5-Fu诱导人结肠癌HCT-116细胞自噬与凋亡的关系及机制研究
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摘要
结直肠癌是临床上常见的消化道恶性肿瘤之一,近年来其发病率有明显上升的趋势,严重威胁着人类的健康。结直肠癌的发生、发展是多途径、多因素共同作用的复杂过程。当前以外科手术为中心的多学科综合治疗是结直肠癌最有效的治疗手段,但这些疗法都存在着一定的局限性,不能达到彻底根治肿瘤的目的。特别是在化疗过程中常会出现肿瘤细胞耐受的现象,逃逸凋亡而达不到理想的治疗效果。自噬是区别于细胞凋亡的另一种细胞程序性死亡路径,当前自噬与5-Fu诱导结肠癌细胞凋亡之间的关系及作用机制正成为结肠癌治疗的一个新热点。自噬和凋亡作为两种不同程序性细胞死亡途径,二者的信号通路在某些环节可能存在交叉,因此对其机制的研究,可为结肠癌化疗治疗敏感性药物开发和有效利用提供新的线索和途径。
一、5-Fu诱导人结肠癌细胞HCT-116自噬与凋亡
目的：研究5-Fu诱导结肠癌HCT-116细胞系自噬和凋亡的可能性。
方法：培养结肠癌HCT-116细胞,分为对照组及不同时点的5-Fu处理组(6h,12h,24h),通过MTT法检测不同处理组细胞增殖情况,通过MDC染色法检测HCT-116细胞自噬的水平,通过western blot法检测自噬相关基因beclin1、LC3B的表达水平,评价5-Fu诱导HCT-116细胞自噬的能力；通过DAPI染色法观察5-Fu诱导HCT-116细胞凋亡情况,流式细胞术定量5-Fu处理细胞后凋亡水平,结合western blot检测凋亡相关基因p53的表达情况,评价5-Fu诱导HCT-116细胞凋亡的能力。
结果：随着5-Fu剂量的增加,与对照组比较,各组吸收光密度值明显低于对照组(P<0.05),随着5-Fu作用时间的延长,各组细胞增殖抑制率显著上升(P<0.05)。5-Fu处理HCT-116细胞后,经MDC染色,于荧光显微镜下可见点状分布的自噬囊泡。自噬囊泡数目在12h达到最大值,与对照组相比有显著性差异(P<0.05)。自噬相关基因beclin1及LC3B蛋白表达水平逐渐升高,至12h达到最大值,与对照组相比显著升高。DAPI染色可见5-Fu作用细胞后细胞核染色质发生形态学改变；5-Fu作用细胞后6h凋亡率9.1%,12h组凋亡率19.5%,24h组凋亡率44.0%,显著高于对照组细胞凋亡率4.0%(P<0.05)；凋亡相关基因p53蛋白水平表达,随时间延长而逐渐升高,至24h达到最大值,与对照组相比具有显著性差异。
结论：5-Fu处理人结肠癌细胞HCT-116后可诱导自噬与凋亡
二、自噬在5-Fu诱导人结肠癌细胞HCT-116凋亡中的作用
目的：研究5-Fu联合自噬抑制剂(3-MA)、自噬诱导剂(Rapamycin)及泛凋亡抑制剂(Z-VAD-FMK)作用HCT-116细胞后,对细胞增殖、凋亡及自噬、凋亡相关基因水平表达的影响。
方法：采用MTT法检测不同处理组细胞增殖的情况,通过流式细胞术检测各组细胞凋亡的水平,荧光实时定量PCR法检测不同处理组细胞自噬和凋亡相关基因mRNA的表达水平。
结果：5-Fu联合3-MA、Rapamycin及Z-VAD-FMK分别作用HCT-116细胞,与对照组相比,随着作用时间延长(6h,12h,24h,48h),细胞吸光度显著降低(P<0.05),细胞增殖抑制率显著增高(P<0.05)。流式细胞术检测细胞凋亡率,与对照组(0.73±0.59%)相比,Rapamycin组(13.20±0.78%)凋亡率显著增高(P<0.05),不同用药方式间比较,Rapamycin组(13.20±0.78%)凋亡率显著高于3-MA组(5.25±1.77%)及Z-VAD-FMK组(2.83±0.97%)；与对照组相比,5-Fu组(21.76±2.14%)、5-Fu联合3-MA组(15.45±1.57%)、5-Fu联合Rapamycin组(28.37±1.72%)及5-Fu联合Z-VAD-FMK组(10.70±1.04%)凋亡率均显著增高(P<0.05)；不同处理方式间比较,5-Fu联合Rapamycin组凋亡率显著高于单独应用5-Fu组(P<0.05),5-Fu联合3-MA组及5-Fu联合Z-VAD-FMK组凋亡率显著低于单独应用5-Fu组(P<0.05)。与对照组比较,其它各组beclinl mRNA转录水平均显著增高(P<0.05)；4种不同处理方式之间,除5-Fu组和5-Fu联合Z-VAD-FMK组无显著差异外,余各组间均具有显著性差异(P<0.05)。