학술논문
顺铂对肿瘤坏死因子相关凋亡诱导配体逆转胃癌多药耐药现象的增敏作用及机制 / Molecular mechanism of cisplatin to enhance the ability of TRAIL in reversing multidrug resistance in gastric cancer cells
Document Type
Academic Journal
Author
朱邢超; 张开光; 王巧民; 陈思; 苟雅雯; 崔喻芳; 李琴; Zhu Xingchao; Zhang Kaiguang; Wang Qiaomin; Chen Si; Gou Yawen; Cui Yufang; Li Qin
Source
中华肿瘤杂志 / Chinese Journal of Oncology. (6):404-411
Subject
Language
Chinese
ISSN
0253-3766
Abstract
目的：研究顺铂（DDP）对肿瘤坏死因子相关凋亡诱导配体（TRAIL）逆转人胃腺癌耐长春新碱（ VCR） SGC7901?VCR细胞多药耐药现象的增敏作用，以及可能的分子机制。方法采用四甲基偶氮唑蓝（ MTT）法检测不同化疗药物处理组SGC7901和SGC7901?VCR细胞的半数抑制浓度（ IC50）和细胞存活率。以不同浓度的DDP和TRAIL单独或联合处理SGC7901?VCR细胞，采用逆转录PCR、荧光定量PCR和Western blot法检测不同处理组中多种基因的mRNA和蛋白表达。以c?myc?siRNA干扰、重组c?myc质粒转染SGC7901?VCR细胞，采用逆转录PCR和Western blot法检测不同处理组SGC7901?VCR细胞中死亡受体（ DR）4、DR5、c?myc基因的mRNA和蛋白表达。结果与单用TRAIL组和空白对照组比较，联合DDP后，VCR、DDP、阿霉素和氟尿嘧啶对SGC7901?VCR细胞的IC50均明显降低（均P＜0．05）。 TRAIL联合DDP时，对SGC7901?VCR细胞中caspase?3的激活作用以及对DNA?PKcs／Akt／GSK?3β信号通路、耐药基因MDR1、MRP1的抑制程度明显高于TRAIL单独作用时（均P＜0．01）。以siRNA干扰沉默c?myc后，SGC7901?VCR细胞中c?myc、DR4和DR5的表达明显减低（均P＜0．01）；以重组 c?myc质粒转染高表达 c?myc后，SGC7901?VCR细胞中 c?myc、DR4和DR5的表达明显增加（均P＜0．01）。10 ng／ml TRAIL组、0．25μg／ml DDP＋10 ng／ml TRAIL组和0．5μg／ml DDP＋10 ng／ml TRAIL组c?myc蛋白的相对表达量分别为0．314±0．012、0．735±0．026和0．876±0．028，细胞色素C（cyt C）蛋白的相对表达量分别为0．339±0．036、0．593±0．020和0．735±0．031，差异有统计学意义（P＜0．05）；DR4、DR5、活性caspase?9和活性caspase?3蛋白的相对表达量也随着DDP 浓度的增加而增加。结论 SGC7901?VCR的多药耐药性与DNA?PKcs／Akt／GSK?3β信号通路的激活以及耐药基因MDR1和MRP1的高表达有关。与TRAIL 联合时，DDP能够通过增加c?myc的表达促进DR4和DR5的高表达，并促进线粒体释放cyt C进一步激活TRAIL通路的caspase阶梯反应，来增强TRAIL对SGC7901?VCR细胞多药耐药特性的逆转作用。
Objective To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor?related apoptosis?inducing ligand ( TRAIL ) in reversing multidrug resistance in vincristine?resistant human gastric cancer SGC7901/VCR cells. Methods MTT assay was used to measure the 50% inhibiting concentration( IC50 ) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments.SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT?PCR, RT?qPCR and Western?blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c?myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c?myc were determined by RT?PCR and Western?blot analysis. Results After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC50 of VCR, DDP, ADM, and 5?Fu treatment was significantly decreased compared with the control group or TRAIL?treated group (P<0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0. 4 μg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down?regulation of DNA?PKcs/Akt/GSK?3β signaling pathway, and higher inhibition of MDR1(P?gp) and MRP1 than those treated with TRAIL alone (P<0.01 for all). The mRNA and protein levels of DR4, DR5, c?myc were significantly decreased after silencing c?myc with specific siRNA in the SGC7901/VCR cells ( P<0. 01 for all ) , and were significantly increased after transfection of recombinant plasmid c?myc into the SGC7901/VCR cells (P<0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0. 25 μg/ml DDP+10 ng/ml TRAIL and 0. 5 μg/ml DDP+10 ng/ml TRAIL, the relative expression level of c?myc protein in the SGC7901/VCR cells was 0.314±0.012, 0.735±0.026, and 0.876±0.028, respectively, and the relative expression of cytochrome C was 0.339±0.036, 0.593±0.020 and 0.735±0.031, respectively, and the relative expression levels of DR4, DR5, active?caspase 3 and active?caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations. Conclusions The activation of DNA?PKcs/Akt/GSK?3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi?drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up?regulating c?myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP?induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.
Objective To study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor?related apoptosis?inducing ligand ( TRAIL ) in reversing multidrug resistance in vincristine?resistant human gastric cancer SGC7901/VCR cells. Methods MTT assay was used to measure the 50% inhibiting concentration( IC50 ) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments.SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT?PCR, RT?qPCR and Western?blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c?myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c?myc were determined by RT?PCR and Western?blot analysis. Results After combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC50 of VCR, DDP, ADM, and 5?Fu treatment was significantly decreased compared with the control group or TRAIL?treated group (P<0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0. 4 μg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down?regulation of DNA?PKcs/Akt/GSK?3β signaling pathway, and higher inhibition of MDR1(P?gp) and MRP1 than those treated with TRAIL alone (P<0.01 for all). The mRNA and protein levels of DR4, DR5, c?myc were significantly decreased after silencing c?myc with specific siRNA in the SGC7901/VCR cells ( P<0. 01 for all ) , and were significantly increased after transfection of recombinant plasmid c?myc into the SGC7901/VCR cells (P<0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0. 25 μg/ml DDP+10 ng/ml TRAIL and 0. 5 μg/ml DDP+10 ng/ml TRAIL, the relative expression level of c?myc protein in the SGC7901/VCR cells was 0.314±0.012, 0.735±0.026, and 0.876±0.028, respectively, and the relative expression of cytochrome C was 0.339±0.036, 0.593±0.020 and 0.735±0.031, respectively, and the relative expression levels of DR4, DR5, active?caspase 3 and active?caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations. Conclusions The activation of DNA?PKcs/Akt/GSK?3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi?drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up?regulating c?myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP?induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.