NS-398逆转结肠癌细胞多药耐药性的作用

NS-398 Reverses Multi-drug Resistance of Colorectal Carcinoma Cells

目的观察COX-2选择性抑制剂NS-398对结肠癌耐药细胞HCT-8/Fu多药耐药的逆转作用及其对P-糖蛋白(P-glycoprotein,P-gp)、多药耐药相关蛋白(multi-drug resistance related protein,MRP)的调控,以初步探讨其作用机制。方法 (1)CCK-8法检测NS-398对HCT-8/Fu的毒性作用及对HCT-8/Fu细胞多药耐药(multi-drug resistance,MDR)的逆转作用;(2)将NS-398、5-Fu、NS-398+5-Fu分别作用于HCT-8/Fu 24 h,酶标仪检测Caspase-3活性、Hoechst33342染色观察药物诱导细胞凋亡情况;(3)0、10、20μmol/L NS-398分别作用癌细胞24 h后ELISA法检测培养液中PGE2含量;0、20μmol/L NS-398作用癌细胞24 h后,免疫细胞化学检测癌细胞P-gp、MRP的表达。结果 (1)10、20μmol/LNS-398作用癌细胞24 h,抑制率分别为6%、8%,与各浓度5-Fu联用后,耐药逆转倍数分别为3.42、7.50,且呈剂量依赖性;(2)20μmol/L NS-398+320μg/ml 5-Fu组Caspase-3活性增加百分比为386.11%,较320μg/ml 5-Fu组的179.94%、20μmol/L N S-3 9 8组的1 2 5.2 3%明显增高;Hoechst33342染色从形态学上也得出相一致的结论;(3)20μmol/L NS-398作用24 h后,癌细胞P-gp、MRP表达较0μmol/L NS-398显著减少;同时在10、20μmol/L NS-398作用24 h,细胞培养上清液中PGE2含量分别为189.50、151.25ng/L,较0μmol/L NS-398时的340.13 ng/L明显下降。结论 COX-2选择性抑制剂NS-398对结肠癌耐药性有显著逆转作用,并呈剂量依赖性,其逆转机制可能是通过抑制P-gp、MRP的表达及抑制PGE2生成而发挥逆转作用的。

Objective To observe the reversal effects of cyclooxygenase-2(COX-2) selective inhibitor NS-398 on the multi-drug resistance of colorectal cancer cell line HCT-8/Fu and its regulatory effects on the expression of P-glycoprotein(P-gp), multi-drug resistance related protein(MRP), in order to explore its mechanism. Methods(1)The inhibition rate and the reversal of multi-drug resistance(MDR) of HCT-8/Fu cells by NS-398 were detected by CCK-8 kit;(2)NS-398, 5-Fu or NS-398 combined with 5-Fu were added on HCT-8/Fu cells for 24 h respectively. Caspase-3 activity was analysed by automatic fluorescence enzymelinked immunoassay detector; the cells were stained by Hoechst33342 and observed under fluorescent inverted microscope;(3)HCT-8/Fu cells were treated by NS-398(0, 10 and 20μmol/L) for 24 h, ELISA analysis were performed to detect PGE2 concentration in the culture supernatant; and the expression of P-gp, MRP before and after 20μmol/L NS-398 treatment were detected by immunocytochemical staining. Results(1)The inhibition rates were 6% and 8% respectively after HCT-8/Fu cells were treated by 10 and 20 μmol/L NS-398 for 24 h; when combined with various concentrations of 5-Fu, the reversal effects were 3.42 and 7.50 times respectively, in a dose-dependent manner.(2)Caspase-3 activity of the combined group(20μmol/L NS-398+320μg/ml 5-Fu) was increased significantly by 386.11%, compared with 179.94% in 320 μg/ml 5-Fu group and 125.23% in 20 μmol/L NS-398 group; the morphological changes by Hoechst33342 staining were consistent with this result;(3) The expression of P-gp and MRP were decreased significantly after treated by 20 μmol/L NS-398 for 24 h detected by immunocytochemical staining. Meanwhile, when HCT-8/Fu cells were treated with 10 and 20 μmol/L NS-398 for 24 h, the levels of PGE2 were 189.50 and 151.25 ng/L respectively, significantly lower than 340.13 ng/L PGE2 treated with 0μmol/L NS-398. Conclusion COX-2 selective inhibitor NS-398 could reverse multi-drug resistance of colorectal HCT-8/Fu cells, in a dose-dependent manner. The reversal mechanism may be through inhibiting the expression of P-gp, MRP and the level of PGE2.