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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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Celastrol-induced vacuoles are derived from mitochondria and the ER(A) YFP-Mito or YFP-ER cells treated with 2 μM celastrol for 3 h were observed under the phase contrast and fluorescence microscope. (B) MDA-MB 435S cells were treated with or without 2 μM celastrol for 3 h. Immunocytochemistry using anti-SDHA (green) and anti-PDI (red) antibodies was performed and the representative phase contrast and fluorescence microscopic images of cells are shown. (C) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points and observed by transmission electron microscopy. Arrowheads indicate mitochondria and arrows denote the ER. Swelling and fusion of mitochondria and the ER are seen after celastrol treatment. Bars, 2 μm.
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Figure 4: Celastrol-induced vacuoles are derived from mitochondria and the ER(A) YFP-Mito or YFP-ER cells treated with 2 μM celastrol for 3 h were observed under the phase contrast and fluorescence microscope. (B) MDA-MB 435S cells were treated with or without 2 μM celastrol for 3 h. Immunocytochemistry using anti-SDHA (green) and anti-PDI (red) antibodies was performed and the representative phase contrast and fluorescence microscopic images of cells are shown. (C) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points and observed by transmission electron microscopy. Arrowheads indicate mitochondria and arrows denote the ER. Swelling and fusion of mitochondria and the ER are seen after celastrol treatment. Bars, 2 μm.

Mentions: Next, to test whether the celastrol-induced vacuoles might originate from mitochondria and/or the ER, we used MDA-MB 435S sublines stably expressing fluorescence selectively in mitochondria or the ER, YFP-Mito cells and YFP-ER cells [21]. As shown in Figure 4A, mitochondria in untreated YFP-Mito cells exhibited a filamentous, elongated morphology, whereas the ER in untreated YFP-ER cells formed a reticulate structure. At 3 h after treatment with 2 μM celastrol, numerous fluorescent vacuoles were observed in both YFP-Mito and YFP-ER cells. Immunocytochemistry using specific antibodies against the subunit A of succinate dehydrogenase (SDHA), a mitochondrial protein, and protein disulfide-isomerase (PDI), an ER-resident protein, showed many small rings of SDHA expression and larger rings of PDI expression in MDA-MB 435S cells treated with celastrol for 3 h (Figure 4B). A more detailed electron microscopy revealed swollen mitochondria and the ER structures in MDA-MB 435S cells treated with 2 μM celastrol for 3 h. At 6 h of celastrol treatment, fusion among swollen mitochondria and the swollen ER had further progressed (Figure 4C). In contrast, untreated MDA-MB 435S cells possessed mitochondria with intact cristae and the ER structures with a reticular morphology. Collectively, these results indicate that celastrol induces paraptotic morphologies. We next sought biochemical evidence for the induction of paraptosis by celastrol. We first tested the effect of the protein synthesis inhibitor, cycloheximide, on celastrol-induced cell death. Pretreatment with cycloheximide (CHX) very effectively blocked cell death in celastrol-treated MDA-MB 435S cells (Figure 5A), and prevented mitochondrial/ER dilation in celastrol-treated YFP-Mito and YFP-ER cells (Figure 5B). Recently, we showed that proteasome dysfunction is required for dilation of mitochondria/ER and consequent paraptotic cell death [21,22]. Thus, we asked whether celastrol also impairs proteasome activity. We found that celastrol-treated cells progressively accumulated poly-ubiquitinated proteins (Figure 5C). Furthermore, celastrol treatment markedly increased the protein levels of ATF4, CHOP, and KDEL in MDA-MB 435S and MCF-7 cells, indicating that celastrol induces ER stress. When we examined the activity changes in MAP kinases, we found that ERKs were progressively activated in MDA-MB 435S cells treated with celastrol. In contrast, the activities of ERKs increased with a peak at 2 h of celastrol treatment and then slowly reduced in MCF-7 cells (Figure 5D). In both MDA-MB 435S and MCF-7 cells, JNKs demonstrated biphasic activation patterns following celastrol treatment, whereas p38 showed transient activation with a peak at 2 h after celastrol treatment. When we investigated the importance of these signals, we found that celastrol-induced cell death was significantly inhibited by SP600125-induced inhibition of JNK, inhibited by PD98059-induced inhibition of ERK at lesser extent, and unaffected by SB203580-induced inhibition of p38 (Figure 5E). Taken together, our results indicate that celastrol may induce paraptosis in the tested breast cancer cell lines.


