Inflammatory cell death PANoptosis is induced by the anti-cancer curaxin CBL0137 via eliciting the assembly of ZBP1-associated PANoptosome.

OBJECTIVE: PANoptosis, a new form of regulated cell death, concomitantly manifests hallmarks for pyroptosis, apoptosis, and necroptosis. It has been usually observed in macrophages, a class of widely distributed innate immune cells in various tissues, upon pathogenic infections. The second-generation curaxin, CBL0137, can trigger necroptosis and apoptosis in cancer-associated fibroblasts. This study aimed to explore whether CBL0137 induces PANoptosis in macrophages in vitro and in mouse tissues in vivo. METHODS: Bone marrow-derived macrophages and J774A.1 cells were treated with CBL0137 or its combination with LPS for indicated time periods. Cell death was assayed by propidium iodide staining and immunoblotting. Immunofluorescence microscopy was used to detect cellular protein distribution. Mice were administered with CBL0137 plus LPS and their serum and tissues were collected for biochemical and histopathological analyses, respectively. RESULTS: The results showed that CBL0137 alone or in combination with LPS induced time- and dose-dependent cell death in macrophages, which was inhibited by a combination of multiple forms of cell death inhibitors but not each alone. This cell death was independent of NLRP3 expression. CBL0137 or CBL0137 + LPS-induced cell death was characterized by simultaneously increased hallmarks for pyroptosis, apoptosis and necroptosis, indicating that this is PANoptosis. Induction of PANoptosis was associated with Z-DNA formation in the nucleus and likely assembly of PANoptosome. ZBP1 was critical in mediating CBL0137 + LPS-induced cell death likely by sensing Z-DNA. Moreover, intraperitoneal administration of CBL0137 plus LPS induced systemic inflammatory responses and caused multi-organ (including the liver, kidney and lung) injury in mice due to induction of PANoptosis in these organs. CONCLUSIONS: CBL0137 alone or plus inflammatory stimulation induces PANoptosis both in vitro and in vivo, which is associated with systemic inflammatory responses in mice.

Blocking reverse electron transfer-mediated mitochondrial DNA oxidation rescues cells from PANoptosis.

PANoptosis is a new type of cell death featured with pyroptosis, apoptosis and necroptosis, and is implicated in organ injury and mortality in various inflammatory diseases, such as sepsis and hemophagocytic lymphohistiocytosis (HLH). Reverse electron transport (RET)-mediated mitochondrial reactive oxygen species (mtROS) has been shown to contribute to pyroptosis and necroptosis. In this study we investigated the roles of mtROS and RET in PANoptosis induced by TGF-ß-activated kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (Oxo) plus lipopolysaccharide (LPS) as well as the effects of anti-RET reagents on PANoptosis. We showed that pretreatment with anti-RET reagents 1-methoxy PMS (MPMS) or dimethyl fumarate (DMF) dose-dependently inhibited PANoptosis in macrophages BMDMs and J774A.1 cells induced by Oxo/LPS treatment assayed by propidium iodide (PI) staining. The three arms of the PANoptosis signaling pathway, namely pyroptosis, apoptosis and necroptosis signaling, as well as the formation of PANoptosomes were all inhibited by MPMS or DMF. We demonstrated that Oxo/LPS treatment induced RET and mtROS in BMDMs, which were reversed by MPMS or DMF pretreatment. Interestingly, the PANoptosome was co-located with mitochondria, in which the mitochondrial DNA was oxidized. MPMS and DMF fully blocked the mtROS production and the formation of PANoptosome induced by Oxo plus LPS treatment. An HLH mouse model was established by poly(I:C)/LPS challenge. Pretreatment with DMF (50 mg·kg-1·d-1, i.g. for 3 days) or MPMS (10 mg·kg-1·d-1, i.p. for 2 days) (DMF i.g. MPMS i.p.) effectively alleviated HLH lesions accompanied by decreased hallmarks of PANoptosis in the liver and kidney. Collectively, RET and mtDNA play crucial roles in PANoptosis induction and anti-RET reagents represent a novel class of PANoptosis inhibitors by blocking oxidation of mtDNA, highlighting their potential application in treating PANoptosis-related inflammatory diseases. PANoptotic stimulation induces reverse electron transport (RET) and reactive oxygen species (ROS) in mitochondia, while 1-methoxy PMS and dimethyl fumarate can inhibit PANoptosis by suppressing RETmediated oxidation of mitochondrial DNA.

