Resveratrol induces apoptosis in human esophageal carcinoma cells

Abstract

AIM: To investigate the apoptosis in esophageal cancer cells induced by resveratrol, and the relation between this apoptosis and expression of Bcl-2 and Bax.

METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of esophageal cancer cell line EC-9706 before and after the resveratrol treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax.

RESULTS: Resveratrol inhibited the growth of esophageal cancer cell line EC-9706 in a dose-and time-dependent manner. Resveratrol induced EC-9706 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. TUNEL assay showed that after the treatment of EC-9706 cells with resveratrol (10 mmol·L^-1) for 24 to 96 hs, the AIs were apparently increased with treated time (P < 0.05). Immunohistochemical staining showed that after the treatment of EC-9706 cells with resveratrol (10 mmol·L^-1) for 24 to 96 hs, the PRs of Bcl-2 proteins were apparently reduced with treated time (P < 0.05) and the PRs of Bax proteins were apparently increased with treated time (P < 0.05).

CONCLUSION: Resveratrol is able to induce the apoptosis in esophageal cancer. This apoptosis may be mediated by down-regulating the apoptosis-regulated gene Bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.

Key Words: $[Keywords]

INTRODUCTION

The Bcl-2 family plays a crucial role in the control of apoptosis. The family includes a number of proteins which have homologous amino acid sequence, including anti-apoptotic members such as Bcl-2 and Bcl-x_L, as well as pro-apoptotic members including Bax and Bad. In in vitro experiments, overexpression of Bcl-2 has been shown to inhibit apoptosis, but overexpression of Bax has been shown to promote apoptosis.

Resveratrol, a phytoalexin found in grapes, fruits, and root extracts of the weed Polygonum cuspidatum, is an important constituent of Chinese folk medicine. Indirect evidence suggests that the presence of resveratrol in white and rose wine may be helpful to reduce risks of coronary heart disease which would be achieved by moderate wine consumption. This effect has been attributed to the inhibition of platelet aggregation and coagulation, in addition to the antioxidant and anti-inflammatory activity of resveratrol. Moreover, a recent report shows that resveratrol is a potent cancer chemopreventive agent in three major stages of carcinogenesis. The anti-tumor activity of resveratrol might be related to induce the apoptosis of tumor cells but the precise mechanism of antitumor activity is not well understood.

MATERIALS AND METHODS

Materials

Resveratrol and MTT were obtained from Sigma Chemical Co. Ltd. In situ cell detection kit, anti-Bcl-2 monoclonal antibody and anti-Bax monoclonal antibody were purchased from Beijing Zhongshan biotechnology Co. Ltd. Stock solution of resveratrol was made in dimethylsulfoxide (DMSO) at a concentration of 100 mmol·L^-1. Working dilutions were directly made in the cell culture medium. Human esophageal carcinoma cell line EC-9706 was obtained from Professor Ming-Rong Wang in Chinese Scientific Academy.

Methods

Cell culture EC-9706 cells were incubated in RPMI 1640 supplemented with 100 ml·L^-1 fetal bovine serum, 100 kU·L^-1 penicillin, 100 mg·L^-1 streptomycin and 2 mmol·L^-1 L-glutamine under 50 ml·L^-1 CO₂ in a humidified incubator at 37 °C. EC-9706 cells were incubated for different time periods in the presence of resveratrol at 0.1, 1, 10 and 100 mmol·L^-1.

MTT assay 1 × 10⁵ cells/well in a 96-well plate after incubation for 24 hs were treated with different concentrations of resveratrol (0.1, 1, 10, 100 mmol·L^-1)for 24, 48, 72, 96 hs respectively. 10 µL of 5 g·L^-1 of MTT was added to the medium in three wells at every dose and incubated for 4 hs at 37 °C. Culture media were discarded followed by adding 0.2 ml DMSO and vibrating for 10 min. The absorbance (OD) was measured at 570 nm using a microplate reader. The cell growth inhibitory rate was calculated as follows: [(OD of control group -OD of experimental group)/(OD of control group-OD of blank group)] × 100%.

