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Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells.

Yoon MJ, Lee AR, Jeong SA, Kim YS, Kim JY, Kwon YJ, Choi KS - Oncotarget (2014)

Bottom Line: Celastrol treatment markedly increased mitochondrial Ca2+ levels and induced ER stress via proteasome inhibition in these cells.Inhibition of the IP3 receptor (IP3R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca2+ accumulation and subsequent paraptotic events.Collectively, our results show that the IP3R-mediated release of Ca2+ from the ER and its subsequent MCU-mediatedinflux into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Department of Biomedical Sciences, Ajou University School of Medicine, Suwon , Korea. These authors contributed equally to this work. .

ABSTRACT
Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine", is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca2+ levels and induced ER stress via proteasome inhibition in these cells. Both MCU (mitochondrial Ca2+ uniporter) knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca2+ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP3 receptor (IP3R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca2+ accumulation and subsequent paraptotic events. Collectively, our results show that the IP3R-mediated release of Ca2+ from the ER and its subsequent MCU-mediatedinflux into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.

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IP3R-mediated Ca2+ release from the ER is critical for celastrol-induced paraptosis(A) MDA-MB 435S cells were pretreated with the indicated concentrations of extracellular Ca2+ chelators (EGTA and BAPTA), 2-APB, and dantrolene for 30 min and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (B) YFP-Mito cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 2 h. Cells were stained with Rhod-2 and processed for the phase contrast and fluorescence microscopy. (C) YFP-Mito and YFP-ER cells were pretreated with 20 μM 2-APB, further treated with 2 μM celastrol for 3 h, and observed under the phase contrast and fluorescence microscope. (D) MDA-MB 435S cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 24 h followed by Western blotting. β-actin was used as a loading control in Western blots. The relative phosphorylation levels of the respective MAP kinase were determined by the fold changes of densitometric values in treated groups to the values in the control group. Densitometric values for the phospho-proteins of interest were normalized for protein loading with their total proteins. The relative expression levels of CHOP and ubiquitin were determined using densitometric analysis compared to untreated control. (E) MDA-MB 435S cells were pretreated with the indicated concentrations of adenophostin A and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (F) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points (left), 2 μM MG132 or 40 nM bortezomib for 24 h (right) and Western blotting of IP3R, MCU, and β-actin was performed. Compared to control (untreated cells), the fold change of protein levels was determined by a densitometric analysis.
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Figure 9: IP3R-mediated Ca2+ release from the ER is critical for celastrol-induced paraptosis(A) MDA-MB 435S cells were pretreated with the indicated concentrations of extracellular Ca2+ chelators (EGTA and BAPTA), 2-APB, and dantrolene for 30 min and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (B) YFP-Mito cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 2 h. Cells were stained with Rhod-2 and processed for the phase contrast and fluorescence microscopy. (C) YFP-Mito and YFP-ER cells were pretreated with 20 μM 2-APB, further treated with 2 μM celastrol for 3 h, and observed under the phase contrast and fluorescence microscope. (D) MDA-MB 435S cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 24 h followed by Western blotting. β-actin was used as a loading control in Western blots. The relative phosphorylation levels of the respective MAP kinase were determined by the fold changes of densitometric values in treated groups to the values in the control group. Densitometric values for the phospho-proteins of interest were normalized for protein loading with their total proteins. The relative expression levels of CHOP and ubiquitin were determined using densitometric analysis compared to untreated control. (E) MDA-MB 435S cells were pretreated with the indicated concentrations of adenophostin A and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (F) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points (left), 2 μM MG132 or 40 nM bortezomib for 24 h (right) and Western blotting of IP3R, MCU, and β-actin was performed. Compared to control (untreated cells), the fold change of protein levels was determined by a densitometric analysis.

