Apoptosis of human primary gastric carcinoma cells induced by genistein

Abstract

AIM: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and the relationship between this apoptosis and expression of bcl-2 and bax.

METHODS: MTT assay was used to determine the cell growth inhibitory rate in vitro. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect the apoptosis of primary gastric cancer cells before and after genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-associated genes bcl-2 and bax.

RESULTS: Genistein inhibited the growth of primary gastric cancer cells in dose-and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the apoptotic rates of primary gastric cancer cells increased time-dependently. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the positivity rates of Bcl-2 proteins were apparently reduced with time and the positivity rates of Bax proteins were apparently increased with time. After exposed to genistein at 20 μmol/L for 24, 48, 72 and 96 respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time.

CONCLUSION: Genistein is able to induce the apoptosis in primary gastric cancer cells. This apoptosis may be mediated by down-regulating the apoptosis- associated bcl-2 gene and up-regulating the expression of apoptosis-associated bax gene.

Key Words: $[Keywords]

INTRODUCTION

Genistein is a planar molecule with an aromatic A-ring, has a second oxygen atom 11.5 Å from the one in the A ring, a molecular weight similar to those of the steroidal estrogens. It has estrogenic properties in receptor binding assays[1,2], cell culture[3,4], and uterine weight assays[5-7]. Genistein inhibits topoisomerase II[8], platelet-activating factor- and epidermal growth factor-induced expression of c-fos[9], diacylglycerol synthesis[10], and tyrosine kinases[11]. It also inhibits microsomal lipid peroxidation[12] and angiogenesis[13]. Genistein exhibits antioxidant properties[14-16] and was reported to induce differentiation of numerous cell types[17-19]. Moreover, a recent report shows that genistein is a potent cancer chemopreventive agent[20-22]. The anti-tumor activity of genistein might be related to induce the apoptosis of tumor cells but the precise mechanism of antitumor activity is not well understood.

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 like Bax and Bad[23-26]. Overexpression of Bax has the effect of promoting cell death[27-31]. Conversely, Overexpression 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 cell apoptosis[37].

In this study, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitatively detect the apoptosis status of primary gastric cancer cells before and after the genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-associated genes bcl-2 and bax.

MATERIALS AND METHODS

Materials

Genistein 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 genistein was made in dimethylsulfoxide (DMSO) at a concentration of 40 µmol/ L. Working dilutions were directly made in the cell culture medium.

Methods

Cell culture Fresh sample from a patient with gastric cancer was obtained in operating room. A single-cell suspension of tumor cells with the concentration of 5×10⁵/mL was prepared for seeding. Primary gastric cancer cells were purified after culture.

MTT assay Cells 1×10⁵ /well in a 96-well plate after incubation for 24 h were treated with different concentrations of genistein (5, 10, 20, 40 µmol/ L) for 24, 48 and 72 h respectively. A 10 µL of 5 g/L of MTT was added to the medium triplicate at each dose and incubated for 4 h at 37 °C. Culture media were discarded followed by addition of 0.2 mL DMSO and vibration for 10 min. The absorbance (A) was measured at 570 nm using a microplate reader. The cell growth inhibitory rate was calculated as follows: {(A of control group -A of experimental group)/(A of control group- A of blank group)} ×100%.

