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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 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.
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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.

Mentions: Since mitochondria and the ER are major reservoirs of intracellular Ca2+, we next tested whether their dilation following celastrol treatment was associated with disruptions in intracellular Ca2+ homeostasis. Flow cytometry using Fluo-3 (a cell-permeable Ca2+-indicator dye) demonstrated that treatment of MDA-MB 435S cells with celastrol dramatically increased the intracellular Ca2+ levels ([Ca2+]i), which peaked at 3 h post-treatment (Figure 6A). Furthermore, flow cytometry using Rhod-2 (an indicator dye for mitochondrial Ca2+) showed that celastrol treatment also increased the mitochondrial Ca2+ levels ([Ca2+]m), which peaked at 2 h post-treatment (Figure 6B). Fluorescence microscopy further confirmed the celastrol-induced increase in Rhod-2 staining intensity in mitochondria of YFP-Mito cells (Figure 6C). Moreover, flow cytometry using Rhod-2 showed that the celastrol-induced increase in [Ca2+]m is common to MCF-7, DLD-1, and RKO cells (Figure 6D).


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 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.
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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.
Mentions: Since mitochondria and the ER are major reservoirs of intracellular Ca2+, we next tested whether their dilation following celastrol treatment was associated with disruptions in intracellular Ca2+ homeostasis. Flow cytometry using Fluo-3 (a cell-permeable Ca2+-indicator dye) demonstrated that treatment of MDA-MB 435S cells with celastrol dramatically increased the intracellular Ca2+ levels ([Ca2+]i), which peaked at 3 h post-treatment (Figure 6A). Furthermore, flow cytometry using Rhod-2 (an indicator dye for mitochondrial Ca2+) showed that celastrol treatment also increased the mitochondrial Ca2+ levels ([Ca2+]m), which peaked at 2 h post-treatment (Figure 6B). Fluorescence microscopy further confirmed the celastrol-induced increase in Rhod-2 staining intensity in mitochondria of YFP-Mito cells (Figure 6C). Moreover, flow cytometry using Rhod-2 showed that the celastrol-induced increase in [Ca2+]m is common to MCF-7, DLD-1, and RKO cells (Figure 6D).

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