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Keisuke Miyazawa - One of the best experts on this subject based on the ideXlab platform.
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Concomitant Targeting Aggresome Formation and IntraCellar Proteolytic Pathways Enhances ER-Stress Mediated Cell Death In Myeloma Cells
Blood, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Keisuke MiyazawaAbstract:ER-stress is caused by an imbalance between the amount of unfolded or misfolded protein in the ER lumen and the capacity of the ER machinery to refold these proteins. This stress induces a coordinated Cellular response known as unfolded protein response (UPR). The main functions of UPR are to reduce the amount of protein that enters the ER by suppressing the translational rate and to increase the folding capacity of the ER via translational activation of chaperon proteins. Additionally, if proteins cannot be folded correctly in the ER, they are retrotranslocated to the cytoplasm for degradation via the ubiquitin-proteasome pathway for adaptation. However, if these adaptation strategies fail, apoptosis is triggered with the induction of the pro-apoptotic transcription factor CHOP/GADD153 and with the IRE1 involved in signaling via caspase-12. Autophagy is a highly conserved Cellular process in eukaryotes. IntraCellular proteins and organelles, including ER, are engulfed in a double-membrane vesicle known as an autophagosome, and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, evidence indicates a selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1. Thus, the two major intraCellular protein degradation systems are directly linked. We previously reported that macrolide antibiotics including clarithromycin (CAM) block autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in myeloma and breast cancer Cells, whereas treatment with CAM alone results in almost no cytotoxicity (Moriya S, et al . Int J Oncol. 2013, Komatsu S, et al . Biochem Biophys Res Commun. 2013). HDAC6, a microtubule-associated deacetylase, is a component of the aggresome and has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors. HDAC6 recruits misfolded protein cargo to dynein motors for transport to aggresomes, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy. Therefore, evidence suggests the existence of an elaborate intraCellular network system for processing unfolded proteins. In the present study, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6 (IC 50 37 nM), with CAM and BZ in myeloma 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. Concomitant treatment with CAM and BZ for blocking autophagy flux and proteasome activity dramatically enhanced aggresome formation in perinuclear region, whereas this aggresome formation was almost completely eliminated in the presence of SAHA. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP, were maximally induced by the simultaneous combination of three reagents. Like myeloma 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 KO 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 myeloma Cells, and also provide novel insight into “ER-stress loading therapy” for myeloma patients. Disclosures: Miyazawa: Taisho Toyama Phamaceutical Co. Ltd.: Research Funding.
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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.
Biochemical and Biophysical Research Communications, 2013Co-Authors: Seiichiro Komatsu, Shota Moriya, Norio Kohno, Tomohisa Yokoyama, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
International Journal of Oncology, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
Blood, 2012Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract:Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved Cellular process of self-degradation in eukaryotes. IntraCellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intraCellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) Cell Lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM Cell Lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), 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. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. 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 up-regulation 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. Disclosures: No relevant conflicts of interest to declare.
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Abstract LB-157: Combined treatment with clarithromycin and bortezomib enhances cytotoxicity via endplasmic reticulum stress-meidated CHOP induction and autophagy in breast cancer Cells
Cancer Research, 2012Co-Authors: Seiichiro Komatsu, Shota Moriya, Masato Inazu, Keisuke Miyazawa, Akiko Takase, Munekazu Naito, Norio Kohno, Masahiro Itoh, Akio TomodaAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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
Shota Moriya - One of the best experts on this subject based on the ideXlab platform.
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Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines.
PLOS ONE, 2016Co-Authors: Kazuhiro Hirasawa, Shota Moriya, Ayako Hirota, Masato Inazu, Masaki Hiramoto, Kana Miyahara, Hiromi Kazama, Jun Takemura, Kiyoaki TsukaharaAbstract:Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for Cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer Cells require a larger metabolic demand than normal Cells because of their exuberant growth, upregulated autophagy in tumor Cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer Cell Lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced Cell death under the amino acid-depleted (AAD) culture condition in these Cell Lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 Cells significantly suppressed macrolide-induced Cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) Cell Lines also attenuated AZM-induced Cell death compared with CHOP+/+ MEF Cell Lines. Using a tet-off atg5 MEF Cell Line, knockout of atg5, an essential gene for autophagy, also induced Cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced Cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely canCelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for "tumor-starving therapy".
