调控Smac-IAPs凋亡通路对胃癌细胞化疗敏感性的影响
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摘要
第一部分XIAP、Smac、Caspase-3在胃癌组织中的表达及其相关性研究
目的探讨凋亡相关基因XIAP、Smac和Caspase-3在人胃癌组织中的表达及其与临床病理特征的相关性。
方法采用免疫组织化学SP法检测80例胃癌组织和40例癌旁正常胃粘膜中XIAP、Smac、Caspase-3的表达情况。
结果80例胃癌组织中XIAP、Smac、Caspase-3的阳性率分别为90%(72/80)、67.5%(54/80)和62.5%(50/80)。XLAP阳性表达与胃癌分化程度负相关(P＜0.05);Smac、Caspase-3与分化程度正相关(P＜0.05)。XLAP、Smac、Caspase-3表达在肠型和弥漫型胃癌之间有显著性差异(P＜0.05),与淋巴结转移相关(P＜0.05),但与胃癌部位、大小、浸润深度无关。胃癌组织中XIAP、Smac的表达分别与Caspase-3呈负相关、正相关。40例正常胃粘膜中XIAP弱阳性表达6例(15%),而Smac、Caspase-3的阳性率分别高达92.5%(37/40)、90%(36/40)。
结论随胃癌恶性程度的增加,XIAP的表达增高,而Smac和Caspase-3表达降低,可能参与胃癌的发生、发展,是评估胃癌预后的潜在指标。
第二部分瞬时转染外源性Smac基因对胃癌细胞株化疗敏感性的调控作用
目的细胞凋亡异常是肿瘤耐药性产生的关键因素之一。Smac是新近发现的一种凋亡调节基因,在介导化疗药物对肿瘤细胞的诱导凋亡效应中起重要作用。本研究旨在观察Smac基因过表达对胃癌细胞株化疗敏感性的影响,为进一步改善胃癌化疗效果奠定基础。
方法采用脂质体GeneSHUTTLE-40介导的方法,将Smac基因转入胃癌细胞株MKN-45,RT-PCR和western blot法检测癌细胞中Smac的表达;选用顺铂、丝裂霉素分别处理转染前后的胃癌细胞,四甲基偶氮唑盐(MTF)比色法检测细胞增殖活性,倒置显微镜下观察细胞形态变化并摄影,Annexin V-FITC和碘化丙锭双染色流式细胞术(FCM)检测细胞凋亡。
结果同未转染对照组比较,转染外源性Smac基因后MKN-45细胞Smac的mRNA和蛋白表达水平显著增高(P＜0.01),各浓度顺铂、丝裂霉素处理24h后细胞生长抑制率分别增加10.10%～23.80%(P＜0.01)、10.01%～15.86%(P＜0.01),细胞明显变圆、折光增强、漂浮细胞增多,细胞凋亡比率分别增加6.7%～20.2%(P＜0.01)、5.4%～13.2%(P＜0.01)。
结论转染外源性Smac基因并使其在胃癌细胞中过表达,能提高MKN-45对化疗药物的敏感性,是改善胃癌化疗效果的潜在途径。
第三部分稳定过表达Smac基因胃癌细胞株的建立及其化疗敏感性研究
目的探讨稳定过表达Smac基因对胃癌细胞株化疗敏感性的影响。
方法采用脂质体将Smac基因真核表达载体pcDNA3.1-Smac及空白载体pcDNA3.1转染入胃癌细胞株MKN-45,G_(418)筛选获得抗性亚克隆细胞株,RT-PCR和western blot检测癌细胞Smac基因表达,四甲基偶氮唑盐(MTT)比色法、克隆形成实验检测丝裂霉素(MMC)对癌细胞的生长抑制效率。
结果建立分别稳定表达Smac基因、新霉素抗性基因(neo)的胃癌亚克隆细胞株MKN-45/Smac、MKN-45/neo。RT-PCR和western blot证实MKN-45/Smac细胞的Smac mRNA及蛋白表达水平均显著高于MKN-45、MKN-45/neo(P＜0.01)。10μg/ml MMC作用24h后,MKN-45、MKN-45/neo细胞生长抑制率分别为27.85%、28.12%,而MKN-45/Smac则高达43.71%(P＜0.01);同MKN-45、MKN-45/neo细胞株比较,MKN-45/Smac的克隆形成能力分别降低了14.07%(P＜0.01)、15.13%(P＜0.01)。
结论稳定转染Smac基因使其在胃癌细胞株中过表达,能显著提高癌细胞对MMC的敏感性,为改善胃癌化疗效果奠定了实验基础。
第四部分稳定转染外源性Smac基因对胃癌细胞凋亡通路的影响
目的探讨稳定过表达Smac基因对胃癌细胞凋亡的影响。
