人食管癌细胞多药耐药的机制及药物干预的研究
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摘要
第一部分人食管癌多药耐药细胞株的建立及其生物学特性
目的：
建立人食管癌细胞多药耐药细胞株(YES-2/DDP),并分析其生物学特性。
方法：
本实验应用顺铂浓度递增和间歇诱导法建立YES-2/DDP耐药株。采用CCK-8法检测不同抗肿瘤药物对人食管癌野生型和耐药型细胞的增殖作用影响,计算半数抑制率(IC50)和耐药指数(RI),并观察冻存、撤药对耐药稳定性的影响。进一步比较野生与耐药细胞株细胞贴壁率、生长曲线、群体倍增时间的差异,并用双层软琼脂实验观察YES-2/WT(人食管癌野生型)和YES-2/DDP克隆形成率。流式细胞仪检测细胞周期、罗丹明外排、细胞凋亡。免疫印迹法检测耐药相关蛋白(MDR-1,MRP-1和LRP)的表达。
结果：
历时九个月诱导的人食管癌YES-2/DDP耐药细胞株对顺铂的耐药指数为16.4,并且YES-2/DDP对其它不同类型的抗肿瘤药物均产生不同程度的耐药,冻存和撤药对YES-2/DDP的耐药指数无明显影响。相对于YES-2/WT细胞株,YES-2/DDP生长曲线缓慢,细胞群体倍增时间延长,细胞克隆形成率显著增加,细胞周期中G1和S期所占比例增加而G2期减少。免疫印迹发现YES-2/DDP耐药细胞株MRP1表达明显增强,而MDR1和LRP表达无明显变化。细胞内罗丹明外排出实验发现YES-2/DDP细胞内罗丹明明显减少。用10μg/ml的顺铂分别处理YES-2/WT和YES-2/DDP细胞72小时后,流式细胞和荧光显微镜分析表明DDP对耐药细胞YES-2/DDP的凋亡率明显减少。
结论：
成功建立人食管癌多药耐药细胞株YES-2/DDP,其耐药机制可能与MRP1的表达增加,导致抗癌药物摄入减少,外排增多。减少药物对肿瘤细胞的凋亡作用相关。
第二部分人食管癌细胞多药耐药机制的初步研究
目的：
研究COX-2、PPAR-β在人食管癌细胞多药耐药中的作用,分析COX-2、PPAR-β、MRP-1三者之间的关系,揭示人食管癌细胞发生多药耐药的机制。
方法：
1、首先用免疫印迹法检测YES-2/WT和YES-2/DDP细胞中COX-2、 PPAR-β的表达,用酶联免疫法和荧光素酶检测仪测定PGE2的生成和PPRE报告基因活性,以评价COX-2、PPAR-β、的活性。
2、分别用25μM的COX-2特异性抑制剂NS398或1μM的PGE2干预YES-2/DDP耐药细胞株72小时,免疫印迹法检测MRP-1和PPAR-β的表达,荧光素酶检测仪测定PPRE报告基因活性,流式细胞仪检测细胞内罗丹明浓度以明确细胞外排药物能力,CCK-8检测不同浓度顺铂处理不同干预组细胞48小时后细胞增殖能力,以评价顺铂的半数抑制率(IC50)和耐药指数(RI)。进一步用110μg/ml的顺铂分别处理上述干预组的YES-2/DDP细胞株,高效液相法测定顺铂处理1小时后细胞内顺铂浓度,流式细胞仪检测顺铂处理72小时后细胞凋亡率。
3、分别用2μM的PPAR-β特异性抑制剂GSK0660或转染5gg的PPAR-β表达质粒干预YES-2/DDP耐药细胞株72小时,免疫印迹法检测MRP-1和COX-2的表达,酶联免疫法测定细胞上清中PGE2含量,流式细胞仪检测细胞内罗丹明浓度以明确细胞外排药物能力,CCK-8检测不同浓度顺铂处理不同干预组细胞48小时后细胞增殖能力,以评价顺铂的半数抑制率(IC50)和耐药指数(RI)。进一步用10μg/ml的顺铂分别处理上述干预组的YES-2/DDP细胞株,高效液相法测定顺铂处理1小时后细胞内顺铂浓度,流式细胞仪检测顺铂处理72小时后细胞凋亡率。
结果：
1、与YES-2/WT细胞相比,YES-2/DDP细胞COX-2蛋白表达和PGE2生成量增加,PPAR-β蛋白表达和PPRE活性也增加,同时MDR1蛋白表达增加,细胞内顺铂浓度明显降低,10μg/ml顺铂处理细胞后凋亡率也明显减少。
2、与YES-2/DDP细胞相比,25μM的COX-2特异性抑制剂NS398干预YES-2/DDP后,MRP-1和PPAR-β蛋白表达明显降低,PPRE活性减少,细胞内罗丹明浓度增高,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显减少；10μg/ml顺铂分别处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度增高,72小时凋亡率也明显增加(均相对于YES-2/DDP细胞,P<0.05)。
