Microsomal glutathione transferase 1 in anti-cancer drug resistance and protection against oxidative stress

Cancer is the second leading cause of death in privileged countries. Tremendous efforts have been done to improve the efficiency of cancer treatment. Anti-cancer drug resistance is still a major problem in failure of cancer therapy. Microsomal glutathione transferase 1 (MGST1) is a membrane protein located in the endoplasmic reticulum and the outer membrane of mitochondria. MGST1 is involved in the direct conjugation of glutathione to endogenous and exogenous toxic electrophilic hydrophobic compounds. The enzyme is often upregulated in tumours, and has been suggested to be an early marker of tumorigenesis. Furthermore, MGST1 has been connected to drug resistance in the clinic. Herein we show that MGST1 plays a potential role in inducing resistance against some commonly used anti-cancer drugs including chlorambucil, melphalan, carmustine, doxorubicin and cisplatin as examined in a human breast carcinoma cell line (MCF7) overexpressing MGST1. Further, we show that MGST1 dependent resistance against cisplatin can be reversed by ethacraplatin, a combination of the drug with a GST inhibitor (ethacrynic acid, EA). Upon cellular uptake, this compound is cleaved and the EA moiety is released. When cisplatin and ethacrynic acid were added together as such they were not as effective as ethacraplatin. Ethacraplatin can therefore be a useful alternative to overcome drug resistance by MGST1. Instead of inhibition of GSTs to reverse resistance, an alternate strategy is to use prodrugs that release cytotoxic molecules catalysed by GSTs. Here we show that prodox, a doxorubicin derivative, releases doxorubicin upon MGST1 and GSTP dependent catalysis. In MGST1 overexpressing cells the protection against prodox was lower compared to doxorubicin. With GSTP a dramatic effect was observed. Prodox was considerably more toxic to GSTP overexpressing than control cells. Thus prodrugs such as prodox, that are preferentially cleaved in GST overexpressing tumours, could be a useful therapeutic approach. We investigated whether MGST1 can protect cells against oxidative stress. Exposing MCF7 cells overexpressing MGST1 to several agents that induce oxidative stress and lipid peroxidation (hydrogen peroxide, cumene hydroperoxide and tert-butyl hydroperoxide) resulted in significant protection. Microsomal GST1 also protected significantly against 4-hydroxynonenal, a highly reactive and toxic end-product of lipid peroxidation. Vitamin E increases the MGST1-dependent protection when toxicity is mediated by oxidative stress (hydroperoxides) but not when direct alkylation is the dominant mechanism (4-hydroxynonenal). Further, measurements of respiration and calcium buffering capacity of mitochondria revealed that MGST1 significantly protects mitochondria against oxidative insult. Taken together the results show that MGST1 protects cells via multiple mechanisms including direct conjugation and reduction as well as downstream protection against lipid peroxidation. As vitamin E did not augment MGST1 resistance against cisplatin we believe that the major toxic mechanism is not related to oxidative stress. The mechanism of the strong protection against cisplatin remains to be determined and a hitherto unknown function of MGST1 is indicated. In summary, these investigations clearly show that MGST1 is an important contributor in cellular protection against oxidative stress and anti-cancer drugs. LIST OF PUBLICATIONS I. Johansson K, Ahlen K, Rinaldi R, Sahlander K, Siritantikorn A, Morgenstern R, (2006), Microsomal glutathione transferase 1 in anti-cancer drug resistance, Carcinogenesis 28, 465-70. II. Johansson K, Matthew S, Heuser-Dahlstrom, V, Zhang J, Shimoji M, Ang WH, Dyson P, Ito M, Abe H, Shibata A, Ito Y and Morgenstern R, (2010), Characterization of two new potential anti-cancer drugs designed to overcome glutathione transferase mediated resistance, Manuscript III. Siritantikorn A, Johansson K, Ahlen K, Rinaldi R, Suthiphongchai T, Wilairat P, Morgenstern R, (2007), Protection of cells from oxidative stress by microsomal glutathione transferase 1. Biochem Biophys Res Commun. 355, 592-6. IV. Johansson K, Jarvliden J, Gogvadze V and Morgenstern R, (2010), Multiple roles of Microsomal Glutathione Transferase 1 in protection against cellular lipid peroxidation, a mechanistic study, Submitted. Additional publications not included in the thesis: Alander J, Johansson K, Dahlstrom Heuser V, Farebo H, Jarvliden J, Abe H, Shibata A, Ito Y and Morgenstern R, (2009), Characterisation of a New Fluorogenic Substrate for Microsomal Glutathione Transferase 1, Anal Biochem, 390,52-6. Staab CA, Vondrachek M, Custodio H, Johansson K, Nilsson JA, Morgan P, Hoog JO, Cotgreave I, Grafstrom RC, (2004), Modelling of normal and premalignant oral tissue by using the immortalised cell line, SVpgC2a: a review of the value of the model, Altern Lab Anim, 32, 401-5.