Transmission electron microscopy Cells treated with 20 µmol/L genistein were harvested after incubation for 24 h. Subsequently the cells were fixed in 40 g/L glutaral and immersed with Epon 821, imbedded for 72 h at 60 °C. After that 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 primary gastric cancer cells was evaluated by using an in situ cell detection kit. The cells were treated in the presence or absence of 20 µmol/L genistein for 24 to 96 h and fixed in ice-cold 800 mL/L ethanol for up to 24 h, treated with proteinase K and then 3 mL/L H₂O₂, and labeled with fluorescein dUTP in a humid box for 1 h at 37 °C. Cells were then combined with POD-horseradish peroxidase, colored with DAB and counterstained with methyl green. Controls received the same treatment except the labeling of omission of fluorescein dUTP. Cells were visualized under 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. Primary gastric cancer cells treated in the presence or absence of 20 µmol/L genistein for 24 to 96 h were grown on six-well glass plates and were fixed by acetone. After washed in PBS, the cells were incubated in 3 mL/L 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 wash of cells in PBS, the second antibody, biotinylated antirat IgG was added and the cells were incubated at room temperature for 1 h. After wash of cells in PBS, ABC compound was added and the cells were 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 treated the same as the experimental group except the incubation of the primary antibody instead of second antibody. The positive rate (PR) was calculated as follows: PR = (Number of positive cells/Total number)×100%.

RT-PCR The primary gastric cancer cells were treated in the presence or absence of 20 µmol/L genistein for 24 to 96 h and total RNA was extracted. The concentration of RNA was determined by absorption at 260 nm. The primers for Bcl-2, Bax and β-actin were as follows: β-actin (500 bp): 5’ GTGGG GCGCCCCAGGCACCA 3’, 5’ CTCCTTAATGTCACG CACGATTTC 3’; bcl-2 (716 bp): 5’ GGAAATATGGCGC ACGCT 3’, 5’ TCACTTGTGGCCCAGAT 3’; bax (508 bp): 5’ CCAGCTCTGAGCAGATCAT 3’, 5’ TATCAGCCCA TCTTCTTCC 3’. Polymerase chain reactions were performed in a 25 μL reaction volume. PCR for Bcl-2 and β-actin was run in the following procedures: at 94 °C for 7 min, 1 circle; at 94 °C for 1 min, at 72 °C for 1 min, 30 cycles; at 72 °C for 7 min, 1 circle. PCR for Bax was run in the following procedure: 94 °C for 1 min, 60 °C for 45 s, 72 °C for 45 s, 35 cycles. Ten μL PCR product was placed onto 15 g/L agarose gel and observed by EB staining using Gel-Pro analyzer.

Statistical analysis

Data were analyzed by the paired two-tailed Student’s t test, and significance was considered when P < 0.05.

RESULTS

MTT assay

Primary gastric cancer cells were exposed to increasing concentrations (5 µmol/L to 40 µmol/L) of genistein for 24 to 72 h, respectively. Primary gastric cancer cells showed death in a dose- and time-dependent manner. The data are summarized in Table 1.

Table 1 A value of primary gastric cancer cells treated with different concentrations of genistein.

	24 h	48 h	72 h
Control	0.400 ± 0.008	0.406 ± 0.007	0.404 ± 0.008
5 μmol/L genistein	0.361 ± 0.002a	0.334 ± 0.012b	0.305 ± 0.004b
10 μmol/L genistein	0.325 ± 0.004d	0.313 ± 0.003b	0.248 ± 0.004d
20 μmol/L genistein	0.308 ± 0.003d	0.249 ± 0.002d	0.206 ± 0.003d
40 μmol/L genistein	0.265 ± 0.004d	0.215 ± 0.004d	0.159 ± 0.002d

^aP < 0.05;

^bP < 0.01;

^dP < 0.001 vs control group.

Morphological changes

After treatment of primary gastric cancer cells with genistein (20 µmol/L) for 24 h, some cells presented apoptotic characteristics including chromatin condensation, appearance of chromatin crescent, nuclear fragmentation that could be seen by transmission electron microscope (Figure 1).

Figure 1 Ultra-microscopic structure of gastric cancer cells in-duced by genistein. A: Primary gastric cancer cells; B: apoptotic gastric cancer cells (Original magnification: ×4 800).

TUNEL assay

Apoptotic cells were determined by TUNEL assay according to the manufacture’s instructions. Positive staining located in the nucleus. After treatment with genistein (20 µmol/L) for 24 to 96 h, Apoptotic index of the cells was apparently increased with the increase of treatment time (P < 0.05) (Table 2).