Triptolide induces PANoptosis in macrophages and causes organ injury in mice.

Macrophages represent the first lines of innate defense against pathogenic infections and are poised to undergo multiple forms of regulated cell death (RCD) upon infections or toxic stimuli, leading to multiple organ injury. Triptolide, an active compound isolated from Tripterygium wilfordii Hook F., possesses various pharmacological activities including anti-tumor and anti-inflammatory effects, but its applications have been hampered by toxic adverse effects. It remains unknown whether and how triptolide induces different forms of RCD in macrophages. In this study, we showed that triptolide exhibited significant cytotoxicity on cultured macrophages in vitro, which was associated with multiple forms of lytic cell death that could not be fully suppressed by any one specific inhibitor for a single form of RCD. Consistently, triptolide induced the simultaneous activation of pyroptotic, apoptotic and necroptotic hallmarks, which was accompanied by the co-localization of ASC specks respectively with RIPK3 or caspase-8 as well as their interaction with each other, indicating the formation of PANoptosome and thus the induction of PANoptosis. Triptolide-induced PANoptosis was associated with mitochondrial dysfunction and ROS production. PANoptosis was also induced by triptolide in mouse peritoneal macrophages in vivo. Furthermore, triptolide caused kidney and liver injury, which was associated with systemic inflammatory responses and the activation of hallmarks for PANoptosis in vivo. Collectively, our data reveal that triptolide induces PANoptosis in macrophages in vitro and exhibits nephrotoxicity and hepatotoxicity associated with induction of PANoptosis in vivo, suggesting a new avenue to alleviate triptolide's toxicity by harnessing PANoptosis.

Dimethyl fumarate inhibits necroptosis and alleviates systemic inflammatory response syndrome by blocking the RIPK1-RIPK3-MLKL axis.

Necroptosis has been implicated in various inflammatory diseases including tumor-necrosis factor-α (TNF-α)-induced systemic inflammatory response syndrome (SIRS). Dimethyl fumarate (DMF), a first-line drug for treating relapsing-remitting multiple sclerosis (RRMS), has been shown to be effective against various inflammatory diseases. However, it is still unclear whether DMF can inhibit necroptosis and confer protection against SIRS. In this study, we found that DMF significantly inhibited necroptotic cell death in macrophages induced by different necroptotic stimulations. Both the autophosphorylation of receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3 and the downstream phosphorylation and oligomerization of MLKL were robustly suppressed by DMF. Accompanying the suppression of necroptotic signaling, DMF blocked the mitochondrial reverse electron transport (RET) induced by necroptotic stimulation, which was associated with its electrophilic property. Several well-known anti-RET reagents also markedly inhibited the activation of the RIPK1-RIPK3-MLKL axis accompanied by decreased necrotic cell death, indicating a critical role of RET in necroptotic signaling. DMF and other anti-RET reagents suppressed the ubiquitination of RIPK1 and RIPK3, and they attenuated the formation of necrosome. Moreover, oral administration of DMF significantly alleviated the severity of TNF-α-induced SIRS in mice. Consistent with this, DMF mitigated TNF-α-induced cecal, uterine, and lung damage accompanied by diminished RIPK3-MLKL signaling. Collectively, DMF represents a new necroptosis inhibitor that suppresses the RIPK1-RIPK3-MLKL axis through blocking mitochondrial RET. Our study highlights DMF's potential therapeutic applications for treating SIRS-associated diseases.

Theaflavin mitigates acute gouty peritonitis and septic organ injury in mice by suppressing NLRP3 inflammasome assembly.

Activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays important role in defending against infections, but its aberrant activation is causally linked to many inflammatory diseases, thus being a therapeutic target for these diseases. Theaflavin, one major ingredient of black tea, exhibits potent anti-inflammatory and anti-oxidative activities. In this study, we investigated the therapeutic effects of theaflavin against NLRP3 inflammasome activation in macrophages in vitro and in animal models of related diseases. We showed that theaflavin (50, 100, 200 µM) dose-dependently inhibited NLRP3 inflammasome activation in LPS-primed macrophages stimulated with ATP, nigericin or monosodium urate crystals (MSU), evidenced by reduced release of caspase-1p10 and mature interleukin-1ß (IL-1ß). Theaflavin treatment also inhibited pyroptosis as shown by decreased generation of N-terminal fragment of gasdermin D (GSDMD-NT) and propidium iodide incorporation. Consistent with these, theaflavin treatment suppressed ASC speck formation and oligomerization in macrophages stimulated with ATP or nigericin, suggesting reduced inflammasome assembly. We revealed that theaflavin-induced inhibition on NLRP3 inflammasome assembly and pyroptosis resulted from ameliorated mitochondrial dysfunction and reduced mitochondrial ROS production, thereby suppressing interaction between NLRP3 and NEK7 downstream of ROS. Moreover, we showed that oral administration of theaflavin significantly attenuated MSU-induced mouse peritonitis and improved the survival of mice with bacterial sepsis. Consistently, theaflavin administration significantly reduced serum levels of inflammatory cytokines including IL-1ß and attenuated liver inflammation and renal injury of mice with sepsis, concomitant with reduced generation of caspase-1p10 and GSDMD-NT in the liver and kidney. Together, we demonstrate that theaflavin suppresses NLRP3 inflammasome activation and pyroptosis by protecting mitochondrial function, thus mitigating acute gouty peritonitis and bacterial sepsis in mice, highlighting a potential application in treating NLRP3 inflammasome-related diseases.

Gout-associated monosodium urate crystal-induced necrosis is independent of NLRP3 activity but can be suppressed by combined inhibitors for multiple signaling pathways.

Monosodium urate (MSU) crystals, the etiological agent of gout, are formed in joints and periarticular tissues due to long-lasting hyperuricemia. Although MSU crystal-triggered NLRP3 inflammasome activation and interleukin 1ß (IL-1ß) release are known to have key roles in gouty arthritis, recent studies revealed that MSU crystal-induced necrosis also plays a critical role in this process. However, it remains unknown what forms of necrosis have been induced and whether combined cell death inhibitors can block such necrosis. Here, we showed that MSU crystal-induced necrosis in murine macrophages was not dependent on NLRP3 inflammasome activation, as neither genetic deletion nor pharmacological blockade of the NLRP3 pathway inhibited the necrosis. Although many cell death pathways (such as ferroptosis and pyroptosis) inhibitors or reactive oxygen species inhibitors did not have any suppressive effects, necroptosis pathway inhibitors GSK'872 (RIPK3 inhibitor), and GW806742X (MLKL inhibitor) dose-dependently inhibited MSU crystal-induced necrosis. Moreover, a triple combination of GSK'872, GW806742X, and IDN-6556 (pan-caspase inhibitor) displayed enhanced inhibition of the necrosis, which was further fortified by the addition of MCC950 (NLRP3 inhibitor), suggesting that multiple cell death pathways might have been triggered by MSU crystals. Baicalin, a previously identified inhibitor of NLRP3, inhibited MSU crystal-induced inflammasome activation and suppressed the necrosis in macrophages. Besides, baicalin gavage significantly ameliorated MSU crystal-induced peritonitis in mice. Altogether, our data indicate that MSU crystals induce NLRP3-independent necrosis, which can be inhibited by combined inhibitors for multiple signaling pathways, highlighting a new avenue for the treatment of gouty arthritis.

Chemotherapeutic paclitaxel and cisplatin differentially induce pyroptosis in A549 lung cancer cells via caspase-3/GSDME activation.