与对照组比较,5-Fu组及5-Fu联合Rapamycin组LC3B mRNA转录水平均显著增高(P<0.05)；4种不同处理方式之间,与单独应用5-Fu组相比,5-Fu联合Rapamycin组LC3B mRNA转录水平显著增高(P<0.05),5-Fu联合3-MA组及5-Fu联合Z-VAD-FMK组LC3B mRNA转录水平均显著降低(P<0.05)。与对照组比较,5-Fu组及5-Fu联合Rapamycin组p53mRNA转录水平显著增高(P<0.05),4种不同处理方式之间,与单独应用5-Fu组相比,5-Fu联合Rapamycin组p53mRNA转录水平显著增高(P<0.05),5-Fu联合3-MA组及5-Fu联合Z-VAD-FMK组转录水平显著降低(P<0.05)。
结论：与单独应用5-Fu相比,5-Fu联合自噬诱导剂能够抑制HCT-116细胞增殖,同时促进细胞凋亡,5-Fu联合自噬抑制剂产生的生物学效应与之相反,5-Fu联合自噬诱导剂及自噬抑制剂不仅可影响自噬通路,同时也对凋亡通路起作用。
三、beclin1基因在5-Fu诱导HCT-116细胞自噬与凋亡中的作用
目的：建立beclin1基因过表达模型和干扰模型,探讨beclin1基因过表达及干扰下对HCT-116细胞增殖、凋亡、自噬与凋亡基因mRNA转录水平及蛋白表达水平的影响。
方法：通过构建重组表达质粒pcDNA3.1-beclin1及pGenesil1.3-beclin1Ri,用脂质体将重组质粒转染到HCT-116细胞中,采用western blot检测beclin1蛋白表达情况。利用MTT法检测各组间细胞增殖的情况,通过流式细胞术检测各组间细胞凋亡水平,荧光定量PCR法检测各组间自噬与凋亡相关基因mRNA转录水平变化,、vestern blot检测各组间自噬与凋亡蛋白表达水平的变化。
结果：随着作用时间的延长,与对照组相比,各组细胞OD值明显降低,细胞增殖抑制率显著增高(P<0.05)；与HCT-116+5-Fu组、beclin1Ri+5-Fu组比较,beclin1过表达+5-Fu组抑制率显著增高(P<0.05)。流式细胞术检测细胞凋亡,与对照组比较,6h、24h各组凋亡率均显著上升(P<0.05)；与HCT-116+5-Fu组比较,beclin1过表达+5-Fu组凋亡率显著上升(P<0.05)。5-Fu作用beclin1过表达HCT-116细胞,beclin1蛋白表达水平随时间延长而逐渐增加,48h达到最高值；5-Fu作用beclin1干扰HCT-116细胞,beclin1蛋白表达水平在6h达到最低值后,12h开始上升,24h达到最大值,48h表达下降,但仍高于对照组；5-Fu作用HCT-116细胞,beclin1蛋白表达水平6h出现升高,12h表达量至最大值,24h及48h表达量下降,但仍高于对照组。与对照组比较,5-Fu作用后各时点各组LC3B mRNA转录水平均显著增高(P<0.05)；5-Fu作用后12h、24h及48h, beclin1基因过表达组与其他各组比较,LC3B mRNA转录水平均显著增高(P<0.05)；5-Fu作用beclin1过表达HCT-116细胞,LC31Ⅱ/LC3Ⅰ蛋白表达比随时间延长而逐渐增加,48h达到最高值；5-Fu作用beclin1干扰HCT-116细胞,LC3Ⅱ/LC3Ⅰ蛋白表达比在6h达到最低值后,12h开始上升,24h达到最大值,48h表达下降,仍高于对照组；5-Fu作用HCT-116细胞,LC3Ⅱ/LC3Ⅰ蛋白表达比6h出现升高,12h至最大值,24h及48h比值下降,但仍高于对照组。5-Fu作用后6h、12h、24h及48h,各处理组p53mRNA的转录水平均显著高于对照组；beclinl基因过表达组与其他各组比较,各时点p53mRNA转录水平均显著增高(P<0.05)。5-Fu作用beclin1过表达HCT-116细胞,p53蛋白表达量随时间延长而逐渐增加,至48h达到最大值；5-Fu作用beclinl干扰HCT-116细胞,p53蛋白表达量在6h后开始升高,至24h达到最高值,48h表达量减少,仍高于6h表达水平；5-Fu作用HCT-116细胞,p53蛋白表达量在6h后开始升高,随着时间的延长表达量逐渐升高,在48h达到最大值。
