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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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The RIPK/NF-κB pathway mediates NA-induced TNFα production and cell death.cAC666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA for 8 h and relative levels of the TNFα transcript were determined and compared with β-actin and the fold change was calculated by comparing with DMSO-treated cells. B. C666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. C. The effect of NAI and Bay117082 (Bay) on NA-induced TNFα transcription. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). TNFα mRNA level was analyzed by quantitative-real time-PCR. D. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). Cell viability was estimated by MTS assay. E. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with NA. TNFα mRNA level was analyzed by quantitative-real time-PCR. F. C666-1 cells transfected for 48 h with siRNA targeting IKKα or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. G. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with different doses of NA. Viability of C666-1 cells was analyzed by MTS assay. Data are shown as means ± S.D. of values from three independent experiments. *p<0.05. **p<0.001. ***p<0.0001
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Figure 4: The RIPK/NF-κB pathway mediates NA-induced TNFα production and cell death.cAC666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA for 8 h and relative levels of the TNFα transcript were determined and compared with β-actin and the fold change was calculated by comparing with DMSO-treated cells. B. C666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. C. The effect of NAI and Bay117082 (Bay) on NA-induced TNFα transcription. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). TNFα mRNA level was analyzed by quantitative-real time-PCR. D. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). Cell viability was estimated by MTS assay. E. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with NA. TNFα mRNA level was analyzed by quantitative-real time-PCR. F. C666-1 cells transfected for 48 h with siRNA targeting IKKα or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. G. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with different doses of NA. Viability of C666-1 cells was analyzed by MTS assay. Data are shown as means ± S.D. of values from three independent experiments. *p<0.05. **p<0.001. ***p<0.0001

Mentions: Researchers reported that RIPK1 and JNKs mediated TNFα production in response to zVAD-fmk and IAP antagonists [8, 9]. Thus, the roles of RIPK1 and JNKs were analyzed to further investigate the molecular mechanisms of TNFα production in NA-treated cells. To determine how TNFα production is activated, we used real-time PCR to measure TNFα mRNA levels after NA treatment. Stimulation with NA increased TNFα mRNA expression, and knockdown of RIPK1, blocked the increase in TNFα mRNA level (Figure 4A). Thus, RIPK1 activates the transcription of TNFα after NA treatment. In a previous study, we found that NA treatment could induce JNKs activation[24]. SP600125 (SP), a small molecular inhibitor of JNKs, was used to verify the possibility that JNKs activation may trigger autocrine production of TNFα. SP treatment could not reduce TNFα transcription level induced by NA (Supplementary Figure 2A). Furthermore, results showed that knockdown of RIPK1 inhibited NA-induced TNFα secretion (Figure 4B), demonstrating that RIPK1 is required for TNFα production. In addition, inhibition of JNKs by SP treatment failed to block NA-induced TNFα production (Supplementary Figure 2B). Although JNKs were phosphorylated and activated by NA treatment, NA-induced TNFα secretion occurred independently of JNKs activation. JNKs activation might be a side effect of NA stimulation. Therefore, these results implied that RIPK1, but not JNKs, contributes to NA-induced TNFα production.


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)

The RIPK/NF-κB pathway mediates NA-induced TNFα production and cell death.cAC666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA for 8 h and relative levels of the TNFα transcript were determined and compared with β-actin and the fold change was calculated by comparing with DMSO-treated cells. B. C666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. C. The effect of NAI and Bay117082 (Bay) on NA-induced TNFα transcription. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). TNFα mRNA level was analyzed by quantitative-real time-PCR. D. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). Cell viability was estimated by MTS assay. E. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with NA. TNFα mRNA level was analyzed by quantitative-real time-PCR. F. C666-1 cells transfected for 48 h with siRNA targeting IKKα or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. G. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with different doses of NA. Viability of C666-1 cells was analyzed by MTS assay. Data are shown as means ± S.D. of values from three independent experiments. *p<0.05. **p<0.001. ***p<0.0001
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Figure 4: The RIPK/NF-κB pathway mediates NA-induced TNFα production and cell death.cAC666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA for 8 h and relative levels of the TNFα transcript were determined and compared with β-actin and the fold change was calculated by comparing with DMSO-treated cells. B. C666-1 cells transfected for 48 h with siRNA targeting RIPK1 or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. C. The effect of NAI and Bay117082 (Bay) on NA-induced TNFα transcription. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). TNFα mRNA level was analyzed by quantitative-real time-PCR. D. Cells were pre-treated with NAI (40 μM) and Bay117082 (Bay) (5 μM) for 1 h, and then treated or not treated with NA (40 μM). Cell viability was estimated by MTS assay. E. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with NA. TNFα mRNA level was analyzed by quantitative-real time-PCR. F. C666-1 cells transfected for 48 h with siRNA targeting IKKα or an empty vector control were treated or not treated with 40 μM NA and harvested at the indicated time points. The presence of TNFα in conditioned cell culture media was measured by Elisa assay. G. IKKα in C666-1 cells was knocked down with siRNA, and then cells were treated with different doses of NA. Viability of C666-1 cells was analyzed by MTS assay. Data are shown as means ± S.D. of values from three independent experiments. *p<0.05. **p<0.001. ***p<0.0001
Mentions: Researchers reported that RIPK1 and JNKs mediated TNFα production in response to zVAD-fmk and IAP antagonists [8, 9]. Thus, the roles of RIPK1 and JNKs were analyzed to further investigate the molecular mechanisms of TNFα production in NA-treated cells. To determine how TNFα production is activated, we used real-time PCR to measure TNFα mRNA levels after NA treatment. Stimulation with NA increased TNFα mRNA expression, and knockdown of RIPK1, blocked the increase in TNFα mRNA level (Figure 4A). Thus, RIPK1 activates the transcription of TNFα after NA treatment. In a previous study, we found that NA treatment could induce JNKs activation[24]. SP600125 (SP), a small molecular inhibitor of JNKs, was used to verify the possibility that JNKs activation may trigger autocrine production of TNFα. SP treatment could not reduce TNFα transcription level induced by NA (Supplementary Figure 2A). Furthermore, results showed that knockdown of RIPK1 inhibited NA-induced TNFα secretion (Figure 4B), demonstrating that RIPK1 is required for TNFα production. In addition, inhibition of JNKs by SP treatment failed to block NA-induced TNFα production (Supplementary Figure 2B). Although JNKs were phosphorylated and activated by NA treatment, NA-induced TNFα secretion occurred independently of JNKs activation. JNKs activation might be a side effect of NA stimulation. Therefore, these results implied that RIPK1, but not JNKs, contributes to NA-induced TNFα production.

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