与YES-2/DDP细胞相比,1μM的PGE2干预YES-2/DDP后,MRP-1和PPAR-β蛋白表达明显增加,PPRE活性也增加,细胞内罗丹明浓度减少,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显增加；10μg/ml顺铂处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度减少,72小时凋亡率也明显减少(均相对于YES-2/DDP细胞,P<0.05)。
3、与YES-2/DDP细胞相比,2μM的PPAR-β特异性抑制剂GSK0660干预YES-2/DDP后,COX-2表达和PGE2含量无明显变化,但MRP-1蛋白表达明显降低,细胞内罗丹明浓度增高,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显减少；10μg/ml顺铂分别处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度增高,72小时凋亡率也明显增加(均相对于YES-2/DDP细胞,P<0.05)。
与YES-2/DDP细胞相比,转染5μg的PPAR-β表达质粒干预YES-2/DDP后,COX-2表达和PGE2含量无明显变化,但MRP-1蛋白表达明显增加,细胞内罗丹明浓度减少,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显增加；10μg/ml顺铂处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度减少,72小时凋亡率也明显减少(均相对于YES-2/DDP细胞,P<0.05)。
结论：
1、COX-2蛋白表达和PGE2生成介导MRP1的表达,促进细胞内顺铂的外排,减少抗肿瘤药物对人食管癌细胞的毒性作用。
2、PPAR-β蛋白表达和激活也介导MRPl的表达,促进细胞内顺铂的外排,减少抗肿瘤药物对人食管癌细胞的毒性作用。
3、人食管癌细胞的耐药形成可能是通过促进COX-2/PGE2系统,上调PPAR-β蛋白表达和活性,进一步介导MDR1的表达,促进抗肿瘤药物的外排,减少肿瘤细胞内抗肿瘤药物的浓度,从而减少细胞对药物的毒性作用,产生多药耐药。
第三部分粉防己碱对YES-2/DDP耐药细胞株的干预作用及机制
目的：
研究粉防已碱对人食管癌YES-2/DDP耐药细胞株MRP-1的影响,并进一步探讨其可能的机制及生物学效应,为人食管癌多药耐药的干预寻找有效的治疗药物。
方法：
以人食管癌耐药细胞株YES-2/DDP为多药耐药细胞模型,实验分为四组,用不同剂量的粉防已碱干预耐药细胞株YES-2/DDP,分别为对照组(无粉防已碱干预)、10μM粉防已碱干预组、3.0gM粉防已碱干预组、100μM粉防已碱干预组。在粉防已碱干预72小时后,免疫印迹法检测COX-2、PAR-β和MRP的表达,酶联免疫法检测PGE2含量,荧光素酶法检测PPRE的活性,流式细胞仪检测细胞内罗丹明浓度。进一步用10μg/ml浓度的顺铂继续处理上述各组细胞,分别在1小时后高效液相仪测细胞内顺铂浓度,48小时后用CCK-8检测各组对顺铂的半数抑制率(IC50)和耐药指数(RI)。
结果：
1、与YES-2/DDP对照组细胞相比,粉防已碱呈剂量信赖性地抑制COX-2的表达,PGE2的生成,PPAR-β蛋白表达和PPRE的激活。同时也抑制MRP的表达和细胞内罗丹明的蓄积。
2、用10μg/ml浓度的顺铂处理不同干预组细胞后,粉防已碱呈剂量依赖性地增加细胞内顺铂浓度,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显减少。
结论：
粉防己碱可逆转YES-2/DDP耐药细胞株中耐药性的产生,其机制有可能与其抑制COX-2的表达后,依次下调细胞中PGE2、PPAR-β与MRP的表达水平,从而减少药物外排有关。
Part I. Establishment of human esophageal cancer cells mult id rug resistant cell line YES-2/DDP and its biological characteristics
Objective To establish human esophageal cancer cells multidrug resistant cell line YES-2/DDP and to analyze its biological characteristics.
