Downregulation of thymidylate synthase and E2F1 by arsenic trioxide in mesothelioma

Abstract

Malignant pleural mesothelioma (MPM) is a global health issue. Arsenic trioxide (ATO) has been shown to suppress thymidylate synthase (TYMS) in lung adenocarcinoma and colorectal cancer, and induce apoptosis in acute promyelocytic leukemia (APL). With TYMS as a putative therapeutic target, the effect of ATO in mesothelioma was therefore studied. A panel of 5 mesothelioma cell lines was used to study the effect of ATO on cell viability, protein expression, mRNA expression and TYMS activity by MTT assay, Western blot, qPCR and tritium-release assay respectively. TYMS and E2F1 knockdown were performed with specific siRNA. Phosphatidylserine externalization and mitochondrial membrane depolarization were measured by annexin-V and JC-1 staining respectively. The in vivo effect of ATO was studied using a nude mice xenograft model. Application of ATO demonstrated anti-cancer effects in the cell line model with clinically achievable concentrations. Downregulation of TYMS protein and mRNA expression, pRB1 and E2F1 and TYMS activity were also evident. E2F1 knockdown decreased cell viability more significantly than TYMS knockdown. In general, thymidine kinase 1, ribonucleotide reductase M1, c-myc and skp2 were downregulated by ATO. p-c-Jun was downregulated in H28 cells while upregulated in 211H cells. Phosphatidylserine externalization, mitochondrial membrane depolarization, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bak and cleaved caspase-3 were displayed. In the H226 xenograft model, the relative tumor growth was aborted, and E2F1 was downregulated while cleaved caspase-3 was elevated and localized to the nucleus in the ATO treatment group. ATO has potent antiproliferative and cytotoxic effects in mesothelioma in vitro and in vivo, partially mediated through E2F1 targeting (less effect through TYMS targeting). There is sound scientific evidence to support the clinical application of ATO in treatment of mesothelioma.