美洲大蠊提取物逆转人肝癌细胞HepG2/ADM多药耐药的机制研究(1)
摘 要 目的：研究美洲大蠊提取物脫脂膏及CⅡ-3(分别简称“脱脂膏”“CⅡ-3”)逆转耐阿霉素(ADM)人肝癌细胞HepG2/ADM多药耐药的作用机制。方法：采用MTT法考察不同质量浓度索拉非尼(阳性对照)、脱脂膏和CⅡ-3对HepG2/ADM细胞的毒性作用，并计算20%抑制浓度(IC20)。试验设置敏感组、耐药组、索拉非尼组、脱脂膏组和CⅡ-3组，敏感组使用HepG2细胞，后4组均使用HepG2/ADM细胞。敏感组和耐药组细胞给予常规培养基，其余3组细胞给予相应药物(浓度均为IC20)。采用激光扫描共聚焦显微技术测定细胞中ADM含量;采用Western blotting法测定细胞中凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、剪切型胱天蛋白酶9 p37 (Cleaved-Caspase-9 p37)]的表达水平;分别采用实时荧光定量-聚合酶链式反应法和免疫细胞化学染色法检测细胞中多药耐药相关基因mRNA及蛋白[P-糖蛋白(P-gp)(MDR1基因表达产物)、肺耐药蛋白(LRP)、乳腺癌耐药相关蛋白(BCRP)]和酶介导多药耐药途径中相关基因mRNA及蛋白[谷胱甘肽转移酶(GST-π)、DNA拓扑异构酶Ⅱ(Topo Ⅱ)]的表达水平。结果：索拉非尼、脱脂膏、CⅡ-3对HepG2/ADM细胞的IC20分别为(2.40±0.16)、(200.44±27.52)、(18.00±1.82) μg/mL。与敏感组比较，耐药组细胞中Bcl-2、P-gp、LRP、BCRP、Topo Ⅱ蛋白表达水平以及MDR1、LRP、BCRP、GST-π mRNA表达水平均显著升高(P<0.05或P<0.01)。与耐药组比较，脱脂膏组和 CⅡ-3组细胞中ADM含量显著增加(P<0.05或P<0.01)，MDR1 mRNA表达水平和LRP、BCRP、GST-π mRNA及其蛋白表达水平均显著降低(P<0.05或P<0.01);CⅡ-3组细胞中Bcl-2蛋白表达水平、Topo Ⅱ mRNA表达水平均显著降低(P<0.01)，Cleaved-Caspase-9 p37蛋白表达水平显著升高(P<0.05)。结论：脱脂膏、CⅡ-3可通过减少药物外排、促进细胞凋亡、减少多药耐药相关基因和酶介导多药耐药途径中相关基因的mRNA及其蛋白的表达等方式，逆转HepG2/ADM细胞的多药耐药性，且CⅡ-3的效果优于脱脂膏。
关键词 美洲大蠊;脱脂膏;CⅡ-3;肝癌;多药耐药;HepG2/ADM细胞;机制
ABSTRACT OBJECTIVE： To study the mechanism of Periplaneta americana extract degreasing cream and CⅡ-3 (shorted for “degreasing cream” and “CⅡ-3”) reversing the multi-drug resistance of human HepG2/ADM cells. METHODS： MTT assay was used to investigate the toxicity effects of different concentrations of sorafenib (positive control)， degreasing cream and CⅡ-3 on HepG2/ADM cells， then IC20 was calculated. The experiment was divided into sensitivity drug， drug-resistance group， sorafenib group， degreasing cream group and CⅡ-3 group. HepG2 cells were included in sensitivity group， and HepG2/ADM cells were included in the latter 4 groups. Sensitivity group and drug-resistance group were treated with routine medium， and other 3 groups were treated with relevant medicine (IC20 as drug concentration). The content of ADM in HepG2/ADM cells was determined by Laser scanning confocal microscopy. The expression of apoptosis-related protein as Bcl-2 and Cleaved-Caspase-9 p37 were detected by Western blotting assay. RT-qPCR and immunocytochemistry were adopted to detect mRNA and protein expressions that related to multidrug resistance [P-gp (expression produce of MDR1 gene)， LRP， BCRP] and that related to enzyme-mediated multidrug resistance pathway (GST-π and Topo Ⅱ). RESULTS： The IC20 of degreasing cream， CⅡ-3 and sorafenib were (2.40±0.16)， (200.44±27.52)， (18.00±1.82) μg/mL， respectively. Compared with sensitivity group， the protein expressions of Bcl-2， P-gp， LRP， BCRP and Topo Ⅱ， the mRNA expressions of MDR1， LRP， BCRP and GST-π were increased significantly in drug resistance group (P<0.05 or P<0.01). Compared with drug-resistance group， the mRNA and protein expression of MDR1 mRNA and LRP，BCRP，GST-π were significantly decreased in degreasing cream group and CⅡ-3 group (P<0.05 or P<0.01); the protein expression of Bcl-2 and the mRNA expression of Topo