丹皮酚联合5-FU对食管癌EC9706细胞增殖及凋亡的影响

Effects of paeonol and 5-FU on the proliferation and apoptosis of human esophageal carcinoma EC9706 cells

目的:探讨丹皮酚(paeonol,Pae)单独及联合5-FU对人食管癌EC9706细胞的增殖抑制及凋亡诱导作用.方法:采用6种浓度的Pae(7.81、15.63、31.25、62.50、125.00、250.00mg/L)、3种浓度的5-FU(12.50、25.00、50.00mg/L)及Pae(31.25mg/L)和5-FU(12.50mg/L)联合分别处理EC9706细胞24、48、72h,同时设对照组(细胞不做处理),采用MTT法检测各个时间段细胞的增殖情况;采用流式细胞术检测4种浓度的Pae(31.25、62.50、125.00、250.00mg/L)处理EC9706细胞72h后细胞周期的变化;倒置显微镜下观察各Pae组细胞各时间段形态学变化,HE染色光镜下观察凋亡细胞;采用免疫细胞化学法检测经Pae(31.25mg/L)、5-FU(12.50mg/L)单独和联合作用48h后细胞中凋亡相关蛋白Bcl-2及Bax的表达.结果:Pae、5-FU可明显抑制EC9706细胞增殖,并随着浓度的增加和作用时间的延长而增强(P<0.05),Pae与5-FU联合用药比单用Pae或5-FU抑制效果更明显(P<0.05);Pae作用后EC9706细胞中G0/G1期和G2/M期细胞比例下降,S期细胞比例上升(Pae125.00mg/L组:G0/G1期21.18%±2.28%vs62.17%±5.23%、G2/M期0.76%±0.54%vs9.92%±3.10%、S期78.06%±2.82%vs27.91%±2.13%,均P<0.05);HE染色光镜下可见典型的肿瘤细胞凋亡改变;Pae、5-FU可下调EC9706细胞中Bcl-2蛋白表达,同时增强EC9706细胞中Bax蛋白的表达,联合用药组较单药组作用更为明显(2.21±0.14vs5.67±0.30,4.22±0.34;8.55±0.33vs3.90±0.27,6.28±0.26,均P<0.05).结论:Pae可明显抑制人食管癌EC9706细胞的增殖,促进其凋亡,Pae联合5-FU作用更为明显.

AIM： To investigate the effects of paeonol alone or in combination with 5-fluorouracil （5-FU） on the proliferation and apoptosis of human esophageal carcinoma EC9706 cells. METHODS： Six different concentrations of paeonol （7.81, 15.63, 31.25, 62.50, 125.00 and 250.00 mg/L, respectively）, three different concentrations of 5-FU （12.50, 25.00 and 50.00 mg/L, respectively）, and paeonol （31.25 mg/L） in combination with 5-FU （12.50 mg/L） were used to treat EC9706 cells for different durations （24, 48 and 72 h）. Untreated EC9706 cells were used as the control group. The proliferation of EC9706 cells was detected by methyl thiazolyl tetrazolium （MTT） assay after treatment for different durations. After treatment of EC9706 cells with paeonol at concentrations of 31.25, 62.50, 125.00and 250.00 mg/L for 72 hours, the cell cycle was analyzed by flow cytometry; cell morphological changes were observed using an inverted microscope; the morphology of apoptotic cells was observed by HE staining and light microscopy. The expression of apoptosis-associated proteins Bcl-2 and Bax was detected by immunocytochemistry after treatment of EC9706 cells with paeonol （31.25 mg/L） and 5-FU （12.50 mg/L）, alone or in combination, for 48 hours. RESULTS： Paeonol or 5-FU could signifi cantly inhibit the proliferation of EC9706 cells in a concentrationand time-dependent manner （both P 0.05）. Paeonol in combination with 5-FU showed more significant inhibitory effects on the proliferation of EC9706 cells when compared with paeonol or 5-FU alone （both P 0.05）. Paeonol （125.00 mg/L） treatment altered the cell cycle distribution of EC9706 cells： the percentages of cells in G0/G1 and G2/M phases decreased, while that of cells in S phase increased （G0/G1 phase： 21.18% ± 2.28% vs 62.17% ± 5.23%; G2/M phase： 0.76% ± 0.54% vs 9.92% ± 3.10%; S phase 78.06% ± 2.82% vs 27.91% ± 2.13%; all P 0.05）. Typical apoptotic changes were observed in EC9706 cells treated with paeonol. Both paeonol and 5-FU down-regulated the expression of Bcl-2 and up-regulated the expression of Bax, which was especially prominent in the combination group （2.21 ± 0.14 vs 5.67 ± 0.30 and 4.22 ± 0.34; 8.55 ± 0.33 vs 3.90 ± 0.27 and 6.28 ± 0.26, all P 0.05）. CONCLUSION： Paeonol can significantly inhibit the proliferation and induce the apoptosis of human esophageal carcinoma EC9706 cells. Paeonol in combination with 5-FU shows a synergistic effect in suppressing the proliferation and promoting the apoptosis of EC9706 cells.