The modulation of necroptosis and its therapeutic potentials

Kim Chun 대한독성 유전단백체 학회 2021 Molecular & cellular toxicology Vol.17 No.2

Purpose of review Necroptosis is a form of cell death regulated by specific cellular protein machinery. Although the cell death is tightly controlled like apoptosis, another type of programed cell death, the biological features of necroptosis rather resemble necrosis that is defined as an uncontrolled accidental cell death. The pathway executing necroptosis relies on a protein kinase, RIPK3, and its downstream effector molecule, MLKL. Upon necroptosis initiating signals, both RIPK3 and MLKL undergo extensive post-translation modifications to construct a death complex called necrosome, finally leading to lysis of cell membrane. Preclinical mouse models demonstrated the physiological importance of necroptosis in the progress of various inflammation-associated diseases. The objective of this brief review is to introduce a new emerging concept in cell death biology and to provide a first entry into the research field of necroptosis. Recent findings The uncovering of necroptosis pathway brought a fundamental change in the basic concept that necrotic cell death is passive and unregulated. Currently, multiple small molecules that can target necrotic cell death are under development and some of them are under clinical trials to evaluate their therapeutic potentials. Better understanding of the molecular mechanism leveraging necroptosis will provide an unprecedented opportunity to pathological necrosis-driven human diseases. Purpose of review Necroptosis is a form of cell death regulated by specific cellular protein machinery. Although the cell death is tightly controlled like apoptosis, another type of programed cell death, the biological features of necroptosis rather resemble necrosis that is defined as an uncontrolled accidental cell death. The pathway executing necroptosis relies on a protein kinase, RIPK3, and its downstream effector molecule, MLKL. Upon necroptosis initiating signals, both RIPK3 and MLKL undergo extensive post-translation modifications to construct a death complex called necrosome, finally leading to lysis of cell membrane. Preclinical mouse models demonstrated the physiological importance of necroptosis in the progress of various inflammation-associated diseases. The objective of this brief review is to introduce a new emerging concept in cell death biology and to provide a first entry into the research field of necroptosis. Recent findings The uncovering of necroptosis pathway brought a fundamental change in the basic concept that necrotic cell death is passive and unregulated. Currently, multiple small molecules that can target necrotic cell death are under development and some of them are under clinical trials to evaluate their therapeutic potentials. Better understanding of the molecular mechanism leveraging necroptosis will provide an unprecedented opportunity to pathological necrosis-driven human diseases.

Oxidative Stress by Mitochondrial Dysfunction Contributes to the Activation of Necroptosis in Acute Respiratory Distress Syndrome

( Su Hwan Lee ),( Ju Hye Shin ),( Ah Young Leem ),( Kyung Soo Chung ),( Jin Hwa Lee ),( Moo Suk Park ),( Jong-seok Moon ),( Young Sam Kim ) 대한결핵 및 호흡기학회 2021 대한결핵 및 호흡기학회 추계학술대회 초록집 Vol.129 No.-

Background Necroptosis is linked to cell death in Acute respiratory distress syndrome (ARDS). And oxidative stress has also been implicated in the pathogenesis of ARDS. Although the effect of necroptosis through oxidative stress is identified in the cellular system, the mechanisms for the activation of necroptosis in ARDS remain unclear. This study aims to confirm whether oxidative stress contributes to the receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis associated with ARDS. Methods The seven human lung tissues with ARDS were investigated for the involvement of RIPK3-mediated necroptosis and oxidative stress using immunoblot and immunostaining. We also investigated whether the oxidative stress due to mitochondrial dysfunction caused by oligomycin, an inhibitor of ATP synthase, might exacerbate the RIPK3-mediated necroptosis in the lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. Results We found that RIPK3-mediated necroptosis was significantly elevated in patients with ARDS compared to the non-ARDS subjects. Oxidative stress was increased in patients with ARDS due to the accumulation of 4-HNE, a marker for oxidative stress. Both RIPK3-mediated necroptosis and oxidative stress were elevated in the lung epithelial cells of the patients with ARDS. The oxidative stress significantly increased lung injury in the LPS-induced ALI mouse model compared to the ALI model without oligomycin treatment. Moreover, the oxidative stress significantly increased the levels of RIPK3 and the release of damageassociated molecular patterns by necroptosis in the lung during ALI compared to the group administered the dose without treatment of oligomycin. Conclusion The Results showed that RIPK3-mediated necroptosis and oxidative stress were increased in human ARDS, and the LPS lung injury mouse model with oxidative stress had increased RIPK3-mediated necroptosis, lung injury, and DAMPs release. Our Results suggest that oxidative stress due to mitochondrial dysfunction might contribute to a critical mechanism for RIPK3- mediated necroptosis-induced lung injury during ARDS.

