Elsevier

Toxicology Letters

Volume 191, Issues 2–3, 15 December 2009, Pages 158-166
Toxicology Letters

5-Azacytidine prevents cisplatin induced nephrotoxicity and potentiates anticancer activity of cisplatin by involving inhibition of metallothionein, pAKT and DNMT1 expression in chemical induced cancer rats

https://doi.org/10.1016/j.toxlet.2009.08.018Get rights and content

Abstract

5-Azactydine inhibits cell growth by direct cytotoxic action as well as by inhibition of DNA methyl transferase enzyme. Inhibitors of DNMT have been reported to potentiate the therapeutic activity of cisplatin in vitro. Dose dependent bone marrow toxicity, neurotoxicity and nephrotoxicity are the major side effects of cisplatin, limiting its use as an effective chemotherapeutic agent. The present study was aimed to reduce the nephrotoxic potential of cisplatin without compensating its potency. To best of our knowledge, this is the first report which shows that the combination of 5-azacytidine with cisplatin leads to remarkable reduction in nephrotoxicity, by involving inhibition of cisplatin induced metallothionein expression. 5-Azacytidine treatment with cisplatin leads to maximum reduction in tumor size in DMH induced colon cancer and tumor volume in DMBA induced breast cancer bearing SD rats. This combination regimen prevents phosphorylation and acetylation of histone H3 which may be involved in inhibition of aberrant gene expression in colon tumors. Further, 5-azacytidine potentiated cisplatin induced antitumor activity by involving decreased expression of pAKT, DNMT1 and an increased expression of p38 in colon tumors. Thus, combination of 5-azactydine with cisplatin attenuates the cisplatin induced nephrotoxicity and potentiates the anti-cancer activity which can have profound clinical implications.

Introduction

Colorectal and breast cancer are the most common causes of cancer related deaths in United States of America (Boring et al., 1994). It is also estimated that half of the western population can expect to develop at least one colorectal tumor by the age of 70 (Kinzler and Vogelstein, 1996). Colon cancer involves several step processes of genetic mutations combined with undefined environmental factors where normal epithelium undergoes dysplastic transformation, proliferation and eventual histological progression to neoplasia. This transition requires multiple genetic alterations in the adenomatous polyposis coli gene (Fang et al., 2003).

Apart from the genetic factors, epigenetic factors including the methylation of the CpG promoter genes, which leads to the repression of the tumor suppressor genes, have also been associated to the etiology of cancers including breast and colon cancer (Chen et al., 2004, Choi and Wu, 2005, Zingg and Jones, 1997). Inhibition of DNMTs has been involved in the reduction of tumorogenicity and increased expression of tumor suppressor genes (Suzuki and Miyata, 2006). 5-Azacytidine (5-AZA), a DNMT inhibitor, incorporates into DNA and results in rapid loss of DNA methyltransferase activity leading to increased expression of tumor suppressor genes (Esteller, 2006). It has been reported that DNMT1 has a potential role in initiating CpG island hypermethylation in human cancer cells (Jair et al., 2006). Being a nucleoside analogue, incorporation of 5-azacytidine into DNA also causes direct cytotoxicity and anti-proliferative effects on tumor cells. Several reports also suggest that DNMT inhibitors sensitize cancer cells and make them more vulnerable to antitumor action of cisplatin (Abbruzzese and Frost, 1992, Suzuki et al., 2007). DNA demethylation can also prevent the development of resistance to chemotherapeutic agents like cisplatin in the cancer cells including the ovarian cancer and SW48 colon cancer cell lines (Plumb et al., 2000).

Cisplatin is one of the most common chemotherapeutic drug used against colon and breast cancer (Rosenberg et al., 1969). It is also a drug of choice in the treatment of other cancers such as testicular cancer, lung cancer, endometrial cancer, head and neck cancer and ovarian cancer (Aisner et al., 1994). Despite its therapeutic benefits, its use in clinical practice is often limited because of dose related toxicity (Campbell et al., 1983). The nephrotoxic potential of cisplatin has been ascribed to its accumulation in the renal tubular cells generating reactive oxygen species (ROS), activation of Bax (Lee et al., 2001), increased secretion of TNFα (Dong and Atherton, 2007) and activation of certain inflammatory mediators like cytokines (Faubel et al., 2007).

