Reverse of the resistance to paclitaxel of the heparin binding-epidermal growth factor-like growth factor inhibitor in ovarian cancer / 中华妇产科杂志

ABSTRACT

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 ofovarian cancer to paclitaxel. Methods (1)The effect of CRM197 on the 50% inhibitory concentrations (IC50) 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 IC50 of A2780/Taxol cells to paclitaxel in A2780/Taxol+CRM197 group [(6.4±0.3)μmol/L] was significantly lower than the IC50 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.