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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 inhibits autophagy(A) MDA-MB 435S cells transiently transfected with mRFP-GFP-LC3 plasmid for 24 h were further treated with 10 nM bafilomycin A1 (Baflo. A1), 1 μM Torin1, or 2 μM celastrol for 8 h. Representative fluorescence microscopic images are shown. Arrow heads: yellow dots (RFP(+)/GFP(+)-LC3 puncta), arrows: RFP-LC3-only dots (RFP(+)/GFP(-)-LC3 puncta). (B,C) Total, RFP(+)/GFP(+)-LC3, and RFP(+)/GFP(-)-LC3 dots were quantified and their percentages were calculated (>20 cells were counted in each experiment from at least three independent experiments. (D) Cells were treated with 2 μM celastrol for the indicated time points (left) or 10 nM bafilomycin A1 for 24 h (right). Whole cell extracts were prepared from the treated cells and subjected to Western blotting. β-actin was used as a loading control in Western blots. The relative expression levels were determined by the fold change of densitometric values in treated groups to the values in the control (untreated) group.
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Figure 3: Celastrol inhibits autophagy(A) MDA-MB 435S cells transiently transfected with mRFP-GFP-LC3 plasmid for 24 h were further treated with 10 nM bafilomycin A1 (Baflo. A1), 1 μM Torin1, or 2 μM celastrol for 8 h. Representative fluorescence microscopic images are shown. Arrow heads: yellow dots (RFP(+)/GFP(+)-LC3 puncta), arrows: RFP-LC3-only dots (RFP(+)/GFP(-)-LC3 puncta). (B,C) Total, RFP(+)/GFP(+)-LC3, and RFP(+)/GFP(-)-LC3 dots were quantified and their percentages were calculated (>20 cells were counted in each experiment from at least three independent experiments. (D) Cells were treated with 2 μM celastrol for the indicated time points (left) or 10 nM bafilomycin A1 for 24 h (right). Whole cell extracts were prepared from the treated cells and subjected to Western blotting. β-actin was used as a loading control in Western blots. The relative expression levels were determined by the fold change of densitometric values in treated groups to the values in the control (untreated) group.

Mentions: When we examined the cellular morphologies following celastrol treatment, we found that marked cellular vacuolation commonly preceded cell death in MDA-MB 435S, MCF-7, DLD-1 and RKO cells (Figure 2A). Therefore, we examined whether celastrol-induced vacuolation and subsequent cell death were associated with autophagy. First, we tested the possibility that celastrol induces lysosomal activation, a late step in autophagy, by staining with LysoTracker-Red. Treatment of MDA-MB 435S cells with Torin1, an autophagy inducer with mTOR (mechanistic target of rapamycin) inhibitory activity [28], markedly increased LysoTracker-Red staining, whereas bafilomycin A1, an autophagy inhibitor, substantially reduced it (Figure 2B). Interestingly, celastrol treatment, like bafilomycin A1 treatment, also inhibited LysoTracker-Red staining. We further measured autophagic flux activity in MDA-MB 435S cells employing the tandem fluorescent construct, mRFP/GFP-LC3 [29]. In this assay, RFP fluorescence is relatively stable in acidic compartments, whereas GFP fluorescence is rapidly quenched in such environments. Accordingly, mRFP/GFP-LC3 in mature autolysosomes will be detected as red puncta, whereas blocking autophagosome-lysosome fusion or suppressing lysosomal degradation (i.e., through an increase in lysosomal pH) will increase the number of yellow puncta [29]. We found that celastrol treatment increased the number of yellow puncta (RFP(+)/GFP(+)-LC3) similar to bafilomycin A1 treatment, whereas Torin1 treatment increased red puncta (RFP(+)/GFP(-)-LC3 puncta) (Figure 3A-3C). Time-course experiments showed that LC3 (both I and II form), as well as p62 [30] and NBR1 (Neighbor of Braca1 gene) [31], the substrates of autophagy, progressively accumulated in both MDA-MB 435S and MCF-7 cells treated with celastrol, similar to those obtained with bafilomycin A1 treatment (Figure 3D). Also similar to bafilomycin A1 treatment, celastrol inhibited the proteolytic processing of cathepsin L, a major lysosomal protease. These results would seem to suggest that celastrol might inhibit autophagy, possibly at the lysosomal step. However, we found that neither celastrol-induced cell death nor cellular vacuolation was affected not only by pretreatment with the autophagy inhibitors, 3-MA, bafilomycin A1, and chloroquine (CQ) but also by knockdown of ATG5, Beclin-1 and LAMP2 (Supplementary Figure 2A-2D). Furthermore, celastrol-induced cell death was not affected by the pretreatment with necrostatin-1, an inhibitor of necroptosis (Supplementary Figure 2E and 2F). Collectively, these results suggest that celastrol-induced vacuolation and subsequent cell death in MDA-MB 435S cells do not involve modulation of autophagy or necroptosis.


