer; HCT116; P53, Pifithrin-α, Bax,

P21

INTRODUCTION

Hesperidin is considered as one of the most abundant flavonoids in

citrus fruits such as lemons and oranges (Gil-Izquierdo et al., 2001; Aranganathan and Nalini, 2009). Hesperidin (Fig. 1) is a flavanone

glycoside consisting of the flavone hesperitin bound to the

disaccharide rutinose; this sugar causes hesperidin to be more soluble

than hesperitin . It is also called Hesperetin 7-rhamnoglucoside, and

hesperetin-7-rutinoside, and present in nature as hesperidin (glycoside

form), and is deglycosylated to hesperitin (aglycone form) by

intestinal bacteria prior to absorption (Ameer et al., 1996). Hesperidin

has a wide range of pharmacological effects as potential anti-

inflamatory, and anti-cancer drug candidate in several kinds of cells

(Garg et al., 2001; Ou, 2002; Benavente-Garcia and Castillo, 2008).

Also, Hesperidin has been reported to exhibit significant anti-

inflammatory activity by modulating the prostaglandin synthesis and

COX-2 gene expression (Hirata et al., 2005). Colon cancer is a serious

health problem in most of the countries and is considered the second

leading cause of cancer mortality throughout the world (Jemal et al.,

2009; Jemal et al., 2010). Epidemiological studies and experimental

investigations suggested factors related to socioeconomic and dietary

conditions that may be important to colorectal cancer development.

Significant risk factors include lower fiber intake, high fat diet and

low calcium micronutrient intake may increase colon cancer incidence

(Park and Conteas, 2010). Frequent consumption of fruits, vegetables

and plant fiber is associated with a decrease in colorectal cancer

incidence (Steinmetz and Potter, 1991; Block et al., 1992). A number

of effective preventive and therapeutic natural product candidates

against colon cancer have been tested to reduce both cancer incidence

and mortality. The effects of hesperidin on several cancers, including

colon cancer have been received a considerable research attention

(Montanari et al., 1998; Park et al., 2008; Lee et al., 2010; Ghorbani et al., 2011). Hesperidin showed antioxidant effects in several cancer

cells (Al-Ashaal and El-Sheltawy, 2011). On the other hand, it is

stated that hesperidin induces oxidative stress and inhibits cell

P53 SENSITIZES HUMAN COLON CANCER CELLS ……..


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proliferation of breast cancer cells (So et al., 1996; Choi, 2007; Kamaraj et al., 2010), colon cancer cells (Park et al., 2008), and

prostate cancer (Lee et al., 2010). However, the precise role of p53 in

the hesperidin action is not yet fully understood. In the current study,

the effect of hesperidin on p53-expressing (HCT116 p53+/+

) and p53-

knockout (HCT116 p53-/-

) colon cancer cells was investigated to

evaluate the role of p53 in its action. To evaluate either p53 is

involved or not in the action of hesperidin and does p53 is essential

for induction of apoptosis and cell cycle arrest by hesperidin, we

investigated the regulatory effect of hesperidin on the p53 downstream

targets proapoptotic Bax and the cyclin-dependent kinase inhibitor

p21 using real time PCR. Next we evaluated the role of p53 in the

action of hesperidin in colon cancer cells via investigating the

attenuative effect of the p53 transcriptional inhibitor pifithrin-α on the

cell proliferation inhibition induced by hesperidin.

MATERIALS AND METHODS

Chemicals and reagents: Hesperidin, Pifithrin-α, MTT (3-[4,5-

dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide), 2’,7’-

dichlorodihydrofluorescein diacetate (DCFH2-DA), DMSO

(Dimethylesulphoxide), and propidium iodide were bought from

Sigma-Aldrich (St. Louis, MO. USA). RPMI cell culture media, fetal

bovine serum (FBS), and penicillin/streptomycin antibiotic mixture

were purchased from Gibco (Invitrogen cooperation, CA, USA).

