Limits...
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: 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).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.

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

NA treatment abolishes RIPK1 ubiquitination and activates the NF-κB non-canonical pathwayA. C666-1 cells were treated or not treated with 40 μM NA for 8 h, and RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. B. Cells were transfected with the His-Ub (wt, K48, K63) plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. C. The effect of increasing doses of NA (0-40 μM) treatment for 8 or 24 h on the expression level of IKKα, IKKβ and NIK and downstream molecules, p65, p100, p52 and IκBα was analyzed by immunoblotting. β-Actin served as a loading control. D. NA inhibits NF-κB reporter gene expression. C666-1 cells were transiently transfected with an NF-κB-containing plasmid for 24 h. After transfection, cells were treated with the indicated concentrations of NA for 24 h. Gene expression was assayed by measuring luciferase activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385831&req=5

Figure 3: NA treatment abolishes RIPK1 ubiquitination and activates the NF-κB non-canonical pathwayA. C666-1 cells were treated or not treated with 40 μM NA for 8 h, and RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. B. Cells were transfected with the His-Ub (wt, K48, K63) plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. C. The effect of increasing doses of NA (0-40 μM) treatment for 8 or 24 h on the expression level of IKKα, IKKβ and NIK and downstream molecules, p65, p100, p52 and IκBα was analyzed by immunoblotting. β-Actin served as a loading control. D. NA inhibits NF-κB reporter gene expression. C666-1 cells were transiently transfected with an NF-κB-containing plasmid for 24 h. After transfection, cells were treated with the indicated concentrations of NA for 24 h. Gene expression was assayed by measuring luciferase activity.

Mentions: Previous studies showed that triggering proteasomal degradation of cIAP1/2 could activate RIPK1 de-ubiquitination to induce cell death [12, 31]. Having established that NA could down-regulate cIAP1 and cIAP2, we examined the RIPK1 ubquitination level in cells treated or not treated with NA. Results showed that NA exposure led to a reduction in RIPK1 ubiquitination in C666-1 cells (Figure 3A). In cells treated with NA, the endogenous levels of cIAP1 and cIAP2 were decreased by co-immunoprecipitation with an anti-RIPK1 antibody (Figure 3A). Different types of ubiquitination modifications of RIPK1 can lead to different functions. To determine which ubiquitination modification of RIPK1 was affected by NA, Wt-Ub, His-UbK48 or His-UbK63 plasmids were introduced into C666-1 cells. NA treatment reduced the ubiquitination of the K63-linked modification compared with the control, whereas the ubiquitination of the K48-linked modification was not affected (Figure 3B).


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 treatment abolishes RIPK1 ubiquitination and activates the NF-κB non-canonical pathwayA. C666-1 cells were treated or not treated with 40 μM NA for 8 h, and RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. B. Cells were transfected with the His-Ub (wt, K48, K63) plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. C. The effect of increasing doses of NA (0-40 μM) treatment for 8 or 24 h on the expression level of IKKα, IKKβ and NIK and downstream molecules, p65, p100, p52 and IκBα was analyzed by immunoblotting. β-Actin served as a loading control. D. NA inhibits NF-κB reporter gene expression. C666-1 cells were transiently transfected with an NF-κB-containing plasmid for 24 h. After transfection, cells were treated with the indicated concentrations of NA for 24 h. Gene expression was assayed by measuring luciferase activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385831&req=5

Figure 3: NA treatment abolishes RIPK1 ubiquitination and activates the NF-κB non-canonical pathwayA. C666-1 cells were treated or not treated with 40 μM NA for 8 h, and RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. B. Cells were transfected with the His-Ub (wt, K48, K63) plasmids for 48 h, then treated or not treated with 40 μM NA for 8 h. RIPK1 was immunoprecipitated and immunoblotted. β-Actin served as a loading control. C. The effect of increasing doses of NA (0-40 μM) treatment for 8 or 24 h on the expression level of IKKα, IKKβ and NIK and downstream molecules, p65, p100, p52 and IκBα was analyzed by immunoblotting. β-Actin served as a loading control. D. NA inhibits NF-κB reporter gene expression. C666-1 cells were transiently transfected with an NF-κB-containing plasmid for 24 h. After transfection, cells were treated with the indicated concentrations of NA for 24 h. Gene expression was assayed by measuring luciferase activity.
Mentions: Previous studies showed that triggering proteasomal degradation of cIAP1/2 could activate RIPK1 de-ubiquitination to induce cell death [12, 31]. Having established that NA could down-regulate cIAP1 and cIAP2, we examined the RIPK1 ubquitination level in cells treated or not treated with NA. Results showed that NA exposure led to a reduction in RIPK1 ubiquitination in C666-1 cells (Figure 3A). In cells treated with NA, the endogenous levels of cIAP1 and cIAP2 were decreased by co-immunoprecipitation with an anti-RIPK1 antibody (Figure 3A). Different types of ubiquitination modifications of RIPK1 can lead to different functions. To determine which ubiquitination modification of RIPK1 was affected by NA, Wt-Ub, His-UbK48 or His-UbK63 plasmids were introduced into C666-1 cells. NA treatment reduced the ubiquitination of the K63-linked modification compared with the control, whereas the ubiquitination of the K48-linked modification was not affected (Figure 3B).

Bottom Line: 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).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.

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