Objective: To investigate the effect and mechanism of CRM197, the heparin binding-epidermal growth factor-like growth factor (HB-EGF) inhibitor, on the reverse of the resistance of ovarian cancer to paclitaxel. Methods: (1)The effect of CRM197 on the 50% inhibitory concentrations (IC(50)) of human ovarian carcinoma cell line A2780 and paclitaxel-resistant ovarian carcinoma cell line A2780/Taxol was tested by methyl thiazolyl tetrazolium (MTT) assay. Western blot was used to detect the effect of CRM197 on the expression of HB-EGF, epidermal growth factor receptor (EGFR) and plasma membrane glycoprotein (P-gp) protein in A2780 and A2780/Taxol cells. Real-time PCR was used to examine the MDR1 mRNA expression in these cells. (2) A2780/Taxol cells were divided into 4 groups, including the cells transfected with empty vector and saline treatment (empty vector group), MDR1 small interference RNA (siRNA) vector and saline treatment (MDR1 siRNA group), empty vector and CRM197 treatment (empty vector+CRM197 group) and MDR1 siRNA vector and CRM197 treatment (MDR1 siRNA+CRM197 group), respectively. Flow cytometry was used to detecte the effect of intracellular rhodomine 123 (Rh123) accumulation, and caspase-3 activity assay was used to test the effect of apoptosis in four groups of A2780/Taxol cells. (3) In experiments in vivo, A2780/Taxol cells were inoculated to nude mouse subcutaneously to determine the EGFR and P-gp protein expression following CRM197 treatment by immunohistochemistry. Results: (1) In vitro, MTT examination showed that the IC(50) of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3) μmol/L] was significantly lower than the IC(50) in A2780/Taxol group [ (34.1±0.5) μmol/L, P<0.01], and the reveral fold of CRM197 was 5.3. The expression level of HB-EGF protein in A2780/Taxol+CRM197 group (1.44±0.29) was significantly lower than HB-EGF protein in A2780/Taxol group (2.72±0.32), respectively (P<0.05). The expression level of EGFR protein (0.71±0.25) and P-gp protein (0.82±0.19) in A2780/Taxol+CRM197 group was significantly lower than EGFR protein (1.87±0.31) and P-gp protein (1.84±0.27) of A2780/Taxol group (P<0.05). Compared with A2780/Taxol group (1.78±0.27) , MDR1 mRNA was significantly down-regulated in A2780/Taxol+CRM197 group (0.79±0.13, P<0.05). (2) The fluorescence intensity of Rh123 of the A2780/Taxol cells in empty vector group, MDR1 siRNA group,empty vector+CRM197 group, MDR1 siRNA+CRM197 group was 33.4±1.6, 56.3±3.3, 43.5±3.1,100.4±7.4, and the pNA of the A2780/Taxol cells was (11.4±1.2) , (52.8±0.9) , (71.2±3.6) , (82.7±3.8) μmol/L. The expression levels in MDR1 siRNA+CRM197 group were both higher than the expression levels in empty vector+CRM197 group, and the expression levels in empty vector+CRM197 group, MDR1 siRNA group were both higher than the expression levels in empty vector group (P<0.05). (3) In vivo, the expression scores of EGFR protein in A2780/Taxol+CRM197 tumors (4.4±1.4) were lower than that in A2780/Taxol tumors (10.2±3.1, P<0.05). The expression scores of P-gp protein in A2780/Taxol+CRM197 tumors (3.8±1.1) were lower than that in A2780/Taxol tumors (8.8±2.7, P<0.05). Conclusion: CRM197 reverses the resistance of ovarian cancer to paclitaxel by increasing caspase-3 activity to advance apoptosis via EGFR/MDR1/P-gp pathway.
目的： 探讨肝素结合表皮生长因子（HB-EGF）抑制剂CRM197对卵巢上皮性癌（卵巢癌）紫杉醇耐药的逆转作用及可能机制。 方法： （1）四甲基偶氮唑蓝（MTT）比色法检测CRM197作用后卵巢癌紫杉醇耐药细胞系A2780/Taxol细胞对紫杉醇的50%抑制浓度（IC(50)）；蛋白印迹法及实时PCR技术分别检测CRM197作用后A2780/Taxol细胞中HB-EGF、表皮生长因子受体（EGFR）、P糖蛋白（P-gp）蛋白及多药耐药基因（MDR1）mRNA的表达。（2）构建MDR1小分子RNA（siRNA）质粒并转染A2780/Taxol细胞，实验分为4组，即空载体组、MDR1 siRNA组、空载体+CRM197组、MDR1 siRNA+CRM197组，检测4组A2780/Taxol细胞中P-gp功能[以P-gp的底物若丹明123（Rh123）的荧光强度表示]和半胱氨酸天冬氨酸蛋白酶3（caspase-3）蛋白活性[以4硝基苯胺（pNA）浓度表示]。（3）建立卵巢癌A2780/Taxol细胞的裸鼠移植瘤模型，免疫组化SP法检测CRM197作用后裸鼠移植瘤组织中EGFR和P-gp蛋白的表达。 结果： （1）MTT比色法检测显示，CRM197作用后A2780/Taxol细胞对紫杉醇的IC(50)为（6.4±0.3）μmol/L，明显低于未经CRM197作用的A2780/Taxol细胞[（34.1±0.5）μmol/L；P<0.01]，CRM197对紫杉醇的耐药逆转倍数为5.3。蛋白印迹法检测显示，CRM197作用后A2780/Taxol细胞中HB-EGF、EGFR、P-gp蛋白的表达水平（分别为1.44±0.29、0.71±0.25、0.82±0.19）均明显低于未经CRM197作用的A2780/Taxol细胞（分别为2.72±0.32、1.87±0.31、1.84±0.27，P均<0.05）；实时PCR技术检测显示，CRM197作用后A2780/Taxol细胞中MDR1 mRNA的表达水平（0.79±0.13）也明显低于未经CRM197作用的A2780/Taxol细胞（1.78±0.27，P<0.05）。（2）空载体组、MDR1 siRNA组、空载体+CRM197组、MDR1 siRNA+CRM197组A2780/Taxol细胞中的Rh123平均荧光强度分别为33.4±1.6、56.3±3.3、43.5±3.1、100.4±7.4，细胞中pNA浓度分别为（11.4±1.2）、（52.8±0.9）、（71.2±3.6）、（82.7±3.8）μmol/L，上述指标均为MDR1 siRNA+CRM197组高于空载体+CRM197组，空载体+CRM197组、MDR1 siRNA组均高于空载体组，分别比较，差异均有统计学意义（P<0.05）。（3）免疫组化SP法检测显示，CRM197作用后的A2780/Taxol细胞裸鼠移植瘤组织中EGFR、P-gp蛋白的表达水平分别为（4.4±1.4）、（3.8±1.1）分，明显低于未经CRM197作用的A2780/Taxol细胞裸鼠移植瘤组织[分别为（10.2±3.1）、（8.8±2.7）分，P<0.05]。 结论： HB-EGF抑制剂CRM197可逆转卵巢癌紫杉醇耐药，其作用机制可能与CRM197抑制EGFR/MDR1/P-gp通路、增加caspase-3活性，从而促进耐药细胞凋亡相关。.
Keywords: Bacterial proteins; Drug resistance, bacterial; Heparin-binding EGF-like growth factor; Ovarian neoplasms; Paclitaxel.