RNase-A was from USB (Cleveland, Ohio, USA). All other chemicals

were obtained from standard chemical sources.

Cell lines and cell culture: Human colon cancer HCT116 p53+/+

(p53-

expressing) and HCT116 p53-/-

(p53 knockout) cell lines were

obtained as a gift from Mr. kang Keusung (Department of Pathology,

Kyungpook National University, Republic of Korea). Cells were

originally maintained in RPMI media containing 10% fetal bovine

serum (FBS) and 1% penicillin/streptomycin antibiotic. The cells were

grown at 37o C in a 5% CO2 incubator to be used in the following

experiments. Hesperidin was also dissolved in DMSO at a final

concentration of 100 mM as a stock solution.

Cell viability assay: The effect of hesperidin on the colon cancer

HCT116 p53+/+

and HCT116 p53-/-

cell growth and the attenuative

effect of Pifithrin-α (2.5 µM) on the hesperidin action were




188

investigated using MTT cell viability assay as previously described

(Park et al., 2008).

Cellular morphological changes: The morphological changes after

hesperidin treatment in colon cancer HCT116 p53+/+

and HCT116

p53-/-

cells were investigated as previously described (Ismail et al.,

2011).

Estimation of intracellular ROS level: The effect of hesperidin on the

intracellular ROS accumulation was detected using the H2O2-sensitive

fluorescent dye 2’,7’-dichlorodihydrofluorescein diacetate (DCFH2-

DA) as previously described (Cabello et al., 2009).

Hoechst staining and nuclear fragmentation: The effect of hesperidin

on the DNA damage and nuclear fragmentation of colon cancer

HCT116 p53+/+

and HCT116 p53-/-

cells was investigated using

Hoechst staining as previously described (Ismail et al., 2011).

Trypan blue and induction of apoptosis: The effect of herperidin on

cell viability of colon cancer HCT116 p53+/+

and HCT116 p53-/-

cells

was investigated using trypan blue exclusion method as previously

described (Shapiro et al., 1999). Trypan blue positive cells were

considered as died apoptotic cell fraction.

Cell cycle analysis: The effect of hesperidin on DNA content and cell-

cycle progression in colon cancer cells were estimated as previously

described (Ismail et al., 2011).

Gene expression analysis: To investigate the involvement of p53 and

its downstream targets in the action of hesperidin in the colon cancer

HCT116 cells, real time quantitative RT-PCR analysis was performed

as previously described (Ismail, 2009). The real time RT-PCR primers

were designed by the Primer Express 1.5 software (Applied

Biosystems) as follows: GAPDH forward,

5´-AGATCATCAGCAATGCCTCCTG-3´ and GAPDH reverse,

5´-ATGGCATGGACTGTGGTCATG-3´ and Bax forward,

5 َ◌–ATGGAGCTGCAGAGGATGATTG-3 َ◌ and reverse,

5 َ◌–CAGTTGAAGTTGCCATCAGCAA-3 َ◌, and p21 forward,

5 َ◌–CAAAGGCCCGCTCTACATCTT-3 َ◌ and reverse,

5 َ◌–AGGAACCTCTCATTCAACCGC-3 َ◌. ◌َThe expression of p21 and bax

were normalized using GAPDH as a house keeping gene.

Statistical analysis: MTT, Trypan blue, and ROS data are expressed as

means ± SEMs. Averages were calculated and graphed using

Microsoft Excel 2003. Calculations of real time PCR data were

performed by estimating the values of ∆cycle threshold (∆Ct) by




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normalizing the average Ct value of each treatment compared to its

opposite endogenous control (GAPDH) and then calculating 2–∆∆Ct

for

each treatment and statistical analysis for data as previously described

(Livak and Schmittgen, 2001). Data then were graphed and

statistically analyzed by the student t-test using Sigma Plot 8.0

software.

