Knockdown Of RIP1 Research Articles

Abstract 2765: Novel WNT-RIP1-GDF15 pathway promotes migration and invasion of colorectal cancer cells

Abstract Growth-differentiation factor 15 (GDF15) is a member of the transforming growth factor β (TGF-β) family protein, also known as NSAID-activated gene 1 (NAG-1) or macrophage inhibitory cytokine 1 (MIC-1), and is a major cause of cancer cachexia, cardiovascular disease, and various inflammatory diseases. Roles of GDF15 as major inflammatory response factor also have been demonstrated in animal models, and it has also showed various malignant functions such as regulating metabolism, promoting metastasis by inducing EMT in cancer. Here, we revealed a novel potential role for RIP1 in colorectal cancer (CRC) in enhancing metastasis by controlling WNT-RIP1-GDF15 signaling. We showed that WNT3A treatment increased the expression of GDF15 and GFRAL. Immunohistochemical analysis of human CRC tissue arrays consisting of primary cancer and metastatic cancer also confirmed that the expression of GDF15 and GFRAL was increased in metastatic cancer. Human XL cytokine assay, western blot, and ELISA results confirmed that GDF15 expression was reduced in cell lines in which RIP1 was knocked down. Reduction of GDF15 expression and secretion by RIP1 knockdown showed suppression of EMT induction resulting to decrease of migration and invasion of CRC cells. Taken together, these results suggest a novel EMT-enhancing role for RIP1 in the WNT pathway and suggest a novel WNT-RIP1-GDF15 pathway contributing to CRC malignancy by promoting EMT. Citation Format: A-Ram Kang, Do Hoon Kim, Jong Kuk Park. Novel WNT-RIP1-GDF15 pathway promotes migration and invasion of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2765.

Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts

Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.

The apoptosis and GLP-1 hyposecretion induced by LPS via RIP/ROS/mTOR pathway in GLUTag cells

Lipopolysaccharide (LPS) as a component of the outer structure of cell wall of gram-negative bacteria, could induce apoptosis in the intestinal endocrine cell line STC-1. However, the signaling cascades involved in this process have not been elucidated. Hence, we investigated the mechanism of cell apoptosis and hyposecretion of glucagon-like peptide 1 (GLP-1) induced by LPS in the GLUTag enteroendocrine cell line. LPS decreased the cell viability of GLUTag cells, up-regulated the TNF-α level, induced the apoptosis and down-regulated the mRNA and protein levels of GLP-1. In addition, TNF-α promoted LPS-induced apoptosis of GLUTag cells through mediating the formation of the RIP1/RIP3 necrosome. RIP1 and RIP3 knockdown increased cell viability, the mRNA and protein levels of GLP-1 and the mTOR signaling pathway-related proteins (p-mTOR and p-S6), and decreased the relative caspase 3/7 activity, cell apoptosis and ROS production. Further studies showed that ROS inhibited the mTOR signaling pathway. Moreover, the antioxidant N-acetyl-l-cysteine increased cell viability, GLP-1 expressions and the mTOR signaling pathway-related proteins, and inhibited the ROS production. However, the mTOR specific inhibitor (Rapa) reversed all these above effects. Taken together, our result revealed that LPS induced the apoptosis of GLUTag cells and GLP-1 hyposecretion through the RIP/ROS/mTOR pathway.

Receptor interacting protein kinase 3 promotes cisplatin-induced necroptosis in apoptosis-resistant HepG2/DDP cells.