Methods In this study, increasing concentrations and intermittent of cisplatin was applied to establish YES-2/DDP resistant strains. The half of inhibition concentrations (IC50) and resistance indexs (RI) of different anticancer drugs were detected by CCK-8in wild-type and drug resistance human esophageal cancer cells. Further the cell adherence rate, growth curve, doubling time differences, and double-layer soft-agar colony formation rate were observed in the YES-2/WT and YES-2/DDP cells. Cell cycle, rhodamine efflux, apoptosis were detected by flow cytometry, respectively. The MDR-1, MRP-1and LRP protein expression were detected by Western blotting.
Results During the nine months, human esophageal cancer YES-2/DDP resistance cells line was induced and its resistance index to cisplatin was16.4, and YES-2/DDP cells for other types of anticancer drugs are different degrees of resistance, Freezing and withdraw of drug had not affect to the RI of YES-2/DDP. Compared to YES-2/WT Cell line, YES-2/DDP showed a slow growth curve, increased cell population doubling time and cell colony formation, decreased the cell cycle in G1and S phase and increased the proportion of G2phase. Western blot result showed that in the resistant cell line YES-2/DDP, MRP1protein expression was significantly increased, while the expression of MDR1and LRP was no significant changed. The concentration of rhodamine was markedly decreased in the YES-2/DDP cells compared with YES-2/WT cells. Flow cytometry and fluorescence microscopy analysis showed that72h after treatment with cisplatin (10μg/ml), the number of cell apoptosis was significantly in YES-2/DDP cells compared with YES-2/WT cells.
Conclusion We successfully established a human esophageal cancer multidrug resistance cell line YES-2/DDP. The resistance may be related to increased MRP1expression, resulting in reducing intake and increasing efflux of anti-cancer drugs, which inhibit the effect of anticancer drugs on tumor cell apoptosis.
Part Ⅱ Study on the mechanisms of multidrug resistance in human esophageal cancer cells
Objective To explore the relationship of COX-2, PPAR-β and MRP-1in the YES-2/DDP cells and reveal the molecular mechanism of multidrug resistance in human esophageal cancer cells.
Methods
1. The expression of COX-2, PPAR-β protein in the YES-2/WT and YES-2/DDP cells were determined by western blotting, and further the production of PGE2and PPRE report gene activity were assayed by enzyme-linked immunosorbent assay and luciferase assay, respectively.
2. YES-2/DDP cells were administrated with NS398(25μM), or PGE2(μM) for72h, respectively, the expression of MRP-1and PPAR-β, PPRE luciferase reporter gene activity, the concentration of cellular rhodamine, the IC50and RI of cisplatin at48h were determined, respectively. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were assayed.
3. YES-2/DDP cells were administrated with GSK0660(2μM) or transfer5μg of PPAR-(3expression plasmid for72hours, respectively, the expression of MRP-1and COX-2, PGE2production, the concentration of cellular rhodamine, the IC50and RI of cisplatin at48h were determined, respectively. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were assayed.
