Alteration of p53 and Bax/ Bcl-2 ratio by fotemustine and proton irradiation

Deregulation of apoptosis commonly occurs in melanoma cells and could be a reason for resistance. The effectiveness of different treatments depends on their ability to activate this process. In this study the effects of combined treatments with fotemustine (FM) and proton irradiation on the regulators of apoptosis were analyzed. Sub-confluent HTB140 human melanoma cells were treated with FM (100, 250 µM) 24 h prior to irradiation (12, 16 Gy). Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak. Flow cytometric analysis of apoptosis and the Western blot analysis of apoptotic regulators were performed 6 or 48 h after treatments. Percent of apoptotic nuclei increased after applied treatments, reaching the level of 4 to 41 %. Induction of apoptosis was associated with p53 and Bax up regulation and Bcl-2 down regulation. The obtained results imply that analyzed treatments induce apoptosis through the activation of the mitochondrial apoptotic pathway, with better pro-apoptotic effects achieved by combined treatments