达玛-24-烯-3β-乙酰氧基-20s-醇对胃癌细胞增殖的抑制作用

摘要

目的 前期研究发现中药连翘醇提物具有明显体外抑制肿瘤细胞的作用,文章进一步探讨从中药连翘提取的化合物达玛-24-烯-3β-乙酰氧基-20s-醇(Dammar-24-ene-3β-acetate-20S-ol,DM)体外抗肿瘤作用及其机制. 方法 采用MTT法检测DM对胃癌细胞株SGC-7901、BGC-823和MKN-45体外抑制作用.实验分为MKN-45对照组及其低、高剂量组,BGC-823对照组及其低、高剂量组,SGC-7901对照组及其低、高剂量组,对照组均为新鲜培养基,低剂量DM为10、50μg/mL,流式细胞仪检测凋亡细胞比例.应用CellQuest软件分析结果,记录细胞不同周期的比例.采用DCFH-DA探针检测ROS水平,实验分为空白对照组,多西紫杉醇组和DM组.配置微管聚集实验反应体系,其中多西紫杉醇10μmol/L,DM 终浓度选择50或100μmol/L,空白对照组不加药物,采用微管聚集实验和微管蛋白免疫荧光染色研究DM对于微管系统的影响. 结果 50μg/mL对于3株胃癌细胞抑制率均在80%以上,IC50 分别为:MKN-45 (11.72±1.35)μg/mL;BGC-823 (17.19±0.82)μg/mL;SGC-7901 (7.55±0.79)μg/mL.2μg/mL DM处理48h后再以低密度培养8d,细胞克隆较对照组明显减少.与MKN-45对照组凋亡细胞[(21.1±2.5)%]相比,MKN-45低剂量组和高剂量组凋亡细胞比率[(25.1±1.3)%和(55.2±2.3)%]均有明显升高(P<0.01).BGC-823对照组凋亡细胞百分率(13.2±2.4)%比较,BGC-823低、高剂量组凋亡细胞百分率[(18.2±2.1)%、(41.8±2.8)%]明显升高(P<0.01).与SGC-7901对照组凋亡细胞率[(10.5±1.8)%]比较,SGC-7901高剂量DM组凋亡细胞率[(21.1±1.9)%]升高(P<0.05),而与SGC-7901低剂量DM组[(12.3±1.6)%]差异无统计学意义(P>0.05).与MKN-45对照组比较,MKN-45低剂量组S期细胞百分率降低[(14.5±2.7)% vs (12.3±3.3)%,P>0.05].与BGC-823对照组比较,BGC-823低剂量组细胞S期百分率增加[(12.2±5.4)% vs (20.2±2.1)%,P<0.05].与SGC-7901对照组比较,SGC-7901低剂量组S期细胞百分率增加[(21.5±3.8)% vs (31.3±2.6%),P<0.05].检测DM处理后细胞内活性氧水平研究发现,10、50μg/mL DM处理48h 3株细胞均可见剂量依赖性ROS水平上升.微管蛋白免疫荧光染色可见采用IC50浓度的多西紫杉醇和10μg/mL DM处理48h后,MKN-45荧光信号均表现为局部浓聚、紊乱. 结论 达玛-24-烯-3β-乙酰氧基-20s-醇能够将胃癌细胞周期阻滞于S期,诱导细胞凋亡,从而抑制细胞增殖作用.%Objective Based on the previous research that the ethanolic extract from traditional Chinese medicine fructus forsythiae (Lianqiao) can obviously inhibit cancer cells in vitro, the article aimed to investigate the anti-proliferation effects of dammar-24-ene-3β-acetate-20S-ol (DM) extracted from fructus forsythiae on gastric cancer cells and its mechanism.Methods MTT assay was used to assess the anti-proliferation effects of DM on gastric cancer cells including SGC-7901, BGC-823, and MKN-45 in vitro.There were MKN-45 control group and its low dose and high dose groups, BGC-823 control group and its low dose and high dose groups, SGC-7901 control group and its low dose and high dose groups in the experiment.Flow cytometry was used to analyze the cell apoptosis rate.Cellquest software was used to analyze the results and record the ratio of cells at different cycles.DCFH-DA probe was applied to detect the ROS levels of blank control group, docetaxol group and DM group.The reaction system of microtubule assembly test was set with 10?mol/L docetaxol, 50 or 100 μmol/L DM final density and no medicine in blank control group.The readings of UV spectrophotometer were recorded.Microtubule assembly assay and microtubule immunofluorescence staining were applied to investigate the effects of DM on microtubule system.Results The inhibition ratio of 50 μg/L DM on the proliferation three gastric cell lines were all above 80%, with IC50s of MKN-45 11.72±1.35 μg/mL, BGC-823 17.19±0.82 μg/mL, SGC-7901 7.55±0.79 μg/mL.8 days′ low density culturing at 48 hours after 2 μg/mL DM treatment, compared with control group, the number of cell clones significantly reduced without much change in clone size, while 48 hours after 10 μg/mL DM treatment, besides a few clones of BGC-823, there were just several megascopic clones of SGC-7901 and MKN-45.In comparison with apoptotic cell ratio in MKN-45 control group[(21.1±2.5)%], its low dose group and high dose group resulted in significant rise of apoptotic cell ratio[(25.1±1.3)% and (55.2±2.3)%] (P<0.01).Compared with apoptotic cell ratio in BGC-823 control group[(13.2±2.5)%], its low dose group and high dose group resulted in significant rise of apoptotic cell ratio[(18.2±2.1)% and (41.8±2.1)%] (P<0.01).Compared with apoptotic cell ratio in SGC-7901 control group[(10.5±1.8)%], significant rise was observed in its high dose group[(41.8±2.1)%] (P<0.05) while no statistical significance was found in its low dose group[(12.3±1.6)%] (P>0.05).In comparison with MKN-45 control group, the ratio of cells at S phase decreased in its low dose group[(14.5±2.7)% vs (12.3±3.3)%,P>0.05].In comparison with BGC-823 control group, the ratio of cells at S phase increased in its low dose group[(12.2±5.4)% vs (20.2±2.1)%,P<0.05].In comparison with SGC-7901 control group, the ratio of cells at S phase increased in its low dose group[(21.5±3.8)% vs (31.3±2.6)%,P<0.05].From the detection of intracellular active oxygen after DM treatment, dose-dependent ROS level increased in all three cell lines 48 hours after 10μg/mL and 50μg/mL DM treatment.From the results of microtubule immunofluorescence staining, 48 hours after the treatment of IC50 docetaxol and 10μg/mL DM, the fluorescence signals were in local concentration and disorder.Conclusion Dammar-24-ene-3β-acetate-20S-ol demonstrated anti-proliferation effects due to the apoptosis induced by cell cycle arrest at S phase.