Induction of senescence in cancer cells by 5′-Aza-2′-deoxycytidine: Bioinformatics and experimental insights to its targets

Highlights

• Demethylating drug, 5-Aza-dC targets HDM2 yielding activation of p53 pathway and growth arrest of cancer cells.

• Cells treated with 5-AZA-dC in combination with three other drugs showed drug-resistance characteristics.

• 5-Aza-dC is a multi-module drug. Further studies are warranted to dissect its mechanism(s) of action beyond demethylation.

Abstract

5′-Aza-2′-deoxycytidine (5-Aza-dC) is a demethylating drug that causes genome-wide hypomethylation resulting in the expression of several tumor suppressor genes causing growth arrest of cancer cells. Cancer is well established as a multifactorial disease and requires multi-module therapeutics. Search for new drugs and their approval by FDA takes a long time. Keeping this in view, research on new functions of FDA-approved anticancer drugs is desired to expand the list of multi-module functioning drugs for cancer therapy. In this study, we conducted an analysis for new functions of 5-Aza-dC by applying bio-chemo-informatics approach. The potential of 5-Aza-dC bioactivity was analyzed by PASS online and Molinspiration. Target proteins were predicted by SuperPred. The protein networks and biological processes were analyzed by Biological Networks using Gene Ontology tool, BINGO, based on BIOGRID database. Interactions between 5-Aza-dC and targeted proteins were examined by Autodoc Vina integrated into pyrx software. Induction of p53 by 5-Aza-dC was tested in vitro using cancer cells. Bioinformatics analyses predicted that 5-Aza-dC functions as a p53 inducer, radiosensitizer, and inhibitor of some enzymes. It was predicted to target proteins including MDM2, POLA1, POLB, and CXCR4 that are involved in the induction of DNA damage response and p53-HDM2-p21 signaling. In this study, we provide experimental evidence showing HDM2 is one of the targets of 5-AZA-dC leading to activation of p53 pathway and growth arrest of cells. Furthermore, we found that the combinatorial treatment of 5-AZA-dC with three other drugs caused drug resistance. We discuss that 5-Aza-dC-induced senescence is a multi-module drug that controls cell proliferation phenotype not only by proteins but also by noncoding miRNAs. Further studies are warranted to dissect these mechanisms and establish 5-Aza-dC as an effective multi-module anticancer reagent.

Introduction

Cancer incidence is increasing rapidly worldwide and has prioritized anticancer drug discovery and development. In spite of remarkable progress in this field, only a small percent of drugs reach to clinical trials and even a smaller fraction of them develop to the drugs. This is because cancer is a diversive in its etiology and progression. Large databases on cancer genomics, transcriptomics and proteomic have established that although all cancers are accepted as a proliferative disease that resists apoptosis, they require multi-module treatment. All cancers escape controls on normal cell proliferation due to lack of one or more functional tumor suppressor proteins; p53 is inactivated in the vast majority of cancers (Vogelstein et al., 2000, Bally et al., 2014, Muller and Vousden, 2014). Besides the local surgery, radiotherapy and chemotherapy are the two mainstream treatments for cancer. Since the new drugs have to undergo lengthy process of approval for safety or side effects, we set out in search of multi-module functions of established cancer drugs so that they could be put into wider use in cancer therapeutics.

Acetylsalicylic acid known as aspirin, commonly used to treat pain, fever, and inflammation, has been shown to prevent heart attacks, strokes, and blood clot formation. It has also been reported to possess anti-hypertension and anticancer activities (Barnett et al., 1996, Berger et al., 2008, Rothwell et al., 2012, Alfonso et al., 2014, Yiannakopoulou, 2014, Al-Nimer et al., 2015, Jung et al., 2015, Burn and Sheth, 2016, Dzeshka et al., 2016, Welsh et al., 2016, Zong et al., 2016). Such studies have initiated a new field of cross-functional research. An identification of new functionality of an existing drug is possible by Bio-Chem information technology. This approach has been widely used to analyze activities of a small molecule for the development of new drugs (Gond et al., 2013, Khademvatan et al., 2013, Deng et al., 2014).

