GSTA1对结肠癌细胞增殖和凋亡的影响研究
摘要
神经酰胺是细胞内重要的脂质第二信使,可介导多种细胞生物学效应,其最主要的作用是抑制细胞的增殖和诱导细胞凋亡(包括肿瘤细胞)。鉴于神经酰胺可诱导肿瘤细胞凋亡的作用,增加内源性神经酰胺浓度或导入外源性类似物已成为肿瘤治疗研究的新靶点。研究证实外源性具有渗透性的神经酰胺类似物(C2-神经酰胺)可通过多种途径诱导HT-29、Lovo、HCT-116等结肠癌细胞的调亡,关于神经酰胺对Caco-2结肠癌细胞凋亡的影响未见报道。
应用化疗药物是治疗结肠癌最常用的方法之一,但是化疗耐药性的产生常造成肿瘤治疗的失败。GSTs是一组多功能Ⅱ相药物代谢酶,它的过表达是导致癌细胞产生化疗耐药性的因素之一,肿瘤细胞可通过表达GSTs而保护自身免受抗肿瘤药物的损害,从而降低抗肿瘤药物疗效。有研究表明GSTs家族中GSTA的过表达与氮芥、蒽醌类抗肿瘤药物的耐药性密切相关,GSTA1是GSTA的一个非常重要的亚型,因此对GSTA1表达调节及其在癌细胞中增殖和凋亡作用的研究具有非常重要的意义。
本研究以结肠癌细胞株Caco-2和HT-29为试验对象,检测了外源性C2-神经酰胺对两种结肠癌细胞系增殖和凋亡的影响,为后续的试验研究GSTA1在细胞增殖抑制和凋亡中的作用奠定了基础。本试验对结肠癌细胞不同生长时期GSTA1表达与活性进行了研究,检测了外源性C2-神经酰胺处理的结肠癌细胞中GSTA1表达与活性的变化。采用基因转染技术使GSTA1沉默或过表达,探讨GSTA1表达对神经酰胺引起的细胞增殖抑制和凋亡的影响,旨在揭示GSTA1能否保护结肠癌细胞免受由神经酰胺引起的增殖抑制和诱导凋亡,对于阐明GSTA1与结肠癌细胞增殖抑制、凋亡及化疗耐药性之间的关系具有重要意义,研究结果可用于指导临床合理用药,提高抗肿瘤药物治疗效果和降低耐药性的产生。
本研究采用MTS法检测外源性C2-神经酰胺对Caco-2和HT-29细胞增殖的影响,分别采用western blotting和real-time PCR技术检测激活型caspase-3蛋白和Bcl-2家族基因表达情况,以评价神经酰胺对细胞凋亡的影响。然后再以western blotting、real-time PCR和酶活性分析法分别检测两个细胞系不同生长时期GSTAl蛋白、mRNA表达与活性变化以及C2-神经酰胺处理对结肠癌细胞GSTA1蛋白、mRNA表达及活性的影响。采用基因转染的方法来双向验证GSTA1在整个过程的功能,一方面采用RNA干扰技术使Caco-2细胞中GSTA1沉默,检测GSTA1沉默对神经酰胺介导的细胞增殖和凋亡的影响。另一方面将含有GSTA1的质粒转染HT-29细胞,检测GSTA1过表达对神经酰胺介导的细胞增殖和凋亡的影响。
外源性C2-神经酰胺对结肠癌细胞增殖影响的结果表明,随着神经酰胺浓度升高,两种结肠癌细胞的存活率逐渐降低。对结肠癌细胞凋亡影响的结果表明,神经酰胺不能诱导Caco-2细胞中激活型caspase-3蛋白的表达,同时抗凋亡基因Bcl-2和促凋亡基因BaxmRNA表达均无明显变化(P>0.05),而神经酰胺处理HT-29细胞后,与对照组相比较,处理组激活型caspase-3蛋白表达量增加7.142倍(P<0.01),Bcl-2mRNA表达降低0.466倍(P<0.001),BaxmRNA表达升高5.938倍(P<0.001)。
对Caco-2和HT-29细胞不同生长时期GSTA1表达及活性检测的结果表明,Caco-2细胞中GSTA1蛋白、mRNA表达与活性随着细胞的汇合程度逐渐升高。与汇合前期相比较,汇合期GSTA1蛋白、mRNA及酶活性分别升高了1.888、3.018和1.994倍(P<0.05或P<0.01),在6d汇合后期GSTA1蛋白、mRNA及活性分别升高了2.988、7.135和6.508倍(P<0.001)。而HT-29细胞的各个生长时期均未检测到GSTA1蛋白和mRNA的表达。