方法体外培养胃癌细胞株MKN-45、MKN-45/neo、MKN-45/Smac,以丝裂霉素(MMC)作为凋亡诱导因子,采用台盼兰活细胞拒染法检测癌细胞体外生长活性,透射电镜、吖啶橙-溴化乙啶荧光染色法、末端TdT酶标记技术(TUNEL)观察癌细胞凋亡及比率;western blot、比色法检测细胞内caspase-3表达和活性。
结果10μg/ml MMC作用6～24 h后,与MKN-45细胞比较,MKN-45/Smac细胞生长活性减弱10.0%～30.8%(P＜0.01),部分MKN-45/Smac细胞发生典型的凋亡形态学改变,凋亡率增高21.2%(P＜0.01)。MMC作用后,MKN-45/Smac细胞内caspase-3的表达和活性均较MKN-45细胞显著增强(P＜0.01)。
结论胃癌细胞中Smac基因的过表达,能提高MMC作用后细胞中caspase-3的表达和活性,显著诱导癌细胞凋亡,为调控胃癌细胞凋亡活性提供了新的途径。
第五部分下调XIAP表达对胃癌细胞株化疗敏感性的影响
目的观察下调XIAP基因表达对胃癌细胞化疗敏感性的影响。
方法构建XIAP基因反义真核表达载体,稳定转染胃癌细胞株MKN-45,RT-PCR和western blot法检测癌细胞XIAP基因表达。选用顺铂、丝裂霉素分别处理转染前后的胃癌细胞,采用MTT比色法、克隆形成抑制实验检测癌细胞体外生长活性;透射电镜、流式细胞术、TUNEL检测癌细胞凋亡及比率;western blot和比色法检测细胞内caspase-3蛋白表达和活性水平。
结果RT-PCR和western blot证实,稳定转染反义XIAP基因的胃癌细胞MKN-45的XIAP mRNA和蛋白表达水平分别降低84.75%(P＜0.01)、89.75%(P＜0.01)。各浓度顺铂、丝裂霉素处理24h后,转染反义XIAP基因的MKN-45细胞生长抑制率分别增加7.3%～25.3%(P＜0.01),12.3%～16.3%(P＜0.01)。透射电镜下可见部分细胞发生典型的凋亡形态学改变,凋亡率分别为34.1%和32.5%,显著高于未转染对照组MKN-45细胞(凋亡率为14.2%,P＜0.05)。与MKN-45细胞比较,稳定转染反义XIAP基因的MKN-45细胞内caspase-3表达水平增高2.45倍(P＜0.01),活性水平提高3.68倍(P＜0.01)。
结论通过反义RNA技术下调XIAP基因表达,能提高癌细胞中caspase-3的表达和活性,增强化疗药物对癌细胞的诱导凋亡作用。
第六部分姜黄素对胃癌细胞株IAP家族及Smac基因表达的影响
目的观察姜黄素(Curcumin)对人胃癌细胞株IAP家族和Smac基因表达的影响,探讨姜黄素诱导胃癌细胞凋亡的分子机制。
方法10～40μmol/L姜黄素分别处理胃癌细胞株MKN-45 6～24 h后,MTF比色法检测癌细胞生长活性,末端TdT酶标记技术和DNA Ladder检测细胞凋亡;逆转录聚合酶链反应fRT-PCR)和western blot法检测IAP家族(Survivin、XIAP)和Smac基因表达;比色法检测癌细胞Caspase-3活性改变。
结果同未加药对照组比较,各浓度姜黄素作用后癌细胞生长明显减慢,抑制率为12.18%～68.15%(P＜0.01),部分细胞呈现典型凋亡形态学改变,凝胶电泳可见“梯形”条带,凋亡率为9.24%～28.12%(P＜0.01);Survivin、XIAP基因mRNA和蛋白水平显著下调(P＜0.01),而Smac基因表达增强(P＜0.01),癌细胞Caspase-3活性增强2.27～6.67倍(P＜0.01)。
结论姜黄素可显著诱导胃癌MKN-45细胞凋亡,通过上调Smac、下调Survivin和XIAP基因表达,进而活化Caspase-3是其作用机制之一。
Part One Expression and correlation of XIAP,Smac and caspase-3 in gastric cancer
Objective To study the expression of XIAP,Smac and caspase-3 in human gastric cancer tissues and its correlation with clinical and pathological characteristics.
Methods Immunohistochemical staining was applied to detect the expression of XIAP,Smac and caspase-3 in 80 cases of gastric cancer and 40 cases of adjacent normal gastric mucosa.
