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. 2014 Aug 30;5(16):6816-31.
doi: 10.18632/oncotarget.2256.

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

Mi Jin Yoon  1 , A Reum Lee  2 , Soo Ah Jeong  3 , You-Sun Kim  3 , Jin Yeop Kim  4 , Yong-Jun Kwon  5 , Kyeong Sook Choi  3
Affiliations

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

Mi Jin Yoon et al. Oncotarget. .

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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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Apoptosis is not critically involved in the celastrol-induced cancer cell death
(A) The chemical structure of celastrol. (B) Two breast cancer cell lines (MDA-MB 435S and MCF-7) and two colon cancer cell lines (DLD-1 and RKO) were treated with celastrol at the indicated concentrations for 24 h. Cellular viability was assessed using calcein-AM and EthD-1 to detect live and dead cells, respectively. (C) MDA-MB 435S cells were pretreated with the indicated concentrations of z-VAD-fmk for 30 min and further treated with 0.2 μg/ml TRAIL or 2 μM celastrol for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. (D) MDA-MB 435S cells were treated with 0.2 μg/ml TRAIL for 24 h or 2 μM celastrol for the indicated time points. 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 fold change of protein levels compared to control (untreated cells) was determined by a densitometric analysis. (E) Cells were pretreated with the indicated concentrations of z-VAD-fmk for 30 min and further treated with or without 2 μM celastrol for 24 h. Cellular viability was assessed using calcein-AM and EthD-1.
Figure 2
Figure 2. Celastrol induces vacuolation, but not lysosomal activation
(A) Cells were treated with 2 μM celastrol for 8 h and observed under the phase contrast microscope. (B) MDA-MB 435S cells were left untreated or treated with 10 nM bafilomycin A1 (Baflo. A1), 1 μM Torin1, or 2 μM celastrol for 8 h, stained with 50 nM LysoTracker-Red for 20 min, and then observed under the phase contrast and a fluorescence microscope.
Figure 3
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.
Figure 4
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.
Figure 5
Figure 5. Celastrol induces paraptosis
(A) MDA-MB 435S cells were left untreated or were pretreated with cycloheximide (CHX) at the indicated concentrations for 30 min and then treated with 2 μM celastrol for an additional 24 h in the continued presence of CHX. Cellular viability was assessed using calcein-AM and EthD-1. (B) YFP-Mito or YFP-ER cells were left untreated or were pretreated with 1 μM CHX and further treated with 2 μM celastrol for an additional 3 h in the continued presence of CHX. Cells were observed under the phase contrast and fluorescence microscope. (C) Cells were treated with 2 μM celastrol for the indicated time points and Western blotting was performed. β-actin was used as a loading control in Western blots. The relative expression levels were determined by the fold changes of densitometric values in treated groups to the values in the control (untreated) group. (D) Cells were treated with 2 μM celastrol for the indicated time points and Western blotting was performed. 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. Similar results were obtained from three independent experiments. (E) MDA-MB 435S cells were untreated or pretreated with the indicated specific inhibitors (SP600125, PD98059, and SB203580) at the indicated concentrations for 30 min and further treated with 2 μM celastrol for 24 h. Cellular viability was assessed using calcein-AM and EthD-1.
Figure 6
Figure 6. Celastrol induces mitochondrial Ca2+ uptake
(A) MDA-MB 435S cells treated with 2 μM celastrol for the indicated time points were stained with 2.5 μM Fluo-3 and processed for FACS analysis. Fluo-3 fluorescence intensities (FI) in cells treated with 2 μM celastrol were compared with that of untreated cells and denoted in the graph (left). Histogram for the cells treated with 2 μM celastrol for 3 h is shown (right). X axis, fluorescence intensity, Y axis, relative number of cells. (B) MDA-MB 435S cells treated with or without 2 μM celastrol for the indicated time points were stained with 2.5 μM Rhod-2 and processed for FACS analysis. Rhod-2 fluorescence intensities (FI) were compared with that of untreated cells and denoted in the graph (left). Histogram for the cells treated with 2 μM celastrol for 2 h is shown (right). (C) YFP-Mito cells treated with or without 2 μM celastrol for 2 h were stained with 2.5 μM Rhod-2 and then observed under the phase contrast and fluorescence microscopy. (D) MCF-7, DLD-1 and RKO cells treated with 2 μM celastrol for 4 h. Treated cells were stained with 2.5 μM Rhod-2 and processed for FACS analysis. The representative histograms are shown. X axis, fluorescence intensity, Y axis, relative number of cells.
Figure 7
Figure 7. MCU knockdown inhibits celastrol-induced paraptosis
(A) MDA-MB 435S cells were transfected with MCU siRNA and further treated with or without 2 μM celastrol for 24 h. Knockdown of MCU was confirmed by Western blotting using anti-MCU antibody. β-actin was used as a loading control in Western blots (upper panel). Cellular viability was assessed using calcein-AM and EthD-1 (lower panel). (B) MDA-MB 435S cells were transfected with MCU siRNA and further treated with or without 2 μM celastrol for 2 h. Cells were stained with 2.5 μM Rhod-2 and processed for FACS analysis. The fold changes of Rhod-2 fluorescence intensities (FI) are shown in the graph. (C) YFP-Mito cells were transfected with MCU siRNA and further treated with or without 2 μM celastrol for 2 h. Treated cells were stained with Rhod-2 and processed for the phase contrast and fluorescence microscopy.
Figure 8
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.
Figure 9
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.
Figure 10
Figure 10. Celastrol induces paraptosis via IP3R-mediated Ca2+ release from the ER and MCU-mediated mitochondrial Ca2+ influx in cancer cells
(A) Cells were pretreated with the indicated concentrations of RR or 2-APB for 30 min and further treated with 2 μM celastrol for 24 h. Cellular viability was assessed using calcein-AM and EthD-1. (B) Hypothetical scheme of celastrol-induced paraptosis. Celastrol triggers IP3R-mediated Ca2+ release from the ER and subsequently MCU-mediated Ca2+ influx into mitochondria, leading to the dilations of mitochondria/ER and cell death.
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