对外源性C2-神经酰胺处理的Caco-2和HT-29细胞中GSTA1表达及活性检测结果表明,神经酰胺处理的Caco-2细胞中GSTA1蛋白、mRNA表达及活性分别增加了1.850、3.093和1.764倍,与对照组比较差异显著(p<0.01或p<0.001)。而神经酰胺处理HT-29细胞后,仍未能检测到GSTA1蛋白和mRNA的表达。
采用western blotting方法确定转染剂Lipofectamine2000为3μl/mL、40nM siRNA、转染48h为最佳的siRNA转染条件。在此基础上研究GSTA1沉默对神经酰胺介导的Caco-2细胞增殖和凋亡的影响。结果显示,对照组、siRNA转染组和阴性对照组细胞存活率差异不显著(P>0.05)。神经酰胺处理后,3个组细胞存活率均降低,但各组间并无统计学差异(P>0.05)。对照组、siRNA转染组和阴性对照组均无激活型caspase-3蛋白表达,神经酰胺处理后,3个组的caspase-3蛋白也均未被激活。
双酶切和序列测定表明本试验成功构建了重组真核表达载体GSTA1-pcDNA3.1/V5-His TOPO并将其瞬时转染至HT-29细胞中。采用western blotting方法确定转染剂Lipofectamine2000为3μl/mL、3μg质粒、转染48h为最佳的质粒转染条件。在此基础上研究GSTA1过表达对神经酰胺介导的HT-29细胞增殖和凋亡的影响。结果表明,对照组、质粒转染组和阴性对照组细胞存活率差异不显著(P>0.05),神经酰胺处理后,3个组细胞存活率均降低,但各组间并无统计学差异(P>0.05)。对照组、质粒转染组和阴性对照组均无激活型caspase-3蛋白表达,神经酰胺处理后,3个组的激活型caspase-3蛋白表达均增加,但各组间差异不显著(P>0.05)。
外源性C2-神经酰胺可显著抑制Caco-2和HT-29结肠癌细胞的增殖,不能诱导Caco-2凋亡,但可诱导HT-29凋亡。Caco-2细胞中GSTA1蛋白、mRNA表达量与活性随细胞汇合程度逐渐增加,而HT-29细胞各生长时期中GSTA1表达量极低或不表达。外源性C2-神经酰胺处理的Caco-2细胞中GSTA1蛋白、mRNA表达量与活性显著升高,而神经酰胺处理的HT-29细胞仍然检测不到GSTA1的表达。基因转染试验证实了GSTA1的沉默或过表达对神经酰胺介导的结肠癌细胞增殖抑制和凋亡无影响,以上结果提示GSTA1不具有保护结肠癌细胞免受神经酰胺引起的增殖抑制和凋亡的作用。
Ceramide is important second messenger in the cells, take part in modulating multiple biological effects. The most well-known roles of ceramide are proliferation inhibition and apoptosis (including tumour cells). In view of its role in inducing apoptsis, enhancing the concentration of endogenous ceramide or introducing exogenous analogues are the new targets of oncotherapy. Studies have shown that an exogenous ceramide analogue (C2-ceramide) can induce apoptosis in colon cancer cell lines HT-29, Lovo, and HCT-116through multiple pathways; however, there is no reports about the affect of ceramide on apoptosis of Caco-2cells.