DNA methylation is an important key of epigenetic modification to maintain genomic stability required in many biological steps including genomic imprinting, regulation of gene and chromatin remodeling. An abnormal pattern of DNA methylation has been reported in many tumor-derived cells leading to silencing of many tumor suppressor genes. 5′-Aza-2′-deoxycytidine (5-AZA-dC) is a FDA-approved widely used hypomethylating anticancer drug (Adès et al., 2012). It causes global demethylation of cytosine in the C-5 position of CpG rich promoters of tumor suppressor genes. Many studies have reported that 5-AZA-dC induces DNA damage response signaling through activation of hallmark proteins including ATM, ATR (ATM-Rad3-related), checkpoint kinase 1 (CHK1), BRCA1, NBS1, and RAD51 (Palii et al., 2008). In an earlier study, we reported that 5-AZA-dC interacts with mortalin and Pex3P causing activation of growth arrest signaling by inhibition of these proteins that occurred independently of its demethylation effect (Widodo et al., 2007). It has also been shown that demethylation causes activation of miRNAs, the noncoding regulators that have been implicated in wide cellular processes including differentiation, proliferation, and carcinogenesis (Asuthkar et al., 2012, Wang et al., 2014, Wang et al., 2015, He et al., 2015, Long et al., 2015; Yu et al., 2015). Of note, hundred target genes can be silenced by a single miRNA and hence, miRNA deregulation has been closely and complexly related to the process of carcinogenesis (Hagan and Croce, 2007, Cheng et al., 2014, Mizuguchi et al., 2016, Wang, 2016).

Recently, we recruited 5-AZA-dC-induced senescence in miR-induced loss-of-function screening and identified miRNA-335 as a methylation regulatory miR and its role in tumor suppression through targeting CARF, a regulator of p53-HDM2-p21 pathway, through mechanism independent of its demethylation effect (Hasan et al., 2009, Cheung et al., 2014, Sato et al., 2015, Fan et al., 2016, Yu et al., 2016). These reports have reflected the complexity of action of 5-AZA-dC. Carcinogenesis, tumor progression and metastasis have also been established as multifactorial phenomena involving loss and gain of function of tumor suppressor and oncogenes, respectively, by multiple and cross-talking pathways (Carroll et al., 1993, Vogelstein et al., 2000, Kaul et al., 2005, Lu et al., 2011, Muller and Vousden, 2014, Deben et al., 2016, Ozaki et al., 2016)

It has been established that p53 is degraded by HDM2 by proteasomal degradation pathway and inactivated by binding to mortalin (Wadhwa et al., 1998, Kashuba et al., 2003, Linares et al., 2003, Kaul et al., 2005). Mortalin is widely distributed in the cytoplasm of normal cells, whereas the cancer cells show perinuclear localization (Wadhwa et al., 1993, Kaul et al., 2005). Human osteosarcoma cell lines when treated with 5-AZA-dC showed shift in mortalin staining pattern, from perinuclear to pancytoplasmic, and an upregulation and nuclear translocation of p53. Upregulation of HDM2 and mortalin has been reported in cancers that account for loss-of-function of p53 (Meek and Knippschild, 2003, Wadhwa et al., 2006, Widodo et al., 2007, Lu et al., 2011, Sane et al., 2014) In agreement with these reports, drugs that abrogate p53-HDM2 and p53-mortalin interactions have been shown to cause growth arrest of cancer cells (Widodo et al., 2007, Grover et al., 2012, Vaishnavi et al., 2012, Leao et al., 2013, Saxena et al., 2013, Goyal et al., 2014, Nigam et al., 2015, Shah et al., 2015). In the present study, we conducted an analysis in search of new functions of 5-AZA-dC using bio-chemo-informatics approach. The data suggested that 5-AZA-dC, independent to its demethylation activity, is a potential inducer of p53-p21 pathway and inhibits the growth of cancer cells.