Results In 80 cases of gastric cancer,the expression rates of XIAP,Smac and caspase-3 were 90%(72/80),67.5%(54/80),and 62.5%(50/80),respectively.The XIAP expression was negatively correlated with tissue differentiation(P＜0.05),while expression of Smac and caspase-3 was positively correlated with tissue differentiation(P＜0.05).The expression of XIAP,Smac and caspase-3 was significantly different between intestinal-type and diffuse-type gastric cancer(P＜0.05),and correlated with lymph node metastasis (P＜0.05),but not with location,volume and invasion.The expression of XIAP and Smac in gastric cancer was negatively and positively correlated with caspase-3,respectively.In 40 cases of normal gastric mucosa,6 cases(15%) presented weak expression of XIAP,and the expression rates of Smac and caspase-3 were 92.5%(37/40) and 90%(36/40),respectively.
Conclusions XIAP expression increased along with the malignant degree of gastric cancer,while expression of Smac and caspase-3 decreased,which may participate in the development and progression of gastric cancer.These proteins may serve as potential biomarkers for evaluating the prognosis of gastric cancer.
Part Two Effects of transient transfection of Smac on chemotherapeutic sensitivities of gastric cancer cell line
Objective Abnormal apoptosis is one of key factors for the drug resistance of neoplasms.Smac is a novel gene involved in the regulation of apoptosis,which plays an important role in the inducing apoptosis effects of chemotherapeutic drugs on tumor cells. This study was designed to explore the effects of over-expression of Smac gene on chemotherapeutic sensitivities of gastric cancer cell line,in order to establish a basis for further improving chemotherapy of gastric cancer.
Methods Under induction of liposome GeneSHUTTLE-40,Smac gene was transfected into gastric cancer cell line MKN-45.Cellular Smac gene expression was determined by reverse transcription-polymerase chain reaction(RT-PCR) and western blot analysis.Cisplatin and MMC were administrated to un-transfeted and transfected MKN-45 cells.Cellular proliferation activities were assayed by tetrazolium bromide(MTT) colorimetry.The morphological changes of cancer cells were observed under an inversion microscope.Cellular apoptosis was determined by Annexin V-FITC and propidium iodide staining flow cytometry.
Results Compared with un-transfected control group,Smac mRNA and protein levels in transfected MKN-45 cells were significantly improved(P＜0.01).The growth inhibition rates of various concentrations of cisplatin and MMC on MKN-45 cells were improved by 10.10%-23.80%(P＜0.01) and 10.01%-15.86%(P＜0.01),respectively,with the cells presenting roundness,obvious refraction,and cellular fragment.Cellular apoptosis rates were improved by 6.7%-20.2%(P＜0.01) and 5.4%-13.2%(P＜0.01),respectively.
Conclusions Transfection of extrinsic Smac gene resulted in its over-expression in gastric cancer cells,which could improve the chemotherapeutic sensitivities of MKN-45 cells.This is a potential strategy for ameliorating chemotherapeutic effects of gastric cancer.
Part Three Establishment of gastric cancer cell line stably overexpressing Smac gene and its chemotherapeutic sensitivities
Objective To explore the effects of stable over-expression of Smac gene on chemotherapeutic sensitivities of gastric cancer cell line.
Methods Under the induction of liposome,the eukaryotic expression vector pcDNA3.1-Smac for Smac gene and its control vector pcDNA3.1 were transfected into gastric cancer cell line MKN-45.The subclone cell lines were obtained by persistent G_(418) selection.Smac gene expression of cancer cells was detected by RT-PCR and western blot methods.The growth inhibition effects of MMC on cancer cells were also observed by MTT colorimetry and clone formation assay.
Results The subclone gastric cancer cell lines,stably expressing Smac and neo gene respectively,were successfully selected,and named as MKN-45/Smac and MKN-45/neo. RT-PCR and western blot results demonstrated that Smac mRNA and protein levels of MKN-45/Smac cells were significantly higher than those of MKN-45 and MKN-45/neo (P＜0.01).After incubation with 10μg/ml MMC for 24 h,the growth inhibition rates of MKN-45 and MKN-45/neo were 27.85%,28.12%respectively,while that of MKN-45/Smac cells was 43.71%(P＜0.01).When compared with MKN-45 and MKN-45/neo cells,the clone formation abilities of MKN-45/Smac were reduced by 14.07%(P＜0.01) and 15.13%(P＜0.01),respectively.
Conclusions Stable transfection of Smac gene and its over-expression in gastric cancer cell line could significantly improve their chemotherapeutic sensitivities to MMC, which established an experimental basis for ameliorating chemotherapy of gastric cancer.
Part Four Regulatory effects of over-expression of Smac gene on apoptosis activities of gastric cancer cell line
Objective To explore the effects of over-expression of Smac gene on apoptosis activities of gastric cancer cell line.