Chemotherapy has been widely used in colon cancer treatment, but the oncotherapy are often failed due to chemoresistance. GSTs are a group of phase Ⅱ drug metabolism enzymes. Overexpression of GSTs is one of the important factor for producing chemoresistance, tumour cells can protect themselves against anti-tumour drugs and reduce curative effect of anti-tumour drugs by express GST. Evidence shows that GSTA expression is strongly related to the resistance of anti-cancer drugs, such as nitrogen mustard and anthraquinone. GSTA1is an important member of the GSTA family. Study in this enzyme will advance our understanding in the proliferation and apoptosis of the cancer cells.
This study detected the effects of exogenous C2-ceramide on proliferation and apoptosis using Caco-2and HT-29cell lines, this research will provide fundamental knowledge for our future study on GSTA1-associated proliferation inhibition and apoptosis of the cell. This study investigated the expression and activity of GSTA1at different stages of colon cancer cells and determined the change of GSTA1expression and activity during exogenous ceramide-modulated proliferation inhibition and apoptosis of colon cancer cells. Gene transfection techniques were used to silence or overexpress GSTA1, and to explore how ceramides affects cell proliferation and apoptosis. This study will reveal if GSTA1expression in colon cancer cells can protect themselves against the effects of ceramide-modulated cell proliferation inhibition and apoptosis and useful to clarify the relationship between GSTA1, proliferation inhibiton/apoptosis of colon cancer cells and chemoresistance. Results can be translated to guide clinical application, and enhance effects of anti-cancer drugs and reduce the drug resistance in new drug development.
MTS assay was employed to determine the effects of C2-ceramide on the cell proliferation in in vitro cell lines Caco-2and HT-29. Western blotting and real-time PCR were used to determine the effects of ceramides on apoptosis by measuring the expression levels of actived caspase-3and Bcl-2gene family. Furthermore, Western blotting, real-time PCR, and enzyme activity assay technologies were employed to determine the expression of GSTA1protein, mRNA and activity at different stages of colon cancer cells, effects of ceramide on GSTA1protein expression, mRNA expression, and activity were also detected. In addition, gene transfection techniques were used to confirm the functions of GSTA1in ceramide-modulated cell proliferation and apoptosis. The first approach is to use siRNA technique to knockdown GSTA1gene in Caco-2cells in order to detect the effect of GSTA1silencing on ceramide-mediated proliferation inhibition and apoptosis; and the second was to transfect HT-29cells with plasmid in order to detect the effect of GSTA1overexpression on ceramide-mediated proliferation inhibition and apoptosis.
The results of effect of exogenous C2-ceramide on proliferation showed that the survival rate of colon cancer cell progressively decreased with the concentration of ceramide. The results of effect of ceramide on apoptosis showed that ceramide did not induce the expression of actived caspase-3protein. The expression of anti-apoptotic Bcl-2mRNA and pro-apoptotic Bax mRNA did not show any significant difference. Treatment with ceramide resulted in7.142-fold increase of the actived caspase-3protein expression in HT-29cells comparing with control group (P<0.01), accompanied with0.466-fold decrease of Bcl-2mRNA (P<0.001)and6.938-fold increase Bax mRNA levels (P<0.001).
The changes of GSTA1protein levels, mRNA expression and activity in different stages of Caco-2and HT-29were detected. Results showd that GSTA1protein levels, mRNA expression, and enzyme activity in Caco-2cells were progressively increased with the confluency. Compared with preconfluent, GSTA1protein, mRNA levels and activity rose to1.888,3.018,1.994-fold in confluent respectively (P<0.05or P<0.01), and rose to2.988,7.135,6.598-fold in6d postconfluent respectively (P<0.001). There were no expression of GSTA1protein and mRNA of all the stages in HT-29cells.
The effect on GSTA1protein levels, mRNA expression and activitiy in Caco-2and HT-29cells induced by exogenous C2-ceramide were detected. Results showed that GSTA1protein, mRNA expression and activity induced by ceramide of Caco-2cells increased1.850,3.093,1.764-fold respectively, significant difference comparing with control group (p<0.01or p<0.001), but no expression of GSTA1protein and mRNA expression were seen in HT-29cells.