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Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress‑mediated Cell death in multiple myeloma Cells.
International Journal of Oncology, 2014Co-Authors: Shota Moriya, Seiichiro Komatsu, Kaho Yamasaki, Yusuke Kawai, Hiroko Kokuba, Ayako Hirota, Masato Inazu, Akihiko Gotoh, Masaki HiramotoAbstract: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 autophagy-lysosome 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 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome 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 wild-type murine embryonic fibroblast (MEF) Cell Line; however, a CHOP-deficient MEF Cell Line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced 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 stress-mediated apoptosis in MM Cells.
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Concomitant Targeting Aggresome Formation and IntraCellar Proteolytic Pathways Enhances ER-Stress Mediated Cell Death In Myeloma Cells
Blood, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Keisuke MiyazawaAbstract:ER-stress is caused by an imbalance between the amount of unfolded or misfolded protein in the ER lumen and the capacity of the ER machinery to refold these proteins. This stress induces a coordinated Cellular response known as unfolded protein response (UPR). The main functions of UPR are to reduce the amount of protein that enters the ER by suppressing the translational rate and to increase the folding capacity of the ER via translational activation of chaperon proteins. Additionally, if proteins cannot be folded correctly in the ER, they are retrotranslocated to the cytoplasm for degradation via the ubiquitin-proteasome pathway for adaptation. However, if these adaptation strategies fail, apoptosis is triggered with the induction of the pro-apoptotic transcription factor CHOP/GADD153 and with the IRE1 involved in signaling via caspase-12. Autophagy is a highly conserved Cellular process in eukaryotes. IntraCellular proteins and organelles, including ER, are engulfed in a double-membrane vesicle known as an autophagosome, and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, evidence indicates a selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1. Thus, the two major intraCellular protein degradation systems are directly linked. We previously reported that macrolide antibiotics including clarithromycin (CAM) block autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in myeloma and breast cancer Cells, whereas treatment with CAM alone results in almost no cytotoxicity (Moriya S, et al . Int J Oncol. 2013, Komatsu S, et al . Biochem Biophys Res Commun. 2013). HDAC6, a microtubule-associated deacetylase, is a component of the aggresome and has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors. HDAC6 recruits misfolded protein cargo to dynein motors for transport to aggresomes, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy. Therefore, evidence suggests the existence of an elaborate intraCellular network system for processing unfolded proteins. In the present study, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6 (IC 50 37 nM), with CAM and BZ in myeloma 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. Concomitant treatment with CAM and BZ for blocking autophagy flux and proteasome activity dramatically enhanced aggresome formation in perinuclear region, whereas this aggresome formation was almost completely eliminated in the presence of SAHA. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP, were maximally induced by the simultaneous combination of three reagents. Like myeloma 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 KO 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 myeloma Cells, and also provide novel insight into “ER-stress loading therapy” for myeloma patients. Disclosures: Miyazawa: Taisho Toyama Phamaceutical Co. Ltd.: Research Funding.
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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.
Biochemical and Biophysical Research Communications, 2013Co-Authors: Seiichiro Komatsu, Shota Moriya, Norio Kohno, Tomohisa Yokoyama, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
International Journal of Oncology, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract: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.
Seiichiro Komatsu - One of the best experts on this subject based on the ideXlab platform.
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Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress‑mediated Cell death in multiple myeloma Cells.
International Journal of Oncology, 2014Co-Authors: Shota Moriya, Seiichiro Komatsu, Kaho Yamasaki, Yusuke Kawai, Hiroko Kokuba, Ayako Hirota, Masato Inazu, Akihiko Gotoh, Masaki HiramotoAbstract: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 autophagy-lysosome 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 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome 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 wild-type murine embryonic fibroblast (MEF) Cell Line; however, a CHOP-deficient MEF Cell Line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced 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 stress-mediated apoptosis in MM Cells.