Gasdermin E (GSDME) has an important role in inducing secondary necrosis/pyroptosis. Upon apoptotic stimulation, it can be cleaved by activated caspase-3 to generate its N-terminal fragment (GSDME-NT), which executes pyroptosis by perforating the plasma membrane. GSDME is expressed in many human lung cancers including A549 cells. Paclitaxel and cisplatin are two representative chemotherapeutic agents for lung cancers, which induce apoptosis via different action mechanisms. However, it remains unclear whether they can induce GSDME-mediated secondary necrosis/pyroptosis in lung A549 cancer cells. Here we showed that both paclitaxel and cisplatin evidently induced apoptosis in A549 cells as revealed by the activation of multiple apoptotic markers. Notably, some of the dying cells displayed characteristic morphology of secondary necrosis/pyroptosis, by blowing large bubbles from the cellular membrane accompanied by caspase-3 activation and GSDME-NT generation. But the ability of cisplatin to induce this phenomenon was much stronger than that of paclitaxel. Consistent with this, cisplatin triggered much higher activation of caspase-3 and generation of GSDME-NT than paclitaxel, suggesting that the levels of secondary necrosis/pyroptosis correlated with the levels of active caspase-3 and GSDME-NT. Supporting this, caspase-3 specific inhibitor (Ac-DEVD-CHO) suppressed cisplatin-induced GSDME-NT generation and concurrently reduced the secondary necrosis/pyroptosis. Besides, GSDME knockdown significantly inhibited cisplatin- but not paclitaxel-induced secondary necrosis/pyroptosis. These results indicated that cisplatin induced higher levels of secondary necrosis/pyroptosis in A549 cells than paclitaxel, suggesting that cisplatin may provide additional advantages in the treatment of lung cancers with high levels of GSDME expression.

ATP induces caspase-3/gasdermin E-mediated pyroptosis in NLRP3 pathway-blocked murine macrophages.

ATP acts as a canonical activator to induce NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome activation in macrophages, leading to caspase-1/gasdermin D (GSDMD)-mediated pyroptosis. It remains unclear whether ATP can induce pyroptosis in macrophages when the NLRP3 pathway is blocked by pathogenic infection. In this study, we used cellular models to mimic such blockade of NLRP3 activation: bone marrow-derived macrophages (BMDMs) treated with NLRP3-specific inhibitor MCC950 and RAW264.7 cells deficient in ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression. The results showed that ATP treatment induced lytic cell death morphologically resembling canonical pyroptosis in both MCC950-treated BMDMs and RAW264.7 cells, but did not cause the activation of caspase-1 (by detecting caspase-1p10 and mature interleukin-1ß) and cleavage of GSDMD. Instead, both apoptotic initiator (caspase-8 and -9) and executioner (caspase-3 and -7) caspases were evidently activated and gasdermin E (GSDME) was cleaved to generate its N-terminal fragment (GSDME-NT) which executes pyroptosis. The GSDME-NT production and lytic cell death induced by ATP were diminished by caspase-3 inhibitor. In BMDMs without MCC950 treatment, ATP induced the formation of ASC specks which were co-localized with caspase-8; with MCC950 treatment, however, ATP did not induced the formation of ASC specks. In RAW264.7 cells, knockdown of GSDME by small interfering RNA attenuated ATP-induced lytic cell death and HMGB1 release into culture supernatants. Collectively, our results indicate that ATP induces pyroptosis in macrophages through the caspase-3/GSDME axis when the canonical NLRP3 pathway is blocked, suggestive of an alternative mechanism for combating against pathogen evasion.

Chemotherapeutic agent CPT-11 eliminates peritoneal resident macrophages by inducing apoptosis.

CPT-11 (Irinotecan) is a first-line chemotherapeutic agent in clinic, but it may induce side effects including diarrhea and enteritis in patients. The underlying mechanism of CPT-11's intestinal toxicity is unclear. Peritoneal resident macrophages have been reported to be important for the maintenance of intestinal homeostasis. In this study, we evaluated the cytotoxic effects of CPT-11 on mouse peritoneal resident macrophages. CPT-11 was administered intraperitoneally to mice and their peritoneal exudate cells were isolated for evaluation. CPT-11 treatment strikingly decreased the ratio of F4/80(hi)MHCII(low) large peritoneal macrophages (LPMs), which are regarded as prenatally-originated peritoneal resident macrophages. Consistent with this, the transcription factor GATA6 specifically expressed in LPMs was barely detectable in the macrophages from CPT-11-treated mice, indicative of elimination of LPMs. Such elimination of LPMs was at least partly due to CPT-induced apoptosis in macrophages, because inhibition of apoptosis by caspase-3 inhibitor z-DEVD-fmk significantly diminished the loss of GATA6(+) LPMs. As GATA6 is a transcription factor that controls expression of multiple genes regulating peritoneal B-1 cell development and translocation, elimination of GATA6(+) LPMs led to a great reduction in B-1 cells in the peritoneal cavity after CPT-11 treatment. These results indicated that CPT-11-induced apoptosis contributed to the elimination of peritoneal resident macrophages, which might in turn impair the function of peritoneal B-1 cells in maintaining intestinal homeostasis. Our findings may at least partly explain why CPT-11 treatment in cancer patients induces diarrhea and enteritis, which may provide a novel avenue to prevent such side effects.