Methods The gastric cancer cell lines MKN-45,MKN-45/neo and MKN-45/Smac were cultured in vitro.After teatment with MMC as an apoptosis inducer,cellular growth activities were investigated by trypan blue staining method.Apoptosis of cancer cells was detected by electronic microscopy,AO-EB fluorescent staining and TUNEL methods. Cellular protein levels and its activities of caspase-3 were assayed by western blot and colorimetry.
Results After incubation with 10μg/ml MMC for 24 h,growth activities of MKN-45/Smac were reduced by 10.0%～30.8%(P＜0.01),when compared with those of MKN-45. Partial MKN-45/Smac cells presented characteristic morphological changes of apoptosis under the electric and fluorescence microscopes,with apoptosis rates being 36.4%,which was significantly higher than that of MKN-45(15.2%,P＜0.01).After treatment with MMC, caspase-3 expression levels in MKN-45/Smac cells were significantly improved than those of MKN-45(P＜0.01),with caspase-3 activities enhanced by 3.25 times(P＜0.01).
Conclusions Transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line could significantly improve expression and activity levels of caspase-3 induced by MMC,resulting in obvious apoptosis-inducing effects,which established a novel strategy for regulating apoptosis activities of gastric cancer.
Part Five The effects of downregulating XIAP expression on the apoptosis of gastric cancer cells induced by chemotherapeutic drugs
Objective To observe the effects of downregulating XIAP expression on the chemotherapeutic sensitivities of gastric cancer cells.
Methods The antisense eukaryotic vector for XIAP was constructed and stably transferred into gastric cancer cell line MKN-45.RT-PCR and western blot were applied to detect the XIAP gene expression.Cisplatin and MMC were administrated to untransfected and transfected gastric cancer cells.MTT colorimetry and clone formation assay were performed to measure the in vitro cell viability.Apoptosis and its rates were detected by electronic microscopy,AO-EB fluorescent staining and TUNEL.Cellular caspase-3 protein expression and its activities were assayed by western blot and colorimetry.
Results RT-PCR and western blot indicated that the mRNA and protein levels of XIAP within gastric cancer MKN-45 cells stably transfected with antisense XIAP vector were significantly decreased by 84.75%(P＜0.01) and 89.75%(P＜0.01),respectively.After treatment with various concentrations of cisplatin and MMC for 24 hours,the cell growth inhibition of MKN-45 cells stably transfected with antisense XIAP was enhanced by 7.3%～25.3%(P＜0.01) and 12.3%～16.3%(P＜0.01),respectively.Partial cancer cells presented characteristic changes of apoptosis under an electronic microscope,while the apoptosis rates were 34.1%and 32.5%,respectively,which were significantly higher than that of untransfected control MKN-45 cells(14.2%,P＜0.05).Compared with MKN-45 cells,the caspase-3 expression within cells stably transfected with antisense XIAP was significantly increased by 2.45 times(P＜0.01),while the activity of caspase-3 was enhanced by 3.68 times(P＜0.01).
Conclusions Downregulation of XIAP expression via antisense RNA could increase the expression and activity of caspase-3,enhance the apoptosis of cancer cells induced by chemotherapeutic drugs.
Part Six Effects of curcumin on IAPs and Smac gene expression of gastric cancer cell line
Objective To explore the effects of curcumin on gene expression of IAPs and Smac in gastric cancer cell line,in order to explore the inducing apoptosis mechanisms of curcumin.
Methods After being treated with 10～40μmol/L curcumin for 6～24 h,growth activities of MKN-45 cells were detected by MTT colorimetry.Cellular apoptosis was assayed by TUNEL and DNA Ladder methods.IAPs(Survivin,XIAP) and Smac gene expression was detected by RT-PCR and western blot.Cellular caspase-3 activities were detected by colorimetry method. expression was detected by RT-PCR and western blot.Cellular caspase-3 activities were detected by colorimetry method.
Results When compared with untreated control group,cellular growth of antisense XIAP-transfected cells was significantly inhibited by various concentrations of curcumin, with inhibitory ratios being 12.18%～68.15%(P＜0.01).Partial MKN-45 cells presented characteristic morphological changes of apoptosis,with trapezia bands on gel electrophoresis.The apoptosis rates were 9.24%～28.12%(P＜0.01).Cellular mRNA and protein levels of Survivin and XIAP were significantly down-regulated(P＜0.01),with those of Smac being up-regulated(P＜0.01).Cellular caspase-3 activities were improved by 2.27～6.67 times(P＜0.01).
Conclusions Curcumin could significantly induce apoptosis of gastric cancer cell line MKN-45,one of its mechanisms was through up-regulating Smac and down-regulating Survivin and XIAP expression,leading to activation of caspase-3.