Transmission electron microscopy Cells treated with 10 mmol·L^-1 resveratrol were harvested after incubation for 24 hs. Subsequently the cells were fixed in 4% glutaral and immersed with Epon 821, imbedded for 72 hs at 60 °C, the cells were prepared into ultrathin section (60 nm) and stained with uranyl acetate and lead citrate. Cell morphology was observed by transmission electron microscopy.

TUNEL assay Apoptosis of EC-9706 cells was evaluated by using an in situ cell detection kit. The cells were treated in the presence or absence of 10 mmol·L^-1 resveratrol for 24 to 96 hs and fixed in ice-colded 80% ethanol for up to 24 hs, treated with proteinase K and then 0.3% H₂O₂, labeled with fluorescein dUTP in a humid box for 1 h at 37 °C. The cells were then combined with POD-Horseradish peroxidase, colorized with DAB and counterstained with methyl green. Controls were received the same management except the labeling of omission of fluorescein dUTP. Cells were visualized with light microscope. The apoptotic index (AI) was calculated as follows: AI = (Number of apoptotic cells/Total number) × 100%.

Immunohistochemical staining Immunohistochemical staining was done by an avidinbiotin technique. EC-9706 cells treated in the presence or absence of 10 mmol·L^-1 resveratrol for 24 to 96 hs were grew six-well glass slides and were fixed by acetone. After washing in PBS, the cells were incubated in 0.3% H₂O₂ solution at room temperature for 5 min. The cells were then incubated with anti-Bcl-2 or anti-Bax monoclonal antibody at a 1:300 dilution at 4 °C overnight. After washing in PBS, the second antibody, biotinylated antirat IgG, was added and the cells were incubated at room temperature for 1 h. After washing in PBS, ABC compound was added and then incubated at room temperature for 10 min. DAB was used as the chromagen. After 10 min, the brown color signifying the presence of antigen bound to antibodies was detected by light microscopy and photographed at × 200. Controls were managed the same as the experimental group except the incubation of the primary antibody. The positive rate (PR) was calculated as follows: PR = (Number of positive cells/Total number) × 100 %.

Statistical analysis

Datas were analyzed by the paired two-tailed Student t test, and significance was considered when P < 0.05.

RESULTS

MTT assay

EC-9706 cells were exposed to increasing concentrations (0.1 mmol·L^-1 to 100 mmol·L^-1) of resveratrol for 24 to 96 hs, respectively. EC-9706 cells showed the mortality in a dose- and time-dependent manner. The data were summarised in Table 1.

Table 1 The inhibitory effect of resveratrol on EC-9706 cells.

Time(h)	Inhibitory rate(%)
	RPMI-1640	Resveratrol (mmol.L-1)
		0.1	1	10	100
24	0.0	9.2a	18.4b	21.8b	34.4b
48	0.0	17.1b	21.3b	36.7b	45.5b
72	0.0	23.9b	37.5b	48.6b	59.9b
96	0.0	36.6b	45.7b	56.6b	88.8b

^aP < 0.01,

^bP < 0.001 vs the control group.

Morphological changes

After treatment of EC-9706 cells with resveratrol (10 mmol·L^-1) for 24 hs, some cells appeared apoptotic characteristics including chromatin condensation, appearance of chromatin crescent, nucleus fragmentation and of formation apoptotic body which could be seen by transmission electron microscope.

TUNEL assay

Apoptotic cell death was determined by TUNEL assay according to the manufacture’s instructions. Positive staining located in the nucleus (Figure 1). After treatment with resveratrol (10 mmol·L^-1)for 24 to 96 hs, AIs were apparently increased with treated time (P < 0.05) (Table 2).

Figure 1 TUNEL assay of apoptotic cells induced by resveratrol with ( × 200).

