Cancer Biology and Signal Transduction Metallothionein 1 G and Zinc Sensitize Human Colorectal Cancer Cells to Chemotherapy

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-kB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing ofMTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhanceMT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, asmeasured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-kB. Addition of zinc enhanced growth inhibition by OXA and 5FU, andwas also capable of resensitizing 5-FU–resistant cell lines to levels comparablewith sensitive cell lines. This effect wasMT independent because silencingMTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents. Mol Cancer Ther; 13(5); 1369–81. 2014 AACR. Introduction Colorectal cancer is the third most frequent cancer worldwide, having a mortality rate near 50% (1). Current therapeutic strategies rely heavily on complete surgical removal of the tumor, despitewhich 40%of patients recur. Chemotherapeutic adjuvant treatment in stage II disease is controversial and improves overall survival by 22% in stage III. In the metastatic setting, overall 5-year survival rates for stage IV patients are less than 10% (2). Therapeutic regimens are mainly based on 5-fluorouracil (5FU), oxaliplatin (OXA), and irinotecan, all of which produce considerable side effects. Thus, it is of paramount importance to develop new therapies or to improve currently available agents. Metallothioneins (MT) are a family of low molecular weightproteins that share significant sequencehomology, and are involved in zinc and redoxmetabolism (3) as well as in many aspects of cancer biology (4, 5). The human genome contains at least 11 functional MT genes that may be divided into four subgroups (MT1-4). There are several MT1 isoforms each encoded by its own gene and along with MT2A are ubiquitously expressed. Given their stress-inducible nature and their capacity to chelate toxic metals and electrophiles, many studies have proposed MT expression to confer resistance to many toxic drugs (6, 7). On the other hand, given their capacity to influence zinc metabolism and this metal’s availability to many zinc-dependent proteins and transcription factors, other studies have associated them with chemosensitivity (8, 9). Indeed, MTs either donate or take away zinc ions from several zinc-dependent proteins, including p53 (10, 11), thereby regulating their function. We and others have previously demonstrated that these proteins are progressively silenced during colorectal cancer progression, and that this is associated to poorer patient survival. (12–14). Zinc is a requirednutrient forproliferation, but elevated concentrations are known to promote cell death by many different mechanisms (15). Free zinc ions exist in the picomolar range and may be considered negligible due to tight regulation by zinc transporters, MTs, and organelle sequestration (16). Intracellular zinc pools consist mainly of tightly bound, unexchangeable zinc bound to proteins (the "immobile" pool), and of the exchangeable, loosely bound zinc termed the "labile" pool, which is complexed to low molecular weight ligands and MTs (17). The latter represents about 5% of the total Authors' Affiliations: Centro de Investigaciones Oncol ogicas de la Fundaci on C ancer (CIO-FUCA); Laboratorio de Cancerología, Fundaci on Instituto Leloir, IIBBA-CONICET; Instituto Alexander Fleming, Buenos Aires, Argentina; and Operative Unit 'Molecular Mechanisms of Cancer Growth and Progression,' Department of Experimental Oncology, Fondazione IRCCS 'Istituto Nazionale dei Tumori,' Milan, Italy Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Michele Bianchini, Centro de Investigaciones Oncol ogicas de la Fundaci on C ancer (CIO-FUCA), Zabala 2836, (1426), Buenos Aires, Argentina. Phone: 54-11-3221-8900; Fax: 54-11-32218900; E-mail: mbianchini@conicet.gov. ar doi: 10.1158/1535-7163.MCT-13-0944 2014 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals. org 1369 on June 23, 2017. © 2014 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Published OnlineFirst March 14, 2014; DOI: 10.1158/1535-7163.MCT-13-0944