目的探讨凋亡相关基因XIAP、Smac和Caspase-3在人胃癌组织中的表达及其与临床病理特征的相关性。
方法采用免疫组织化学SP法检测80例胃癌组织和40例癌旁正常胃粘膜中XIAP、Smac、Caspase-3的表达情况。
结果80例胃癌组织中XIAP、Smac、Caspase-3的阳性率分别为90%(72/80)、67.5%(54/80)和62.5%(50/80)。XLAP阳性表达与胃癌分化程度负相关(P＜0.05);Smac、Caspase-3与分化程度正相关(P＜0.05)。XLAP、Smac、Caspase-3表达在肠型和弥漫型胃癌之间有显著性差异(P＜0.05),与淋巴结转移相关(P＜0.05),但与胃癌部位、大小、浸润深度无关。胃癌组织中XIAP、Smac的表达分别与Caspase-3呈负相关、正相关。40例正常胃粘膜中XIAP弱阳性表达6例(15%),而Smac、Caspase-3的阳性率分别高达92.5%(37/40)、90%(36/40)。
结论随胃癌恶性程度的增加,XIAP的表达增高,而Smac和Caspase-3表达降低,可能参与胃癌的发生、发展,是评估胃癌预后的潜在指标。
第二部分瞬时转染外源性Smac基因对胃癌细胞株化疗敏感性的调控作用
目的细胞凋亡异常是肿瘤耐药性产生的关键因素之一。Smac是新近发现的一种凋亡调节基因,在介导化疗药物对肿瘤细胞的诱导凋亡效应中起重要作用。本研究旨在观察Smac基因过表达对胃癌细胞株化疗敏感性的影响,为进一步改善胃癌化疗效果奠定基础。
方法采用脂质体GeneSHUTTLE-40介导的方法,将Smac基因转入胃癌细胞株MKN-45,RT-PCR和western blot法检测癌细胞中Smac的表达;选用顺铂、丝裂霉素分别处理转染前后的胃癌细胞,四甲基偶氮唑盐(MTF)比色法检测细胞增殖活性,倒置显微镜下观察细胞形态变化并摄影,Annexin V-FITC和碘化丙锭双染色流式细胞术(FCM)检测细胞凋亡。
结果同未转染对照组比较,转染外源性Smac基因后MKN-45细胞Smac的mRNA和蛋白表达水平显著增高(P＜0.01),各浓度顺铂、丝裂霉素处理24h后细胞生长抑制率分别增加10.10%～23.80%(P＜0.01)、10.01%～15.86%(P＜0.01),细胞明显变圆、折光增强、漂浮细胞增多,细胞凋亡比率分别增加6.7%～20.2%(P＜0.01)、5.4%～13.2%(P＜0.01)。
结论转染外源性Smac基因并使其在胃癌细胞中过表达,能提高MKN-45对化疗药物的敏感性,是改善胃癌化疗效果的潜在途径。
第三部分稳定过表达Smac基因胃癌细胞株的建立及其化疗敏感性研究
目的探讨稳定过表达Smac基因对胃癌细胞株化疗敏感性的影响。
方法采用脂质体将Smac基因真核表达载体pcDNA3.1-Smac及空白载体pcDNA3.1转染入胃癌细胞株MKN-45,G_(418)筛选获得抗性亚克隆细胞株,RT-PCR和western blot检测癌细胞Smac基因表达,四甲基偶氮唑盐(MTT)比色法、克隆形成实验检测丝裂霉素(MMC)对癌细胞的生长抑制效率。
结果建立分别稳定表达Smac基因、新霉素抗性基因(neo)的胃癌亚克隆细胞株MKN-45/Smac、MKN-45/neo。RT-PCR和western blot证实MKN-45/Smac细胞的Smac mRNA及蛋白表达水平均显著高于MKN-45、MKN-45/neo(P＜0.01)。10μg/ml MMC作用24h后,MKN-45、MKN-45/neo细胞生长抑制率分别为27.85%、28.12%,而MKN-45/Smac则高达43.71%(P＜0.01);同MKN-45、MKN-45/neo细胞株比较,MKN-45/Smac的克隆形成能力分别降低了14.07%(P＜0.01)、15.13%(P＜0.01)。
结论稳定转染Smac基因使其在胃癌细胞株中过表达,能显著提高癌细胞对MMC的敏感性,为改善胃癌化疗效果奠定了实验基础。
第四部分稳定转染外源性Smac基因对胃癌细胞凋亡通路的影响
目的探讨稳定过表达Smac基因对胃癌细胞凋亡的影响。
方法体外培养胃癌细胞株MKN-45、MKN-45/neo、MKN-45/Smac,以丝裂霉素(MMC)作为凋亡诱导因子,采用台盼兰活细胞拒染法检测癌细胞体外生长活性,透射电镜、吖啶橙-溴化乙啶荧光染色法、末端TdT酶标记技术(TUNEL)观察癌细胞凋亡及比率;western blot、比色法检测细胞内caspase-3表达和活性。
