Steroids affect collateral sensitivity to gemcitabine of multidrug-resistant human lung cancer cells

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Abstract

Gemcitabine is phosphorylated by deoxycytidine kinase and thymidine kinase 2 and during S-phase incorporated into DNA. The steroids cortisol and dexamethasone, which regulate cell proliferation and gene expression, are pumped out of the cell by the membrane efflux pumps P-glycoprotein and multidrug resistance-associated protein (MRP), which are blocked by verapamil. In parental non-small cell lung cancer (NSCLC) cells (SW1573), 5 μM cortisol and 100 nM dexamethasone decreased sensitivity to gemcitabine. However, both cortisol and dexamethasone only decreased sensitivity with verapamil in MRP (2R120) and P-glycoprotein (2R160) overexpressing variants. Cortisol decreased deoxycytidine kinase activity in SW1573 cells and cortisol with verapamil in 2R120 and 2R160 cells. Dexamethasone with verapamil decreased deoxycytidine kinase activity in 2R160. Cortisol decreased thymidine kinase 2 activity in 2R120 and 2R160 cells. Dexamethasone decreased thymidine kinase 2 activity in SW1573, 2R120 and 2R160 cells. In conclusion, since dexamethasone is frequently used to treat side effects of oncolytic therapy, a decrease of sensitivity to gemcitabine by steroids might be clinically relevant.

Introduction

2′,2′-Difluorodeoxycytidine (gemcitabine) is a deoxycytidine analog which is active against non-small cell lung cancer (NSCLC), in patients and in tumor models, both in vitro and in vivo Hertel et al., 1990, Boven et al., 1993, Abratt et al., 1994. Gemcitabine and deoxycytidine are phosphorylated by deoxycytidine kinase to their monophosphates. Also the mitochondrial enzyme thymidine kinase 2 phosphorylates deoxycytidine and gemcitabine, however, to a lesser extent than deoxycytidine kinase Bergman et al., 1999, Eriksson et al., 1991. The monophosphates of gemcitabine and deoxycytidine are subsequently phosphorylated to their triphosphates, which are incorporated into DNA, resulting in DNA damage (Huang et al., 1991). Since gemcitabine requires passage through the S-phase in order to be incorporated, this antimetabolite is predominantly active in fast growing tumors (Huang and Plunkett, 1995).

Steroids, like the natural corticosteroid cortisol and the synthetic steroid dexamethasone, inhibit the proliferation of many cell types including lung cancer cell lines Nakane et al., 1990, Croxtall and Flower, 1992. Although steroids are known to induce discrete changes in gene expression and protein synthesis (Ivarie and O'Farrell, 1978), the molecular mechanisms involved in cell growth control have not been clarified yet. Several studies report a decrease in thymidine kinase activity, most likely the cytosolic thymidine kinase 1, as a result of corticosteroid exposure in rat and chicken Tesoriere et al., 1989, Herzfeld and Raper, 1980, Naray et al., 1977, Sakata, 1975. Corticosteroids might have a similar effect on deoxycytidine kinase activity.

In patients, side effects of gemcitabine treatment include nausea and vomiting. The adverse effects of treatment of lung carcinoma is prevented by combinations of anti-emetics and dexamethasone (Cleri et al., 1995). However, not much is known about interaction of cytostatic therapy and dexamethasone. Since steroids inhibit cell proliferation and gemcitabine is S-phase dependent, theoretically, steroids would decrease sensitivity to gemcitabine.

It is known that some of the steroid hormones such as cortisol, progesterone and aldosterone are substrates for the plasma membrane drug efflux pumps P-glycoprotein and multidrug resistance-associated protein (MRP) Endicott and Ling, 1989, Grant et al., 1994, Van Kalken et al., 1993, Mulder et al., 1996. This pump function can be blocked by verapamil (Cornwell et al., 1987). In our previous study an increased sensitivity to gemcitabine was found in P-glycoprotein and MRP overexpressing NSCLC cells, which was related to an increased deoxycytidine kinase activity (Bergman et al., 1998). It was hypothesized that a decrease of intracellular cortisol and dexamethasone concentration as a result of P-glycoprotein or MRP activity might result in an increase in gemcitabine phosphorylation and eventually sensitivity.

In this study we determined whether steroids interact with sensitivity to gemcitabine in cells with a MRP and P-glycoprotein overexpression and whether changes in deoxycytidine kinase and thymidine kinase 2 activity are involved.

Section snippets

Chemicals and reagents

Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Flow Laboratories (Irvine, UK) and fetal calf serum from Gibco (New York, USA), trichloroacetic acid, glutamine and gentamicin from Merck (Darmstadt, Germany), trypsin, sulforhodamine B, cortisol and dexamethasone from Sigma (St. Louis, USA). [5-3H]deoxycytidine (21.9 Ci/mmol) was from Moravek, Brea, CA. All other chemicals were of analytical grade and commercially available.

Cell culture

The in vitro experiments were performed with the human NSCLC

Effect of cortisol, dexamethasone and verapamil on sensitivity to gemcitabine and proliferation rate of SW1573 and its MDR variants

To investigate the effect of cortisol and dexamethasone on sensitivity to gemcitabine, cells were exposed to gemcitabine with or without corticosteroids. Sensitivities of the cells relative to their sensitivities to gemcitabine alone are depicted in Fig. 1. Cortisol (5 μM) decreased sensitivity to gemcitabine 1.8- and 1.5-fold in SW1573 and 2R160 cells, respectively. However, 2R120 cells were two-fold more sensitive to gemcitabine during cortisol exposure. Cortisol did not affect doubling time

Discussion

In this paper, we describe a decrease in gemcitabine sensitivity by steroid exposure in wild-type NSCLC cells. This is in agreement with a study of Rieger et al. (1999) who described a reduction of gemcitabine sensitivity of cultured human glioma cells by clinically relevant concentrations of dexamethasone. However, in the P-glycoprotein and MRP overexpressing variants no effect was found, which might be mediated by a decrease of steroid concentrations by the action of the membrane efflux

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