The best transfection conditions,3μl/mL Lipofectamine2000,40nM siRNA, transfected48h,were confirmed by western blotting analysis. These GSTA1-silenced Caco-2cells were then used to study the effect of ceramide on cell proliferation and apoptosis in Caco-2cells. Results showed that there was no significant difference in survival rate between control, siRNA transfection, negative control group (P>0.05), the survival rate decreased of the3groups with the ceramide treatment, but no statistics difference between groups (P>0.05). There was no detection of activated caspase-3protein expression in control, siRNA transfection, negative control group, activated caspase-3protein expression was not detected in3groups with ceramide treatment.
The construction of recombinant plasmid GSTA1-pcDNA3.1/V5-His TOPO was verified by double-enzyme cleavage and gene sequence analysis. Recombinant plasmids were then transiently transfected into HT-29cells. The best transfection conditions,3μl/mL Lipofectamine2000,3μg DNA, transfected48h,were confirmed by western blotting analysis. These GSTAl-overexpressed Caco-2cells were then used to study the effect of ceramide on cell proliferation and apoptosis in HT-29cells. Results showed that there was no significant difference in survival rate between control, plasmid transfection, negative control group (P>0.05), the survival rate decreased of the3groups with the ceramide treatment, but no statistics difference between groups (P>0.05). There was no detection of activated caspase-3protein expression in control, plasmid transfection, negative control group, activated caspase-3protein expression were induced with ceramide treatment, but no statistics difference between groups(P>0.05).
In conclusion, ceramides inhibits the proliferations of two colon cancer cells. Ceramides can induce apoptosis of HT-29but not Caco-2cells. The protein levels, mRNA expression, and enzymes activities of Caco-2cells are increased in correlation with cell confluency, the GSTA1expression is very low or not existed in HT-29cells. Treatment with ceramides can increase GSTA1protein levels, mRNA expression, and activity in Caco-2cells, but the protein and mRNA expression is not seen in HT-29cells. Gene transfection experiments manifest that GSTA1does not affect the ceramide-modulated proliferation inhibition and anti-apoptosis, these results suggested no protective role for GSTA1in ceramide-modulated proliferation inhibition and apoptosis.
应用化疗药物是治疗结肠癌最常用的方法之一,但是化疗耐药性的产生常造成肿瘤治疗的失败。GSTs是一组多功能Ⅱ相药物代谢酶,它的过表达是导致癌细胞产生化疗耐药性的因素之一,肿瘤细胞可通过表达GSTs而保护自身免受抗肿瘤药物的损害,从而降低抗肿瘤药物疗效。有研究表明GSTs家族中GSTA的过表达与氮芥、蒽醌类抗肿瘤药物的耐药性密切相关,GSTA1是GSTA的一个非常重要的亚型,因此对GSTA1表达调节及其在癌细胞中增殖和凋亡作用的研究具有非常重要的意义。
