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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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Inhibition of MCU blocks celastrol-induced paraptosis(A) YFP-Mito cells were pretreated with 4 μM ruthenium red (RR) 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. (B) MDA-MB 435S cells were pretreated with the indicated concentrations of ruthenium red and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (C) YFP-Mito and YFP-ER cells were pretreated with 4 μM ruthenium red (RR), 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 4 μM RR 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.
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Figure 8: Inhibition of MCU blocks celastrol-induced paraptosis(A) YFP-Mito cells were pretreated with 4 μM ruthenium red (RR) 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. (B) MDA-MB 435S cells were pretreated with the indicated concentrations of ruthenium red and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (C) YFP-Mito and YFP-ER cells were pretreated with 4 μM ruthenium red (RR), 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 4 μM RR 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.

Mentions: Ca2+ reportedly enters mitochondria via the MCU when [Ca2+]i are high [32]. Thus, we next tested whether inhibition of MCU affected celastrol-induced paraptosis. The functional role of the MCU in celastrol-induced cell death was investigated by knocking it down using small interfering RNA. We found that the cell death in YFP-Mito cells induced by 2 μM celastrol was significantly attenuated by transfection with MCU siRNA, despite the incomplete knockdown of MCU (Figure 7A). In addition, FACS analysis and fluorescence microscopy using Rhod-2 in these cells showed that mitochondrial Ca2+ accumulation and cellular vacuolation induced by treatment with 2 μM celastrol for 2 h were also markedly reduced by MCU knockdown (Figures 7B and 7C). Pretreatment with ruthenium red (RR), an inhibitor of uniporter-mediated mitochondrial Ca2+ uptake [33,34], also effectively blocked the celastrol-induced increase in [Ca2+]m in YFP-Mito cells (Figure 8A) and celastrol-induced death of MDA-MB 435S cells (Figure 8B). RR pretreatment also inhibited the dilation of mitochondria and the ER in YFP-Mito and YFP-ER cells (Figure 8C). Furthermore, RR pretreatment markedly inhibited celastrol-induced the accumulations of poly-ubiquitinated proteins, CHOP, activated ERK, and activated JNK (Figure 8D). We further tested whether treatment with kaempferol, an activator of the MCU [35], could sensitize MDA-MB 435S cells treated with the low-dose celastrol to paraptotic cell death. Compared to treatment with subtoxic dose (20 μM) of kaempferol or low-dose (1 μM) celastrol alone, combined treatment with kaempferol and celastrol for 4 h markedly increased [Ca2+]m and cellular vacuolation in YFP-Mito cells (Supplementary Figure 3A). In addition, co-treatment with kaempferol dose-dependently enhanced the death of MDA-MB 435S cells treated with 1 μM celastrol, compared with cells treated with 1 μM celastrol alone (Supplementary Figure 3B). Collectively, these observations indicate that MCU-mediated mitochondrial Ca2+ uptake may play a critical role in celastrol-induced paraptosis.


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)

Inhibition of MCU blocks celastrol-induced paraptosis(A) YFP-Mito cells were pretreated with 4 μM ruthenium red (RR) 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. (B) MDA-MB 435S cells were pretreated with the indicated concentrations of ruthenium red and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (C) YFP-Mito and YFP-ER cells were pretreated with 4 μM ruthenium red (RR), 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 4 μM RR 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.
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Figure 8: Inhibition of MCU blocks celastrol-induced paraptosis(A) YFP-Mito cells were pretreated with 4 μM ruthenium red (RR) 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. (B) MDA-MB 435S cells were pretreated with the indicated concentrations of ruthenium red and further treated with or without 2 μM celastrol for 24 h. Cellular viability was measured using calcein-AM and EthD-1. (C) YFP-Mito and YFP-ER cells were pretreated with 4 μM ruthenium red (RR), 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 4 μM RR 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.
Mentions: Ca2+ reportedly enters mitochondria via the MCU when [Ca2+]i are high [32]. Thus, we next tested whether inhibition of MCU affected celastrol-induced paraptosis. The functional role of the MCU in celastrol-induced cell death was investigated by knocking it down using small interfering RNA. We found that the cell death in YFP-Mito cells induced by 2 μM celastrol was significantly attenuated by transfection with MCU siRNA, despite the incomplete knockdown of MCU (Figure 7A). In addition, FACS analysis and fluorescence microscopy using Rhod-2 in these cells showed that mitochondrial Ca2+ accumulation and cellular vacuolation induced by treatment with 2 μM celastrol for 2 h were also markedly reduced by MCU knockdown (Figures 7B and 7C). Pretreatment with ruthenium red (RR), an inhibitor of uniporter-mediated mitochondrial Ca2+ uptake [33,34], also effectively blocked the celastrol-induced increase in [Ca2+]m in YFP-Mito cells (Figure 8A) and celastrol-induced death of MDA-MB 435S cells (Figure 8B). RR pretreatment also inhibited the dilation of mitochondria and the ER in YFP-Mito and YFP-ER cells (Figure 8C). Furthermore, RR pretreatment markedly inhibited celastrol-induced the accumulations of poly-ubiquitinated proteins, CHOP, activated ERK, and activated JNK (Figure 8D). We further tested whether treatment with kaempferol, an activator of the MCU [35], could sensitize MDA-MB 435S cells treated with the low-dose celastrol to paraptotic cell death. Compared to treatment with subtoxic dose (20 μM) of kaempferol or low-dose (1 μM) celastrol alone, combined treatment with kaempferol and celastrol for 4 h markedly increased [Ca2+]m and cellular vacuolation in YFP-Mito cells (Supplementary Figure 3A). In addition, co-treatment with kaempferol dose-dependently enhanced the death of MDA-MB 435S cells treated with 1 μM celastrol, compared with cells treated with 1 μM celastrol alone (Supplementary Figure 3B). Collectively, these observations indicate that MCU-mediated mitochondrial Ca2+ uptake may play a critical role in celastrol-induced paraptosis.

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