Mentions: Next, we assessed the contribution of extracellular Ca2+ and intracellular Ca2+ stores to celastrol-induced mitochondrial Ca2+ accumulation. Since pretreatment with extracellular Ca2+ chelators, including EGTA or BAPTA, did not appear to alter celastrol-induced cell death (Figure 9A), we tested whether Ca2+ release from the ER contributed to celastrol-induced paraptosis. Experiments using specific inhibitors of two major Ca2+ release receptors in the ER, the IP3 receptor (IP3R) and the ryanodine receptor (RyR) [36], showed that celastrol-induced cell death was very effectively inhibited by 2-APB, a specific inhibitor of IP3R [37], but not by dantrolene, a specific inhibitor of the RyR [38] (Figure 9A). Staining of YFP-Mito cells with Rhod-2 also showed that 2-APB markedly reduced the celastrol-induced increase in [Ca2+]m (Figure 9B). Furthermore, 2-APB effectively inhibits celastrol-induced dilation of mitochondria and the ER in YFP-Mito and YFP-ER cells, respectively (Figure 9C). These results suggest that pretreatment with 2-APB effectively inhibits the celastrol-induced cell death by inhibiting the IP3R-mediated release of Ca2+ from the ER and the subsequent MCU-mediated influx of Ca2+ into mitochondria. In addition, 2-APB pretreatment markedly inhibited celastrol-induced the accumulations of poly-ubiquitinated proteins, CHOP, activated ERK, and activated JNK (Figure 9D). The importance of IP3R-mediated Ca2+ release from the ER in celastrol-induced paraptosis was further tested using adenophostin A, an agonist of IP3R [39]. We found that co-treatment of MDA-MB 435S cells with adenophostin A dose-dependently enhanced cell death, when combined with 2 μM celastrol (Figure 9E). Fluorescence microscopy using Rhod-2 showed that co-treatment with 10 μM adenophostin A accelerated and enhanced celastrol-induced increase in [Ca2+]m in cells treated with 2 μM celastrol (Supplementary Figure 4). Notably, the levels of IP3R and MCU protein increased following celastrol treatment (Figure 9F) and similar to celastrol, MG132 or bortezomib also increased IP3R and MCU protein levels in MDA-MB 435S cells (Figure 9F). These results suggest that the upregulation of IP3R and MCU as a consequence of celastrol-induced proteasome inhibition may contribute to the release of Ca2+ from the ER, Ca2+ influx into mitochondria, and subsequent paraptotic events. Finally, we found that pretreatment with RR or 2-APB significantly and dose-dependently inhibited celastrol-induced cell death also in MCF-7, DLD-1, and RKO cells (Figure 10A). In sum, we herein show for the first time that celastrol-induced paraptotic cell death in cancer cells is triggered by IP3R-mediated Ca2+ release from the ER and uniporter-mediated mitochondrial Ca2+ influx which collectively trigger dilation of mitochondria and the ER (Figure 10B).


Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells.

Yoon MJ, Lee AR, Jeong SA, Kim YS, Kim JY, Kwon YJ, Choi KS - Oncotarget (2014)