HS-173 as a novel inducer of RIP3-dependent necroptosis in lung cancer

Park, Jung Hee,Jung, Kyung Hee,Kim, Soo Jung,Yoon, Young-Chan,Yan, Hong Hua,Fang, Zhenghuan,Lee, Ji Eun,Lim, Joo Han,Mah, Shinmee,Hong, Sungwoo,Kim, You-Sun,Hong, Soon-Sun Elsevier 2019 Cancer letters Vol.444 No.-

<P><B>Abstract</B></P> <P>Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting kinase 3 (RIP3). Recently, necroptosis has gained attention as a novel alternative therapy to target cancer cells. In this study, we screened several chemotherapeutics used in preclinical and clinical studies, and identified a drug HS-173 that induces RIP3-mediated necroptosis. HS-173 decreased the cell survival in a dose-dependent manner in RIP3-expressing lung cancer cells, compared to the cells lacking RIP3. Also, the cell death induced by HS-173 was rescued by specific necroptosis inhibitors such as necrostatin-1 and dabrafenib. Additionally, HS-173 increased the phosphorylation of RIP3 and MLKL, which was decreased by necroptosis inhibitors, indicating that HS-173 activates RIP3/MLKL signaling in lung cancer cells. HS-173 increased the necroptotic events, as observed by the increased levels of HMGB1 and necroptotic morphological features. Furthermore, HS-173 inhibited the tumor growth by stimulation of necroptosis in mouse xenograft models. Our findings offer new insights into the role of HS-173 in inducing necroptosis by enhancing RIP3 expression and activating the RIP3/MLKL signaling pathway in lung cancer cells.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Necroptosis is a novel alternative therapy to target cancer cells. </LI> <LI> HS-173 induced necroptosis in lung cancer cells by enhancing the expression of RIP3 and activating RIP3/MLKL signaling pathway. </LI> <LI> The necroptotic events induced by HS-173 has been observed by the increased levels of HMGB1 and necroptotic morphological features. </LI> <LI> HS-173 inhibited the tumor growth by stimulation of necroptosis in mouse xenograft models. </LI> <LI> Our findings offer new insights into the role of HS-173 on inducing necroptosis through RIP3 activation in lung cancer cells. </LI> </UL> </P>

Necroptosis: an emerging type of cell death in liver diseases.

Saeed, Waqar Khalid,Jun, Dae Won WJG Press 2014 WORLD JOURNAL OF GASTROENTEROLOGY Vol.20 No.35

<P>Cell death has been extensively evaluated for decades and it is well recognized that pharmacological interventions directed to inhibit cell death can prevent significant cell loss and can thus improve an organ's physiological function. For long, only apoptosis was considered as a sole form of programmed cell death. Recently necroptosis, a RIP1/RIP3-dependent programmed cell death, has been identified as an apoptotic backup cell death mechanism with necrotic morphology. The evidences of necroptosis and protective effects achieved by blocking necroptosis have been extensively reported in recent past. However, only a few studies reported the evidence of necroptosis and protective effects achieved by inhibiting necroptosis in liver related disease conditions. Although the number of necroptosis initiators is increasing; however, interestingly, it is still unclear that what actually triggers necroptosis in different liver diseases or if there is always a different necroptosis initiator in each specific disease condition followed by specific downstream signaling molecules. Understanding the precise mechanism of necroptosis as well as counteracting other cell death pathways in liver diseases could provide a useful insight towards achieving extensive therapeutic significance. By targeting necroptosis and/or other parallel death pathways, a significant cell loss and thus a decrement in an organ's physiological function can be prevented.</P>

Inhibitory Role of TRIP-Br1/XIAP in Necroptosis under Nutrient/Serum Starvation

Sandag, Zolzaya,Jung, Samil,Quynh, Nguyen Thi Ngoc,Myagmarjav, Davaajargal,Anh, Nguyen Hai,Le, Dan-Diem Thi,Lee, Beom Suk,Mongre, Raj Kumar,Jo, Taeyeon,Lee, MyeongSok Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.3

