Original Contribution
A copper chelate of thiosemicarbazone NSC 689534 induces oxidative/ER stress and inhibits tumor growth in vitro and in vivo

https://doi.org/10.1016/j.freeradbiomed.2010.10.696Get rights and content

Abstract

In this study, a Cu2+ chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu2+ complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu2+ mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu2+ effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu2+-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu2+-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu2+ was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu2+ is a potent oxidative stress inducer worthy of further preclinical investigation.

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Cell lines and reagents

NSC 689534 was obtained from the Drug Synthesis and Chemistry Branch of the Developmental Therapeutics Program, National Cancer Institute (Rockville, MD, USA). All cell lines were from the Division of Cancer Treatment and Diagnosis Tumor Repository (Frederick, MD, USA). Materials were from the following: BCA protein assay, Pierce (Rockford, IL, USA); nitrocellulose membranes and polyacrylamide gels, Invitrogen (Carlsbad, CA, USA); Complete protease inhibitor tablets, Roche (Mannheim, Germany).

Cytotoxicity of the thiosemicarbazone NSC 689534 is differentially modulated by copper and iron

NSC 689534 (2-pyridinecarbaldehyde N,N-bis(2-pyridinylmethyl)thiosemicarbazone) is a member of the heterocyclic thiosemicarbazone family of compounds (for structure see Fig. 1A). This agent was first evaluated for cytotoxicity against HL60 (monocytic leukemia) and PC3 (prostate carcinoma) cell lines over the course of 3 days, using the [14C]leucine viability assay (Supplementary Fig. 1). Results showed that HL60 cells were more sensitive to NSC 689534 than PC3 cells after the first 2 days of

Discussion

Thiosemicarbazones, such as NSC 689534, are high-affinity metal-ion chelators, whose biological activity is markedly different between unconjugated and metal-bound forms [33], [34], [35]. In this study, we provide evidence that a copper chelate of NSC 689534 has properties worthy of further preclinical evaluation. Although NSC 689534 binds iron and copper, only the Cu2+ chelate has activity in the nanomolar range. Furthermore, once copper is chelated, exogenous free iron, transferrin, heme, or

Acknowledgments

We gratefully acknowledge the excellent technical assistance of Ms. Sherry Yu. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. This

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