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Neoalbaconol induces cell death through necroptosis by regulating RIPK-dependent autocrine TNFα and ROS production.

Yu X, Deng Q, Li W, Xiao L, Luo X, Liu X, Yang L, Peng S, Ding Z, Feng T, Zhou J, Fan J, Bode AM, Dong Z, Liu J, Cao Y - Oncotarget (2015)

Bottom Line: Necroptosis/regulated necrosis is a caspase-independent, but receptor interacting protein kinase (RIPK)-dependent form of cell death.The molecular mechanism of NA-induced necroptosis is described in this research study.Moreover, we also found that NA caused RIPK3-mediated reactive oxygen species (ROS) production and contribution to cell death.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, Xiangya School of Medicine, Central South University, Hunan, China.

ABSTRACT
Necroptosis/regulated necrosis is a caspase-independent, but receptor interacting protein kinase (RIPK)-dependent form of cell death. In previous studies, neoalbaconol (NA), a constituent extracted from Albatrellus confluens, was demonstrated to induce necroptosis in some cancer cell lines. The molecular mechanism of NA-induced necroptosis is described in this research study. We determined that NA-induced cell death is partly dependent on tumor necrosis factor α (TNFα) feed-forward signaling. More importantly, NA abolished the ubiquitination of RIPK1 by down-regulating E3 ubiquitin ligases, cellular inhibitors of apoptosis protein 1/2 (cIAP1/2) and TNFα receptor-associated factors (TRAFs). The suppression of RIPK1 ubiquitination induced the activation of the non-canonical nuclear factor-κB (NF-κB) pathway and stimulated the transcription of TNFα. Moreover, we also found that NA caused RIPK3-mediated reactive oxygen species (ROS) production and contribution to cell death. Taken together, these results suggested that two distinct mechanisms are involved in NA-induced necroptosis and include RIPK1/NF-κB-dependent expression of TNFα and RIPK3-dependent generation of ROS.

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NA induces auto-ubiquitination and proteasomal degradation of cIAP1/2A. NA causes loss of cIAP1 and cIAP2. C666-1 and HK1 cells were treated with NA at the indicated dose for 8 h and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. β-Actin is shown as a loading control. B. NA-induced degradation of cIAP1 and cIAP2 is dependent on proteasomal machinery. C666-1 and HK1 cells were treated with NA (40 μM) for 1 h in the absence or presence of proteasome inhibitor (2 mM MG132) and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. C. NA stimulates auto-ubiquitination of c-IAP1 and cIAP2. C666-1 cells were transfected with the cIAP1-myc or cIAP2-flag plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. cIAP1 and cIAP2 were immunoprecipitated and immunoblotted. β-Actin served as a loading control. D. C666-1 and HK1 cells were exposed to increasing concentrations of NA for 8 h, then lysed and immunoblotted. β-Actin served as a loading control.
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Figure 2: NA induces auto-ubiquitination and proteasomal degradation of cIAP1/2A. NA causes loss of cIAP1 and cIAP2. C666-1 and HK1 cells were treated with NA at the indicated dose for 8 h and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. β-Actin is shown as a loading control. B. NA-induced degradation of cIAP1 and cIAP2 is dependent on proteasomal machinery. C666-1 and HK1 cells were treated with NA (40 μM) for 1 h in the absence or presence of proteasome inhibitor (2 mM MG132) and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. C. NA stimulates auto-ubiquitination of c-IAP1 and cIAP2. C666-1 cells were transfected with the cIAP1-myc or cIAP2-flag plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. cIAP1 and cIAP2 were immunoprecipitated and immunoblotted. β-Actin served as a loading control. D. C666-1 and HK1 cells were exposed to increasing concentrations of NA for 8 h, then lysed and immunoblotted. β-Actin served as a loading control.

Mentions: Cells exposed to IAP antagonists can induce cIAP1/2 degradation, leading to RIPK1 de-ubquitination and autocrine TNFα to trigger either apoptotic or necroptotic cell death [12, 26, 27]. To determine whether cIAP1/2 can be affected by NA, we examined the cIAP1/2 protein level in cells incubated with or without NA. Results demonstrated that NA caused loss of cIAP1 and cIAP2 proteins and the XIAP protein level was also reduced at the same time in these cell lines (Figure 2A). IAP antagonists can induce loss of cIAP1 and cIAP2 by inducing their auto-ubiquitination and proteosomal degradation. To address possible mechanisms of NA-induced loss of cIAP1 and 2, C666-1 and HK1 cells were treated with NA in the absence or presence of the proteasome inhibitor, MG132. The presence of MG132 efficiently blocked the NA-dependent decreases in c-IAP1/2 protein levels (Figure 2B). NA exposure leading to enhancement in cIAP1 and cIAP2 ubiquitination further indicated that loss of cIAP1 and cIAP2 involves their auto-ubiquitination (Figure 2C). TRAF2, an E3 ubiquitin ligase, was originally identified as a cIAP1/2-binding protein mediating IAP antagonist-induced degradation of cellular cIAP1 [28-30]. Knockdown of TRAF2 in L929 cells activated RIPK1-dependent TNFα production and induced necroptosis. The protein levels of TRAF2 and TRAF6 were reduced in cancer cell lines treated with NA, but not TRAF3, TRADD, and FADD (Figure 2D). Collectively, these results demonstrate that NA triggers auto-ubiquitination and subsequent proteasomal degradation of cIAP1 and cIAP2.


Neoalbaconol induces cell death through necroptosis by regulating RIPK-dependent autocrine TNFα and ROS production.