Currently, many available anti-cancer therapies are targeting apoptosis. However, many cancer cells have acquired resistance to apoptosis. To overcome this problem, simultaneous induction of other types of programmed cell death in addition to apoptosis of cancer cells might be an attractive strategy. For this purpose, we initially investigated the inhibitory role of TRIP-Br1/XIAP in necroptosis, a regulated form of necrosis, under nutrient/serum starvation. Our data showed that necroptosis was significantly induced in all tested 9 different types of cancer cell lines in response to prolonged serum starvation. Among them, necroptosis was induced at a relatively lower level in MCF-7 breast cancer line that was highly resistant to apoptosis than that in other cancer cell lines. Interestingly, TRIP-Br1 oncogenic protein level was found to be very high in this cell line. Up-regulated TRIP-Br1 suppressed necroptosis by repressing reactive oxygen species generation. Such suppression of necroptosis was greatly enhanced by XIAP, a potent inhibitor of apoptosis. Our data also showed that TRIP-Br1 increased XIAP phosphorylation at serine87, an active form of XIAP. Our mitochondrial fractionation data revealed that TRIP-Br1 protein level was greatly increased in the mitochondria upon serum starvation. It suppressed the export of CypD, a vital regulator in mitochondria-mediated necroptosis, from mitochondria to cytosol. TRIP-Br1 also suppressed shikonin-mediated necroptosis, but not TNF-α-mediated necroptosis, implying possible presence of another signaling pathway in necroptosis. Taken together, our results suggest that TRIP-Br1/XIAP can function as onco-proteins by suppressing necroptosis of cancer cells under nutrient/serum starvation.

Expression of necroptosis effectors in mouse oocytes

Jeong Min An,Dayoung Park,Juhee Lee,Haengseok Song,Hyunjung J. Lim 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Necroptosis is one of the regulated cell death pathways, which shares several upstream signaling elements with apoptosis. It and can be triggered by tumor necrosis factor α (TNFα) and other death ligands. Activation of necroptosis involves sequential actions of cytoplasmic effectors, receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL). Necroptosis leads to plasma membrane rupture and release of danger associated molecular patterns (DAMPS) which includes various inflammatory chemokines. In this study, we examined whether major necroptosis effectors are expressed in mouse oocytes and whether oocytes respond to necroptosis-inducing signal. We examined expression of RIPK1, RIPK3, and MLKL in mouse oocytes by RT-PCR and immunofluorescence staining. All three factors are expressed in germinal vesicle (GV) and polar body (PB) stage oocytes. RIPK3 and MLKL are localized within the nucleus of GV oocytes when the nuclear membrane is intact, but do not show distinct patterns after GV breakdown. We then investigated whether GSK"872 and dabrafenib, both of which are RIPK3 inhibitors, affect oocyte maturation in vitro. Dabrafenib-treated oocytes showed maturation arrest (54.7% arrested at MI stage) while GSK’872 did not show such effect. We next examined if treatment of TSZ (necroptosis-inducing cocktail containing TNFα, Smac mimetics, and zVAD-FMK) causes necroptotic activation in oocytes. Expression of two representative DAMP genes, Cxcl1 and Cxcl10, were examined in TSZ-treated oocytes by RT-PCR. Unlike L929 cells which expressed both genes upon TSZ treatment, oocytes did not show induction of these two DAMP genes, suggesting that oocytes may respond differently to exogenous necroptosis-inducing signals. Collectively, the results show that mouse oocytes harbor necroptosis machinery which may influence maturation and development.- This work was supported by the National Research Foundation of Korea (NRF) grant 2020R1A2C100412 funded by the Korea government.

New role of E3 ubiquitin ligase in the regulation of necroptosis

( Jinho Seo ),( Eun-woo Lee ),( Jaewhan Song ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.5

Necroptosis is a well-known form of caspase-independent cell death. Necroptosis can be triggered by various extrinsic stimuli, including death ligands in the presence of receptorinteracting protein kinase 3 (RIPK3), a key mediator of necroptosis induction. Our recent studies have revealed that C-terminus HSC-70 interacting protein (CHIP), an E3 ligase, can function as an inhibitor of necroptosis. CHIP-/- mouse embryonic fibroblast showed higher sensitivity to necrotic stimuli than wild-type mouse embryonic fibroblast cells. Deleterious effects of CHIP knockout MEFs were retrieved by RIPK3 depletion. We found that CHIP negatively regulated RIPK3 and RIPK1 by ubiquitylation- and lysosome- dependent degradation. In addition, CHIP-/- mice showed postnatal lethality with intestinal defects that could be rescued by crossing with RIPK3-/- mice. These results suggest that CHIP is a negative regulator of RIPK1 and RIPK3, thus inhibiting necroptosis. [BMB Reports 2016; 49(5): 247-248]

New role of E3 ubiquitin ligase in the regulation of necroptosis

( Jin Ho Seo ),( Eun-woo Lee ),( Jae Whan Song ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.6

