Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells

Abstract

The specific 26S proteasome inhibitor bortezomib (BZ) potently induces autophagy, endoplasmic reticulum (ER) stress and apoptosis in multiple myeloma (MM) cell lines (U266, IM-9 and RPMI8226). The macrolide antibiotics including concanamycin A, erythromycin (EM), clarithromycin (CAM) and azithromycin (AZM) all blocked autophagy flux, as assessed by intracellular accumulation of LC3B-II and p62. Combined treatment of BZ and CAM or AZM enhanced cytotoxicity in MM cell lines, although treatment with either CAM or AZM alone exhibited almost no cytotoxicity. This combination also substantially enhanced aggresome formation, intracellular ubiquitinated proteins and induced the proapoptotic transcription factor CHOP (CADD153). Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (BIM, BAX, DR5 and TRB3) were all enhanced by combined treatment with BZ plus CAM, compared with treatment with each reagent alone. Like the MM cell lines, the CHOP+/+ murine embryonic fibroblast (MEF) cell line exhibited enhanced cytotoxicity and upregulation of CHOP and its transcriptional targets with a combination of BZ and one of the macrolides. In contrast, CHOP−/− MEF cells exhibited resistance against BZ and almost completely canceled enhanced cytotoxicity with a combination of BZ and a macrolide. These data suggest that ER stress-mediated CHOP induction is involved in pronounced cytotoxicity. Simultaneously targeting two major intracellular protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances ER stress-mediated apoptosis in MM cells. This result suggests the therapeutic possibility of using a macrolide antibiotic with a proteasome inhibitor for MM therapy.

Clarithromycin Synergistically Enhances Thalidomide Cytotoxicity in Myeloma Cells

“…Data from Nakamura et al [13 ]indicated that 18 h of CAM (50 μg/ml) incubation increased the apoptosis of 12PE cells. However, Moriya et al [20 ]demonstrated that 48 h of CAM (50 μg/ml) treatment did not affect the apoptosis of IM-9 cells, similar to our data. The differences between studies might be due to the use of 3 different myeloma cell lines.…”

“…The differences between studies might be due to the use of 3 different myeloma cell lines. Moriya et al [20] also found that CAM enhanced the effect of bortezomib on inducing apoptosis. However, we failed to demonstrate the synergistic effect of CAM and thalidomide.…”

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

“…The proteostat aggresome detection assay (Enzo Life Sciences) was used [38] to determine whether ESE-16 causes aggresome formation within the SNO cells as an indication of autophagic cell death [39][40][41]. The assay was developed to detect aggregated proteins by means of a fluorescent molecular rotor dye.…”

Endoplasmic reticulum stress and autophagy participate in apoptosis induced by bortezomib in cervical cancer cells

“…Increasing evidence demonstrates that, in addition to mitochondrial apoptosis, anti-tumor agents can induce apoptosis by up-regulating ER stress-associated apoptosis protein expression, such as CHOP (Zhong et al 2012). Bortezomib can induce ERassociated apoptotic cell death in myeloma cells (Moriya et al 2013). To investigate the relationship between ER stress and apoptosis induced by bortezomib in human cervical carcinoma, we detected the expression of ER stress-related proteins in human cervical carcinoma cells treated with bortezomib.…”