Yu X, Deng Q, Li W, Xiao L, Luo X, Liu X, Yang L, Peng S, Ding Z, Feng T, Zhou J, Fan J, Bode AM, Dong Z, Liu J, Cao Y - Oncotarget (2015)

NA induces auto-ubiquitination and proteasomal degradation of cIAP1/2A. NA causes loss of cIAP1 and cIAP2. C666-1 and HK1 cells were treated with NA at the indicated dose for 8 h and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. β-Actin is shown as a loading control. B. NA-induced degradation of cIAP1 and cIAP2 is dependent on proteasomal machinery. C666-1 and HK1 cells were treated with NA (40 μM) for 1 h in the absence or presence of proteasome inhibitor (2 mM MG132) and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. C. NA stimulates auto-ubiquitination of c-IAP1 and cIAP2. C666-1 cells were transfected with the cIAP1-myc or cIAP2-flag plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. cIAP1 and cIAP2 were immunoprecipitated and immunoblotted. β-Actin served as a loading control. D. C666-1 and HK1 cells were exposed to increasing concentrations of NA for 8 h, then lysed and immunoblotted. β-Actin served as a loading control.
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Related In: Results  -  Collection

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Figure 2: NA induces auto-ubiquitination and proteasomal degradation of cIAP1/2A. NA causes loss of cIAP1 and cIAP2. C666-1 and HK1 cells were treated with NA at the indicated dose for 8 h and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. β-Actin is shown as a loading control. B. NA-induced degradation of cIAP1 and cIAP2 is dependent on proteasomal machinery. C666-1 and HK1 cells were treated with NA (40 μM) for 1 h in the absence or presence of proteasome inhibitor (2 mM MG132) and cell lysates were examined by Western blotting using antibodies against cIAP1 and cIAP2. C. NA stimulates auto-ubiquitination of c-IAP1 and cIAP2. C666-1 cells were transfected with the cIAP1-myc or cIAP2-flag plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. cIAP1 and cIAP2 were immunoprecipitated and immunoblotted. β-Actin served as a loading control. D. C666-1 and HK1 cells were exposed to increasing concentrations of NA for 8 h, then lysed and immunoblotted. β-Actin served as a loading control.
Mentions: Cells exposed to IAP antagonists can induce cIAP1/2 degradation, leading to RIPK1 de-ubquitination and autocrine TNFα to trigger either apoptotic or necroptotic cell death [12, 26, 27]. To determine whether cIAP1/2 can be affected by NA, we examined the cIAP1/2 protein level in cells incubated with or without NA. Results demonstrated that NA caused loss of cIAP1 and cIAP2 proteins and the XIAP protein level was also reduced at the same time in these cell lines (Figure 2A). IAP antagonists can induce loss of cIAP1 and cIAP2 by inducing their auto-ubiquitination and proteosomal degradation. To address possible mechanisms of NA-induced loss of cIAP1 and 2, C666-1 and HK1 cells were treated with NA in the absence or presence of the proteasome inhibitor, MG132. The presence of MG132 efficiently blocked the NA-dependent decreases in c-IAP1/2 protein levels (Figure 2B). NA exposure leading to enhancement in cIAP1 and cIAP2 ubiquitination further indicated that loss of cIAP1 and cIAP2 involves their auto-ubiquitination (Figure 2C). TRAF2, an E3 ubiquitin ligase, was originally identified as a cIAP1/2-binding protein mediating IAP antagonist-induced degradation of cellular cIAP1 [28-30]. Knockdown of TRAF2 in L929 cells activated RIPK1-dependent TNFα production and induced necroptosis. The protein levels of TRAF2 and TRAF6 were reduced in cancer cell lines treated with NA, but not TRAF3, TRADD, and FADD (Figure 2D). Collectively, these results demonstrate that NA triggers auto-ubiquitination and subsequent proteasomal degradation of cIAP1 and cIAP2.

Bottom Line: Necroptosis/regulated necrosis is a caspase-independent, but receptor interacting protein kinase (RIPK)-dependent form of cell death.The molecular mechanism of NA-induced necroptosis is described in this research study.Moreover, we also found that NA caused RIPK3-mediated reactive oxygen species (ROS) production and contribution to cell death.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, Xiangya School of Medicine, Central South University, Hunan, China.

ABSTRACT
Necroptosis/regulated necrosis is a caspase-independent, but receptor interacting protein kinase (RIPK)-dependent form of cell death. In previous studies, neoalbaconol (NA), a constituent extracted from Albatrellus confluens, was demonstrated to induce necroptosis in some cancer cell lines. The molecular mechanism of NA-induced necroptosis is described in this research study. We determined that NA-induced cell death is partly dependent on tumor necrosis factor α (TNFα) feed-forward signaling. More importantly, NA abolished the ubiquitination of RIPK1 by down-regulating E3 ubiquitin ligases, cellular inhibitors of apoptosis protein 1/2 (cIAP1/2) and TNFα receptor-associated factors (TRAFs). The suppression of RIPK1 ubiquitination induced the activation of the non-canonical nuclear factor-κB (NF-κB) pathway and stimulated the transcription of TNFα. Moreover, we also found that NA caused RIPK3-mediated reactive oxygen species (ROS) production and contribution to cell death. Taken together, these results suggested that two distinct mechanisms are involved in NA-induced necroptosis and include RIPK1/NF-κB-dependent expression of TNFα and RIPK3-dependent generation of ROS.

Show MeSH
Related in: MedlinePlus