结果10μg/ml MMC作用6～24 h后,与MKN-45细胞比较,MKN-45/Smac细胞生长活性减弱10.0%～30.8%(P＜0.01),部分MKN-45/Smac细胞发生典型的凋亡形态学改变,凋亡率增高21.2%(P＜0.01)。MMC作用后,MKN-45/Smac细胞内caspase-3的表达和活性均较MKN-45细胞显著增强(P＜0.01)。
结论胃癌细胞中Smac基因的过表达,能提高MMC作用后细胞中caspase-3的表达和活性,显著诱导癌细胞凋亡,为调控胃癌细胞凋亡活性提供了新的途径。
第五部分下调XIAP表达对胃癌细胞株化疗敏感性的影响
目的观察下调XIAP基因表达对胃癌细胞化疗敏感性的影响。
方法构建XIAP基因反义真核表达载体,稳定转染胃癌细胞株MKN-45,RT-PCR和western blot法检测癌细胞XIAP基因表达。选用顺铂、丝裂霉素分别处理转染前后的胃癌细胞,采用MTT比色法、克隆形成抑制实验检测癌细胞体外生长活性;透射电镜、流式细胞术、TUNEL检测癌细胞凋亡及比率;western blot和比色法检测细胞内caspase-3蛋白表达和活性水平。
结果RT-PCR和western blot证实,稳定转染反义XIAP基因的胃癌细胞MKN-45的XIAP mRNA和蛋白表达水平分别降低84.75%(P＜0.01)、89.75%(P＜0.01)。各浓度顺铂、丝裂霉素处理24h后,转染反义XIAP基因的MKN-45细胞生长抑制率分别增加7.3%～25.3%(P＜0.01),12.3%～16.3%(P＜0.01)。透射电镜下可见部分细胞发生典型的凋亡形态学改变,凋亡率分别为34.1%和32.5%,显著高于未转染对照组MKN-45细胞(凋亡率为14.2%,P＜0.05)。与MKN-45细胞比较,稳定转染反义XIAP基因的MKN-45细胞内caspase-3表达水平增高2.45倍(P＜0.01),活性水平提高3.68倍(P＜0.01)。
结论通过反义RNA技术下调XIAP基因表达,能提高癌细胞中caspase-3的表达和活性,增强化疗药物对癌细胞的诱导凋亡作用。
第六部分姜黄素对胃癌细胞株IAP家族及Smac基因表达的影响
目的观察姜黄素(Curcumin)对人胃癌细胞株IAP家族和Smac基因表达的影响,探讨姜黄素诱导胃癌细胞凋亡的分子机制。
方法10～40μmol/L姜黄素分别处理胃癌细胞株MKN-45 6～24 h后,MTF比色法检测癌细胞生长活性,末端TdT酶标记技术和DNA Ladder检测细胞凋亡;逆转录聚合酶链反应fRT-PCR)和western blot法检测IAP家族(Survivin、XIAP)和Smac基因表达;比色法检测癌细胞Caspase-3活性改变。
结果同未加药对照组比较,各浓度姜黄素作用后癌细胞生长明显减慢,抑制率为12.18%～68.15%(P＜0.01),部分细胞呈现典型凋亡形态学改变,凝胶电泳可见“梯形”条带,凋亡率为9.24%～28.12%(P＜0.01);Survivin、XIAP基因mRNA和蛋白水平显著下调(P＜0.01),而Smac基因表达增强(P＜0.01),癌细胞Caspase-3活性增强2.27～6.67倍(P＜0.01)。
结论姜黄素可显著诱导胃癌MKN-45细胞凋亡,通过上调Smac、下调Survivin和XIAP基因表达,进而活化Caspase-3是其作用机制之一。
Part One Expression and correlation of XIAP,Smac and caspase-3 in gastric cancer
Objective To study the expression of XIAP,Smac and caspase-3 in human gastric cancer tissues and its correlation with clinical and pathological characteristics.
Methods Immunohistochemical staining was applied to detect the expression of XIAP,Smac and caspase-3 in 80 cases of gastric cancer and 40 cases of adjacent normal gastric mucosa.
