三羟基异黄酮诱导胃癌原代细胞凋亡的分子机制

Genistein induces apoptosis in human primary gastric carcinoma cells

目的:探讨三羟基异黄酮诱导胃癌原代细胞发生凋亡的可能性,揭示该凋亡发生与基因bcl-2和bax之间的关系. 方法:在体外实验中,采用MTT比色法测定三羟基异黄酮对胃癌原代细胞的生长抑制率;以透射电镜和TUNEL染色法,定性、定量地研究三羟基异黄酮与胃癌原代细胞凋亡的关系;通过免疫组织化学法和RT—PCR 法检测基因bcl-2和bax的表达. 结果:三羟基异黄酮对胃癌原代细胞有明显抑制作用,随三羟基异黄酮浓度增加和培养时间的延长而增强;三羟基异黄酮诱导胃癌原代细胞出现凋亡细胞形态; TUNEL染色法可见,经三羟基异黄酮处理24,48,72, 96 h后,胃癌原代细胞凋亡数明显随时间增加(1.25±0.30%→4.97±0.80%,18.44±1.92%,35.18±0.35%, 43.93±1.11%,P<0.05).免疫组织化学发现经三羟基异黄酮处理24,48,72,96 h后,胃癌原代细胞的Bcl-2蛋白阳性率减少(36.34±0.72%→21.62±0.08%,10.60±0.49%,7.21±0.45%,4.54±0.36%, P<0.01),Bax蛋白阳性率增加(10.73±0.57%→20.63 ±0.86%,34.3±0.81%,45.96±0.42%,58.61±1.46%, P<0.01).RT—PCR也发现经三羟基异黄酮处理24,48, 72,96 h后,胃癌原代细胞的bcl-2 mRNA条带密度降低,bax mRNA条带密度加强. 结论:三羟基异黄酮可能通过下调bcl-2的表达和上调bax的表达而诱导胃癌原代细胞发生凋亡.

AIM: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and its relationship with bcl-2 and bax. METHODS: MTT assay was used to determine cell growth inhibition. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect apoptosis. Immunohistochemistry and RT-PCR were used to detect the expression of apoptosis-regulated genes bcl-2 and bax. RESULTS: Genistein inhibited the growth of primary gastric cancer cells in a dose- and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that the percentage of apoptotic cells was increased gradually along with the time of genistein treatment(1.25±0.30%, 4.97±0.80%, 18.44±1.92%, 35.18±0.35%, and 43.93±1.11% at 0,24,48,72 and 96 h after treatment, respectively, P<0.05). The percentage of bcl-2 protein positive cells was significantly reduced (36.34±0.72%, 21.62±0.08%, 10.60±0.49%,7.21±0.45%, and 4.54±0.36% at 0,24,48,72 and 96 h after treatment, respectively, P<0.01), whereas the percentage of bax protein positive cells was markedly increased (10.73±0.57%, 20.63±0.86%, 34.3±0.81%, 45.96±0.42%, and 58.61±1.46%, at 0, 24, 48, 72 and 96 h after treatment, respectively, P<0.01). After exposed to 20 μmol/L genistein for 24, 48,72 and 96 h, bcl-2 mRNA was decreased, while bax mRNA was increased progressively with elongation of genistein treatment time. CONCLUSION: Genistein can induce apoptosis in primary gastric cancer, which may be mediated by down-regulating the apoptosis-regulated gene bcl-2 and up-regulating the apoptosis-regulated gene bax.