本研究以结肠癌细胞株Caco-2和HT-29为试验对象,检测了外源性C2-神经酰胺对两种结肠癌细胞系增殖和凋亡的影响,为后续的试验研究GSTA1在细胞增殖抑制和凋亡中的作用奠定了基础。本试验对结肠癌细胞不同生长时期GSTA1表达与活性进行了研究,检测了外源性C2-神经酰胺处理的结肠癌细胞中GSTA1表达与活性的变化。采用基因转染技术使GSTA1沉默或过表达,探讨GSTA1表达对神经酰胺引起的细胞增殖抑制和凋亡的影响,旨在揭示GSTA1能否保护结肠癌细胞免受由神经酰胺引起的增殖抑制和诱导凋亡,对于阐明GSTA1与结肠癌细胞增殖抑制、凋亡及化疗耐药性之间的关系具有重要意义,研究结果可用于指导临床合理用药,提高抗肿瘤药物治疗效果和降低耐药性的产生。
本研究采用MTS法检测外源性C2-神经酰胺对Caco-2和HT-29细胞增殖的影响,分别采用western blotting和real-time PCR技术检测激活型caspase-3蛋白和Bcl-2家族基因表达情况,以评价神经酰胺对细胞凋亡的影响。然后再以western blotting、real-time PCR和酶活性分析法分别检测两个细胞系不同生长时期GSTAl蛋白、mRNA表达与活性变化以及C2-神经酰胺处理对结肠癌细胞GSTA1蛋白、mRNA表达及活性的影响。采用基因转染的方法来双向验证GSTA1在整个过程的功能,一方面采用RNA干扰技术使Caco-2细胞中GSTA1沉默,检测GSTA1沉默对神经酰胺介导的细胞增殖和凋亡的影响。另一方面将含有GSTA1的质粒转染HT-29细胞,检测GSTA1过表达对神经酰胺介导的细胞增殖和凋亡的影响。
外源性C2-神经酰胺对结肠癌细胞增殖影响的结果表明,随着神经酰胺浓度升高,两种结肠癌细胞的存活率逐渐降低。对结肠癌细胞凋亡影响的结果表明,神经酰胺不能诱导Caco-2细胞中激活型caspase-3蛋白的表达,同时抗凋亡基因Bcl-2和促凋亡基因BaxmRNA表达均无明显变化(P>0.05),而神经酰胺处理HT-29细胞后,与对照组相比较,处理组激活型caspase-3蛋白表达量增加7.142倍(P<0.01),Bcl-2mRNA表达降低0.466倍(P<0.001),BaxmRNA表达升高5.938倍(P<0.001)。
对Caco-2和HT-29细胞不同生长时期GSTA1表达及活性检测的结果表明,Caco-2细胞中GSTA1蛋白、mRNA表达与活性随着细胞的汇合程度逐渐升高。与汇合前期相比较,汇合期GSTA1蛋白、mRNA及酶活性分别升高了1.888、3.018和1.994倍(P<0.05或P<0.01),在6d汇合后期GSTA1蛋白、mRNA及活性分别升高了2.988、7.135和6.508倍(P<0.001)。而HT-29细胞的各个生长时期均未检测到GSTA1蛋白和mRNA的表达。
对外源性C2-神经酰胺处理的Caco-2和HT-29细胞中GSTA1表达及活性检测结果表明,神经酰胺处理的Caco-2细胞中GSTA1蛋白、mRNA表达及活性分别增加了1.850、3.093和1.764倍,与对照组比较差异显著(p<0.01或p<0.001)。而神经酰胺处理HT-29细胞后,仍未能检测到GSTA1蛋白和mRNA的表达。
采用western blotting方法确定转染剂Lipofectamine2000为3μl/mL、40nM siRNA、转染48h为最佳的siRNA转染条件。在此基础上研究GSTA1沉默对神经酰胺介导的Caco-2细胞增殖和凋亡的影响。结果显示,对照组、siRNA转染组和阴性对照组细胞存活率差异不显著(P>0.05)。神经酰胺处理后,3个组细胞存活率均降低,但各组间并无统计学差异(P>0.05)。对照组、siRNA转染组和阴性对照组均无激活型caspase-3蛋白表达,神经酰胺处理后,3个组的caspase-3蛋白也均未被激活。
双酶切和序列测定表明本试验成功构建了重组真核表达载体GSTA1-pcDNA3.1/V5-His TOPO并将其瞬时转染至HT-29细胞中。采用western blotting方法确定转染剂Lipofectamine2000为3μl/mL、3μg质粒、转染48h为最佳的质粒转染条件。在此基础上研究GSTA1过表达对神经酰胺介导的HT-29细胞增殖和凋亡的影响。结果表明,对照组、质粒转染组和阴性对照组细胞存活率差异不显著(P>0.05),神经酰胺处理后,3个组细胞存活率均降低,但各组间并无统计学差异(P>0.05)。对照组、质粒转染组和阴性对照组均无激活型caspase-3蛋白表达,神经酰胺处理后,3个组的激活型caspase-3蛋白表达均增加,但各组间差异不显著(P>0.05)。
外源性C2-神经酰胺可显著抑制Caco-2和HT-29结肠癌细胞的增殖,不能诱导Caco-2凋亡,但可诱导HT-29凋亡。Caco-2细胞中GSTA1蛋白、mRNA表达量与活性随细胞汇合程度逐渐增加,而HT-29细胞各生长时期中GSTA1表达量极低或不表达。外源性C2-神经酰胺处理的Caco-2细胞中GSTA1蛋白、mRNA表达量与活性显著升高,而神经酰胺处理的HT-29细胞仍然检测不到GSTA1的表达。基因转染试验证实了GSTA1的沉默或过表达对神经酰胺介导的结肠癌细胞增殖抑制和凋亡无影响,以上结果提示GSTA1不具有保护结肠癌细胞免受神经酰胺引起的增殖抑制和凋亡的作用。
Ceramide is important second messenger in the cells, take part in modulating multiple biological effects. The most well-known roles of ceramide are proliferation inhibition and apoptosis (including tumour cells). In view of its role in inducing apoptsis, enhancing the concentration of endogenous ceramide or introducing exogenous analogues are the new targets of oncotherapy. Studies have shown that an exogenous ceramide analogue (C2-ceramide) can induce apoptosis in colon cancer cell lines HT-29, Lovo, and HCT-116through multiple pathways; however, there is no reports about the affect of ceramide on apoptosis of Caco-2cells.