结论：自噬相关基因beclin1过表达可抑制HCT-116细胞增殖,同时可促进细胞凋亡,LC3B及p53mRNA转录水平明显升高,beclin1、LC3Ⅱ和p53蛋白表达水平增加,；beclinl基因干扰后,HCT-116细胞增殖增多,抑制细胞凋亡,LC3B及p53mRNA转录水平降低,beclin1、LC3Ⅱ和p53蛋白表达量减少,LC3B及p53mRNA转录水平降低；自噬相关基因beclin1对HCT-116细胞的自噬及凋亡起正调控作用。
Colorectal cancer is one of the common malignant tumor for people. Although surgery resection currently remain the only curative treatment for colorectal cancer, it is always unsatisfactionary. Many people have no effects or only minor ones for the adjuvant drugs. During the process of colorectal cancer cell apoptosis induced by chemotherapy, the tolerance of the cells often appears and escapes from the apoptosis. Now the relationship between autophagy and apoptosis induced by5-Fu is becoming a new hot spot, which will be resulted in an increased response rate in advanced colorectal cancer, and the new therapeutic strategies and a new adjuvant drugs can be explored.
1. Detection of autophagy and apoptosis in HCT-116cells induced by5-Fu.
Objective:Research on the autophagy and apoptosis induced by5-Fu in HCT-116cells.
Methods:Culture the cells and take into control group and induced by5-Fu group. To evaluate autophagy induced by5-Fu, the methods include dyeing with MDC and Western blot. To evaluate apoptosis induced by5-Fu, the methods include dyeing with DAPI, FlowCytometry and Western blot.
Results:By dyed with MDC, The pointlike distribution of autophagy body were observed by fluorescence microscope. By dyed with DAPI, it observed the DNA of chromatin changed as apoptosis, and the apoptosis rate induced by5-Fu group significantly increased than the control group by FlowCytometry. Also the protein level of beclinl, LC3B and p53increased than the control group with Western blot.
Conclusion:The result shows that5-Fu can induce the autophagy and apoptosis of HCT-116cells.
2. The role of autophagy in apoptosis induced by5-Fu in HCT-116cells
Objective:Effects of proliferation, apoptosis and gene expression by inducers or inhibitors for autophagy and apoptosis on HCT-116cells.