Results
1. Compared with YES-2/WT cells, COX-2protein expression and PGE2production were significantly increased in the YES-2/DDP cells, further PPAR-β expression and PPRE activity were also increased.
2. Compared with YES-2/DDP cells, in the25μM NS398-treated YES-2/DDP cells, the expressions of MRP-1and PPAR-β protein, the PPRE activity, IC50and RI of cisplatin were significantly reduced, while the concentrations of intracellular Rhodamine were significantly increased. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly increased. Compared with the YES-2/DDP cells, in the1μM PGE2-treated YES-2/DDP cells, the expressions of MRP-1and PPAR-β protein, the PPRE activity, IC50and RI of cisplatin were significantly increased, while the concentrations of intracellular Rhodamine were significantly reduced. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly reduced.
3. Compared with YES-2/DDP cells, in the2μM GSK0660-treated YES-2/DDP cells, the expression of COX-2protein and PGE2production had no changes, but the MRP-1protein expression, IC50and RI of cisplatin at48h were markedly reduced; the concentrations of intracellular Rhodamine were significantly increased. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly increased.
4. Compared with the YES-2/DDP cells, in the5μg of PPAR-β expression plasmid-transfected YES-2/DDP cells, the expression of COX-2protein and PGE2production had no changes, but the MRP-1protein expression, IC50and RI of cisplatin at48h were markedly increased; the concentrations of intracellular Rhodamine were significantly reduced. Further the concentration of intracellular cisplatin at lh and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly reduced.
Conclusion
1.The COX-2protein expression and PGE2production might mediate the expression of MRP1protein, which reduced the concentration of intracellular cisplatin, resulting in reducing the toxicity and apoptosis of the antitumor drugs in human esophageal cancer cells.
2. The PPAR-β protein expression and activation might mediate expression of MRP1protein, which reduced the concentration of intracellular cisplatin, resulting in reducing the toxicity and apoptosis of the antitumor drugs in human esophageal cancer cells.
3. In conclusion, the mechanism of multidrug resistance in human esophageal cancer cells YES-2/DDP may be through the promotion of COX-2/PGE2system, increasing of PPAR-β protein expression and activity, which mediated MDR1expression, and promoted the efflux of antitumor drugs, lead to reducing the concentrations of anti-tumor drugs, thereby reducing the cellular toxicity of these drug, resulting in multidrug resistance.
Part Ⅲ.Effect and mechanism of tetrandrine on the YES-2/DDP resistant cells
Objective
To research the effect of tetrandrine on human esophageal cancer resistant cell lines YES-2/DDP, and further explore its possible mechanism.
Methods
Human esophageal cancer cells were divided into four groups:the control group (no intervention tetrandrine),10μ.M tetrandrine in the intervention group,30μM tetrandrine in the intervention group,100μM tetrandrine in the intervention group. At72hours after tetrandrine administration, the COX-2, PPAR-β and MRP expression were determined by western blotting, PGE2levels were assayed by enzyme-linked immunosorbent assay, PPRE luciferase enzyme activity was detected, intracellular Rhodamine concentration was analyzed by flow cytometry, the IC50and RI of cisplatin at48h were detected by CCK-8. Further the concentrations of intracellular cisplatin were measured by HPLC at1h after treatment with10μg/ml cisplatin.
Results
1. Compared with YES-2/DDP control cells, tetrandrine dose-dependently inhibited the expression of COX-2protein and the production of PGE2, PPAR-β expression and activation. Further we also showed that tetrandrine inhibited the expression of MRP-1protein and accumulation of cellular rhodamine in a dose-dependent maner.
2. When cells were treated with10μg/ml cisplatin in different intervention groups, tetrandrine concentration-dependently increased the concentration of intracellular cisplatin, decreased the IC50and RI of cisplatin.
Conclusion
Tetrandrine could effectively reverse the resistance of YES-2/DDP resistant cells line; the mechanism may be related to inhibition of COX-2expression and production of PGE2, PPAR-β expression and activity, and the expression of MRP-1.
