Effects of proteosome inhibitor MG-132 in cancer cell lines

In this work, the effects of MG-132, one of the 26S proteasome inhibitors which are used as alternatives of the anti-cancer drugs nowadays, on morphologic changes of cancer cell lines and the role of Hsp70 on eIF2? phosphorylation causing inhibition of protein synthesis following proteasome inhibition are investigated. In our work, in 4T1 breast and B16F10 melanoma cancer cells treated in a time-dependent manner with 50 ?M MG-132, morphologic changes commonly observed in apoptotic cells were observed starting from 8 hour; however, such changes were not observed in control group treated with 0.1% DMSO. Cell rounding, shrinkage, apoptotic vesicules in the membrane are characteristic features of this type of cell death. In addition, in order to determine the cell death, after treating with 0.1% DMSO or 10 ?M MG-132, dead and live cells were counted using typan blue exclusion test with the help of a hemacytometer. When the survival graph is examined, significant differences in MG-132 treated 4T1 and B16F10 cells as compared to the control were obtained (p < 0.05). In order to determine whether this cytotoxic effect is due to the inhibition of the protein synthesis, we examined eIF2? phosphorylation. 4T1 breast cancer cells were lysed after treating with 0.1% DMSO or 50 µM MG132 for 6 hour. Afterwards, we examined the level of eIF2? (P) and total eIF2?, respectively from the total protein using SDS-PAGE and Western-blot techniques. No change was observed in control groups; however, the level of eIF2? (P) was increased in cells treated with 50 µM MG132. No change was observed in the level of total eIF2?. In 4T1 cells treated for a brief time with MG132, a significant increase in Hsp70 level in parallel to eIF2? phosphorylation was observed in response to proteasome inhibition. This result suggests that Hsp70 may play a role in the observed increase in eIF2? after 26S proteasomal inhibition.