Methods:After the HCT-116cells treated with5-Fu in combination with autophagy inducer(rapamycin), autophagy inhibitor(3-MA) or apoptosis inhibitor(Z-VAD-FMK), respectively, cell proliferation observed by MTT, apoptosis rate detected by FlowCytometry and the gene level expression by RT-PCR.
Results:The cell absorbency value reduced significantly, and the inhibit of cell proliferation significantly increased, as compared with in the control. The cell proliferation rates6,12and24h after induced by5-Fu were significantly lower than those at Oh. As compared with that in the controls, the apoptosis percentage of cells treated by using5-Fu and autophagy inducer increased significangtly. The apoptosis percentage of cells treated with5-Fu in combination with autophagy inducer, autophagy inhibitor or apoptosis inhibitor increased as compared with that in the controls.The apoptosis percentage of cells in5-Fu+rapamycin group increased significantly as compared with that in the5-Fu+3-MA and5-Fu+Z-VAD-FMK groups. The relative expressions of beclinl, LC3B and p53mRNA increased in HCT-116cells treated by5-Fu jointing with rapamycin, as compared with5-Fu group. On the contrary, The relative expressions of beclin1, LC3B and p53mRNA decreased in HCT-116cells treated by5-Fu jointing with autophagy inhibitor or apoptosis inhibitor. The relative expressions of p53mRNA increased in HCT-116cells treated by5-Fu and that jointing with rapamycin, as compared with5-Fu jointing with autophagy inhibitor or apoptosis inhibitor.
Conclusion:The result indicate that5-Fu combined with autophagy inducer can inhibit HCT-116cells proliferation, and it combinated with autophagy inhibitor can promote the cell proliferation. And5-Fu combined with autophagy inducer can promote the apoptosis of HCT-116cells. The cell autophagy and apoptosis are two different programmed cell death ways, that have overlap.5-Fu jointing with autophagy inducer affects not only the autophagy pathway but also the apoptosis pathway in the cells.
3.The role of beclinl gene in autophagy and apoptosis in HCT-116cells induced by5-Fu
Objective:Constructing beclinl gene interference and over-expression models, and research on the proliferation, apoptosis, protein level and gene expression effected by5-Fu on beclinl gene interference and over-expression models.
Methods:Constructed pcDNA3.1-beclinland pGenesill.3-beclin1Ri recombinated vector, and transfected into the HCT-116cells by liposome method. To observed cell proliferation by MTT on beclinl gene interference and over expression models in HCT-116cells. And the effects of5-Fu on apoptosis in HCT-116, HCT-116-beclinl and HCT-116-beclinlRi cells by FlowCytometry. Also the methods of RT-PCR and western blot detect the gene expression and protein level respectly.
Results:Cell prolifetation in the5-Fu and HCT-116-beclinl group decreased significantly as compared with the other groups. The apoptosis percentages of the5-Fu group, HCT-116-beclinl and HCT-116-beclinlRi6h and24h increased significantly as the control group. The apoptosis percentage between the HCT-116-beclinl, HCT-116-beclinlRi5-Fu and5-Fu is statistical different. It is also significantly between HCT-116-beclinl and HCT-116-beclinlRi. The cell apoptosis percentage in the HCT-116-beclinl was the highest. The expression of beclinl protein in HCT-116cells increased6h after5-Fu induced, which reached to the greatest12h later, and decreased24,48h, still higher than that in the control. The expression of p53rise6h. which increased gradually with the extension of time. In the HCT-116-beclinlRi cells are lower at6h after induced by5-Fu. which begin to rise12h, and reach to the max in24h. the expression of p53protein begin to rise6h, and reach to the max in24h. The expression of beclinl protein in HCT-116-beclinl increased gradually with time. The relative expression of LC3B/GAPDH gene of HCT-116cells, HCT-116-beclinl and HCT-116-beclinlRi were significantly different. The relative expression of p53/GAPDH in5-Fu group, HCT-116-beclinl and HCT-116-beclinlRi group was significantly higher than that in the HCT-116group. And it was significantly different in expression of p53mRNA between all of the groups.
