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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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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.
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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.

Mentions: Celastrol, a quinone methide triterpene, is a pharmacologically active compound derived from the Chinese medicinal plant, Tripterygium wilfordii [1]. Two carbons of celastrol, C2 of the A-ring and C6 of the B-ring (Figure 1A), reportedly show high susceptibilities for nucleophilic attack [2]. Celastrol can react with the nucleophilic thiol groups of cysteine residues and form covalent Michael adducts [3-6]. This seems to be the major mechanism through which celastrol can alter the functions of various proteins. Celastrol has traditionally been used to treat autoimmune diseases [7], chronic inflammation [8], asthma [9], and neurodegenerative diseases [10]. More recently, it has attracted interest as a potential anti-cancer agent, since it has been shown to inhibit proliferation and suppress the initiation, progression and metastasis of tumors in a wide variety of models in vitro and in vivo [11-14]. To date, the studies on the cancer-killing activity of celastrol have mainly focused on its ability to induce apoptosis [15,16]. In the present study, in contrast, we show that celastrol kills breast and colon cancer cell lines via inducing paraptosis. Despite recent improvements in anti-cancer therapies, inherent or acquired cellular resistance to various pro-apoptotic treatments often leads to therapeutic failure [17]. Thus, a better understanding of alternative, non-apoptotic cell death pathways, including paraptosis, may facilitate the design of novel therapeutics against malignant cancer cells that harbor defective apoptotic machineries. The term “paraptosis” was originally introduced to describe a form of programmed cell death that is morphologically and biochemically distinct from apoptosis [18,19]. It is characterized by: extensive cytoplasmic vacuolization that arises via swelling of the ER [19-21] and/or mitochondria [19,21,22]; the lack of characteristic apoptotic features, such as pyknosis, DNA fragmentation and caspase activation [19,21,23]; insensitivity to caspase inhibitors [18,24]; and overexpression of anti-apoptotic Bcl-2-like proteins [18,21,24]. Therefore, identification of agents that can induce paraptosis by targeting both mitochondria and the ER may provide a rational therapeutic strategy for effectively killing malignant cancer cells that resist apoptosis. However, the mechanisms underlying paraptosis, particularly the signals responsible for triggering dilation of mitochondria and the ER are still poorly defined. Observations that paraptosis can be inhibited by cycloheximide indicate that the paraptotic process requires protein synthesis [19,21,22,25]. MAP kinase activation has been associated with paraptosis induced by insulin-like growth factor I receptor (IGFIR) [18], curcumin [21,22], celastrol [25], and taxol [26], although the importance of the respective MAP kinase differs depending on the stimulus [18,21,22,25,26]. We recently showed that proteasomal dysfunction and the generation of mitochondrial superoxide are critical for the curcumin-induced dilation of mitochondria/ER and subsequent paraptotic cell death in breast cancer cells [21]. We propose here that the IP3R-mediated release of Ca2+ from the ER and its subsequent mitochondrial Ca2+ uniporter-mediated influx into mitochondria may critically contribute to extensive dilation of mitochondria and the ER, leading to celastrol-induced paraptotic cell death.


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)

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.
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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.
Mentions: Celastrol, a quinone methide triterpene, is a pharmacologically active compound derived from the Chinese medicinal plant, Tripterygium wilfordii [1]. Two carbons of celastrol, C2 of the A-ring and C6 of the B-ring (Figure 1A), reportedly show high susceptibilities for nucleophilic attack [2]. Celastrol can react with the nucleophilic thiol groups of cysteine residues and form covalent Michael adducts [3-6]. This seems to be the major mechanism through which celastrol can alter the functions of various proteins. Celastrol has traditionally been used to treat autoimmune diseases [7], chronic inflammation [8], asthma [9], and neurodegenerative diseases [10]. More recently, it has attracted interest as a potential anti-cancer agent, since it has been shown to inhibit proliferation and suppress the initiation, progression and metastasis of tumors in a wide variety of models in vitro and in vivo [11-14]. To date, the studies on the cancer-killing activity of celastrol have mainly focused on its ability to induce apoptosis [15,16]. In the present study, in contrast, we show that celastrol kills breast and colon cancer cell lines via inducing paraptosis. Despite recent improvements in anti-cancer therapies, inherent or acquired cellular resistance to various pro-apoptotic treatments often leads to therapeutic failure [17]. Thus, a better understanding of alternative, non-apoptotic cell death pathways, including paraptosis, may facilitate the design of novel therapeutics against malignant cancer cells that harbor defective apoptotic machineries. The term “paraptosis” was originally introduced to describe a form of programmed cell death that is morphologically and biochemically distinct from apoptosis [18,19]. It is characterized by: extensive cytoplasmic vacuolization that arises via swelling of the ER [19-21] and/or mitochondria [19,21,22]; the lack of characteristic apoptotic features, such as pyknosis, DNA fragmentation and caspase activation [19,21,23]; insensitivity to caspase inhibitors [18,24]; and overexpression of anti-apoptotic Bcl-2-like proteins [18,21,24]. Therefore, identification of agents that can induce paraptosis by targeting both mitochondria and the ER may provide a rational therapeutic strategy for effectively killing malignant cancer cells that resist apoptosis. However, the mechanisms underlying paraptosis, particularly the signals responsible for triggering dilation of mitochondria and the ER are still poorly defined. Observations that paraptosis can be inhibited by cycloheximide indicate that the paraptotic process requires protein synthesis [19,21,22,25]. MAP kinase activation has been associated with paraptosis induced by insulin-like growth factor I receptor (IGFIR) [18], curcumin [21,22], celastrol [25], and taxol [26], although the importance of the respective MAP kinase differs depending on the stimulus [18,21,22,25,26]. We recently showed that proteasomal dysfunction and the generation of mitochondrial superoxide are critical for the curcumin-induced dilation of mitochondria/ER and subsequent paraptotic cell death in breast cancer cells [21]. We propose here that the IP3R-mediated release of Ca2+ from the ER and its subsequent mitochondrial Ca2+ uniporter-mediated influx into mitochondria may critically contribute to extensive dilation of mitochondria and the ER, leading to celastrol-induced paraptotic cell death.

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