Reversal of multidrug resistance by 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide through the reversible inhibition of P-glycoprotein

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Abstract

Overexpression of P-glycoprotein (P-gp) is one of the major obstacles to successful cancer chemotherapy. In this study, we examined the ability of 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide (C-4) to reverse multidrug resistance (MDR) in P-gp expressing KBV20C cells. Treatment of KBV20C cells with C-4 led to a dramatic increase in paclitaxel- or vincristine-induced cytotoxicity without any cytotoxicity by itself. In parallel, C-4 treatment caused an increase in apoptotic cell death by paclitaxel or vincristine. Furthermore, C-4 treatment significantly increases in intracellular accumulation of fluorescent P-gp substrate rhodamine 123, indicating that C-4 treatment leads to reversal of the MDR phenotype resulting from an increased accumulation of anticancer drugs by inhibiting drug efflux function of P-gp. This notion is further supported by the observation that C-4 treatment potentiates paclitaxel-induced G2/M arrest of the cell cycle. In addition, the drug efflux function of P-gp was reversibly inhibited by C-4 treatment, while the expression level of P-gp was not affected. Collectively, our results describe the potential of C-4 to reverse the P-gp-mediated MDR phenotype through reversible inhibition of P-gp function, which may make it an attractive new agent for the chemosensitization of cancer cells.

Section snippets

Materials and methods

Cell culture. P-gp non-expressing KB cells and P-gp expressing KBV20C cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT) and 1% penicillin/streptomycin (Invitrogen). KBV20C cells were grown in the presence of 20 nM vincristine (Sigma Chemical, St. Louis, MO) as described previously [22].

Reagents and cytotoxicity test. Paclitaxel, verapamil, and rhodamine 123 (Rh123) were obtained from Sigma. Compound C-4 (4-chloro-N-(3-((

C-4 treatment increases vincristine- or paclitaxel-induced cytotoxicity in drug-resistant KBV20C cells

In order to explore the molecular mechanism for the MDR modulating activity of curcumin and its analog C-4, we used P-gp expressing KBV20C cells and its parental line of P-gp non-expressing KB cells. We first determined the cytotoxic response of both KBV20C and KB cells to vincristine or paclitaxel. As expected, KBV20C cells were more resistant than KB cells to paclitaxel and vincristine; IC50 in KB cells were 4.2 nM (paclitaxel) and 7.9 nM (vincristine) (data not shown), whereas those in KBV20C

Discussion

Chemotherapy is the most effective treatment for patients who suffer from metastatic cancers. The effectiveness of chemotherapy, however, is seriously limited by MDR which is mainly due to the overexpression of P-gp, an integral membrane protein. P-gp functions as a drug efflux pump which actively transports drugs from the inside to the outside of cancer cells and prevents the intracellular accumulation of anticancer drugs inside cancer cells necessary for cytotoxic activity. Therefore, novel

Acknowledgments

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-003-E00024, to Y.K. Kim).

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