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 inhibits autophagy(A) MDA-MB 435S cells transiently transfected with mRFP-GFP-LC3 plasmid for 24 h were further treated with 10 nM bafilomycin A1 (Baflo. A1), 1 μM Torin1, or 2 μM celastrol for 8 h. Representative fluorescence microscopic images are shown. Arrow heads: yellow dots (RFP(+)/GFP(+)-LC3 puncta), arrows: RFP-LC3-only dots (RFP(+)/GFP(-)-LC3 puncta). (B,C) Total, RFP(+)/GFP(+)-LC3, and RFP(+)/GFP(-)-LC3 dots were quantified and their percentages were calculated (>20 cells were counted in each experiment from at least three independent experiments. (D) Cells were treated with 2 μM celastrol for the indicated time points (left) or 10 nM bafilomycin A1 for 24 h (right). Whole cell extracts were prepared from the treated cells and subjected to Western blotting. β-actin was used as a loading control in Western blots. The relative expression levels were determined by the fold change of densitometric values in treated groups to the values in the control (untreated) group.
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Figure 3: Celastrol inhibits autophagy(A) MDA-MB 435S cells transiently transfected with mRFP-GFP-LC3 plasmid for 24 h were further treated with 10 nM bafilomycin A1 (Baflo. A1), 1 μM Torin1, or 2 μM celastrol for 8 h. Representative fluorescence microscopic images are shown. Arrow heads: yellow dots (RFP(+)/GFP(+)-LC3 puncta), arrows: RFP-LC3-only dots (RFP(+)/GFP(-)-LC3 puncta). (B,C) Total, RFP(+)/GFP(+)-LC3, and RFP(+)/GFP(-)-LC3 dots were quantified and their percentages were calculated (>20 cells were counted in each experiment from at least three independent experiments. (D) Cells were treated with 2 μM celastrol for the indicated time points (left) or 10 nM bafilomycin A1 for 24 h (right). Whole cell extracts were prepared from the treated cells and subjected to Western blotting. β-actin was used as a loading control in Western blots. The relative expression levels were determined by the fold change of densitometric values in treated groups to the values in the control (untreated) group.
Mentions: When we examined the cellular morphologies following celastrol treatment, we found that marked cellular vacuolation commonly preceded cell death in MDA-MB 435S, MCF-7, DLD-1 and RKO cells (Figure 2A). Therefore, we examined whether celastrol-induced vacuolation and subsequent cell death were associated with autophagy. First, we tested the possibility that celastrol induces lysosomal activation, a late step in autophagy, by staining with LysoTracker-Red. Treatment of MDA-MB 435S cells with Torin1, an autophagy inducer with mTOR (mechanistic target of rapamycin) inhibitory activity [28], markedly increased LysoTracker-Red staining, whereas bafilomycin A1, an autophagy inhibitor, substantially reduced it (Figure 2B). Interestingly, celastrol treatment, like bafilomycin A1 treatment, also inhibited LysoTracker-Red staining. We further measured autophagic flux activity in MDA-MB 435S cells employing the tandem fluorescent construct, mRFP/GFP-LC3 [29]. In this assay, RFP fluorescence is relatively stable in acidic compartments, whereas GFP fluorescence is rapidly quenched in such environments. Accordingly, mRFP/GFP-LC3 in mature autolysosomes will be detected as red puncta, whereas blocking autophagosome-lysosome fusion or suppressing lysosomal degradation (i.e., through an increase in lysosomal pH) will increase the number of yellow puncta [29]. We found that celastrol treatment increased the number of yellow puncta (RFP(+)/GFP(+)-LC3) similar to bafilomycin A1 treatment, whereas Torin1 treatment increased red puncta (RFP(+)/GFP(-)-LC3 puncta) (Figure 3A-3C). Time-course experiments showed that LC3 (both I and II form), as well as p62 [30] and NBR1 (Neighbor of Braca1 gene) [31], the substrates of autophagy, progressively accumulated in both MDA-MB 435S and MCF-7 cells treated with celastrol, similar to those obtained with bafilomycin A1 treatment (Figure 3D). Also similar to bafilomycin A1 treatment, celastrol inhibited the proteolytic processing of cathepsin L, a major lysosomal protease. These results would seem to suggest that celastrol might inhibit autophagy, possibly at the lysosomal step. However, we found that neither celastrol-induced cell death nor cellular vacuolation was affected not only by pretreatment with the autophagy inhibitors, 3-MA, bafilomycin A1, and chloroquine (CQ) but also by knockdown of ATG5, Beclin-1 and LAMP2 (Supplementary Figure 2A-2D). Furthermore, celastrol-induced cell death was not affected by the pretreatment with necrostatin-1, an inhibitor of necroptosis (Supplementary Figure 2E and 2F). Collectively, these results suggest that celastrol-induced vacuolation and subsequent cell death in MDA-MB 435S cells do not involve modulation of autophagy or necroptosis.

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