Combined treatment with SAHA, bortezomib, and clarithromycin for concomitant targeting of aggresome formation and intracellular proteolytic pathways enhances ER stress-mediated cell death in breast cancer cells

The ubiquitin-proteasome pathway and the autophagy-lysosome pathway are two major intracellular protein degradation systems. We previously reported that clarithromycin (CAM) blocks autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in breast cancer cells, whereas treatment with CAM alone results in almost no cytotoxicity. Since HDAC6 is involved in aggresome formation, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6, with CAM and BZ in breast cancer cell lines. SAHA exhibited some cytotoxicity along with an increased acetylation level of α-tubulin, a substrate of HDAC6. Combined treatment of SAHA, CAM, and BZ potently enhanced the apoptosis-inducing effect compared with treatment using each reagent alone or a combination of two of the three. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP (GADD153), were maximally induced by the simultaneous combination of three reagents. Like breast cancer cell lines, a wild-type murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and maximally up-regulated Chop after combined treatment with SAHA, CAM, and BZ; however, a Chop knockout MEF cell line almost completely canceled this enhanced effect. The specific HDAC6 inhibitor tubacin also exhibited a pronounced cytocidal effect with a combination of CAM plus BZ. These data suggest that simultaneous targeting of intracellular proteolytic pathways and HDAC6 enhances ER-stress-mediated apoptosis in breast cancer cells.