目的：
建立人食管癌细胞多药耐药细胞株(YES-2/DDP),并分析其生物学特性。
方法：
本实验应用顺铂浓度递增和间歇诱导法建立YES-2/DDP耐药株。采用CCK-8法检测不同抗肿瘤药物对人食管癌野生型和耐药型细胞的增殖作用影响,计算半数抑制率(IC50)和耐药指数(RI),并观察冻存、撤药对耐药稳定性的影响。进一步比较野生与耐药细胞株细胞贴壁率、生长曲线、群体倍增时间的差异,并用双层软琼脂实验观察YES-2/WT(人食管癌野生型)和YES-2/DDP克隆形成率。流式细胞仪检测细胞周期、罗丹明外排、细胞凋亡。免疫印迹法检测耐药相关蛋白(MDR-1,MRP-1和LRP)的表达。
结果：
历时九个月诱导的人食管癌YES-2/DDP耐药细胞株对顺铂的耐药指数为16.4,并且YES-2/DDP对其它不同类型的抗肿瘤药物均产生不同程度的耐药,冻存和撤药对YES-2/DDP的耐药指数无明显影响。相对于YES-2/WT细胞株,YES-2/DDP生长曲线缓慢,细胞群体倍增时间延长,细胞克隆形成率显著增加,细胞周期中G1和S期所占比例增加而G2期减少。免疫印迹发现YES-2/DDP耐药细胞株MRP1表达明显增强,而MDR1和LRP表达无明显变化。细胞内罗丹明外排出实验发现YES-2/DDP细胞内罗丹明明显减少。用10μg/ml的顺铂分别处理YES-2/WT和YES-2/DDP细胞72小时后,流式细胞和荧光显微镜分析表明DDP对耐药细胞YES-2/DDP的凋亡率明显减少。
结论：
成功建立人食管癌多药耐药细胞株YES-2/DDP,其耐药机制可能与MRP1的表达增加,导致抗癌药物摄入减少,外排增多。减少药物对肿瘤细胞的凋亡作用相关。
第二部分人食管癌细胞多药耐药机制的初步研究
目的：
研究COX-2、PPAR-β在人食管癌细胞多药耐药中的作用,分析COX-2、PPAR-β、MRP-1三者之间的关系,揭示人食管癌细胞发生多药耐药的机制。
方法：
1、首先用免疫印迹法检测YES-2/WT和YES-2/DDP细胞中COX-2、 PPAR-β的表达,用酶联免疫法和荧光素酶检测仪测定PGE2的生成和PPRE报告基因活性,以评价COX-2、PPAR-β、的活性。
2、分别用25μM的COX-2特异性抑制剂NS398或1μM的PGE2干预YES-2/DDP耐药细胞株72小时,免疫印迹法检测MRP-1和PPAR-β的表达,荧光素酶检测仪测定PPRE报告基因活性,流式细胞仪检测细胞内罗丹明浓度以明确细胞外排药物能力,CCK-8检测不同浓度顺铂处理不同干预组细胞48小时后细胞增殖能力,以评价顺铂的半数抑制率(IC50)和耐药指数(RI)。进一步用110μg/ml的顺铂分别处理上述干预组的YES-2/DDP细胞株,高效液相法测定顺铂处理1小时后细胞内顺铂浓度,流式细胞仪检测顺铂处理72小时后细胞凋亡率。
3、分别用2μM的PPAR-β特异性抑制剂GSK0660或转染5gg的PPAR-β表达质粒干预YES-2/DDP耐药细胞株72小时,免疫印迹法检测MRP-1和COX-2的表达,酶联免疫法测定细胞上清中PGE2含量,流式细胞仪检测细胞内罗丹明浓度以明确细胞外排药物能力,CCK-8检测不同浓度顺铂处理不同干预组细胞48小时后细胞增殖能力,以评价顺铂的半数抑制率(IC50)和耐药指数(RI)。进一步用10μg/ml的顺铂分别处理上述干预组的YES-2/DDP细胞株,高效液相法测定顺铂处理1小时后细胞内顺铂浓度,流式细胞仪检测顺铂处理72小时后细胞凋亡率。
结果：
1、与YES-2/WT细胞相比,YES-2/DDP细胞COX-2蛋白表达和PGE2生成量增加,PPAR-β蛋白表达和PPRE活性也增加,同时MDR1蛋白表达增加,细胞内顺铂浓度明显降低,10μg/ml顺铂处理细胞后凋亡率也明显减少。
2、与YES-2/DDP细胞相比,25μM的COX-2特异性抑制剂NS398干预YES-2/DDP后,MRP-1和PPAR-β蛋白表达明显降低,PPRE活性减少,细胞内罗丹明浓度增高,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显减少；10μg/ml顺铂分别处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度增高,72小时凋亡率也明显增加(均相对于YES-2/DDP细胞,P<0.05)。
