Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5′-serial truncation analysis of the MDR1 promoter defined a region from position −450 to −193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.

多药耐药性（multidrug resistance, MDR）是导致癌症化疗失败的主要原因之一，抑制该表型可提升肿瘤治疗效果。人类多药耐药基因1（multidrug resistance 1, MDR1）编码细胞膜上的P-糖蛋白（P-glycoprotein, P-gp），该蛋白可将多种抗癌药物泵出癌细胞。R-HepG2与MES-SA/Dx5细胞分别为阿霉素诱导的高表达P-gp的多药耐药亚系，源自人肝细胞癌HepG2细胞与人子宫癌MES-SA细胞。
本研究中，我们观察到从大白桩菇（Leucopaxillus giganteus）中提取的天然化合物离褶伞菌素（clitocine）在多药耐药细胞系中的细胞毒性与其亲本细胞系相近，且可显著抑制R-HepG2与MES-SA/Dx5细胞中P-gp的表达。进一步研究显示，离褶伞菌素可提高R-HepG2细胞对阿霉素的敏感性及细胞内阿霉素积累量，同时下调MDR1 mRNA水平与启动子活性，表明其可逆转多药耐药性。
对MDR1启动子的5'端系列截短分析表明，-450至-193位核苷酸区域是离褶伞菌素抑制MDR1表达的关键区段。该区域内的保守核因子κB（NF-κB）结合位点发生突变，或过表达NF-κB p65，均可抵消离褶伞菌素对MDR1启动子的抑制作用。免疫组织化学实验证实，离褶伞菌素可下调R-HepG2细胞及移植瘤中P-gp与NF-κB p65的蛋白水平；此外，离褶伞菌素还可抑制MES-SA/Dx5细胞中NF-κB p65的表达。更重要的是，即使在阿霉素存在的条件下，离褶伞菌素仍可抑制NF-κB的活化。
综上，本研究结果表明，离褶伞菌素可通过下调核因子κB通路逆转P-gp介导的多药耐药性。