Table 2 Apoptotic index (AI) of primary gastric cancer cells treated by genistein.

	Control	24 h	48 h	72 h	96 h
AI (%)	1.25 ± 0.30	4.97 ± 0.80	18.44 ± 1.92	35.18 ± 0.35	43.93 ± 1.11
t		-6.13	-13.82	-180.16	-52.80
P		P < 0.05	P < 0.05	P < 0.05	P < 0.05

P < 0.05 vs control group; AI: Apoptotic index.

Expression of Bcl-2 proteins

Positive staining located in the cytoplasm. After treatment with genistein (20 µmol/L) for 24 to 96 h, Positivity rate of the cells of Bcl-2 proteins was apparently reduced with the increase of treatment periods (P < 0.05) (Table 3). This suggested that genistein could down-regulate the expression of Bcl-2.

Table 3 Positivity rate (PR) of Bcl-2 in primary gastric cancer cells treated by genistein.

	Control	24 h	48 h	72 h	96 h
PR(%)	36.34 ± 0.72	21.62 ± 0.08	10.60 ± 049	7.21 ± 0.45	4.54 ± 0.36
t		31.93	44.67	73.80	59.32
P		P < 0.01	P < 0.01	P < 0.01	P < 0.01

P < 0.01 vs control group; PR: Positivity rate.

Expression of Bax proteins

Positive staining located in the cytoplasm. After treatment with genistein (20 µmol/L) for 24 to 96 h, PRs of Bax proteins were apparently increased with increase of treated time (P < 0.05) (Table 4). This suggested that genistein could up-regulate the expression of Bax.

Table 4 Positivity rate (PR) of Bax in primary gastric cancer cells treated by genistein.

	Control	24 h	48 h	72 h	96 h
PR(%)	10.73 ± 0.57	20.63 ± 0.86	34.3 ± 0.81	45.96 ± 0.42	58.61 ± 1.46
t		-57.49	-41.23	-69.53	-51.31
P		P < 0.001	P < 0.001	P < 0.001	P < 0.001

P < 0.01 vs control group; PR: Positivity rate.

RT-PCR

After exposed to 20 µmol/L genistein for 24, 48, 72 and 96 h respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR. It suggested that genistein could down-regulate the expression of Bcl-2 and up-regulate the expression of Bax.

DISCUSSION

Populations who consume a diet high in soy are reported to have a lower incidence of breast and prostate cancer. The activity of the soy-derived phytoestrogen, genistein, is thought to account for a portion of the chemopreventative properties of soy; these include an inhibitory effect on tyrosine kinases[11], DNA topoisomerases I and II[8], and ribosomal S6 kinase, antiestrogenicity, antioxidant activity, anti-angiogenesis activity[12], suppression of cell proliferation, induction of differentiation[17-19], and modulation of apoptosis[20-22].

In this study, we found that primary gastric cancer cells showed death in a dose- and time-dependent manner in MTT assay. This suggested genistein was able to restrain the growth of primary gastric cancer cells. It was found that genistein was able to induce the apoptosis of primary gastric cancer cells by transmission electron microscope assay and TUNEL assay. After treatment with 20 µmol/L genistein for 24 to 96 h, PRs of Bcl-2 proteins were apparently reduced and PRs of Bax proteins were apparently increased with increase of treated time by Immunohistochemical staining. After exposed to 20 µmol/L genistein for 24, 48, 72 and 96 h respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.

Genistein could reduce Bcl-2 expression and enhance bax expression. The ratio of Bcl-2/Bax was decreased when primary gastric cancer cells were treated with genistein, which could trigger the apoptosis of primary gastric cancer cells.

The present study demonstrated that genistein was able to induce the apoptosis in primary gastric cancer. This apoptosis may be mediated by down-regulating the expression of apoptosis-associated gene bcl-2 and up-regulating the expression of apoptosis-associated gene bax. Genistein may be used as a potential chemotherapeutic drug in the anti-gastric carcinoma chemotherapy.