Conclusion:The over expression of beclinl gene restrain the cell prolification of HCT-116cells. The expression of beclinl in HCT-116cells induced by gene promoted the increased of p53protein expression, and the autophagy induced by5-Fu can increase cell apoptosis. The beclinl gene in the HCT-116cells can regulated the cell autophagy and apoptosis.
一、5-Fu诱导人结肠癌细胞HCT-116自噬与凋亡
目的：研究5-Fu诱导结肠癌HCT-116细胞系自噬和凋亡的可能性。
方法：培养结肠癌HCT-116细胞,分为对照组及不同时点的5-Fu处理组(6h,12h,24h),通过MTT法检测不同处理组细胞增殖情况,通过MDC染色法检测HCT-116细胞自噬的水平,通过western blot法检测自噬相关基因beclin1、LC3B的表达水平,评价5-Fu诱导HCT-116细胞自噬的能力；通过DAPI染色法观察5-Fu诱导HCT-116细胞凋亡情况,流式细胞术定量5-Fu处理细胞后凋亡水平,结合western blot检测凋亡相关基因p53的表达情况,评价5-Fu诱导HCT-116细胞凋亡的能力。
结果：随着5-Fu剂量的增加,与对照组比较,各组吸收光密度值明显低于对照组(P<0.05),随着5-Fu作用时间的延长,各组细胞增殖抑制率显著上升(P<0.05)。5-Fu处理HCT-116细胞后,经MDC染色,于荧光显微镜下可见点状分布的自噬囊泡。自噬囊泡数目在12h达到最大值,与对照组相比有显著性差异(P<0.05)。自噬相关基因beclin1及LC3B蛋白表达水平逐渐升高,至12h达到最大值,与对照组相比显著升高。DAPI染色可见5-Fu作用细胞后细胞核染色质发生形态学改变；5-Fu作用细胞后6h凋亡率9.1%,12h组凋亡率19.5%,24h组凋亡率44.0%,显著高于对照组细胞凋亡率4.0%(P<0.05)；凋亡相关基因p53蛋白水平表达,随时间延长而逐渐升高,至24h达到最大值,与对照组相比具有显著性差异。
结论：5-Fu处理人结肠癌细胞HCT-116后可诱导自噬与凋亡
二、自噬在5-Fu诱导人结肠癌细胞HCT-116凋亡中的作用
目的：研究5-Fu联合自噬抑制剂(3-MA)、自噬诱导剂(Rapamycin)及泛凋亡抑制剂(Z-VAD-FMK)作用HCT-116细胞后,对细胞增殖、凋亡及自噬、凋亡相关基因水平表达的影响。
方法：采用MTT法检测不同处理组细胞增殖的情况,通过流式细胞术检测各组细胞凋亡的水平,荧光实时定量PCR法检测不同处理组细胞自噬和凋亡相关基因mRNA的表达水平。
结果：5-Fu联合3-MA、Rapamycin及Z-VAD-FMK分别作用HCT-116细胞,与对照组相比,随着作用时间延长(6h,12h,24h,48h),细胞吸光度显著降低(P<0.05),细胞增殖抑制率显著增高(P<0.05)。流式细胞术检测细胞凋亡率,与对照组(0.73±0.59%)相比,Rapamycin组(13.20±0.78%)凋亡率显著增高(P<0.05),不同用药方式间比较,Rapamycin组(13.20±0.78%)凋亡率显著高于3-MA组(5.25±1.77%)及Z-VAD-FMK组(2.83±0.97%)；与对照组相比,5-Fu组(21.76±2.14%)、5-Fu联合3-MA组(15.45±1.57%)、5-Fu联合Rapamycin组(28.37±1.72%)及5-Fu联合Z-VAD-FMK组(10.70±1.04%)凋亡率均显著增高(P<0.05)；不同处理方式间比较,5-Fu联合Rapamycin组凋亡率显著高于单独应用5-Fu组(P<0.05),5-Fu联合3-MA组及5-Fu联合Z-VAD-FMK组凋亡率显著低于单独应用5-Fu组(P<0.05)。与对照组比较,其它各组beclinl mRNA转录水平均显著增高(P<0.05)；4种不同处理方式之间,除5-Fu组和5-Fu联合Z-VAD-FMK组无显著差异外,余各组间均具有显著性差异(P<0.05)。与对照组比较,5-Fu组及5-Fu联合Rapamycin组LC3B mRNA转录水平均显著增高(P<0.05)；4种不同处理方式之间,与单独应用5-Fu组相比,5-Fu联合Rapamycin组LC3B mRNA转录水平显著增高(P<0.05),5-Fu联合3-MA组及5-Fu联合Z-VAD-FMK组LC3B mRNA转录水平均显著降低(P<0.05)。与对照组比较,5-Fu组及5-Fu联合Rapamycin组p53mRNA转录水平显著增高(P<0.05),4种不同处理方式之间,与单独应用5-Fu组相比,5-Fu联合Rapamycin组p53mRNA转录水平显著增高(P<0.05),5-Fu联合3-MA组及5-Fu联合Z-VAD-FMK组转录水平显著降低(P<0.05)。
