Induction of apoptosis by curcumin: mediation by glutathione S-transferase P1-1 inhibition

doi:10.1016/S0006-2952(03)00501-X

Biochemical Pharmacology

Volume 66, Issue 8, 15 October 2003, Pages 1475-1483

https://doi.org/10.1016/S0006-2952(03)00501-X Get rights and content

Abstract

Expression of glutathione S-transferase P1-1 (GSTP1-1) is correlated to carcinogenesis and resistance of cancer cells against chemotherapeutic agents. Curcumin, a natural compound extracted from Curcuma longa, has shown strong antioxidant and anticancer properties and also the ability to regulate a wide variety of genes that require activating protein 1 and nuclear factor κB (NF-κB) activation. In the present study, we examined the inhibitory effect of curcumin on the expression of GSTP1-1 mRNA as well as protein, and we correlated this inhibition with the apoptotic effect of curcumin on K562 leukemia cells. Curcumin efficiently inhibited the tumour necrosis factor α- and phorbol ester-induced binding of AP-1 and NF-κB transcription factors to sites located on the GSTP1-1 gene promoter. TNFα-induced GSTP1-1 promoter activity was also inhibited by curcumin as shown by reporter gene assay. In parallel, curcumin induced pro-caspases 8 and 9 as well as poly ADP ribose polymerase cleavage and thus leading to apoptosis in K562 cells. Our results overall add a novel role for curcumin as this chemoprotective compound could contribute to induce apoptosis by its ability to inhibit the GSTP1-1 expression at the level of transcription.

Introduction

GSTs (E.C. 2.5.1.18) are a multigene superfamily of enzymes that have been classified into eight distinct gene families coding for seven cytosolic isoforms (alpha, mu, pi, theta, omega, kappa and zeta) and one microsomal form. GSTP1-1, or GST pi, is expressed in most human tissues except in adult liver and in cancer cell lines like the Burkitt lymphoma Raji, HepG2 hepatoma and MCF7 breast cancer cells [1]. Its subunits are between 23 and 28 kDa and its gene has a length of 2.8 kbp with 7 exons and 6 introns. Studies of human GSTP1-1 promoter show that the necessary regulatory elements are situated in the region −80 to −8, containing AP-1 and Sp1 sites [2].

GSTs play an important role in detoxification by catalysing the conjugation of electrophilic compounds such as xenobiotic drugs, toxins and carcinogens [3], [4] to glutathione (GSH) allowing the drug to be exported from the cell through the GS-X pump in an ATP-dependent way. However, GSTs also allow the development of resistance to chemotherapy and elevated levels of GSTP1-1 mRNA are found in cell lines resistant to a range of anticancer drugs. Indeed, MCF7, an estrogen-receptor positive breast cancer cell line, was found to develop a resistance to ethacrynic acid [5], doxorubicin and benzopyrene [6] when transfected with GSTP1-1 and multidrug resistant protein 1. Ovarian cancer cell lines overexpressing GSTP1-1 are resistant to doxorubicin or taxol [7]. COS cells become resistant to doxorubicin [8] and CHO cells resist to cisplatine and carboplatine [9] after transfection with the GSTP1-1 gene. Moreover, carcinogenesis is found to be related in many cases to an overexpression of GSTs, in particular GSTP1-1. Indeed, in many human tumours, like prostate carcinoma [10], squamous-cell carcinoma [11], acute lymphoblastic leukemia [12] and chronic lymphoid leukemia [13], GSTP1-1 is overexpressed, even though in the corresponding normal tissues the protein is either absent or expressed at very low levels. GSTP1-1 can thus be used as a valuable prognostic tool in sarcoma [14] or gastric carcinoma [15].

AP-1 [16], [17] and NF-κB activation is inhibited by curcumin (diferuloylmethane) [18], [19], the yellow component of Curcuma longa that gives curry its colour and flavour. Curcumin has shown anti-inflammatory and antioxidant properties [20], [21], [22], [23], [24], [25], [26] due to the inhibition of cyclooxygenase 2 and lipoxygenase [27], [28]. Curcumin inhibits tumour initiation (by benz[a]pyrene and 7,12-dimethylbenz[a]anthracene [29]) and promotion (by phorbol esters) thus showing anticarcinogenic properties [30], [31].

