The specific 26S proteasome inhibitor, bortezomib (BZ) potently induces apoptosis as well as autophagy in metastatic breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of clarithromycin (CAM) and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 μg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 μg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP (CADD153) and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wild-type murine embryonic fibroblast (MEF) cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that endoplasmic reticulum (ER)-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis.

The present study investigated issues regarding cytotoxicity tests using mouse hepatocytes in primary culture. The results indicated that the cytotoxicity of acetaminophen (APAP) markedly varied depending on culture period for up to 5 days. P450 isoforms involved in the activation of APAP, such as CYP1A2, CYP2E1, and CYP3A, showed a marked decrease in expressions of respective mRNA. Their expression levels were a few percent within 24 hr after the start of the culture, and remained low throughout. Meanwhile, the mRNA expression of enzymes involved in the detoxification of APAP, UGT1A6 and SULT1A1, declined moderately and then recovered to approximately 40% of the in vivo level later in the culture. Treatment with APAP at non-cytotoxic concentrations increased expressions of CYP1A2, CYP2E1, CYP3A11 and CYP3A41 mRNA, while it decreased that of SULT1A1 mRNA. Using the lactate dehydrogenase (LDH) assay, the cells treated with 25-mM APAP for 24-48 or 48-72 hr showed higher cytotoxicity values than those for 72-96 or 96-120 hr, but the values after 50-mM treatment were completely inverted. By employing the methylthiazolyl tetrazolium (MTT) assay, culture period-dependent cytotoxicity was also observed on 25-mM APAP treatment, but this was not clear at 50 mM. Lower LDH or higher MTT activities than the control were observed in cells treated with lower concentrations of APAP, becoming the more prominent the longer the culture period. The observations suggested that, since the expression of metabolizing enzymes alters markedly during the primary culture and the range of cytotoxic concentrations of a test compound varies depending on the culture period, it is necessary to take account of these fluctuating parameters when assessing the cytotoxic character of a compound.

Bortezomib (BZ), a selective and potent inhibitor of the 26S proteasome, has been approved for treating multiple myeloma. It was reported that BZ also inhibited breast cancer cell growth. Autophagy is a highly conserved cellular process in eukaryotes. Intracellular proteins and organelles including endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and delivered to lysosomes for degradation by lysosomal hydrolases. However, recent reports focused on that the selective degradation pathway of ubiquitinated protein through autophagy via p62 and the related protein NBR1, which are docking proteins having both a LC3-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. Thus, after binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Clarithromycin (CAM) has been reported to inhibit autophagy flux and to exhibit some cell growth inhibition in myeloma cells. Based on previous data, we attempted to investigate whether combined treatment with CAM and BZ in terms of simultaneous inhibition of two major intracellular protein degradation systems enhances ER stress-mediated cell death in breast cancer cells. BZ potently induces apoptosis as well as autophagy in breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of CAM and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 μg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 μg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wildtype MEF cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that ER-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-157. doi:1538-7445.AM2012-LB-157