Chemotherapy has been widely used in colon cancer treatment, but the oncotherapy are often failed due to chemoresistance. GSTs are a group of phase Ⅱ drug metabolism enzymes. Overexpression of GSTs is one of the important factor for producing chemoresistance, tumour cells can protect themselves against anti-tumour drugs and reduce curative effect of anti-tumour drugs by express GST. Evidence shows that GSTA expression is strongly related to the resistance of anti-cancer drugs, such as nitrogen mustard and anthraquinone. GSTA1is an important member of the GSTA family. Study in this enzyme will advance our understanding in the proliferation and apoptosis of the cancer cells.
This study detected the effects of exogenous C2-ceramide on proliferation and apoptosis using Caco-2and HT-29cell lines, this research will provide fundamental knowledge for our future study on GSTA1-associated proliferation inhibition and apoptosis of the cell. This study investigated the expression and activity of GSTA1at different stages of colon cancer cells and determined the change of GSTA1expression and activity during exogenous ceramide-modulated proliferation inhibition and apoptosis of colon cancer cells. Gene transfection techniques were used to silence or overexpress GSTA1, and to explore how ceramides affects cell proliferation and apoptosis. This study will reveal if GSTA1expression in colon cancer cells can protect themselves against the effects of ceramide-modulated cell proliferation inhibition and apoptosis and useful to clarify the relationship between GSTA1, proliferation inhibiton/apoptosis of colon cancer cells and chemoresistance. Results can be translated to guide clinical application, and enhance effects of anti-cancer drugs and reduce the drug resistance in new drug development.
MTS assay was employed to determine the effects of C2-ceramide on the cell proliferation in in vitro cell lines Caco-2and HT-29. Western blotting and real-time PCR were used to determine the effects of ceramides on apoptosis by measuring the expression levels of actived caspase-3and Bcl-2gene family. Furthermore, Western blotting, real-time PCR, and enzyme activity assay technologies were employed to determine the expression of GSTA1protein, mRNA and activity at different stages of colon cancer cells, effects of ceramide on GSTA1protein expression, mRNA expression, and activity were also detected. In addition, gene transfection techniques were used to confirm the functions of GSTA1in ceramide-modulated cell proliferation and apoptosis. The first approach is to use siRNA technique to knockdown GSTA1gene in Caco-2cells in order to detect the effect of GSTA1silencing on ceramide-mediated proliferation inhibition and apoptosis; and the second was to transfect HT-29cells with plasmid in order to detect the effect of GSTA1overexpression on ceramide-mediated proliferation inhibition and apoptosis.