Gossypol induces pyroptosis in mouse macrophages via a non-canonical inflammasome pathway.

Gossypol, a polyphenolic compound isolated from cottonseeds, has been reported to possess many pharmacological activities, but whether it can influence inflammasome activation remains unclear. In this study, we found that in mouse macrophages, gossypol induced cell death characterized by rapid membrane rupture and robust release of HMGB1 and pro-caspase-11 comparable to ATP treatment, suggesting an induction of pyroptotic cell death. Unlike ATP, gossypol induced much low levels of mature interleukin-1ß (IL-1ß) secretion from mouse peritoneal macrophages primed with LPS, although it caused pro-IL-1ß release similar to that of ATP. Consistent with this, activated caspase-1 responsible for pro-IL-1ß maturation was undetectable in gossypol-treated peritoneal macrophages. Besides, RAW 264.7 cells lacking ASC expression and caspase-1 activation also underwent pyroptotic cell death upon gossypol treatment. In further support of pyroptosis induction, both pan-caspase inhibitor and caspase-1 subfamily inhibitor, but not caspase-3 inhibitor, could sharply suppress gossypol-induced cell death. Other canonical pyroptotic inhibitors, including potassium chloride and N-acetyl-l-cysteine, could suppress ATP-induced pyroptosis but failed to inhibit or even enhanced gossypol-induced cell death, whereas nonspecific pore-formation inhibitor glycine could attenuate this process, suggesting involvement of a non-canonical pathway. Of note, gossypol treatment eliminated thioglycollate-induced macrophages in the peritoneal cavity with recruitment of other leukocytes. Moreover, gossypol administration markedly decreased the survival of mice in a bacterial sepsis model. Collectively, these results suggested that gossypol induced pyroptosis in mouse macrophages via a non-canonical inflammasome pathway, which raises a concern for its in vivo cytotoxicity to macrophages.

Extracellular ATP contributes to the reactive oxygen species burst and exaggerated mitochondrial damage in D-galactosamine and lipopolysaccharide-induced fulminant hepatitis.

Fulminant hepatitis (FH) is a severe clinical syndrome leading to hepatic failure and even mortality. D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) challenge is commonly used to establish an FH mouse model, but the mechanism underlying D-GalN/LPS-induced liver injury is incompletely understood. Previously, it has been reported that extracellular ATP that can be released under cytotoxic and inflammatory stresses serves as a damage signal to induce potassium ion efflux and trigger the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome activation through binding to P2X7 receptor. In this study, we tried to investigate whether it contributed to the fulminant hepatitis (FH) induced by D-GalN plus LPS. In an in vitro cellular model, D-GalN plus extracellular ATP, instead of D-GalN alone, induced pyroptosis and apoptosis, accompanied by mitochondrial reactive oxygen species (ROS) burst, and the oligomerization of Drp1, Bcl-2, and Bak, as well as the loss of mitochondrial membrane potential in LPS-primed macrophages, well reproducing the events induced by D-GalN and LPS in vivo. Moreover, these events in the cellular model were markedly suppressed by both A-804598 (an ATP receptor P2X7R inhibitor) and glibenclamide (an ATP-sensitive potassium ion channel inhibitor); in the FH mouse model, administration of A-804598 significantly mitigated D-GalN/LPS-induced hepatic injury, mitochondrial damage, and the activation of apoptosis and pyroptosis signaling, corroborating the contribution of extracellular ATP to the cell death. Collectively, our data suggest that extracellular ATP acts as an autologous damage-associated molecular pattern to augment mitochondrial damage, hepatic cell death, and liver injury in D-GalN/LPS-induced FH mouse model.

Potassium ion efflux induces exaggerated mitochondrial damage and non-pyroptotic necrosis when energy metabolism is blocked.