结论：与单独应用5-Fu相比,5-Fu联合自噬诱导剂能够抑制HCT-116细胞增殖,同时促进细胞凋亡,5-Fu联合自噬抑制剂产生的生物学效应与之相反,5-Fu联合自噬诱导剂及自噬抑制剂不仅可影响自噬通路,同时也对凋亡通路起作用。
三、beclin1基因在5-Fu诱导HCT-116细胞自噬与凋亡中的作用
目的：建立beclin1基因过表达模型和干扰模型,探讨beclin1基因过表达及干扰下对HCT-116细胞增殖、凋亡、自噬与凋亡基因mRNA转录水平及蛋白表达水平的影响。
方法：通过构建重组表达质粒pcDNA3.1-beclin1及pGenesil1.3-beclin1Ri,用脂质体将重组质粒转染到HCT-116细胞中,采用western blot检测beclin1蛋白表达情况。利用MTT法检测各组间细胞增殖的情况,通过流式细胞术检测各组间细胞凋亡水平,荧光定量PCR法检测各组间自噬与凋亡相关基因mRNA转录水平变化,、vestern blot检测各组间自噬与凋亡蛋白表达水平的变化。
结果：随着作用时间的延长,与对照组相比,各组细胞OD值明显降低,细胞增殖抑制率显著增高(P<0.05)；与HCT-116+5-Fu组、beclin1Ri+5-Fu组比较,beclin1过表达+5-Fu组抑制率显著增高(P<0.05)。流式细胞术检测细胞凋亡,与对照组比较,6h、24h各组凋亡率均显著上升(P<0.05)；与HCT-116+5-Fu组比较,beclin1过表达+5-Fu组凋亡率显著上升(P<0.05)。5-Fu作用beclin1过表达HCT-116细胞,beclin1蛋白表达水平随时间延长而逐渐增加,48h达到最高值；5-Fu作用beclin1干扰HCT-116细胞,beclin1蛋白表达水平在6h达到最低值后,12h开始上升,24h达到最大值,48h表达下降,但仍高于对照组；5-Fu作用HCT-116细胞,beclin1蛋白表达水平6h出现升高,12h表达量至最大值,24h及48h表达量下降,但仍高于对照组。与对照组比较,5-Fu作用后各时点各组LC3B mRNA转录水平均显著增高(P<0.05)；5-Fu作用后12h、24h及48h, beclin1基因过表达组与其他各组比较,LC3B mRNA转录水平均显著增高(P<0.05)；5-Fu作用beclin1过表达HCT-116细胞,LC31Ⅱ/LC3Ⅰ蛋白表达比随时间延长而逐渐增加,48h达到最高值；5-Fu作用beclin1干扰HCT-116细胞,LC3Ⅱ/LC3Ⅰ蛋白表达比在6h达到最低值后,12h开始上升,24h达到最大值,48h表达下降,仍高于对照组；5-Fu作用HCT-116细胞,LC3Ⅱ/LC3Ⅰ蛋白表达比6h出现升高,12h至最大值,24h及48h比值下降,但仍高于对照组。5-Fu作用后6h、12h、24h及48h,各处理组p53mRNA的转录水平均显著高于对照组；beclinl基因过表达组与其他各组比较,各时点p53mRNA转录水平均显著增高(P<0.05)。5-Fu作用beclin1过表达HCT-116细胞,p53蛋白表达量随时间延长而逐渐增加,至48h达到最大值；5-Fu作用beclinl干扰HCT-116细胞,p53蛋白表达量在6h后开始升高,至24h达到最高值,48h表达量减少,仍高于6h表达水平；5-Fu作用HCT-116细胞,p53蛋白表达量在6h后开始升高,随着时间的延长表达量逐渐升高,在48h达到最大值。
结论：自噬相关基因beclin1过表达可抑制HCT-116细胞增殖,同时可促进细胞凋亡,LC3B及p53mRNA转录水平明显升高,beclin1、LC3Ⅱ和p53蛋白表达水平增加,；beclinl基因干扰后,HCT-116细胞增殖增多,抑制细胞凋亡,LC3B及p53mRNA转录水平降低,beclin1、LC3Ⅱ和p53蛋白表达量减少,LC3B及p53mRNA转录水平降低；自噬相关基因beclin1对HCT-116细胞的自噬及凋亡起正调控作用。