Hepatocellular carcinoma is the most common primary malignancy of the liver. The chemotherapeutic drug cisplatin is widely used for advanced liver cancer. However, the development of cisplatin resistance in cancer cells, which is related to the decreased cellular susceptibility to apoptosis, results in a major limitation of cisplatin-based chemotherapy. Recently, triggering necroptosis has been proposed to be a novel therapeutic strategy to eradicate apoptosis-resistant cancer cells. In this study, we provided evidence that cisplatin could induce cell death in HepG2 cells, but not in the apoptosis-resistant HepG2/DDP cells. Ectopic expression of RIP3 promoted cisplatin-induced HepG2/DDP cells death, HMGB1 and LDH release. Moreover, we demonstrated that this type of cell death was necroptosis and depended on RIP1-RIP3-MLKL signaling pathway because inhibition of MLKL activity by necrosulfonamide (NSA) or knockdown of RIP1 significantly attenuated cisplatin-induced cell death in HepG2/DDP-RIP3 cells. Finally, we found that ectopic expression of RIP3 sensitized HepG2/DDP cancer cells to cisplatin treatment in vivo. The findings offer new insights into the molecular mechanisms underlying cisplatin-induced necroptosis in liver cancer cells and suggest that combination of cisplatin with other drugs which can restore RIP3 expression in cancer cells maybe a better choice for therapy of apoptosis-resistant cancer.

Highly Multiplexed CRISPRi Repression of Respiratory Functions Enhances Mitochondrial Localized Ethyl Acetate Biosynthesis in Kluyveromyces marxianus.

The emergence of CRISPR-Cas9 for targeted genome editing and regulation has enabled the manipulation of desired traits and enhanced strain development of nonmodel microorganisms. The natural capacity of the yeast Kluyveromyces marxianus to produce volatile esters at high rate and at elevated temperatures make it a potentially valuable production platform for industrial biotechnology. Here, we identify the native localization of ethyl acetate biosynthesis in K. marxianus and use this information to develop a multiplexed CRISPRi system for redirecting carbon flux along central metabolic pathways, increasing ethyl acetate productivity. First, we identified the primary pathways of precursor and acetate ester biosynthesis. A genetic knockout screen revealed that the alcohol acetyltransferase Eat1 is the critical enzyme for ethyl, isoamyl, and phenylethyl acetate production. Truncation studies revealed that high ester biosynthesis is contingent on Eat1 mitochondrial localization. As ethyl acetate is formed from the condensation of ethanol and acetyl-CoA, we modulated expression of the TCA cycle and electron transport chain genes using a highly multiplexed CRISPRi approach. The simultaneous knockdown of ACO2b, SDH2, RIP1, and MSS51 resulted in a 3.8-fold increase in ethyl acetate productivity over the already high natural capacity. This work demonstrates that multiplexed CRISPRi regulation of central carbon flux, supported by a fundamental understanding of pathway biochemistry, is a potent strategy for metabolic engineering in nonconventional microorganisms.

Therapeutic inhibition of RIP1 improves metabolic dysfunction and inhibits atherosclerosis in mouse models of cardiometabolic diseases

IntroductionChronic activation of the innate immune system drives inflammation and contributes directly to obesity, insulin resistance and atherosclerosis. Previously we showed that necroptosis, a pro‐inflammatory form of programmed cell death, is activated in the vessel wall and drives atherosclerosis via activation of RIP3 and MLKL. We sought to determine upstream genetic regulators of necroptosis in atherosclerosis and metabolic disease, and hypothesized that gene expression of RIP1, a key regulatory kinase upstream of NFkB activation, apoptosis and necroptosis, drives macrophage inflammation in cardiometabolic diseases.MethodsRIP1 anti‐sense oligonucleotides (ASOs, 2 unique sequences) were used to reduce RIP1 expression in 2 mouse models: i) atherosclerotic model [ApoE−/− mice fed a western diet for 8wks] and ii) diet‐induced obesity (DIO) model [C57Bl/6 male mice fed a high fat diet for 24 wks]. Mice were given weekly injections of RIP1 ASOs (50 or 100mg/kg) or control ASO (non‐targeting).ResultsRIP1 ASO treated ApoE−/− mice had a marked reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL‐1a, IL‐17A, p<0.05). RIP1 knockdown in macrophages decreased inflammatory gene expression (NFkB, TNFa, IL‐1a) and in vivo LPS‐induced NFkB activation. In obese mice, RIP1 ASOs strikingly decreased body weight (25% decrease versus control p<0.001) and total fat mass (50–65% decrease versus control, p<0.001). Further, insulin resistance was improved in RIP1 ASO treated mice (fasted blood glucose: 10.9mM in control versus 8.5±mM in RIP1 ASO, p<0.001; GTT and ITT both p<0.001). In humans, we identified 5 novel SNPs in strong linkage disequilibrium in or nearby RIP1 gene exon 5. Notably, in a cohort of >1,800 people, individuals carrying the minor allele of these RIP1 SNPs have a 75–89% increase in the risk of developing obesity (adjusted odds ratios: 1.75–1.89, p<10−5) and a significant increase in RIP1 mRNA expression in adipose tissue (eQTL association in METSIM cohort, p=10−23).Conclusions: We have identified RIP1 as central a driver of insulin resistance, obesity, and atherosclerosis and demonstrate the potential of RIP1 as a novel therapeutic target.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heat Shock Protein 70 (Hsp70) Suppresses RIP1-Dependent Apoptotic and Necroptotic Cascades.

