Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells

The specific 26S proteasome inhibitor, bortezomib (BZ) potently inducesapoptosis as well as autophagy in metastatic breast cancer cell lines such asMDA-MB-231 and MDA-MB-468. The combined treatment of clarithromycin (CAM) andBZ significantly enhances cytotoxicity in these cell lines. Although treatmentwith up to 100 µg/ml CAM alone had little effect on cell growth inhibition, theaccumulation of autophagosomes and p62 was observed after treatment with 25 µg/mlCAM. This result indicated that CAM blocked autophagy flux. However, the combinedtreatment of BZ and CAM resulted in more pronounced autophagy induction, as assessedby increased expression ratios of LC3B-II to LC3B-I and clearance of intracellularp62, than treatment with BZ alone. This combination further enhanced inductionof the pro-apoptotic transcription factor CHOP (CADD153) and the chaperone proteinGRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZin MDA-MB-231 cells. A wild-type murine embryonic fibroblast (MEF) cell line alsoexhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas aChop knockout MEF cell line completely abolished this enhancement and exhibitedresistance to BZ treatment. These data suggest that endoplasmic reticulum (ER)-stressmediated CHOP induction is involved in pronounced cytotoxicity by combining thesereagents. Simultaneously targeting two major intracellular protein degradationpathways such as the ubiquitin-proteasome system by BZ and the autophagy-lysosomepathway by CAM may improve the therapeutic outcome in breast cancer patients viaER-stress mediated apoptosis.