RESULTS

Effect of hesperidin on HCT-116 cell viability

Hesperidin inhibits HCT-116 p53+\+

and HCT116 p53-\-

colon cancer

cell proliferation after 24 hrs of treatment as indicated in a dose-

dependent manner (Fig.1). However, there was a differential

chemosensitivity between HCT-116 p53+\+

and HCT116 p53-\-

cells to

hesperidin. Since these results showed that hesperidin inhibited the

proliferation of p53-expressing (HCT-116 p53+\+

) cells more

effectively compared to p53-knockout (HCT116 p53-\-

) cells with

IC50s 55.8 and 70.8 u µM; respectively (Fig.1). Interestingly, hesperidin at lower concentrations (5 µM) increased cell proliferation

in only p53-knockout cells (Fig.1).

Fig.1 The effect of hesperidin on cell proliferation of HCT116 p53

+/+

and HCT116 p53-/-

colon cancer cells. Cells were treated with

different concentrations of hesperidin as indicated above for 24 hrs




190

and then cell viability was investigated using MTT cell viability assay

as shown in materials and methods. Data from three independent

experiments were analyzed to determine the statistical significances

using student t-test (*p < 0.01, #p < 0.05).

Effect of hesperidin on cell morphology and nuclear fragmentation

When cells were treated with hesperidin (100 µM) for 24hrs, cell

shrinkage was induced, and cell number and size were reduced. Cells

become rounded and lost their processes (Fig.2). In addition,

hesperidin induced cell detachment and a considerable cell ratio

become floating cells; showing a typical appearance of anoikis, when

compared to untreated cells (Fig.2). Hesperidin was also more

effective in p53 expressing cells (Fig.2).

Fig.2 The morphological changes in human HCT116 p53

+/+ and

HCT116 p53-/-

colon cancer cells after treatment with hesperidin.

Cells were treated with hesperidin (100 µM) for 24 hrs and then the

morphological changes were investigated using phase contrast

microscopy.




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Using nuclear Hoechst staining; we found that after treatment with

hesperidin cell number decreased, nuclei showed irregular shapes,

condensed with very small size, disruption of nuclear integrity and

chromatin condensation was also detected with characteristic features

of apoptosis (data not shown).

The effect of hesperidin on the induction of apoptosis

The apoptosis-inductive effect of hesperidin in human colon cancer

HCT116 p53+/+

and HCT116 p53-/-

cell lines was investigated after

treatment with different doses (0, 5, 12.5, 25, 50, and 100 µM) of

hesperidin for 24 hrs using trypan blue exclusion method and

examined using phase contrast microscopy. Total number of died cells

were counted as those took up trypan blue dye and appeared blue

color while viable cells are those excluded the trypan blue dye and

appeared with clear cytoplasm. Hesperidin induced cell death and

reduced cell viability in both HCT116 p53+/+

and HCT116 p53-/-

cells

(Fig.3). Hesperidin showed no effect at low doses (5 µM) in both cells

and then showed increased effect at higher doses (12.5, 25, 50, and

100 µM) in a dose-dependent manner (Fig.3). Also, hesperidin was

more effective in p53-expressing cells compared to p53-knockout

cells (Fig.3).

Fig.3 The effect of Hesperidin on cell death and induction of

apoptosis in HCT116 p53+/+

and HCT116 p53-/-

colon cancer cells was

determined using trypan blue exclusion method. Cells were treated

with hesperidin as indicated for 24 hrs and then harvested and stained




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with trypan blue and examined using phase contrast microscope. Total

number of died cells were counted by trypan blue exclusion method

and these results are averages of three independent experiments.

Statistical differences were investigated using student t-test (*p <

0.01).

The effect of hesperidin on the intracellular reactive oxygen species

(ROS)

The effect of hesperidin on the cellular release of ROS was

investigated by measuring the changes in the fluorescence using

DCFH-DA. Hesperidin treatment markedly increased the DCFH-DA

derived fluorescence at high concentrations (≥ 25 µM) in both cells,

and reduced at low concentrations (≤ 5 µM) in only p53-knockout

cells (Fig.4).