Colorectal cancer is one of the common malignant tumor for people. Although surgery resection currently remain the only curative treatment for colorectal cancer, it is always unsatisfactionary. Many people have no effects or only minor ones for the adjuvant drugs. During the process of colorectal cancer cell apoptosis induced by chemotherapy, the tolerance of the cells often appears and escapes from the apoptosis. Now the relationship between autophagy and apoptosis induced by5-Fu is becoming a new hot spot, which will be resulted in an increased response rate in advanced colorectal cancer, and the new therapeutic strategies and a new adjuvant drugs can be explored.
1. Detection of autophagy and apoptosis in HCT-116cells induced by5-Fu.
Objective:Research on the autophagy and apoptosis induced by5-Fu in HCT-116cells.
Methods:Culture the cells and take into control group and induced by5-Fu group. To evaluate autophagy induced by5-Fu, the methods include dyeing with MDC and Western blot. To evaluate apoptosis induced by5-Fu, the methods include dyeing with DAPI, FlowCytometry and Western blot.
Results:By dyed with MDC, The pointlike distribution of autophagy body were observed by fluorescence microscope. By dyed with DAPI, it observed the DNA of chromatin changed as apoptosis, and the apoptosis rate induced by5-Fu group significantly increased than the control group by FlowCytometry. Also the protein level of beclinl, LC3B and p53increased than the control group with Western blot.
Conclusion:The result shows that5-Fu can induce the autophagy and apoptosis of HCT-116cells.
2. The role of autophagy in apoptosis induced by5-Fu in HCT-116cells
Objective:Effects of proliferation, apoptosis and gene expression by inducers or inhibitors for autophagy and apoptosis on HCT-116cells.
Methods:After the HCT-116cells treated with5-Fu in combination with autophagy inducer(rapamycin), autophagy inhibitor(3-MA) or apoptosis inhibitor(Z-VAD-FMK), respectively, cell proliferation observed by MTT, apoptosis rate detected by FlowCytometry and the gene level expression by RT-PCR.