In order to understand the relationship between apoptosis and transcriptional regulation of the GSTP1-1 gene, we examined the effect of curcumin on the GSTP1-1 expression correlated to cellular viability in cultured human leukemia cells. In this study, we provide evidence that curcumin significantly reduces GSTP1-1 mRNA and protein levels as well as TNFα- or phorbol ester-activated NF-κB and AP-1 transcription factor binding to consensus and GSTP1-1 promoter probes. This inhibition was also confirmed by reporter gene assays demonstrating the effect of curcumin at the molecular level and thus underlining its chemopreventive potential. Furthermore, the appearance of apoptotic cleavage products is correlated to a curcumin-induced reduction of GSTP1-1 expression.

Section snippets

Cells and medium

K562 cells (human chronic myelogenous leukemia) were cultured in RPMI medium (Bio-Whittaker) containing 10% (v/v) fetal calf serum (FCS) (Bio-Whittaker) and 1% (v/v) antibiotic–antimycotic (Bio-Whittaker) at 37°, 5% of CO₂. Before treatments, cells were cultured in RPMI with 0.1% (v/v) FCS and 1% (v/v) antibiotic–antimycotic for 72 hr at 37°.

Compounds

TPA was obtained from ICN, TNFα and curcumin were purchased from Sigma Chemical Co. Curcumin and TPA were dissolved in 100% DMSO at 20, 1 mM, respectively.

Inhibition of the basal GSTP1-1 expression by curcumin

K562 cells were treated with curcumin (0, 1, 5 or 10 μM) for 24 hr at 37°. Figure 1A shows that 10 μM curcumin strongly inhibits (−25%) the expression of the 1.2 kbp transcript corresponding to the human GSTP1-1 mRNA. This inhibition was not due to an unspecific downregulation of RNA synthesis, since curcumin treatment did not significantly modify the expression of GAPDH mRNA. Figure 1B shows that GSTP1-1 protein expression was decreased as well with 10 μM curcumin under low serum conditions.

Inhibition of TNFα-induced GSTP1-1 mRNA expression by curcumin

K562

Discussion

Curcumin is a compound known for its antioxidant and anticarcinogenic properties [20], [21], [22], [23], [24], [25], [26]. It has also been shown to have an inhibitory effect on AP-1 and NF-κB activation. We investigated here its effects on GSTP1-1 expression in leukemia cell lines. Indeed, GSTP1-1 was found to be related to carcinogenesis and resistance to anticancer drugs [38]. The implication of NF-κB and AP-1 [3], [39] in GSTP1-1 regulation was demonstrated and the use of curcumin in

Acknowledgements

The authors thank B. Aggarwal for helpful discussions. This work was supported by the Fondation de Recherche “Cancer et Sang”. A.D., Ma.S. and Mi.S. are supported by fellowships from the Government of Luxembourg. This work was in part supported by the Télévie grant 7.4577.02.

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Induction of apoptosis by curcumin: mediation by glutathione S-transferase P1-1 inhibition

Abstract

Introduction

Section snippets

Cells and medium

Compounds

Inhibition of the basal GSTP1-1 expression by curcumin

Inhibition of TNFα-induced GSTP1-1 mRNA expression by curcumin

Discussion

Acknowledgements

Gene

Biochem. Pharmacol.

Biochem. Biophys. Res. Commun.

J. Urol.

Ann. Oncol.

Oncogene

J. Biol. Chem.

Biochem. Pharmacol.

Prostaglandins Leukot. Med.

Leuk. Res.

J. Biol. Chem.

Cell

J. Biol. Chem.

Am. J. Pathol.

Exp. Cell Res.

Leuk. Res.

Exp. Cell Res.

Toxicol. Lett.

J. Biol. Chem.

Trends Cell Biol.

J. Biol. Chem.

Differences among human tumor cell lines in the expression of glutathione transferases and other glutathione-linked enzymes

Carcinogenesis

The organization of the human GSTP1-1 gene promoter and its response to retinoic acid and cellular redox status

Biochem. J.

Glutathione S-transferases and cancer

Int. J. Oncol.

Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins

Mol. Pharmacol.