This hesperidin-mediated increase in ROS release was preferentially

higher in p53-knockout cells (HCT116 p53-/-

) compared to the p53-

expressing cells (HCT116 p53+/+

) (Fig.4). This high increase in p53-

knockout cells may due to the finding that the basal ROS level is

higher in p53-knockout cells compared to p53-expressing cells (Ismail

et al., 2011).

Fig.4 The effect of hesperidin on the intracellular reactive oxygen

species (ROS) in HCT116 p53+/+

and HCT116 p53-/-

colon cancer

cells. Cells were treated with hesperidin as the indicated

concentrations for 24 hrs and then incubated with 5 µg/ml of DCFH-

DA for 30 minutes and then processed for measuring ROS level as




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indicated in materials and methods. Data from three independent

experiments were analyzed to determine the statistical differences

using student t-test (*p < 0.01, #p < 0.05).

The effect of hesperidin on cell cycle progression

Cells were treated with different concentrations of hesperidin (25, 50,

100 µM), and 0.1%-treated DMSO as a control for 24 hrs as indicated

in materials and methods. In p53-expressing (HCT116p53+\+

) colon

cancer cells, hesperidin reduced cells at S and G2\M phase and

accumulated cells at G1 phase (Table 1), and the accumulation of cells

as G1 phase increased after hesperidin treatment in a concentration-

dependent manner (Table 1). Also, hesperidin induced apoptosis in

HCT116p53+\+

cells in a concentration dependent manner (Table 1).

However under the same experimental conditions, in p53-knockout

(HCT116 p53-\-

) cells, hesperidin showed no effect on cell cycle

progression at all concentration used in this experiment (Table 1).

Also, hesperidin induced apoptosis in HCT116 p53-\-

cells without

affecting cell progression distribution.

The effect of hesperidin on Bax and p21 expression

To test the role of p53 in the action of hesperidin-inducing cell

proliferation inhibition, we investigated the effect of hesperidin on the

p53 activation through investigation of its downstream targets

involved in apoptosis (Bax) and cell cycle arrest (p21). Interestingly,

hesperidin induced bax and p21 mRNA expression levels in only p53-

expressing HCT116 p53+\+

cells without showing any effect in p53-

knockout HCT116 p53-/-

cells (Fig. 5A & 5B). These data suggest that

hesperidin-inducing Bax and p21 takes place via activation of p53. To

confirm these data we blocked p53 transcriptional activity in both

cells via using p53transcriptional inhibitor pifithrin-α, and then

investigate the effect of hesperidin on bax and p21 transcriptional

activation. Interestingly, pifithrin- α was able to repress only p21

transcription without affecting Bax expression in only HCT116 p53+\+

cells. Consistently, we found that, pifithrin-α was able to significantly

inhibit the Bax and p21 transcription induced by hesperidin in only

HCT116 p53+\+

cells without any significant changes in HCT116 p53-

/- cells (Fig. 5A & 5B).




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Fig.5. The effect of hesperidin on the proapoptotic Bax (A) and cyclin

dependent kinase inhibitor p21 (B) transcriptional levels were

investigated using real time PCR. Cells were treated with either 100

µM of hesperidin (H-100), pifithrin-α (2.5 µM), or cotreatment of

hesperidin (100 µM) and pifithrin-α (H-100-α) in HCT116 p53+/+

and

HCT116 p53-/-

colon cancer cells for 24 hrs. For cotreatment, cells

were first pretreated with pifithrin-α for 30 minutes followed by

treatment with pifithrin-α. Bax and p21 mRNA expressions were then

evaluated using real time PCR. Data from three independent

experiments were analyzed and statistical significances were

determined using student t-test (*p < 0.01, #p < 0.05).