Results:The cell absorbency value reduced significantly, and the inhibit of cell proliferation significantly increased, as compared with in the control. The cell proliferation rates6,12and24h after induced by5-Fu were significantly lower than those at Oh. As compared with that in the controls, the apoptosis percentage of cells treated by using5-Fu and autophagy inducer increased significangtly. The apoptosis percentage of cells treated with5-Fu in combination with autophagy inducer, autophagy inhibitor or apoptosis inhibitor increased as compared with that in the controls.The apoptosis percentage of cells in5-Fu+rapamycin group increased significantly as compared with that in the5-Fu+3-MA and5-Fu+Z-VAD-FMK groups. The relative expressions of beclinl, LC3B and p53mRNA increased in HCT-116cells treated by5-Fu jointing with rapamycin, as compared with5-Fu group. On the contrary, The relative expressions of beclin1, LC3B and p53mRNA decreased in HCT-116cells treated by5-Fu jointing with autophagy inhibitor or apoptosis inhibitor. The relative expressions of p53mRNA increased in HCT-116cells treated by5-Fu and that jointing with rapamycin, as compared with5-Fu jointing with autophagy inhibitor or apoptosis inhibitor.
Conclusion:The result indicate that5-Fu combined with autophagy inducer can inhibit HCT-116cells proliferation, and it combinated with autophagy inhibitor can promote the cell proliferation. And5-Fu combined with autophagy inducer can promote the apoptosis of HCT-116cells. The cell autophagy and apoptosis are two different programmed cell death ways, that have overlap.5-Fu jointing with autophagy inducer affects not only the autophagy pathway but also the apoptosis pathway in the cells.
3.The role of beclinl gene in autophagy and apoptosis in HCT-116cells induced by5-Fu
Objective:Constructing beclinl gene interference and over-expression models, and research on the proliferation, apoptosis, protein level and gene expression effected by5-Fu on beclinl gene interference and over-expression models.
Methods:Constructed pcDNA3.1-beclinland pGenesill.3-beclin1Ri recombinated vector, and transfected into the HCT-116cells by liposome method. To observed cell proliferation by MTT on beclinl gene interference and over expression models in HCT-116cells. And the effects of5-Fu on apoptosis in HCT-116, HCT-116-beclinl and HCT-116-beclinlRi cells by FlowCytometry. Also the methods of RT-PCR and western blot detect the gene expression and protein level respectly.
Results:Cell prolifetation in the5-Fu and HCT-116-beclinl group decreased significantly as compared with the other groups. The apoptosis percentages of the5-Fu group, HCT-116-beclinl and HCT-116-beclinlRi6h and24h increased significantly as the control group. The apoptosis percentage between the HCT-116-beclinl, HCT-116-beclinlRi5-Fu and5-Fu is statistical different. It is also significantly between HCT-116-beclinl and HCT-116-beclinlRi. The cell apoptosis percentage in the HCT-116-beclinl was the highest. The expression of beclinl protein in HCT-116cells increased6h after5-Fu induced, which reached to the greatest12h later, and decreased24,48h, still higher than that in the control. The expression of p53rise6h. which increased gradually with the extension of time. In the HCT-116-beclinlRi cells are lower at6h after induced by5-Fu. which begin to rise12h, and reach to the max in24h. the expression of p53protein begin to rise6h, and reach to the max in24h. The expression of beclinl protein in HCT-116-beclinl increased gradually with time. The relative expression of LC3B/GAPDH gene of HCT-116cells, HCT-116-beclinl and HCT-116-beclinlRi were significantly different. The relative expression of p53/GAPDH in5-Fu group, HCT-116-beclinl and HCT-116-beclinlRi group was significantly higher than that in the HCT-116group. And it was significantly different in expression of p53mRNA between all of the groups.
Conclusion:The over expression of beclinl gene restrain the cell prolification of HCT-116cells. The expression of beclinl in HCT-116cells induced by gene promoted the increased of p53protein expression, and the autophagy induced by5-Fu can increase cell apoptosis. The beclinl gene in the HCT-116cells can regulated the cell autophagy and apoptosis.
引文
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