Table 2 Apoptotic Index (AI) of treated EC-9706 cells by resveratrol.

Time(h)	AI(%)
0	1.46 ± 2.51
24	4.96 ± 2.67a
48	16.64 ± 1.87a
72	27.94 ± 3.32b
96	37.46 ± 3.96b

^aP < 0.05,

^bP < 0.01 vs the control group.

Expression of Bcl-2 proteins

Positive staining located in the cytoplasm (Figure 2). After treatment with resveratrol (10 mmol·L^-1)for 24 to 96 hs, PRs of Bcl-2 proteins were apparently reduced with treated time (P < 0.05) (Table 3). This suggested that resveratrol could down-regulate the expression of Bcl-2.

Figure 2 Immunohistochemical staining of the expression of Bcl-2 ( × 200).

Table 3 Positive rate of Bcl-2 on treated EC-9706 cells by resveratrol.

Time (h)	PR(%)
0	35.64 ± 3.95
24	20.50 ± 2.79a
48	10.76 ± 2.46a
72	6.82 ± 1.78b
96	3.88 ± 1.24b

^aP < 0.05,

^bP < 0.01 vs the control group.

Expression of bax proteins

Positive staining located in the cytoplasm (Figure 3). After treatment with resveratrol (10 mmol·L^-1)for 24 to 96 hs, PRs of Bax proteins were apparently increased with treated time (P < 0.05) (Table 4). This suggested that resveratrol could up-regulate the expression of Bax.

Figure 3 Immunohistochemical staining of the expression of Bax ( × 200).

Table 4 Positive rate of Bax on treated EC-9706 cells by resveratrol.

Time (h)	PR(%)
0	10.66 ± 2.26
24	19.68 ± 2.67a
48	30.77 ± 3.76a
72	41.56 ± 6.14a
96	59.96 ± 5.34a

^aP < 0.01 vs the control group.

DISCUSSION

Currently, only few chemotherapeutic drugs take effect in the treatment of human primary esophageal carcinoma and it clearly need to look for new anti-esophageal carcinoma drugs. Resveratrol, a polyphenol found in various fruits and vegetables and is rich in grapes. The root extracts of the weed Polygonum cuspidatum, an important constituent of Chinese folk medicine, is also an ample source of resveratrol[1,2]. Several studys in last several years have shown that resveratrol have cardioprective and chemopreventive effects[3-5]. This constituent may account for the reduced risk of coronary heart disease in humans which may be achieved by moderate wine consumption[6]. Resveratrol is able to inhibit the growth of a wide variety of tumor cells, including leukemic, prostate, breast and hepatic cells[7-11]. The anti-tumor activity of resveratrol might be related to the induction of tumor apoptosis of tumor cells[12-22].

The Bcl-2 family plays a crucial role in the control of apoptosis. The family includes a number of proteins which have homologous amino acid sequences, including anti-apoptotic members such as Bcl-2 and Bcl-x_L, as well as pro-apoptotic members including Bax and Bad[23-26]. Overpression of Bax has the effect of promoting the cell death[27-31]. Conversely, Overpression of antiapoptotic proteins such as Bcl-2 will repress the function of Bax[32-36]. Thus, the ratio of Bcl-2 / Bax appears to be a critical determinant of a cell’s threshold for undergoing apoptosis[37].

In this study resveratrol could reduce Bcl-2 expression and improve Bcl-2 expression. The ratio of Bcl-2 /Bax was decreased when EC-9706 cells were treated with resveratrol. The decreased ratio could triggered the apoptosis of EC-9706 cells.

Our results demonstrated resveratrol is able to induce the apoptosis in esophageal cancer. This apoptosis may be mediated by down-regulating the expression of apoptosis-regulated gene Bcl-2 and up-regulating the expression of apoptosis-regulated gene Bax. Resveratrol may be potentially used as a chemotherapeutic drug in the anti-esophageal carcinoma chemptherapy.