Modulation of P-glycoprotein expression and function by curcumin in multidrug-resistant human KB cells

Abstract

Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in the tumor cells of a patient, resulting from enhanced drug efflux. It is often related to the overexpression of P-glycoprotein (Pgp) on the surface of tumor cells, thereby reducing drug cytotoxicity. In this study, curcumin was tested for its potential ability to modulate the expression and function of Pgp in the multidrug-resistant human cervical carcinoma cell line KB-V1. Western blot analysis and reverse transcription–polymerase chain reaction (RT–PCR) showed that treatment with 1, 5, and 10 μM curcumin for up to 72 hr was able to significantly lower Pgp expression in KB-V1 cells. Curcumin (1–10 μM) decreased Pgp expression in a concentration-dependent manner and was also found to have the same effect on MDR1 mRNA levels. The effect of curcumin on Pgp function was demonstrated by rhodamine 123 (Rh123) accumulation and efflux in Pgp-expressing KB-V1 cells. Curcumin increased Rh123 accumulation in a concentration-dependent manner (1–55 μM) and inhibited the efflux of Rh123 from these cells, but did not affect the efflux of Rh123 from the wild-type drug-sensitive KB-3-1 cells. Treatment of drug-resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with an increased intracellular accumulation of Rh123. In addition, curcumin inhibited verapamil-stimulated ATPase activity and the photoaffinity labeling of Pgp with the prazosin analog [${}^{125}\text{I}$]iodoarylazidoprazosin in a concentration-dependent manner, demonstrating that curcumin interacts directly with the transporter. Thus, curcumin seems to be able to modulate the in vitro expression and function of Pgp in multidrug-resistant human KB-V1 cells. In summary, this study describes the duel modulation of MDR1 expression and Pgp function by the phytochemical curcumin, which may be an attractive new agent for the chemosensitization of cancer cells.

Introduction

When patients with cancer are treated with a cytotoxic agent, the pharmacological goal is to deliver as much active drug as possible to the molecular target in the cancer cells, causing sufficient molecular damage to lead to cell death. On the other hand, the occurrence of drug resistance renders cells resistant not only to the drug used in the chemotherapy, but also to a broad spectrum of unrelated cytotoxic drugs as well. Cancer cells may develop a multidrug-resistant phenotype. When human tumor cells express this phenotype, they often overexpress the drug export protein called plasma membrane Pgp with a molecular mass of approximately 150–170 kDa. This plasma membrane phosphoglycoprotein, which belongs to the superfamily of ATP-binding cassette (ABC) transporters, consists of two homologous halves that share a high degree of sequence similarity [1].

Uptake and/or efflux of isotope-labelled drugs or Rh123 are used frequently for the functional assay of Pgp in tumor cells. Several classes of compounds that inhibit Pgp-mediated efflux and enhance the accumulation and efficacy of anticancer compounds have been identified. MDR-reversing agents include calcium channel blockers (verapamil), calmodulin inhibitors (phenothiazines), indole alkaloids (reserpines), and nonpolar cyclic oligopeptides with immunosuppressant activity (cyclosporin A) [2]. Although Pgp mediates the transport of many structurally and functionally diverse compounds, many potent MDR-inhibiting compounds share common physical characteristics such as cyclicity, lipophilicity, and a positive or neutral charge at physiological pH. Many synthetic MDR modulators, including reversins 121 and 205 [3] and the cyclosporin D analog Valspodar (PSC 833) [4], successfully reverse the MDR phenotype in vitro. However, the efficacy of these compounds in animal studies and clinical trials has been disappointing due to dose-limiting toxicity. Accordingly, much effort is currently being expanded toward identifying natural compounds from plant origins that inhibit Pgp, reverse the MDR phenotype, and sensitize cancer cells to conventional chemotherapy without undesired toxicological effects. The other approach for MDR modulation is the modulation of the MDR1 gene. Studies of the MDR1 gene promoter sequence suggest that modulation of Pgp expression at the genetic level may be possible [5]. These types of MDR modulators may either block the induction of MDR1 gene expression or inhibit its promoter and down-regulate Pgp expression.

Curcumin is a natural phenolic coloring compound found in rhizomes of Curcuma longa Linn., commonly called turmeric. The curcumin content in turmeric is about 1–5%, and it has been identified as the major yellow pigment in turmeric. It has been widely used as a spice, to color cheese and butter, as a cosmetic, and in some medicinal preparations [6], [7]. Curcumin has a wide range of biological and pharmacological activities, including antioxidant [8], [9], [10] and anti-inflammatory properties [7], anti-mutagenic activity in vitro[11], anti-carcinogenic effects [12], [13], [14], hypocholesterolemic effects in rats [15], and hypoglycemic effects in humans [16]. The safety of C. longa and its derivatives has been studied in various animal models [17], and it is clear that turmeric is not toxic even at high doses in laboratory animals. A single feeding of a 30% turmeric diet to rats did not produce any toxic effects. In a 24-hr acute toxicity study, mice were fed dosages of 0.5, 1.0, and 3.0 g/kg of turmeric extract daily. There was no increase in the mortality rate when compared with the respective controls in either study. A 90-day treatment with turmeric extract resulted in no significant weight gain [18].

Due to its wide range of biological and pharmacological effects, lack of toxicity in animal models, cyclicity, and lipophilicity, curcumin was examined in the present study to determine possible interactions with Pgp expression and function. We demonstrated that curcumin down-regulates Pgp expression and reduces Pgp-mediated efflux in drug-resistant human cervical carcinoma cells (KB-V1). In addition, biochemical assays demonstrated that curcumin interacts directly with Pgp. In summary, these results suggest that curcumin may have chemosensitizing properties on the MDR phenotype as a result of its ability to modulate both the expression and function of MDR1.