与YES-2/DDP细胞相比,1μM的PGE2干预YES-2/DDP后,MRP-1和PPAR-β蛋白表达明显增加,PPRE活性也增加,细胞内罗丹明浓度减少,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显增加；10μg/ml顺铂处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度减少,72小时凋亡率也明显减少(均相对于YES-2/DDP细胞,P<0.05)。
3、与YES-2/DDP细胞相比,2μM的PPAR-β特异性抑制剂GSK0660干预YES-2/DDP后,COX-2表达和PGE2含量无明显变化,但MRP-1蛋白表达明显降低,细胞内罗丹明浓度增高,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显减少；10μg/ml顺铂分别处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度增高,72小时凋亡率也明显增加(均相对于YES-2/DDP细胞,P<0.05)。
与YES-2/DDP细胞相比,转染5μg的PPAR-β表达质粒干预YES-2/DDP后,COX-2表达和PGE2含量无明显变化,但MRP-1蛋白表达明显增加,细胞内罗丹明浓度减少,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显增加；10μg/ml顺铂处理NS398干预的YES-2/DDP细胞后,细胞内顺铂浓度减少,72小时凋亡率也明显减少(均相对于YES-2/DDP细胞,P<0.05)。
结论：
1、COX-2蛋白表达和PGE2生成介导MRP1的表达,促进细胞内顺铂的外排,减少抗肿瘤药物对人食管癌细胞的毒性作用。
2、PPAR-β蛋白表达和激活也介导MRPl的表达,促进细胞内顺铂的外排,减少抗肿瘤药物对人食管癌细胞的毒性作用。
3、人食管癌细胞的耐药形成可能是通过促进COX-2/PGE2系统,上调PPAR-β蛋白表达和活性,进一步介导MDR1的表达,促进抗肿瘤药物的外排,减少肿瘤细胞内抗肿瘤药物的浓度,从而减少细胞对药物的毒性作用,产生多药耐药。
第三部分粉防己碱对YES-2/DDP耐药细胞株的干预作用及机制
目的：
研究粉防已碱对人食管癌YES-2/DDP耐药细胞株MRP-1的影响,并进一步探讨其可能的机制及生物学效应,为人食管癌多药耐药的干预寻找有效的治疗药物。
方法：
以人食管癌耐药细胞株YES-2/DDP为多药耐药细胞模型,实验分为四组,用不同剂量的粉防已碱干预耐药细胞株YES-2/DDP,分别为对照组(无粉防已碱干预)、10μM粉防已碱干预组、3.0gM粉防已碱干预组、100μM粉防已碱干预组。在粉防已碱干预72小时后,免疫印迹法检测COX-2、PAR-β和MRP的表达,酶联免疫法检测PGE2含量,荧光素酶法检测PPRE的活性,流式细胞仪检测细胞内罗丹明浓度。进一步用10μg/ml浓度的顺铂继续处理上述各组细胞,分别在1小时后高效液相仪测细胞内顺铂浓度,48小时后用CCK-8检测各组对顺铂的半数抑制率(IC50)和耐药指数(RI)。
结果：
1、与YES-2/DDP对照组细胞相比,粉防已碱呈剂量信赖性地抑制COX-2的表达,PGE2的生成,PPAR-β蛋白表达和PPRE的激活。同时也抑制MRP的表达和细胞内罗丹明的蓄积。
2、用10μg/ml浓度的顺铂处理不同干预组细胞后,粉防已碱呈剂量依赖性地增加细胞内顺铂浓度,同时顺铂的半数抑制率(IC50)和耐药指数(RI)明显减少。
结论：
粉防己碱可逆转YES-2/DDP耐药细胞株中耐药性的产生,其机制有可能与其抑制COX-2的表达后,依次下调细胞中PGE2、PPAR-β与MRP的表达水平,从而减少药物外排有关。
Part I. Establishment of human esophageal cancer cells mult id rug resistant cell line YES-2/DDP and its biological characteristics
Objective To establish human esophageal cancer cells multidrug resistant cell line YES-2/DDP and to analyze its biological characteristics.