The results of effect of exogenous C2-ceramide on proliferation showed that the survival rate of colon cancer cell progressively decreased with the concentration of ceramide. The results of effect of ceramide on apoptosis showed that ceramide did not induce the expression of actived caspase-3protein. The expression of anti-apoptotic Bcl-2mRNA and pro-apoptotic Bax mRNA did not show any significant difference. Treatment with ceramide resulted in7.142-fold increase of the actived caspase-3protein expression in HT-29cells comparing with control group (P<0.01), accompanied with0.466-fold decrease of Bcl-2mRNA (P<0.001)and6.938-fold increase Bax mRNA levels (P<0.001).
The changes of GSTA1protein levels, mRNA expression and activity in different stages of Caco-2and HT-29were detected. Results showd that GSTA1protein levels, mRNA expression, and enzyme activity in Caco-2cells were progressively increased with the confluency. Compared with preconfluent, GSTA1protein, mRNA levels and activity rose to1.888,3.018,1.994-fold in confluent respectively (P<0.05or P<0.01), and rose to2.988,7.135,6.598-fold in6d postconfluent respectively (P<0.001). There were no expression of GSTA1protein and mRNA of all the stages in HT-29cells.
The effect on GSTA1protein levels, mRNA expression and activitiy in Caco-2and HT-29cells induced by exogenous C2-ceramide were detected. Results showed that GSTA1protein, mRNA expression and activity induced by ceramide of Caco-2cells increased1.850,3.093,1.764-fold respectively, significant difference comparing with control group (p<0.01or p<0.001), but no expression of GSTA1protein and mRNA expression were seen in HT-29cells.
The best transfection conditions,3μl/mL Lipofectamine2000,40nM siRNA, transfected48h,were confirmed by western blotting analysis. These GSTA1-silenced Caco-2cells were then used to study the effect of ceramide on cell proliferation and apoptosis in Caco-2cells. Results showed that there was no significant difference in survival rate between control, siRNA transfection, negative control group (P>0.05), the survival rate decreased of the3groups with the ceramide treatment, but no statistics difference between groups (P>0.05). There was no detection of activated caspase-3protein expression in control, siRNA transfection, negative control group, activated caspase-3protein expression was not detected in3groups with ceramide treatment.
The construction of recombinant plasmid GSTA1-pcDNA3.1/V5-His TOPO was verified by double-enzyme cleavage and gene sequence analysis. Recombinant plasmids were then transiently transfected into HT-29cells. The best transfection conditions,3μl/mL Lipofectamine2000,3μg DNA, transfected48h,were confirmed by western blotting analysis. These GSTAl-overexpressed Caco-2cells were then used to study the effect of ceramide on cell proliferation and apoptosis in HT-29cells. Results showed that there was no significant difference in survival rate between control, plasmid transfection, negative control group (P>0.05), the survival rate decreased of the3groups with the ceramide treatment, but no statistics difference between groups (P>0.05). There was no detection of activated caspase-3protein expression in control, plasmid transfection, negative control group, activated caspase-3protein expression were induced with ceramide treatment, but no statistics difference between groups(P>0.05).
In conclusion, ceramides inhibits the proliferations of two colon cancer cells. Ceramides can induce apoptosis of HT-29but not Caco-2cells. The protein levels, mRNA expression, and enzymes activities of Caco-2cells are increased in correlation with cell confluency, the GSTA1expression is very low or not existed in HT-29cells. Treatment with ceramides can increase GSTA1protein levels, mRNA expression, and activity in Caco-2cells, but the protein and mRNA expression is not seen in HT-29cells. Gene transfection experiments manifest that GSTA1does not affect the ceramide-modulated proliferation inhibition and anti-apoptosis, these results suggested no protective role for GSTA1in ceramide-modulated proliferation inhibition and apoptosis.
引文
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