Ⅱ were significantly decreased (P<0.01)， while the protein expression level of Cleaved-Caspase-9 p37 was significantly increased in CⅡ-3 group (P<0.05). CONCLUSIONS： Degreasing cream and CⅡ-3 can reverse multidrug resistance of HepG2/ADM cells by reducing drug efflux，promoting cell apoptosis，reducing the mRNA and protein expression of multi-drug resistance gene as well as gene in enzyme-mediated multi-drug resistance pathway. The effect of CⅡ-3 is better than that of degreasing cream., 百拇医药(李彩琳 吴定宇 吕鸿 张鸿翰 王彦权 Najib Mohammerd 彭芳)
关键词 美洲大蠊;脱脂膏;CⅡ-3;肝癌;多药耐药;HepG2/ADM细胞;机制
ABSTRACT OBJECTIVE： To study the mechanism of Periplaneta americana extract degreasing cream and CⅡ-3 (shorted for “degreasing cream” and “CⅡ-3”) reversing the multi-drug resistance of human HepG2/ADM cells. METHODS： MTT assay was used to investigate the toxicity effects of different concentrations of sorafenib (positive control)， degreasing cream and CⅡ-3 on HepG2/ADM cells， then IC20 was calculated. The experiment was divided into sensitivity drug， drug-resistance group， sorafenib group， degreasing cream group and CⅡ-3 group. HepG2 cells were included in sensitivity group， and HepG2/ADM cells were included in the latter 4 groups. Sensitivity group and drug-resistance group were treated with routine medium， and other 3 groups were treated with relevant medicine (IC20 as drug concentration). The content of ADM in HepG2/ADM cells was determined by Laser scanning confocal microscopy. The expression of apoptosis-related protein as Bcl-2 and Cleaved-Caspase-9 p37 were detected by Western blotting assay. RT-qPCR and immunocytochemistry were adopted to detect mRNA and protein expressions that related to multidrug resistance [P-gp (expression produce of MDR1 gene)， LRP， BCRP] and that related to enzyme-mediated multidrug resistance pathway (GST-π and Topo Ⅱ). RESULTS： The IC20 of degreasing cream， CⅡ-3 and sorafenib were (2.40±0.16)， (200.44±27.52)， (18.00±1.82) μg/mL， respectively. Compared with sensitivity group， the protein expressions of Bcl-2， P-gp， LRP， BCRP and Topo Ⅱ， the mRNA expressions of MDR1， LRP， BCRP and GST-π were increased significantly in drug resistance group (P<0.05 or P<0.01). Compared with drug-resistance group， the mRNA and protein expression of MDR1 mRNA and LRP，BCRP，GST-π were significantly decreased in degreasing cream group and CⅡ-3 group (P<0.05 or P<0.01); the protein expression of Bcl-2 and the mRNA expression of Topo Ⅱ were significantly decreased (P<0.01)， while the protein expression level of Cleaved-Caspase-9 p37 was significantly increased in CⅡ-3 group (P<0.05). CONCLUSIONS： Degreasing cream and CⅡ-3 can reverse multidrug resistance of HepG2/ADM cells by reducing drug efflux，promoting cell apoptosis，reducing the mRNA and protein expression of multi-drug resistance gene as well as gene in enzyme-mediated multi-drug resistance pathway. The effect of CⅡ-3 is better than that of degreasing cream., 百拇医药(李彩琳 吴定宇 吕鸿 张鸿翰 王彦权 Najib Mohammerd 彭芳)