Cisplatin induced toxicity along with the development of resistance provokes the use of a combination therapy with a drug that can reduce its toxicity, at the same time keeping intact or potentiating its efficacy (Goligorsky, 2007). The DNMT inhibitors can be utilized to enhance the potency of cisplatin and may help in reducing its dose without compromising its efficacy. As cisplatin and 5-azacytidine have different mechanisms of antitumor activity, combination of these drugs can lead to additive antitumor activity. A phase II clinical trial of cisplatin and decitabine (2-deoxy-5-azacytidine) had been reported to be moderately active in patients with advanced squamous cell carcinoma of the cervix; however, it also shows significant hematologic toxicity (Pohlmann et al., 2002). Li et al. (2009) had reported that azacytidine sensitizes the platinum-resistant ovarian cancer cells (2008/C13 and Hey) to carboplatin. However, effect of 5-azacytidine on nephrotoxicity induced by cisplatin is not yet reported.

Various histone modifications like histone H3 phosphorylation has been reported to increase in mitogenic and oncogene-transformed cells, leading to an increased level of aberrant global gene expression (Chadee et al., 1999). Increased mitotic Ser-10 phosphorylation is also reported in various colorectal tumor cells and is a major precipitating factor for chromosome instability, playing a critical role in carcinogenesis (Ota et al., 2002). HER2 over-expressing breast cancer cell lines are also reported to show increased acetylation of histone H3 (Mishra et al., 2001). However, effect of 5-azacytidine and cisplatin either alone or in combination on cancer induced histone modifications has not been reported in vivo. Therefore, the present study was designed to explore a combination therapy which can potentiate anticancer activity of cisplatin and prevent its toxicity. Furthermore, we investigated the mechanism underlying the effect of 5-azacytidine on cisplatin induced nephrotoxicity and its chemotherapeutic activity in chemically induced colon and breast cancer model.

Section snippets

Reagents and solutions

Cis-diamminplatinumdichloride (cisplatin) was generously provided by Dabur India Pvt. Ltd. All the other chemicals were purchased from Sigma (St. Louis, MO). Cisplatin and 5-azacytidine solution were made freshly in saline (0.9% NaCl). DMH was prepared in saline and the pH of the solution was adjusted to 7.4 using 0.1N NaOH.

Animals

All the experiments were approved by the Institutional Animal Ethics Committee (IAEC) and were in compliance with the NIH guidelines on handling of experimental animals.

Treatment with 5-azacytidine prevents cisplatin induced nephrotoxicity

Cisplatin treatment has been reported to increase the level of BUN and creatinine through extensive tubular damage (Dickey et al., 2005). Treatment with cisplatin (5 mg/kg) led to loss in the body weight and increase in the levels of blood urea nitrogen and creatinine, as compared to untreated male SD rats (Table 1). 5-Azacytidine (2.5 mg/kg) treated group did not show any significant change in both the parameters, but administration of 5-azacytidine along with cisplatin (5 mg/kg) show significant

Discussion

5-Azacytidine treatment shows significant reduction in the DNMT1 expression in colon tumors. In line with DNMT1 inhibition, 5-azacytidine treatment leads to significant reduction in percentage tumor size in the colon tumor and tumor volume in breast tumor bearing rats, as compared to cancer control animals.

Furthermore, this is the first report which suggests that treatment with 5-azacytidine can prevent cisplatin induced nephrotoxicity. 5-Azacytidne shows reduction in the nephrotoxicity profile

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank Mr. Anil Gaikwad and Mr. Praveen Bhuwal for providing their help in the initial stages of this work. This work was supported by DPRP project grant from Department of Science and Technology and National Institute of Pharmaceutical Education and Research (NIPER). Cis-diamminplatinumdichloride (cisplatin, CP) was generously provided by Dabur India Pvt. Ltd.

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