Abstract 1975: Disulfiram targets hypoxia-induced cancer stem cells and reverses chemoresistance in non-small cell lung cancer
Authors: Butcher Kate , Kannappan Vinodh , Azar Karim , Kilari Rajagopal , Nkeonye Ogechi , Morris Mark R. , McConville Christopher , Wang Yaohe , Wang Weiguang 
Published: August 2018

Abstract
Background: This study aims to repurpose disulfiram (DS), a drug used to treat alcohol dependence, into an effective treatment for non-small cell lung cancer (NSCLC). NSCLC is the leading cause of cancer related death worldwide because of early metastasis and chemoresistance. Cancer stem cells (CSCs) play a key role in resistance and metastasis. Our previous studies indicate that tumor hypoxia induced activation of the nuclear factor-κB (NF-κB) pathway, a pivotal regulator of CSCs. Therefore, development of an NF-κB and CSCs targeting drug will improve NSCLC therapeutic outcomes. New drug development is an expensive and time-consuming procedure. Cytotoxicity of DS is copper (II) (Cu)-dependent. DS/Cu induces reactive oxygen species, inhibits NF-κB activity and demonstrates excellent in vitro anti-CSCs activity in a wide range of cancers. The clinical application of DS as an anticancer drug is impeded by its very short half-life in the bloodstream. To improve the drug delivery efficiency, we developed a poly lactic-co-glycolic-DS (PLGA-DS), which demonstrates strong anti-cancer efficacy in mouse NSCLC models. Methods: NSCLC cell line A549 and H23 cultured as spheroids, hypoxia and PCDNA-3.1 NF-κB p65 transfect A659 cell lines. The cytotoxic effect of DS/Cu analyzed using sphere reformation, clonogenic and MTT assays. Flow cytometric analysis to detect CSC markers ALDH and CD44, and analysis of apoptosis by Annexin V. Western blot analysis to determine expression of embryonic stem cell markers Nanog and OCT4, and identify the importance of the NF-κB pathway together with HIF and including expression of associated apoptotic proteins Bax and Bcl2. Results: CSC and hypoxic cultured cells expressed high levels of CSC markers and were resistant to first and second line NSCLC anticancer drugs (doxorubicin, oxaliplatin, paclitaxel and gemcitabine). High NF-κB expression was detected in CSCs and hypoxia-cultured NSCLC cell lines. After transfection with p65 subunit of NF-κB, A549 cells expressed CSC markers and became resistant to a wide range of anticancer drugs. DS (5-10 nM) supplemented with Cu (10 μM) induced cytotoxicity to hypoxic cultured NSCLC cells, DS (1 μM) in combination with Cu inhibited sphere reformation. DS/Cu effectively inhibited NF-κB activity, abolished the CSC population and synergistically enhanced the cytotoxicity of the above conventional anticancer drugs. Our study also shows that PLGA-DS extends the half-life of free DS in the blood stream from 30 seconds to 7 hours. Intravenous injection of PLG-DS in combination with oral Cu can effectively inhibit the growth of subcutaneous and lung NSCLC xenografts. Conclusion: DS/Cu specifically inhibits NF-κB pathway and targets CSC in NSCLC cell lines. PLGA encapsulation improves delivery of DS which demonstrated very strong anticancer activity in NSCLC xenografts in vivo.
Citation Format: Kate Butcher, Vinodh Kannappan, Karim Azar, Rajagopal Kilari, Ogechi Nkeonye, Mark R. Morris, Christopher McConville, Yaohe Wang, Weiguang Wang. Disulfiram targets hypoxia-induced cancer stem cells and reverses chemoresistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1975.