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Concomitant Targeting Aggresome Formation and IntraCellar Proteolytic Pathways Enhances ER-Stress Mediated Cell Death In Myeloma Cells
Blood, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Keisuke MiyazawaAbstract:ER-stress is caused by an imbalance between the amount of unfolded or misfolded protein in the ER lumen and the capacity of the ER machinery to refold these proteins. This stress induces a coordinated Cellular response known as unfolded protein response (UPR). The main functions of UPR are to reduce the amount of protein that enters the ER by suppressing the translational rate and to increase the folding capacity of the ER via translational activation of chaperon proteins. Additionally, if proteins cannot be folded correctly in the ER, they are retrotranslocated to the cytoplasm for degradation via the ubiquitin-proteasome pathway for adaptation. However, if these adaptation strategies fail, apoptosis is triggered with the induction of the pro-apoptotic transcription factor CHOP/GADD153 and with the IRE1 involved in signaling via caspase-12. Autophagy is a highly conserved Cellular process in eukaryotes. IntraCellular proteins and organelles, including ER, are engulfed in a double-membrane vesicle known as an autophagosome, and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, evidence indicates a selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1. Thus, the two major intraCellular protein degradation systems are directly linked. We previously reported that macrolide antibiotics including clarithromycin (CAM) block autophagy flux, and that combined treatment with CAM and proteasome inhibitor bortezomib (BZ) enhances ER-stress-mediated apoptosis in myeloma and breast cancer Cells, whereas treatment with CAM alone results in almost no cytotoxicity (Moriya S, et al . Int J Oncol. 2013, Komatsu S, et al . Biochem Biophys Res Commun. 2013). HDAC6, a microtubule-associated deacetylase, is a component of the aggresome and has the capacity to bind both polyubiquitinated misfolded proteins and dynein motors. HDAC6 recruits misfolded protein cargo to dynein motors for transport to aggresomes, which is recognized as a cytoprotective response serving to sequester misfolded proteins and facilitate their clearance by autophagy. Therefore, evidence suggests the existence of an elaborate intraCellular network system for processing unfolded proteins. In the present study, we further investigated the combined effect of vorinostat (suberoylanilide hydroxamic acid (SAHA)), which has a potent inhibitory effect for HDAC6 (IC 50 37 nM), with CAM and BZ in myeloma 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. Concomitant treatment with CAM and BZ for blocking autophagy flux and proteasome activity dramatically enhanced aggresome formation in perinuclear region, whereas this aggresome formation was almost completely eliminated in the presence of SAHA. Expression levels of ER-stress-related genes, including the pro-apoptotic transcription factor CHOP, were maximally induced by the simultaneous combination of three reagents. Like myeloma 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 KO 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 myeloma Cells, and also provide novel insight into “ER-stress loading therapy” for myeloma patients. Disclosures: Miyazawa: Taisho Toyama Phamaceutical Co. Ltd.: Research Funding.
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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.
Biochemical and Biophysical Research Communications, 2013Co-Authors: Seiichiro Komatsu, Shota Moriya, Norio Kohno, Tomohisa Yokoyama, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
International Journal of Oncology, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
Blood, 2012Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract:Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved Cellular process of self-degradation in eukaryotes. IntraCellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intraCellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) Cell Lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM Cell Lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), 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. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. 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 up-regulation 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. Disclosures: No relevant conflicts of interest to declare.
Masato Inazu - One of the best experts on this subject based on the ideXlab platform.
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Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines.
PLOS ONE, 2016Co-Authors: Kazuhiro Hirasawa, Shota Moriya, Ayako Hirota, Masato Inazu, Masaki Hiramoto, Kana Miyahara, Hiromi Kazama, Jun Takemura, Kiyoaki TsukaharaAbstract:Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for Cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer Cells require a larger metabolic demand than normal Cells because of their exuberant growth, upregulated autophagy in tumor Cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer Cell Lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced Cell death under the amino acid-depleted (AAD) culture condition in these Cell Lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 Cells significantly suppressed macrolide-induced Cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) Cell Lines also attenuated AZM-induced Cell death compared with CHOP+/+ MEF Cell Lines. Using a tet-off atg5 MEF Cell Line, knockout of atg5, an essential gene for autophagy, also induced Cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced Cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely canCelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for "tumor-starving therapy".
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Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress‑mediated Cell death in multiple myeloma Cells.