Necroptosis is a well-known form of caspase-independent cell death. Necroptosis can be triggered by various extrinsic stimuli, including death ligands in the presence of receptorinteracting protein kinase 3 (RIPK3), a key mediator of necroptosis induction. Our recent studies have revealed that C-terminus HSC-70 interacting protein (CHIP), an E3 ligase, can function as an inhibitor of necroptosis. CHIP-/- mouse embryonic fibroblast showed higher sensitivity to necrotic stimuli than wild-type mouse embryonic fibroblast cells. Deleterious effects of CHIP knockout MEFs were retrieved by RIPK3 depletion. We found that CHIP negatively regulated RIPK3 and RIPK1 by ubiquitylation- and lysosome- dependent degradation. In addition, CHIP-/- mice showed postnatal lethality with intestinal defects that could be rescued by crossing with RIPK3-/- mice. These results suggest that CHIP is a negative regulator of RIPK1 and RIPK3, thus inhibiting necroptosis. [BMB Reports 2016; 49(5): 247-248]

Investigation on the role of necroptosis in alopecia areata: A preliminary study

( Yong Hyun Jang ),( Sun Young Moon ),( Weon Ju Lee ),( Seok Jong Lee ),( Mei Ling Jin ),( Sang Hyun Kim ),( Do Won Kim ) 대한피부과학회 2015 대한피부과학회 학술발표대회집 Vol.67 No.2

Background: There are three modes of programmed cell death, namely, apoptosis, autophagy and necroptosis. Among them, necroptosis shows morphological features similar to necrosis. However, unlike necrosis, cell death in necroptosis is highly regulated by a programmed intracellular platform, as in apoptosis. Objectives: To investigate the occurrence of necroptosis in alopecia areata (AA) patients and its contribution to cell death following autoimmunity in AA. Methods: We collected human scalp skin samples of 15 patients (6 men and 9 women, 36.1【 4.0 years) with AA and scalp samples of 13 healthy persons (9 men and 4 women, 48.4【 5.6 years) as controls. The mRNAexpression of RIP3, RIP1, MLKL and caspase-8 was evaluated by real- time PCR. Protein expression of RIP3 and RIP1 was also examined by western blotting and immunohistochemical staining. Results: RIP3, RIP1, and MLKL mRNA expressions were not upregulated in the lesional scalp skin of AA patients compared to that in controls and the expression level of caspase-8 mRNA was also not significantly different. Moreover, RIP3 and RIP1 protein expressions were not upregulated in the lesional skin of AA patients compared with normal skin. Conclusion: The results of our preliminary study suggest that necroptosis is not involved in the pathogenesis of AA. However, further studies are needed to elucidate the potential role of necroptosis in AA and other inflammatory hair diseases.

Genipin protects d-galactosamine and lipopolysaccharide-induced hepatic injury through suppression of the necroptosis-mediated inflammasome signaling

Seo, M.J.,Hong, J.M.,Kim, S.J.,Lee, S.M. North-Holland ; Elsevier Science Ltd 2017 european journal of pharmacology Vol.812 No.-

<P>Acute liver failure (ALF) is a life-threatening syndrome resulting from massive inflammation and hepatocyte death. Necroptosis, a programmed cell death controlled by receptor-interacting protein kinase (RIP) 1 and RIP3, has been shown to play an important role in regulating inflammation via crosstalk between other intracellular signaling. The inflammasome is a major intracellular multiprotein that induces inflammatory responses by mediating immune cell infiltration, thus potentiating injury. Genipin, a major active compound of the gardenia fruit, exhibits anti-inflammatory, antioxidant, and anti-apoptotic properties. This study investigated the hepatoprotective mechanisms of genipin on D-galactosamine (GalN) and lipopolysaccharide (LPS)induced ALF, particularly focusing on interaction between necroptosis and inflammasome. Mice were given an intraperitoneal injection of genipin (25, 50, and 100 mg/kg) or necrostatin-1 (Nec-1, a necroptosis inhibitor; 1.8 mg/kg) 1 h prior to GalN (800 mg/kg)/LPS (40 mu g/kg) injection and were killed 3 h after GalN/LPS injection. Genipin improved the survival rate and attenuated increases in serum aminotransferase activities and inflammatory cytokines after GalN/LPS injection. Genipin reduced GalN/LPS-induced increases in RIP3, phosphorylated RIP1 and RIP3 protein expression, and RIP1/RIP3 necrosome complex, similar to the effects of Nec-1. GalN/LPS significantly increased serum levels of high-mobility group box 1 and interleukin (IL)-33, which were attenuated by genipin and Nec-1. Moreover, similar to Nec-1, genipin attenuated GalN/LPSinduced increases in the protein expression levels of NLRP3, ASC, and caspase-1, inflammasome components, and levels of liver and serum IL-1 beta. Taken together, our findings suggest that genipin ameliorates GalN/LPSinduced hepatocellular damage by suppressing necroptosis-mediated inflammasome signaling.</P>