The effect of pifithrin-α on the cell viability inhibitory action of

hesperidin

Under the same experimental conditions as in Fig.5, the attenuative

effect of pifithrin-α on the antiproliferative activity of hesperidin was

investigated via cotreatment of pifithrin-α and hesperidin for 24 hrs as

shown in materials and methods. pifithrin-α (2.5 µM) was pretreated

for 30 minutes followed by cotreatment with hesperidin- pifithrin-α

for 24 hrs, and then cell viability assay was investigated using MTT

assay. pifithrin-α significantly reduced the cell proliferation inhibitory

effect of hesperidin in only HCT116 p53+\+

cells without any

significant additive or recovery role in HCT116 p53-/-

cells (Fig.6).




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Fig.6. The attenuative effect of pifithrin-α on cell proliferation

inhibition induced by hesperidin in HCT116 p53+/+

and HCT116 p53-/-

human colon cancer cells was assessed using MTT cell viability assay.

Cells were treated with 100 µM of hesperidin (H-100), or pretreated

for 30 minutes with pifithrin-α and/or followed by 24 hrs cotreatment

with hesperidin- pifithrin-α (H-100-α) as indicated and then cell

viability was measured. Data from three independent experiments

were analyzed and statistical differences were investigated using

student t-test (*p < 0.01).

Table.1. The effect of Hesperidin (hesp) on cell cycle progression in

HCT116 p53+/+

(Fig.6.A) and HCT116 p53-/-

(Fig.6.B) colon cancer

cells using flow cytometric analysis. In p53 expressing (HCT116

p53+/+

) cells hesperidin reduced cells at S and G2/M phase and

accumulated cells at G1 phase; however, in p53 knockout (HCT116 p53

-/-) cells; hesperidin showed no effect on cell cycle progression at

all concentrations used in this experiment.




196

DISCUSSION

According to the world health organization (WHO), about 1.1

million new cases diagnosed with colorectal cancer in 2010. This

estimation is consistent with the study that over one million new cases

of colorectal cancer estimated to be added in 2010 (Jemal et al.,

2009). Recent studies suggest that there is a close relationship

between cancer incidence and our nutritional behavior and nutrient

components (Ames et al., 1995). Also, several epidemiological studies

of colorectal cancer suggested that dietary agents play an important

role in the development of intestinal neoplasia (Ames et al., 1995).

Here we investigated the effect of hesperidin on human colon cancer

p53-expressing (HCT116 p53+\+

) and p53-knockout (HCT116 p53-\-

)

cells. Hesperidin has many biological activities; including anti-

inflammatory, antimicrobial, anticarcinogenic, antioxidant effects and

decreasing capillary fragility (Garg et al., 2001). Our data showed

that, hesperidin inhibits cell proliferation differentially in p53-

expressing and p53-knockout HCT116 cells. Proliferation inhibitory

action induced by hesperidin was more effective in p53-expressing

(HCT116 p53+\+

) cells despite the release of reactive oxygen species

(ROS) in both cells after hesperidin treatment, particularly at higher

concentrations. This ROS release is followed by DNA damage,

chromosomal condensation, and nuclear fragmentation. Also trypan

blue exclusion method showed that hesperidin induced cell death and

apoptosis in both cells. Furthermore, consistently to our results, it is

reported that hesperidin inhibits cell proliferation and induced

apoptosis via bax and caspase 3 activation in p53-expressing human

colon cancer SNU-C4 cells (Park et al., 2008).

On the other hand, hesperidin showed strong antioxidant properties

and less cytotoxicity to the B16 mouse melanoma cells (Zhang et al.,

2007). It is reported also that, hesperidin has powerful protection

effects on DNA damage, reducing the frequency of micronuclei

induced by γ-irradiation in mouse bone marrow cells (Hosseinimehr

and Nemati, 2006).