International Journal of Oncology, 2014Co-Authors: Shota Moriya, Seiichiro Komatsu, Kaho Yamasaki, Yusuke Kawai, Hiroko Kokuba, Ayako Hirota, Masato Inazu, Akihiko Gotoh, Masaki HiramotoAbstract: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 autophagy-lysosome 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 2-reagent combination. CAM/BZ treatment induced vimentin positive-aggresome 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 wild-type murine embryonic fibroblast (MEF) Cell Line; however, a CHOP-deficient MEF Cell Line almost completely canceled this pronounced cytotoxicity. Knockdown of HDAC6 with siRNA exhibited further enhanced CAM/BZ-induced 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 stress-mediated apoptosis in MM Cells.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
International Journal of Oncology, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
Blood, 2012Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract:Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved Cellular process of self-degradation in eukaryotes. IntraCellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intraCellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) Cell Lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM Cell Lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), 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. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. 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 up-regulation 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. Disclosures: No relevant conflicts of interest to declare.
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Abstract LB-157: Combined treatment with clarithromycin and bortezomib enhances cytotoxicity via endplasmic reticulum stress-meidated CHOP induction and autophagy in breast cancer Cells
Cancer Research, 2012Co-Authors: Seiichiro Komatsu, Shota Moriya, Masato Inazu, Keisuke Miyazawa, Akiko Takase, Munekazu Naito, Norio Kohno, Masahiro Itoh, Akio TomodaAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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
Akio Tomoda - One of the best experts on this subject based on the ideXlab platform.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
International Journal of Oncology, 2013Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract: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.
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macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress mediated chop induction in myeloma Cells
Blood, 2012Co-Authors: Shota Moriya, Seiichiro Komatsu, Masato Inazu, Akihiko Gotoh, Akio Tomoda, Tomohiro Kawaguchi, Keisuke MiyazawaAbstract:Abstract 4992 Macroautophagy (hereafter, “autophagy”) is a highly conserved Cellular process of self-degradation in eukaryotes. IntraCellular proteins and organelles including the endoplasmic reticulum (ER) are engulfed in a double-membrane vesicle called an autophagosome and are delivered to lysosomes for degradation by lysosomal hydrolases. Autophagy has been regarded as a bulk non-selective degradation system for long-lived proteins and organelles, in contrast to the specific degradation of polyubiquitinated short-lived proteins by proteasome. However, recent reports revealed the selective degradation pathway of ubiquitinated protein through autophagy via docking proteins such as p62 and the related protein NBR1, having both a microtubule-associated protein 1 light chain 3 (LC3)-interacting region and a ubiquitin-associated domain. LC3 is essential for autophagy and is associated with autophagosome membranes after processing. By binding ubiquitin via their C-terminal ubiquitin-associated domains, p62-mediated degradation of ubiquitinated cargo occurs by selective autophagy. Thus the two major intraCellular degradation systems are directly linked. We have reported on the inhibition of autophagy using the autophagy inhibitor bafilomycin A1enhanced bortezomib (BZ)-induced apoptosis by burdening ER stress in multiple myeloma (MM) Cell Lines. It was also reported that clarithromycin (CAM) attenuated or blocked autophagy flux, probably mediated through inhibiting the lysosomal function. We therefore investigated whether simultaneous inhibition of protein degradation systems such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome system by a macrolide antibiotic enhances the loading of ER-stress and ER–stress-mediated CHOP (CADD153) induction, followed by transcriptional activation for proapoptotic genes. BZ potently induces autophagy, ER–stress, and apoptosis in MM Cell Lines (e. g. U266, IM-9, and RPMI8226). The macrolide antibiotics including CAM, concanamycin A, erythromycin (EM), 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. Expression levels of the proapoptotic genes transcriptionally regulated by CHOP (e. g. 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 up-regulation 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. Disclosures: No relevant conflicts of interest to declare.
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Abstract LB-157: Combined treatment with clarithromycin and bortezomib enhances cytotoxicity via endplasmic reticulum stress-meidated CHOP induction and autophagy in breast cancer Cells
Cancer Research, 2012Co-Authors: Seiichiro Komatsu, Shota Moriya, Masato Inazu, Keisuke Miyazawa, Akiko Takase, Munekazu Naito, Norio Kohno, Masahiro Itoh, Akio TomodaAbstract:Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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
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Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer Cells
International Journal of Oncology, 2011Co-Authors: Seiichiro Komatsu, Shota Moriya, Masato Inazu, Keisuke Miyazawa, Akiko Takase, Munekazu Naito, Norio Kohno, Masahiro Itoh, Akio TomodaAbstract: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.