Abstract
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.
Highlights
Cancer cells can develop resistance against structurally and mechanistically unrelated chemotherapeutic agents, a phenomenon named as multidrug resistance (MDR) [1]
The anti-proliferation effect of clitocine on human cancer cell lines was assessed by the MTT assay, including HepG2, RHepG2, MES-SA, MES-SA/Dx5, SMMC7721, Bcap37, MCF-7, HeLa,and SGC-7901 for 48 h
The treatment of the nine human cancer cell lines with clitocine exhibited a marked inhibition on the survival of these cells dose-dependently
Summary
Cancer cells can develop resistance against structurally and mechanistically unrelated chemotherapeutic agents, a phenomenon named as multidrug resistance (MDR) [1]. The calcium channel blocker verapamil has been well-known to reverse multidrug resistance by directly binding to P-gp protein on special sites [8,9]. Cyclosporine such as cyclosporin A could modulate the efflux function of P-gp by interfering with both the substrate-binding sites and the ATP hydrolysis cycle [10]. Most of these agents necessitated high doses and produced unacceptable toxicity because of their low affinity with P-gp. New generation of P-gp inhibitors with high affinity at very low doses, such as elacridar (GF120918), tariquidar (XR9576) and OC144-093 (ONT-093) have been developed, novel approaches in overcoming P-gp associated MDR are still needed
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