Damage-associated molecular patterns (DAMPs) such as extracellular ATP and nigericin (a bacterial toxin) not only act as potassium ion (K+) efflux inducers to activate NLRP3 inflammasome, leading to pyroptosis, but also induce cell death independently of NLRP3 expression. However, the roles of energy metabolism in determining NLRP3-dependent pyroptosis and -independent necrosis upon K+ efflux are incompletely understood. Here we established cellular models by pharmacological blockade of energy metabolism, followed by stimulation with a K+ efflux inducer (ATP or nigericin). Two energy metabolic inhibitors, namely CPI-613 that targets α-ketoglutarate dehydrogenase and pyruvate dehydrogenase (a rate-limiting enzyme) and 2-deoxy-d-glucose (2-DG) that targets hexokinase, are recruited in this study, and Nlrp3 gene knockout macrophages were used. Our data showed that CPI-613 and 2-DG dose-dependently inhibited NLRP3 inflammasome activation, but profoundly increased cell death in the presence of ATP or nigericin. The cell death was K+ efflux-induced but NLRP3-independent, which was associated with abrupt reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential, and oligomerization of mitochondrial proteins, all indicating mitochondrial damage. Notably, the cell death induced by K+ efflux and blockade of energy metabolism was distinct from pyroptosis, apoptosis, necroptosis or ferroptosis. Furthermore, fructose 1,6-bisphosphate, a high-energy intermediate of glycolysis, significantly suppressed CPI-613+nigericin-induced mitochondrial damage and cell death. Collectively, our data show that energy deficiency diverts NLRP3 inflammasome activation-dependent pyroptosis to Nlrp3-independent necrosis upon K+ efflux inducers, which can be dampened by high-energy intermediate, highlighting a critical role of energy metabolism in cell survival and death under inflammatory conditions.

Anti-Necroptotic Effects of Itaconate and its Derivatives.

Itaconate is an unsaturated dicarboxylic acid that is derived from the decarboxylation of the Krebs cycle intermediate cis-aconitate and has been shown to exhibit anti-inflammatory and anti-bacterial/viral properties. But the mechanisms underlying itaconate's anti-inflammatory activities are not fully understood. Necroptosis, a lytic form of regulated cell death (RCD), is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) signaling. It has been involved in the pathogenesis of organ injury in many inflammatory diseases. In this study, we aimed to explore whether itaconate and its derivatives can inhibit necroptosis in murine macrophages, a mouse MPC-5 cell line and a human HT-29 cell line in response to different necroptotic activators. Our results showed that itaconate and its derivatives dose-dependently inhibited necroptosis, among which dimethyl itaconate (DMI) was the most effective one. Mechanistically, itaconate and its derivatives inhibited necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling and the oligomerization of MLKL. Furthermore, DMI promoted the nuclear translocation of Nrf2 that is a critical regulator of intracellular redox homeostasis, and reduced the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS) that were induced by necroptotic activators. Consistently, DMI prevented the loss of mitochondrial membrane potential induced by the necroptotic activators. In addition, DMI mitigated caerulein-induced acute pancreatitis in mice accompanied by reduced activation of the necroptotic signaling in vivo. Collectively, our study demonstrates that itaconate and its derivatives can inhibit necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling, highlighting their potential applications for treating necroptosis-associated diseases.

Formation of cofilin-actin rods following cucurbitacin-B-induced actin aggregation depends on Slingshot homolog 1-mediated cofilin hyperactivation.

Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation.

LC3B-II deacetylation by histone deacetylase 6 is involved in serum-starvation-induced autophagic degradation.

Autophagy is a conserved mechanism for controlling the degradation of misfolded proteins and damaged organelles in eukaryotes and can be induced by nutrient withdrawal, including serum starvation. Although differential acetylation of autophagy-related proteins has been reported to be involved in autophagic flux, the regulation of acetylated microtubule-associated protein 1 light chain 3 (LC3) is incompletely understood. In this study, we found that the acetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II), which is a critical component of double-membrane autophagosome, were profoundly decreased in HeLa cells upon autophagy induction by serum starvation. Pretreatment with lysosomal inhibitor chloroquine did not attenuate such deacetylation. Under normal culture medium, we observed increased levels of acetylated LC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase 6 (HDAC6). However, tubacin only partially suppressed serum-starvation-induced LC3B-II deacetylation, suggesting that HDAC6 is not the only deacetylase acting on LC3B-II during serum-starvation-induced autophagy. Interestingly, tubacin-induced increase in LC3B-II acetylation was associated with p62/SQSTM1 accumulation upon serum starvation. HDAC6 knockdown did not influence autophagosome formation but resulted in impaired degradation of p62/SQSTM1 during serum starvation. Collectively, our data indicated that LC3B-II deacetylation, which was partly mediated by HDAC6, is involved in autophagic degradation during serum starvation.