Methods In this study, increasing concentrations and intermittent of cisplatin was applied to establish YES-2/DDP resistant strains. The half of inhibition concentrations (IC50) and resistance indexs (RI) of different anticancer drugs were detected by CCK-8in wild-type and drug resistance human esophageal cancer cells. Further the cell adherence rate, growth curve, doubling time differences, and double-layer soft-agar colony formation rate were observed in the YES-2/WT and YES-2/DDP cells. Cell cycle, rhodamine efflux, apoptosis were detected by flow cytometry, respectively. The MDR-1, MRP-1and LRP protein expression were detected by Western blotting.
Results During the nine months, human esophageal cancer YES-2/DDP resistance cells line was induced and its resistance index to cisplatin was16.4, and YES-2/DDP cells for other types of anticancer drugs are different degrees of resistance, Freezing and withdraw of drug had not affect to the RI of YES-2/DDP. Compared to YES-2/WT Cell line, YES-2/DDP showed a slow growth curve, increased cell population doubling time and cell colony formation, decreased the cell cycle in G1and S phase and increased the proportion of G2phase. Western blot result showed that in the resistant cell line YES-2/DDP, MRP1protein expression was significantly increased, while the expression of MDR1and LRP was no significant changed. The concentration of rhodamine was markedly decreased in the YES-2/DDP cells compared with YES-2/WT cells. Flow cytometry and fluorescence microscopy analysis showed that72h after treatment with cisplatin (10μg/ml), the number of cell apoptosis was significantly in YES-2/DDP cells compared with YES-2/WT cells.
Conclusion We successfully established a human esophageal cancer multidrug resistance cell line YES-2/DDP. The resistance may be related to increased MRP1expression, resulting in reducing intake and increasing efflux of anti-cancer drugs, which inhibit the effect of anticancer drugs on tumor cell apoptosis.
Part Ⅱ Study on the mechanisms of multidrug resistance in human esophageal cancer cells
Objective To explore the relationship of COX-2, PPAR-β and MRP-1in the YES-2/DDP cells and reveal the molecular mechanism of multidrug resistance in human esophageal cancer cells.
Methods
1. The expression of COX-2, PPAR-β protein in the YES-2/WT and YES-2/DDP cells were determined by western blotting, and further the production of PGE2and PPRE report gene activity were assayed by enzyme-linked immunosorbent assay and luciferase assay, respectively.
2. YES-2/DDP cells were administrated with NS398(25μM), or PGE2(μM) for72h, respectively, the expression of MRP-1and PPAR-β, PPRE luciferase reporter gene activity, the concentration of cellular rhodamine, the IC50and RI of cisplatin at48h were determined, respectively. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were assayed.
3. YES-2/DDP cells were administrated with GSK0660(2μM) or transfer5μg of PPAR-(3expression plasmid for72hours, respectively, the expression of MRP-1and COX-2, PGE2production, the concentration of cellular rhodamine, the IC50and RI of cisplatin at48h were determined, respectively. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were assayed.
Results
1. Compared with YES-2/WT cells, COX-2protein expression and PGE2production were significantly increased in the YES-2/DDP cells, further PPAR-β expression and PPRE activity were also increased.