IP3R-mediated Ca2+ release from the ER is critical for celastrol-induced paraptosis(A) MDA-MB 435S cells were pretreated with the indicated concentrations of extracellular Ca2+ chelators (EGTA and BAPTA), 2-APB, and dantrolene for 30 min and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (B) YFP-Mito cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 2 h. Cells were stained with Rhod-2 and processed for the phase contrast and fluorescence microscopy. (C) YFP-Mito and YFP-ER cells were pretreated with 20 μM 2-APB, further treated with 2 μM celastrol for 3 h, and observed under the phase contrast and fluorescence microscope. (D) MDA-MB 435S cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 24 h followed by Western blotting. β-actin was used as a loading control in Western blots. The relative phosphorylation levels of the respective MAP kinase were determined by the fold changes of densitometric values in treated groups to the values in the control group. Densitometric values for the phospho-proteins of interest were normalized for protein loading with their total proteins. The relative expression levels of CHOP and ubiquitin were determined using densitometric analysis compared to untreated control. (E) MDA-MB 435S cells were pretreated with the indicated concentrations of adenophostin A and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (F) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points (left), 2 μM MG132 or 40 nM bortezomib for 24 h (right) and Western blotting of IP3R, MCU, and β-actin was performed. Compared to control (untreated cells), the fold change of protein levels was determined by a densitometric analysis.
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Figure 9: IP3R-mediated Ca2+ release from the ER is critical for celastrol-induced paraptosis(A) MDA-MB 435S cells were pretreated with the indicated concentrations of extracellular Ca2+ chelators (EGTA and BAPTA), 2-APB, and dantrolene for 30 min and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (B) YFP-Mito cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 2 h. Cells were stained with Rhod-2 and processed for the phase contrast and fluorescence microscopy. (C) YFP-Mito and YFP-ER cells were pretreated with 20 μM 2-APB, further treated with 2 μM celastrol for 3 h, and observed under the phase contrast and fluorescence microscope. (D) MDA-MB 435S cells were pretreated with 20 μM 2-APB and further treated with 2 μM celastrol for 24 h followed by Western blotting. β-actin was used as a loading control in Western blots. The relative phosphorylation levels of the respective MAP kinase were determined by the fold changes of densitometric values in treated groups to the values in the control group. Densitometric values for the phospho-proteins of interest were normalized for protein loading with their total proteins. The relative expression levels of CHOP and ubiquitin were determined using densitometric analysis compared to untreated control. (E) MDA-MB 435S cells were pretreated with the indicated concentrations of adenophostin A and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (F) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points (left), 2 μM MG132 or 40 nM bortezomib for 24 h (right) and Western blotting of IP3R, MCU, and β-actin was performed. Compared to control (untreated cells), the fold change of protein levels was determined by a densitometric analysis.
Mentions: Next, we assessed the contribution of extracellular Ca2+ and intracellular Ca2+ stores to celastrol-induced mitochondrial Ca2+ accumulation. Since pretreatment with extracellular Ca2+ chelators, including EGTA or BAPTA, did not appear to alter celastrol-induced cell death (Figure 9A), we tested whether Ca2+ release from the ER contributed to celastrol-induced paraptosis. Experiments using specific inhibitors of two major Ca2+ release receptors in the ER, the IP3 receptor (IP3R) and the ryanodine receptor (RyR) [36], showed that celastrol-induced cell death was very effectively inhibited by 2-APB, a specific inhibitor of IP3R [37], but not by dantrolene, a specific inhibitor of the RyR [38] (Figure 9A). Staining of YFP-Mito cells with Rhod-2 also showed that 2-APB markedly reduced the celastrol-induced increase in [Ca2+]m (Figure 9B). Furthermore, 2-APB effectively inhibits celastrol-induced dilation of mitochondria and the ER in YFP-Mito and YFP-ER cells, respectively (Figure 9C). These results suggest that pretreatment with 2-APB effectively inhibits the celastrol-induced cell death by inhibiting the IP3R-mediated release of Ca2+ from the ER and the subsequent MCU-mediated influx of Ca2+ into mitochondria. In addition, 2-APB pretreatment markedly inhibited celastrol-induced the accumulations of poly-ubiquitinated proteins, CHOP, activated ERK, and activated JNK (Figure 9D). The importance of IP3R-mediated Ca2+ release from the ER in celastrol-induced paraptosis was further tested using adenophostin A, an agonist of IP3R [39]. We found that co-treatment of MDA-MB 435S cells with adenophostin A dose-dependently enhanced cell death, when combined with 2 μM celastrol (Figure 9E). Fluorescence microscopy using Rhod-2 showed that co-treatment with 10 μM adenophostin A accelerated and enhanced celastrol-induced increase in [Ca2+]m in cells treated with 2 μM celastrol (Supplementary Figure 4). Notably, the levels of IP3R and MCU protein increased following celastrol treatment (Figure 9F) and similar to celastrol, MG132 or bortezomib also increased IP3R and MCU protein levels in MDA-MB 435S cells (Figure 9F). These results suggest that the upregulation of IP3R and MCU as a consequence of celastrol-induced proteasome inhibition may contribute to the release of Ca2+ from the ER, Ca2+ influx into mitochondria, and subsequent paraptotic events. Finally, we found that pretreatment with RR or 2-APB significantly and dose-dependently inhibited celastrol-induced cell death also in MCF-7, DLD-1, and RKO cells (Figure 10A). In sum, we herein show for the first time that celastrol-induced paraptotic cell death in cancer cells is triggered by IP3R-mediated Ca2+ release from the ER and uniporter-mediated mitochondrial Ca2+ influx which collectively trigger dilation of mitochondria and the ER (Figure 10B).

Bottom Line: Celastrol treatment markedly increased mitochondrial Ca2+ levels and induced ER stress via proteasome inhibition in these cells.Inhibition of the IP3 receptor (IP3R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca2+ accumulation and subsequent paraptotic events.Collectively, our results show that the IP3R-mediated release of Ca2+ from the ER and its subsequent MCU-mediatedinflux into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Department of Biomedical Sciences, Ajou University School of Medicine, Suwon , Korea. These authors contributed equally to this work. .

ABSTRACT
Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine", is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca2+ levels and induced ER stress via proteasome inhibition in these cells. Both MCU (mitochondrial Ca2+ uniporter) knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca2+ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP3 receptor (IP3R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca2+ accumulation and subsequent paraptotic events. Collectively, our results show that the IP3R-mediated release of Ca2+ from the ER and its subsequent MCU-mediatedinflux into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.

Show MeSH
Related in: MedlinePlus