Cucurbitacin E exhibits anti-inflammatory effect in RAW 264.7 cells via suppression of NF-κB nuclear translocation.

OBJECTIVE: Cucurbitacin E (CuE), a triterpenoid compound isolated from Cucurbitaceae plants, possesses a wide range of biological activities including anti-inflammatory properties. The present study aimed to investigate the anti-inflammatory effect of CuE and the underlying mechanism of action. METHODS: The anti-inflammatory effect of CuE was evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Cell proliferation was assessed using a modified MTT assay. Cell cycle distribution was analyzed by propidium iodide staining. The actin cytoskeleton was examined by immunofluorescent staining. The expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß was determined by intracellular cytokine staining. G-actin level and nuclear factor (NF)-κB nuclear translocation were detected by immunoblotting. RESULTS: CuE inhibited cell proliferation and induced cell cycle arrest at G2/M phase in RAW 264.7 cells. CuE also suppressed LPS-induced cell spreading and pseudopodia formation. These effects were associated with decreased G-actin level and severe actin aggregation. Moreover, CuE significantly inhibited both TNF-α and IL-1ß production in LPS-stimulated RAW 264.7 cells. This was likely mediated by suppressing LPS-induced nuclear translocation of NF-κB, a critical transcription factor responsible for pro-inflammatory cytokine expression. CONCLUSION: CuE displayed anti-inflammatory effects through suppression of NF-κB nuclear translocation leading to a decreased expression of TNF-α and IL-1ß in LPS-stimulated RAW 264.7 cells.

Celastrol inhibits necroptosis by attenuating the RIPK1/RIPK3/MLKL pathway and confers protection against acute pancreatitis in mice.

Necroptosis is a necrotic form of regulated cell death, which is primarily mediated by the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) pathway in a caspase-independent manner. Necroptosis has been found to occur in virtually all tissues and diseases evaluated, including pancreatitis. Celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium wilfordii (thunder god vine), possesses potent anti-inflammatory and anti-oxidative activities. Yet, it is unclear whether celastrol has any effects on necroptosis and necroptotic-related diseases. Here we showed that celastrol significantly suppressed necroptosis induced by lipopolysaccharide (LPS) plus pan-caspase inhibitor (IDN-6556) or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). In these in vitro cellular models, celastrol inhibited the phosphorylation of RIPK1, RIPK3, and MLKL and the formation of necrosome during necroptotic induction, suggesting its possible action on upstream signaling of the necroptotic pathway. Consistent with the known role of mitochondrial dysfunction in necroptosis, we found that celastrol significantly rescued TSI-induced loss of mitochondrial membrane potential. TSI-induced intracellular and mitochondrial reactive oxygen species (mtROS), which are involved in the autophosphorylation of RIPK1 and recruitment of RIPK3, were significantly attenuated by celastrol. Moreover, in a mouse model of acute pancreatitis that is associated with necroptosis, celastrol administration significantly reduced the severity of caerulein-induced acute pancreatitis accompanied by decreased phosphorylation of MLKL in pancreatic tissues. Collectively, celastrol can attenuate the activation of RIPK1/RIPK3/MLKL signaling likely by attenuating mtROS production, thereby inhibiting necroptosis and conferring protection against caerulein-induced pancreatitis in mice.

Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS.

