Subtilisin from Bacillus amyloliquefaciens induces apoptosis in breast cancer cells through ubiquitin-proteasome-mediated tubulin degradation

To search for novel proteases from environmental isolates which can induce apoptosis in cancer cells, we have purified subtilisin from Bacillus amyloliquefaciens and studied its anti-cancer properties. Subtilisin induced apoptosis in colon (HT29) and breast (MCF7) cancer cells but showed no effect on mouse peritoneal macrophages and normal breast cells (MCF10A). Western blot analysis showed that Bax, Bcl-2 level remained unchanged but tubulin level decreased significantly. Subtilisin does not induce the intrinsic pathway of apoptosis, rather it induced tubulin degradation in MCF-7 cells, whereas in normal cells (MCF-10A) tubulin degradation was not observed. Subtilisin activates ubiquitination and proteasomal-mediated tubulin degradation which was completely restored in presence of proteasome inhibitor MG-132. We further observed PARKIN, one of the known E3-ligase, is overexpressed and interacts with tubulin in subtilisin treated cells. Knockdown of PARKIN effectively downregulates ubiquitination and inhibits degradation of tubulin. PARKIN activation and tubulin degradation lead to ER-stress which in turn activates caspase-7 and PARP cleavage, thus guiding the subtilisin treated cells towards apoptosis. To our knowledge this is the first report of subtilisin induced apoptosis in cancer cells by proteasomal degradation of tubulin.