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)

Celastrol-induced vacuoles are derived from mitochondria and the ER(A) YFP-Mito or YFP-ER cells treated with 2 μM celastrol for 3 h were observed under the phase contrast and fluorescence microscope. (B) MDA-MB 435S cells were treated with or without 2 μM celastrol for 3 h. Immunocytochemistry using anti-SDHA (green) and anti-PDI (red) antibodies was performed and the representative phase contrast and fluorescence microscopic images of cells are shown. (C) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points and observed by transmission electron microscopy. Arrowheads indicate mitochondria and arrows denote the ER. Swelling and fusion of mitochondria and the ER are seen after celastrol treatment. Bars, 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196165&req=5

Figure 4: Celastrol-induced vacuoles are derived from mitochondria and the ER(A) YFP-Mito or YFP-ER cells treated with 2 μM celastrol for 3 h were observed under the phase contrast and fluorescence microscope. (B) MDA-MB 435S cells were treated with or without 2 μM celastrol for 3 h. Immunocytochemistry using anti-SDHA (green) and anti-PDI (red) antibodies was performed and the representative phase contrast and fluorescence microscopic images of cells are shown. (C) MDA-MB 435S cells were treated with 2 μM celastrol for the indicated time points and observed by transmission electron microscopy. Arrowheads indicate mitochondria and arrows denote the ER. Swelling and fusion of mitochondria and the ER are seen after celastrol treatment. Bars, 2 μm.
Mentions: Next, to test whether the celastrol-induced vacuoles might originate from mitochondria and/or the ER, we used MDA-MB 435S sublines stably expressing fluorescence selectively in mitochondria or the ER, YFP-Mito cells and YFP-ER cells [21]. As shown in Figure 4A, mitochondria in untreated YFP-Mito cells exhibited a filamentous, elongated morphology, whereas the ER in untreated YFP-ER cells formed a reticulate structure. At 3 h after treatment with 2 μM celastrol, numerous fluorescent vacuoles were observed in both YFP-Mito and YFP-ER cells. Immunocytochemistry using specific antibodies against the subunit A of succinate dehydrogenase (SDHA), a mitochondrial protein, and protein disulfide-isomerase (PDI), an ER-resident protein, showed many small rings of SDHA expression and larger rings of PDI expression in MDA-MB 435S cells treated with celastrol for 3 h (Figure 4B). A more detailed electron microscopy revealed swollen mitochondria and the ER structures in MDA-MB 435S cells treated with 2 μM celastrol for 3 h. At 6 h of celastrol treatment, fusion among swollen mitochondria and the swollen ER had further progressed (Figure 4C). In contrast, untreated MDA-MB 435S cells possessed mitochondria with intact cristae and the ER structures with a reticular morphology. Collectively, these results indicate that celastrol induces paraptotic morphologies. We next sought biochemical evidence for the induction of paraptosis by celastrol. We first tested the effect of the protein synthesis inhibitor, cycloheximide, on celastrol-induced cell death. Pretreatment with cycloheximide (CHX) very effectively blocked cell death in celastrol-treated MDA-MB 435S cells (Figure 5A), and prevented mitochondrial/ER dilation in celastrol-treated YFP-Mito and YFP-ER cells (Figure 5B). Recently, we showed that proteasome dysfunction is required for dilation of mitochondria/ER and consequent paraptotic cell death [21,22]. Thus, we asked whether celastrol also impairs proteasome activity. We found that celastrol-treated cells progressively accumulated poly-ubiquitinated proteins (Figure 5C). Furthermore, celastrol treatment markedly increased the protein levels of ATF4, CHOP, and KDEL in MDA-MB 435S and MCF-7 cells, indicating that celastrol induces ER stress. When we examined the activity changes in MAP kinases, we found that ERKs were progressively activated in MDA-MB 435S cells treated with celastrol. In contrast, the activities of ERKs increased with a peak at 2 h of celastrol treatment and then slowly reduced in MCF-7 cells (Figure 5D). In both MDA-MB 435S and MCF-7 cells, JNKs demonstrated biphasic activation patterns following celastrol treatment, whereas p38 showed transient activation with a peak at 2 h after celastrol treatment. When we investigated the importance of these signals, we found that celastrol-induced cell death was significantly inhibited by SP600125-induced inhibition of JNK, inhibited by PD98059-induced inhibition of ERK at lesser extent, and unaffected by SB203580-induced inhibition of p38 (Figure 5E). Taken together, our results indicate that celastrol may induce paraptosis in the tested breast cancer cell lines.

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