Results In 80 cases of gastric cancer,the expression rates of XIAP,Smac and caspase-3 were 90%(72/80),67.5%(54/80),and 62.5%(50/80),respectively.The XIAP expression was negatively correlated with tissue differentiation(P＜0.05),while expression of Smac and caspase-3 was positively correlated with tissue differentiation(P＜0.05).The expression of XIAP,Smac and caspase-3 was significantly different between intestinal-type and diffuse-type gastric cancer(P＜0.05),and correlated with lymph node metastasis (P＜0.05),but not with location,volume and invasion.The expression of XIAP and Smac in gastric cancer was negatively and positively correlated with caspase-3,respectively.In 40 cases of normal gastric mucosa,6 cases(15%) presented weak expression of XIAP,and the expression rates of Smac and caspase-3 were 92.5%(37/40) and 90%(36/40),respectively.
Conclusions XIAP expression increased along with the malignant degree of gastric cancer,while expression of Smac and caspase-3 decreased,which may participate in the development and progression of gastric cancer.These proteins may serve as potential biomarkers for evaluating the prognosis of gastric cancer.
Part Two Effects of transient transfection of Smac on chemotherapeutic sensitivities of gastric cancer cell line
Objective Abnormal apoptosis is one of key factors for the drug resistance of neoplasms.Smac is a novel gene involved in the regulation of apoptosis,which plays an important role in the inducing apoptosis effects of chemotherapeutic drugs on tumor cells. This study was designed to explore the effects of over-expression of Smac gene on chemotherapeutic sensitivities of gastric cancer cell line,in order to establish a basis for further improving chemotherapy of gastric cancer.
Methods Under induction of liposome GeneSHUTTLE-40,Smac gene was transfected into gastric cancer cell line MKN-45.Cellular Smac gene expression was determined by reverse transcription-polymerase chain reaction(RT-PCR) and western blot analysis.Cisplatin and MMC were administrated to un-transfeted and transfected MKN-45 cells.Cellular proliferation activities were assayed by tetrazolium bromide(MTT) colorimetry.The morphological changes of cancer cells were observed under an inversion microscope.Cellular apoptosis was determined by Annexin V-FITC and propidium iodide staining flow cytometry.
Results Compared with un-transfected control group,Smac mRNA and protein levels in transfected MKN-45 cells were significantly improved(P＜0.01).The growth inhibition rates of various concentrations of cisplatin and MMC on MKN-45 cells were improved by 10.10%-23.80%(P＜0.01) and 10.01%-15.86%(P＜0.01),respectively,with the cells presenting roundness,obvious refraction,and cellular fragment.Cellular apoptosis rates were improved by 6.7%-20.2%(P＜0.01) and 5.4%-13.2%(P＜0.01),respectively.
Conclusions Transfection of extrinsic Smac gene resulted in its over-expression in gastric cancer cells,which could improve the chemotherapeutic sensitivities of MKN-45 cells.This is a potential strategy for ameliorating chemotherapeutic effects of gastric cancer.
Part Three Establishment of gastric cancer cell line stably overexpressing Smac gene and its chemotherapeutic sensitivities
Objective To explore the effects of stable over-expression of Smac gene on chemotherapeutic sensitivities of gastric cancer cell line.
Methods Under the induction of liposome,the eukaryotic expression vector pcDNA3.1-Smac for Smac gene and its control vector pcDNA3.1 were transfected into gastric cancer cell line MKN-45.The subclone cell lines were obtained by persistent G_(418) selection.Smac gene expression of cancer cells was detected by RT-PCR and western blot methods.The growth inhibition effects of MMC on cancer cells were also observed by MTT colorimetry and clone formation assay.
Results The subclone gastric cancer cell lines,stably expressing Smac and neo gene respectively,were successfully selected,and named as MKN-45/Smac and MKN-45/neo. RT-PCR and western blot results demonstrated that Smac mRNA and protein levels of MKN-45/Smac cells were significantly higher than those of MKN-45 and MKN-45/neo (P＜0.01).After incubation with 10μg/ml MMC for 24 h,the growth inhibition rates of MKN-45 and MKN-45/neo were 27.85%,28.12%respectively,while that of MKN-45/Smac cells was 43.71%(P＜0.01).When compared with MKN-45 and MKN-45/neo cells,the clone formation abilities of MKN-45/Smac were reduced by 14.07%(P＜0.01) and 15.13%(P＜0.01),respectively.
Conclusions Stable transfection of Smac gene and its over-expression in gastric cancer cell line could significantly improve their chemotherapeutic sensitivities to MMC, which established an experimental basis for ameliorating chemotherapy of gastric cancer.
Part Four Regulatory effects of over-expression of Smac gene on apoptosis activities of gastric cancer cell line
Objective To explore the effects of over-expression of Smac gene on apoptosis activities of gastric cancer cell line.
