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.

Targeting bortezomib-induced aggresome formation using vinorelbine enhances the cytotoxic effect along with ER stress loading in breast cancer cell lines.

The ubiquitin-proteasome and autophagy-lysosome pathways are two major self-digestive systems for cellular proteins. Ubiquitinated misfolded proteins are degraded mostly by proteasome. However, when ubiquitinated proteins accumulate beyond the capacity of proteasome clearance, they are transported to the microtubule-organizing center (MTOC) along the microtubules to form aggresomes, and subsequently some of them are degraded by the autophagy-lysosome system. We previously reported that macrolide antibiotics such as azithromycin and clarithromycin block autophagy flux, and that concomitant treatment with the proteasome inhibitor bortezomib (BZ) and macrolide enhances endoplasmic reticulum (ER) stress-mediated apoptosis in breast cancer cells. As ubiquitinated proteins are concentrated at the aggresome upon proteasome failure, we focused on the microtubule as the scaffold of this transport pathway for aggresome formation. Treatment of metastatic breast cancer cell lines (e.g., MDA-MB­231 cells) with BZ resulted in induction of aggresomes, which immunocytochemistry detected as a distinctive eyeball-shaped vimentin-positive inclusion body that formed in a perinuclear lesion, and that electron microscopy detected as a sphere of fibrous structure with some dense amorphous deposit. Vinorelbine (VNR), which inhibits microtubule polymerization, more effectively suppressed BZ-induced aggresome formation than paclitaxel (PTX), which stabilizes microtubules. Combined treatment using BZ and VNR, but not PTX, enhanced the cytotoxic effect and apoptosis induction along with pronounced ER stress loading such as upregulation of GRP78 and CHOP/GADD153. The addition of azithromycin to block autophagy flux in the BZ plus VNR-containing cell culture further enhanced the cytotoxicity. These data suggest that suppression of BZ-induced aggresome formation using an inhibitory drug such as VNR for microtubule polymerization is a novel strategy for metastatic breast cancer therapy.

Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress­mediated cell death in multiple myeloma cells.

The inhibitory effects of macrolide antibiotics including clarithromycin (CAM) on autophagy flux have been reported. Although a macrolide antibiotic exhibits no cytotoxicity, its combination with bortezomib (BZ), a proteasome inhibitor, for the simultaneous blocking of the ubiquitin (Ub)­proteasome and autophagy­lysosome pathways leads to enhanced multiple myeloma (MM) cell apoptosis induction via stress overloading of the endoplasmic reticulum (ER). As misfolded protein cargo is recruited by histone deacetylase 6 (HDAC6) to dynein motors for aggresome transport, serving to sequester misfolded proteins, we further investigated the cellular effects of targeting proteolytic pathways and aggresome formation concomitantly in MM cells. Pronounced apoptosis was induced by the combination of vorinostat [suberoylanilide hydroxamic acid (SAHA); potently inhibits HDAC6] with CAM and BZ compared with each reagent or a 2­reagent combination. CAM/BZ treatment induced vimentin positive­aggresome formation along with the accumulation of autolysosomes in the perinuclear region, whereas they were inhibited in the presence of SAHA. The SAHA/CAM/BZ combination treatment maximally upregulated genes related to ER stress including C/EBP homologous protein (CHOP). Similarly to MM cell lines, enhanced cytotoxicity with CHOP upregulation following SAHA/CAM/BZ treatment was shown by a wild­type murine embryonic fibroblast (MEF) cell line; however, a CHOP­deficient MEF cell line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ­induced cytotoxicity and CHOP induction along with the cancellation of aggresome formation. Targeting the integrated networks of aggresome, proteasome, and autophagy is suggested to induce efficient ER stress­mediated apoptosis in MM cells.

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

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 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 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.