An investigation of the apoptotic effects of proteasome inhibition in 4T1 breast and B16F10 melanoma cells

It has been releaved that proteasome inhibition affects several cellular functions and induces apoptosis. In this thesis study, the apoptotic effects of proteasome inhibition on 4T1 breast cancer and B16F10 melanoma cells has been investigated. In our study, B16F10 cells treated with different bortezomib concentrations (10 nM, 100 nM, 500 nM, 1 µM) for 48 hours have showed morphological changes such as detaching from petri dish, rounding, shrinkage, seperating from adjacent cells and reflecting more light, which are commonly observed in apoptotic cells. In addition, DNA fragmentation, an important indicator of apoptosis, was observed. In 4T1 cells, apoptotic changes were observed with higher concentrations (100 nM, 500 nM, 1 µM) as compared to B16F10 cells. Furthermore, while apoptotic changes were similary observed in B16F10 cells treated with 0.5 µM MG132 for 48 h, no significant changes were observed in 4T1 cells treated with 0.5 µM MG132 for 48 h. Also, when p53 protein level was investigated with Western blot in 10 µM bortezomib treated B16F10 and 4T1 cells for 6 h, an increase in p53 was observed only in B16F10 cells, but not in 4T1 cells. These results show that B16F10 cells are more sensitive to the proteasome inhibition as compared to 4T1 cells, and in this sensitivity p53 protein plays a significant role. In fallow-up experiments, a significant increase in caspase-3 enzyme activity observed in both 4T1 and B16F10 cells; however, this result was not confirmed by Western blot analyzes. Western blot experiments did not show an increase caspase-3 activity in response to proteasome inhibiton. These results show that the increase in caspase-3 activity observed in the activity experiments may thus result from other caspases (e.g., caspase-7). In addition, Western blot analyzes showed that ubiquitin-protein conjugates are increased in bortezomib-treated 4T1 and B16F10 cells. The results obtained indicated that proteasome inhibition is effective in triggering apoptosis and may be used as an alternative in the cancer therapy.