Methods The gastric cancer cell lines MKN-45,MKN-45/neo and MKN-45/Smac were cultured in vitro.After teatment with MMC as an apoptosis inducer,cellular growth activities were investigated by trypan blue staining method.Apoptosis of cancer cells was detected by electronic microscopy,AO-EB fluorescent staining and TUNEL methods. Cellular protein levels and its activities of caspase-3 were assayed by western blot and colorimetry.
Results After incubation with 10μg/ml MMC for 24 h,growth activities of MKN-45/Smac were reduced by 10.0%～30.8%(P＜0.01),when compared with those of MKN-45. Partial MKN-45/Smac cells presented characteristic morphological changes of apoptosis under the electric and fluorescence microscopes,with apoptosis rates being 36.4%,which was significantly higher than that of MKN-45(15.2%,P＜0.01).After treatment with MMC, caspase-3 expression levels in MKN-45/Smac cells were significantly improved than those of MKN-45(P＜0.01),with caspase-3 activities enhanced by 3.25 times(P＜0.01).
Conclusions Transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line could significantly improve expression and activity levels of caspase-3 induced by MMC,resulting in obvious apoptosis-inducing effects,which established a novel strategy for regulating apoptosis activities of gastric cancer.
Part Five The effects of downregulating XIAP expression on the apoptosis of gastric cancer cells induced by chemotherapeutic drugs
Objective To observe the effects of downregulating XIAP expression on the chemotherapeutic sensitivities of gastric cancer cells.
Methods The antisense eukaryotic vector for XIAP was constructed and stably transferred into gastric cancer cell line MKN-45.RT-PCR and western blot were applied to detect the XIAP gene expression.Cisplatin and MMC were administrated to untransfected and transfected gastric cancer cells.MTT colorimetry and clone formation assay were performed to measure the in vitro cell viability.Apoptosis and its rates were detected by electronic microscopy,AO-EB fluorescent staining and TUNEL.Cellular caspase-3 protein expression and its activities were assayed by western blot and colorimetry.
Results RT-PCR and western blot indicated that the mRNA and protein levels of XIAP within gastric cancer MKN-45 cells stably transfected with antisense XIAP vector were significantly decreased by 84.75%(P＜0.01) and 89.75%(P＜0.01),respectively.After treatment with various concentrations of cisplatin and MMC for 24 hours,the cell growth inhibition of MKN-45 cells stably transfected with antisense XIAP was enhanced by 7.3%～25.3%(P＜0.01) and 12.3%～16.3%(P＜0.01),respectively.Partial cancer cells presented characteristic changes of apoptosis under an electronic microscope,while the apoptosis rates were 34.1%and 32.5%,respectively,which were significantly higher than that of untransfected control MKN-45 cells(14.2%,P＜0.05).Compared with MKN-45 cells,the caspase-3 expression within cells stably transfected with antisense XIAP was significantly increased by 2.45 times(P＜0.01),while the activity of caspase-3 was enhanced by 3.68 times(P＜0.01).
Conclusions Downregulation of XIAP expression via antisense RNA could increase the expression and activity of caspase-3,enhance the apoptosis of cancer cells induced by chemotherapeutic drugs.
Part Six Effects of curcumin on IAPs and Smac gene expression of gastric cancer cell line
Objective To explore the effects of curcumin on gene expression of IAPs and Smac in gastric cancer cell line,in order to explore the inducing apoptosis mechanisms of curcumin.
Methods After being treated with 10～40μmol/L curcumin for 6～24 h,growth activities of MKN-45 cells were detected by MTT colorimetry.Cellular apoptosis was assayed by TUNEL and DNA Ladder methods.IAPs(Survivin,XIAP) and Smac gene expression was detected by RT-PCR and western blot.Cellular caspase-3 activities were detected by colorimetry method. expression was detected by RT-PCR and western blot.Cellular caspase-3 activities were detected by colorimetry method.
Results When compared with untreated control group,cellular growth of antisense XIAP-transfected cells was significantly inhibited by various concentrations of curcumin, with inhibitory ratios being 12.18%～68.15%(P＜0.01).Partial MKN-45 cells presented characteristic morphological changes of apoptosis,with trapezia bands on gel electrophoresis.The apoptosis rates were 9.24%～28.12%(P＜0.01).Cellular mRNA and protein levels of Survivin and XIAP were significantly down-regulated(P＜0.01),with those of Smac being up-regulated(P＜0.01).Cellular caspase-3 activities were improved by 2.27～6.67 times(P＜0.01).
Conclusions Curcumin could significantly induce apoptosis of gastric cancer cell line MKN-45,one of its mechanisms was through up-regulating Smac and down-regulating Survivin and XIAP expression,leading to activation of caspase-3.
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