The chondrogenic potential of multipotent mesenchymal stem cells (MSCs) makes them a promising source for cell-based therapy of cartilage defects; however, the exact intracellular molecular mechanisms of chondrogenesis as well as self-renewal of MSCs remain largely unknown. To gain more insight into the underlying molecular mechanisms, we applied isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/MS/MS technology to identify proteins differentially expressed in an in vitro model for chondrogenesis: chondrogenic differentiation of C3H10T1/2 cells, a murine embryonic mesenchymal cell line, was induced by micromass culture and 100 ng/ml bone morphogenetic protein 2 treatment for 6 days. A total of 1756 proteins were identified with an average false discovery rate <0.21%. Linear regression analysis of the quantitative data gave strong correlation coefficients: 0.948 and 0.923 for two replicate two-dimensional LC/MS/MS analyses and 0.881, 0.869, and 0.927 for three independent iTRAQ experiments, respectively (p < 0.0001). Among 1753 quantified proteins, 100 were significantly altered (95% confidence interval), and six of them were further validated by Western blotting. Functional categorization revealed that the 17 up-regulated proteins mainly comprised hallmarks of mature chondrocytes and enzymes participating in cartilage extracellular matrix synthesis, whereas the 83 down-regulated were predominantly involved in energy metabolism, chromatin organization, transcription, mRNA processing, signaling transduction, and cytoskeleton; except for a number of well documented proteins, the majority of these altered proteins were novel for chondrogenesis. Finally, the biological roles of BTF3l4 and fibulin-5, two novel chondrogenesis-related proteins identified in the present study, were verified in the context of chondrogenic differentiation. These data will provide valuable clues for our better understanding of the underlying mechanisms that modulate these complex biological processes and assist in the application of MSCs in cell-based therapy for cartilage regeneration.

Cucurbitacin B induces cell cycle arrest, apoptosis and autophagy associated with G actin reduction and persistent activation of cofilin in Jurkat cells.

AIM: The present study aimed to explore the antitumor effect and action mechanism of cucurbitacin B (CuB) on human T-cell leukemia Jurkat cells. METHODS: Cell proliferation was measured by the MTS assay. Cell cycle distribution, mitochondrial membrane potential and annexin V staining were analyzed using flow cytometry. Western blotting was used to determine the levels of apoptosis- and autophagy-related proteins. RESULTS: CuB inhibited the proliferation of Jurkat cells in a dose-dependent manner and induced G 2 /M phase arrest as well as formation of tetraploid cells. Accompanied with these effects, the actin dynamics was disrupted, and cofilin, a key regulator of actin dynamics, was persistently activated (dephosphorylated). Although CuB induced around 10% cells undergoing apoptosis, most of the cells were alive after CuB treatment for 24 h. Induction of autophagy was also evident by accumulation of LC3-II. CuB-induced autophagy seemed to be a prosurvival response, since suppression of CuB-induced autophagy significantly increased the activation of caspase-3. CONCLUSION: Our results demonstrated that CuB exhibited antitumor activity in Jurkat cells through induction of cell cycle arrest and apoptosis which was at least partly due to the disruption of actin dynamics.

Baicalin inhibits necroptosis by decreasing oligomerization of phosphorylated MLKL and mitigates caerulein-induced acute pancreatitis in mice.

Necroptosis is a form of regulated necrosis mainly controlled by receptor-interacting protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Necroptosis has important roles in defensing against pathogenic infections, but it is also implicated in various inflammatory diseases including pancreatitis. Baicalin, a flavonoid from Scutellaria baicalensis Georgi, has been shown to possess anti-inflammatory and anti-pyroptosis properties, yet it is unclear whether baicalin can inhibit necroptosis and confer protection against necroptosis-related diseases. Here we reported that baicalin significantly inhibited necroptosis in macrophages induced by lipopolysaccharide plus pan-caspase inhibitor (IDN-6556), or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). Mechanistically, baicalin did not inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, nor membrane translocation of p-MLKL, during necroptotic induction, but instead inhibited p-MLKL oligomerization that is required for executing necroptosis. As intracellular reactive oxygen species (ROS) has been reported to be involved in p-MLKL oligomerization, we assessed the effects of N-acetyl-L-cysteine (NAC), an ROS scavenger, on necroptosis and found that NAC significantly attenuated TSI-induced necroptosis and intracellular ROS production concomitantly with reduced levels of oligomerized p-MLKL, mirroring the effect of baicalin. Indeed, inhibitory effect of baicalin was associated with reduced TSI-induced superoxide (indicating mitochondrial ROS) production and increased mitochondrial membrane potential within cells during necroptosis. Besides, oral administration of baicalin significantly reduced the severity of caerulein-induced acute pancreatitis in mice, an animal model of necroptosis-related disease. Collectively, baicalin can inhibit necroptosis through attenuating p-MLKL oligomerization and confers protection against caerulein-induced pancreatitis in mice.