2. Compared with YES-2/DDP cells, in the25μM NS398-treated YES-2/DDP cells, the expressions of MRP-1and PPAR-β protein, the PPRE activity, IC50and RI of cisplatin were significantly reduced, while the concentrations of intracellular Rhodamine were significantly increased. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly increased. Compared with the YES-2/DDP cells, in the1μM PGE2-treated YES-2/DDP cells, the expressions of MRP-1and PPAR-β protein, the PPRE activity, IC50and RI of cisplatin were significantly increased, while the concentrations of intracellular Rhodamine were significantly reduced. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly reduced.
3. Compared with YES-2/DDP cells, in the2μM GSK0660-treated YES-2/DDP cells, the expression of COX-2protein and PGE2production had no changes, but the MRP-1protein expression, IC50and RI of cisplatin at48h were markedly reduced; the concentrations of intracellular Rhodamine were significantly increased. Further the concentration of intracellular cisplatin at1h and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly increased.
4. Compared with the YES-2/DDP cells, in the5μg of PPAR-β expression plasmid-transfected YES-2/DDP cells, the expression of COX-2protein and PGE2production had no changes, but the MRP-1protein expression, IC50and RI of cisplatin at48h were markedly increased; the concentrations of intracellular Rhodamine were significantly reduced. Further the concentration of intracellular cisplatin at lh and the rate of cell apoptosis at72h after treatment with10μg/ml cisplatin were significantly reduced.
Conclusion
1.The COX-2protein expression and PGE2production might mediate the expression of MRP1protein, which reduced the concentration of intracellular cisplatin, resulting in reducing the toxicity and apoptosis of the antitumor drugs in human esophageal cancer cells.
2. The PPAR-β protein expression and activation might mediate expression of MRP1protein, which reduced the concentration of intracellular cisplatin, resulting in reducing the toxicity and apoptosis of the antitumor drugs in human esophageal cancer cells.
3. In conclusion, the mechanism of multidrug resistance in human esophageal cancer cells YES-2/DDP may be through the promotion of COX-2/PGE2system, increasing of PPAR-β protein expression and activity, which mediated MDR1expression, and promoted the efflux of antitumor drugs, lead to reducing the concentrations of anti-tumor drugs, thereby reducing the cellular toxicity of these drug, resulting in multidrug resistance.
Part Ⅲ.Effect and mechanism of tetrandrine on the YES-2/DDP resistant cells
Objective
To research the effect of tetrandrine on human esophageal cancer resistant cell lines YES-2/DDP, and further explore its possible mechanism.
Methods
Human esophageal cancer cells were divided into four groups:the control group (no intervention tetrandrine),10μ.M tetrandrine in the intervention group,30μM tetrandrine in the intervention group,100μM tetrandrine in the intervention group. At72hours after tetrandrine administration, the COX-2, PPAR-β and MRP expression were determined by western blotting, PGE2levels were assayed by enzyme-linked immunosorbent assay, PPRE luciferase enzyme activity was detected, intracellular Rhodamine concentration was analyzed by flow cytometry, the IC50and RI of cisplatin at48h were detected by CCK-8. Further the concentrations of intracellular cisplatin were measured by HPLC at1h after treatment with10μg/ml cisplatin.
Results
1. Compared with YES-2/DDP control cells, tetrandrine dose-dependently inhibited the expression of COX-2protein and the production of PGE2, PPAR-β expression and activation. Further we also showed that tetrandrine inhibited the expression of MRP-1protein and accumulation of cellular rhodamine in a dose-dependent maner.
2. When cells were treated with10μg/ml cisplatin in different intervention groups, tetrandrine concentration-dependently increased the concentration of intracellular cisplatin, decreased the IC50and RI of cisplatin.
Conclusion
Tetrandrine could effectively reverse the resistance of YES-2/DDP resistant cells line; the mechanism may be related to inhibition of COX-2expression and production of PGE2, PPAR-β expression and activity, and the expression of MRP-1.
引文
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