Abstract 5449: Transfection of connexin 43 enhances sunitinib-induced apoptosis in malignant mesothelioma cells possibly via its interaction with Bax
Authors: Uzu Miaki , Sato Hiromi , Kashiba Tatsuro , Fujiwara Takuya , Shibata Yukihiro , Yamaura Katsunori , Hisaka Akihiro 
Published: August 2015

Introduction: Malignant mesothelioma (MM) is an aggressive tumor arising from mesothelial cells. MM is an increasing tumor worldwide with poor prognosis due to its chemo and radioresistant characteristics. We are aiming to enhance chemosensitivity of MM, and focus our attention on connexin (Cx), which is a component of intercellular channel called gap junction (GJ). Various evidences suggest that cellular physiology is maintained via GJ function. However, in cancer cells, GJ function is often aberrant, associating with the decreased Cx expression. Previously, we have shown that the sensitivity of MM cells to cisplatin was enhanced by increasing expression of Cx43, which is the major Cx subtype in mesothelial cells. Recently, it is suggested that MM is a highly angiogenic tumor and vascular endothelial growth factor (VEGF) signaling pathway plays a key role in pathogenesis of MM. Therefore, tyrosine kinase inhibitors (TKIs) are thought to be new prominent therapeutic agents for this tumor; however, they displayed a limited therapeutic activity in MM patients. Hence, the restoration of Cx expression would be beneficial to treatments with sunitinib (SU), a representative TKI, to exert an antiproliferative effect in MM patients. The present study aims to evaluate the effect of Cx43 transfection on the cytotoxicity of SU in MM cells.

Methods: We used H28, a human MM cell line, to transfect Cx43 gene. The transfected cell was named H28-T. Cell viability was assessed by MTT assay. SubG1 population (suggested apoptotic cell population) was measured by flow cytometry. Protein interaction and expression level were analyzed by western blotting after immunoprecipitation.

Results: At first, sensitivity to SU was compared between H28 and H28-T. Cell viability of H28-T was significantly lower than that of H28 in the presence of 0.38-10 μM SU. Moreover, the rate of SubG1 population was significantly higher in H28-T than in H28 in the presence of 10 μM SU. Next, the mechanism of enhanced cytotoxicity of SU was investigated using H28-T. To examine the involvement of GJ function, the combined treatment with SU and 18β-glycyrrhetinic acid, a GJ inhibitor, was performed; however, cell viability was not altered as compared to SU treatment alone. The interaction between Cx43 and Bax, a proapoptotic factor, was then investigated. It was shown that Bax was immunoprecipitated with an anti-Cx43 antibody. Protein expression of Bax was higher in H28-T than in H28. Furthermore, a cleaved (active) form of Bax was detected by treatment with 10 μM SU only in H28-T.

Conclusion: These findings indicate that Cx43 directly interacts with Bax, and this interaction may be involved in enhancement of SU-induced apoptosis in MM cells through a GJ function-independent mechanism.

Citation Format: Miaki Uzu, Hiromi Sato, Tatsuro Kashiba, Takuya Fujiwara, Yukihiro Shibata, Katsunori Yamaura, Akihiro Hisaka. Transfection of connexin 43 enhances sunitinib-induced apoptosis in malignant mesothelioma cells possibly via its interaction with Bax. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5449. doi:10.1158/1538-7445.AM2015-5449