Role and mechanism of the regulation of nuclear factor-κB by heparin binding-epidermal growth factor-like growth factor in the induction of paclitaxel resistance of ovarian cancer

Abstract

Objective: To investigate the role and mechanism of the regulation of nuclear factor-κB (NF-κB) by heparin binding-epidermal growth factor-like growth factor (HB-EGF) in paclitaxel resistance of ovarian cancer in vitro and in vivo. Methods: (1) The detection of NF-κB expression: parental (A2780) and paclitaxel-resistant (A2780/Taxol) ovarian carcinoma cells were divided into four groups, named A2780 group, A2780+cross-reacting material 197 (CRM197, HB-EGF inhibitor) group, A2780/Taxol group and A2780/Taxol+CRM197 group. Among four groups, the expression level HB-EGF and epidermal growth factor receptor (EGFR) were examined by immunofluorescence double staining on confocal microscopy. Western blot was used to detect the expression level of NF-κB. In vivo, A2780 and A2780/Taxol cells were injected intraperitoneally to nude mouse to determine the expression level of NF-κB of the tumors from these four groups by immunohistochemistry method. (2) The detection on the function of NF-κB: A2780/Taxol cells were divided into four groups, named transfected with empty vector+saline group, NF-κB small interference RNA (siRNA)+saline group, empty vector+CRM197 group and NF-κB siRNA+CRM197 group respectively. Among four groups, the 50% inhibitory concentrations (IC(50)) of A2780/Taxol cells to paclitaxel, the expression level of plasma membrane glycoprotein (P-gp) and the effect of intracellular rhodomine123 (Rh123) accumulation were detected. Results: (1) The detection of NF-κB expression: the expression scores of HB-EGF protein among four groups were 5.6±1.3, 2.1±1.2, 11.7±3.5 and 6.2±1.4; the expression scores of EGFR protein were 5.1±1.6, 2.8±0.6, 10.4±3.1 and 5.6±1.9, respectively. The expression levels of NF-κB protein in the cells of the group named A2780, A2780+CRM197, A2780/Taxol and A2780/Taxol+CRM197 group were 1.89±0.23, 0.74±0.12, 3.45±0.16 and 1.31±0.08, respectively; the expression scores of NF-κB protein in the tissue tumors from four groups were 3.3±1.1, 1.4±0.4, 8.7±2.3 and 3.6±1.2, respectively. The expression level of HB-EGF, EGFR and NF-κB protein between A2780 and A2780/Taxol groups in vivo and in vitro were higher than these in A2780+CRM197 and A2780/Taxol+CRM197 group, while the expression level of HB-EGF, EGFR and NF-κB protein in A2780 group were lower than those in A2780/Taxol groups in vivo and in vitro (P<0.05). (2) The examination of NF-κB function: the IC(50) of A2780/Taxol cells to paclitaxel in groups transfected with empty vector+saline, NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were respectively (39.4±0.8), (7.6±0.6), (6.7±0.5) and (4.2±0.4) μmol/L, while the expression levels of P-gp protein among four groups were respectively 3.11±0.23,1.45±0.16, 1.73±0.21 and 0.68±0.14, the cellular Rh123 accumulation among four groups were respectively 110±15, 246±19, 231±22 and 296±24. The expression levels of IC(50) and P-gp protein in groups transfected with NF-κB siRNA+saline, empty vector+CRM197 and NF-κB siRNA+CRM197 group were significantly higher than those in group transfected with empty vector+saline group (P<0.01), while the cellular Rh123 accumulation among three groups were significantly lower than that in group transfected with empty vector+saline (P<0.01). Conclusions: The expression of NF-κB may contributes to the paclitaxel resistance to ovarian cancer. HB-EGF may induce the paclitaxel resistance of ovarian cancer by the regulation of EGFR/NF-κB/P-gp pathway.