The tumor suppressor p53 protein plays important roles in a diversity

of physiologic functions. Cellular p53 functions as a tumor suppressor

by increasing genomic stability and inhibiting cell transformation. In

accordance with our results of the regulatory effect of hesperidin on




197

p53, it is stated that hesperidin induced p53 and inhibits NF-kB

activation to induce apoptosis in NALM-6 cells via Bax activation and

caspase 3 cleavage (Ghorbani et al., 2011). After DNA damage active

p53 works with p21 to induce cell cycle arrest (Bunz et al., 1998; Fuster et al., 2007). This is consistent with our data that, in only p53-

expressing (HCT116 p53+\+

) cells, hesperidin induced G1 cell cycle

arrest. Under the same experimental conditions, hesperidin induced

p21 expression in only HCT116 p53+\+

cells, suggesting that G1 cell

cycle arrest-induced by hesperidin takes place in a p53/p21-dependent

manner. The p53-regulated pro-apoptotic function is believed to

contribute to the efficacy of the anti-cancer therapy to enhance cellular

chemosensitivity via inducing p53-dependent growth inhibition

(Fuster et al., 2007). Currently, we found that hesperidin induced

apoptosis in both cells while activated Bax expression in only

HCT116 p53+\+

cells, suggesting that bax activation is not required for

hesperidin-inducing apoptosis in HCT116 colon cancer cells.

pifithrin-α, a potent and specific inhibitor of p53, the protective effects

of pifithrin-α involved both down-regulation of the transcriptional

activation of Bax and a direct inhibition of p53 translocation to

mitochondria (Dagher, 2004). The effects of pifithrin-α on p53

includes the inhibition of the transcriptional activation of the p53

downstream targets such as p21 and Bax and inhibition of p53

translocation to the mitochondria. These data were also supported by

knowing that pifithrin-α inhibits p53 function in vivo by blockade its

translocation to the nucleus and mitochondria (Leker et al., 2004).

Also, pifithrin-α exerts an anti-apoptotic effect by inhibiting not only

Bax but also by inhibiting the p53-regulated pro-apoptotic gene

PUMA (Luo et al., 2009). In the current data, pifithrin-α treatment

recovered cell growth inhibition induced by hesperidin in only

HCT116 p53+\+

cells. Therefore, in the present study we found that

pifithrin-α inhibits hesperidin action via inhibiting p53-inducing Bax

and p21 upregulation mediated via hesperidin treatment in HCT116

p53+\+

cells.

Altogether, it could be concluded that, p53 is not essential for

hesperidin-inducing apoptosis but essential for G1 cell cycle arrest in

HCT116 colon cancer cells. Also hesperidin induced Bax-independent

apoptosis in the absence of p53. These data suggest that, p53 has the

ability to sensitize and increase HCT116 cellular chemo-sensitivity to

hesperidin via inducing G1 cell cycle arrest and Bax-dependent




198

apoptosis. Also hesperdin failed to activate bax and p21 in the absence

of p53. Finally, these data suggest that hesperidin inhibits colon

cancer cell growth via several pathways including; p53/p21-dependent

G1 cell cycle arrest, and p53/Bax-dependent apoptosis as well as

p53/bax-independent apoptosis (Fig.7).

Fig.7. Schematic diagram shows the action and pathways involved in

the action of hesperidin in colon cancer HCT116 cells. Hesperidin

inhibits HCT116 cell proliferation in a p53-dependent and –

independent manners with differential chemosensitivity.

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الملخص العربي

يزيد من استجابة خ"يا سرطان القولون للھسبريدين عن طريق زيادة ال P53بروتين الBAX والP21

٢منى عبدالرحمن ابراھيم، ٢شيماء قطب، ٢محمد سيد جبرى، ١اسماعيل أحمد اسماعيل

طجامعة أسيو -كلية العلوم -قسم علم الحيوان –معمل بيولوجيا الخلية الجزيئية -١ جامعة حلوان -كلية العلوم -قسم علم الحيوان -٢

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P53 SENSITIZES HUMAN COLON CANCER CELLS TO … · Ismail A. Ismail 1, Mohamed S. Gabry 2, Shimaa K....

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