PLGA-DS reverses chemoresistance in malignant mesothelioma by targeting hypoxia induced cancer stem cells

A thesis submitted in fulfilment of the requirement of the University of Wolverhampton for the degree Master of Philosophy.Background: Malignant Mesothelioma (MM) is a malignancy related to asbestos exposure which causes a wide variety of molecular aberrations. MM has a very dismal treatment outcome with an overall survival of fewer than 12 months, with less than five drugs available for its treatment. MM recurrence is unavoidable due to chemoresistance. Long-term inflammation triggered by asbestos activates a key transcription factor, nuclear factor-��B (NF-��B), which is further upregulated in cancer stem cells (CSCs) by hypoxia. Both hypoxia and NF-��B pathway plays a pivotal role in the maintenance of stemness in hypoxia-induced CSCs leading to upregulation of anti-apoptotic signalling, chemo-radiation resistance and metastasis. Therefore, the development of drugs targeting hypoxia-NF-��B-CSCs axis is of clinical significance for MM treatment. Our previous studies have shown that Disulfiram (DS), a clinically used anti-alcoholism drug, in combination with Copper (II) (Cu) has substantial toxicity in CSCs in a wide range of cancer types. The clinical application of DS in Cancer is limited by its very short half-life (< 2 minutes) in the bloodstream. MM is Cancer which mainly infiltrates local organs and tissues with rare distant metastasis. Considering this growing feature of MM, we developed a biodegradable and controlled released poly (lactic-co-glycolic acid) microparticle-encapsulate disulfiram (PLGA-DS) for local treatment of MM. This study aims to examine the anti-MM effect of PLGA-DS and elucidate its molecular mechanisms. Methodologies: In order to determine drug sensitivity, stemness, apoptosis, invasiveness and NF��B status, the following methodologies were performed in this study: MTT cytotoxicity assay, flow cytometry, analysis of CSC markers, hypoxic cell cultures, western blot, stable transfection of MM cell line with NF��B, CRISPR-Cas9 knock out of NF-kB-p65, CSC sphere reformation, invasion and migration assay. Results and conclusions: Two MM cell lines were examined and cultured in a hypoxic environment. MM cell lines were highly resistant to Pemetrexed (PMT) and Cisplatin (CIS), the first-line chemotherapeutic agents for MM. Hypoxia cultured MM cells showed high NF-��B activity and CSC markers and manifested strong migration/invasion ability. The NF-��B-p65 over expressed transfected cell lines did not demonstrate CSC traits along with no increase in resistance to first line drugs. PLGA-DS/Cu completely abolished CSC population in a culture which is demonstrated by sphere reformation assays and flow cytometry analysis of CSC markers such as CD24, CD133 and ABCG2. PLGA-DS/Cu also inhibited the hypoxiainduced NF-kB expression and blocked the migration and invasion ability of MM cells. It showed substantial toxicity to MM cell lines and reversed hypoxia-induced chemoresistance. Also, PLGA-DS/Cu potentiated the cytotoxic effect of Cisplatin/Pemetrexed in vitro. Isobologram analysis indicates moderate synergistic effect between PLGA-DS and cisplatin and pemetrexed in MSTO 211 and JU77 cell lines, respectively. As an FDA approved a drug with all preclinical safety data available, further studies may quickly translate it into MM clinical treatment. This is very promising in vitro data and indicate that PLGA-DS could be a promising formulation for localised MM treatment