Hsp70 is a molecular chaperone that binds to "client" proteins and protects them from protein degradation. Hsp70 is essential for the survival of many cancer cells, but it is not yet clear which of its clients are involved. Using structurally distinct chemical inhibitors, we found that many of the well-known clients of the related chaperone, Hsp90, are not strikingly responsive to Hsp70 inhibition. Rather, Hsp70 appeared to be important for the stability of the RIP1 (RIPK1) regulators: cIAP1/2 (BIRC1 and BIRC3), XIAP, and cFLIPS/L (CFLAR). These results suggest that Hsp70 limits apoptosis and necroptosis pathways downstream of RIP1. Consistent with this model, MDA-MB-231 breast cancer cells treated with Hsp70 inhibitors underwent apoptosis, while cotreatment with z-VAD.fmk switched the cell death pathway to necroptosis. In addition, cell death in response to Hsp70 inhibitors was strongly suppressed by RIP1 knockdown or inhibitors. Thus, these data indicate that Hsp70 plays a previously unrecognized and important role in suppressing RIP1 activity.Implications: These findings clarify the role of Hsp70 in prosurvival signaling and suggest IAPs as potential new biomarkers for Hsp70 inhibition. Mol Cancer Res; 16(1); 58-68. ©2017 AACR.

CHAC1 degradation of glutathione enhances cystine-starvation-induced necroptosis and ferroptosis in human triple negative breast cancer cells via the GCN2-eIF2α-ATF4 pathway.

Cancer cells exhibit an abnormal amino acid metabolism and a dependence on specific amino acids, which might provide potential targets for treating cancer patients. In this study, we demonstrated that human triple negative breast cancer (TNBC) cells were highly susceptible to cystine starvation. We found that necrostatin-1 (Nec-1, a RIP1 inhibitor), necrosulfonamide (an MLKL inhibitor), deferoxamine (an ion chelator), ferrostatin-1 (a ferroptosis inhibitor) and RIP1 knockdown can prevent cystine-starvation-induced cell death, suggesting that cystine starvation induces necroptosis and ferroptosis in TNBC cells. Moreover, cystine starvation induced mitochondrial fragmentation, dysfunction, and ROS production. A mitochondrial ROS scavenger, Necrox-5, can prevent cystine-starvation-induced cell death. In addition, cystine starvation was found to activate GCN2, but not PERK, to increase the phosphorylation of eIF2α at serine 51, the protein expression of ATF4, and the expression of ATF4 target genes such as CHAC1, which might be downstream of the RIP1/RIP3-MLKL pathway and contribute to cystine-starvation-induced cell death. Knockdown of CHAC1 rescued the cystine-starvation-induced reduction in glutathione (GSH) levels and cell death. Furthermore, N-acetyl-cysteine (NAC), Trolox, and Nec-1 significantly prevented the cystine-starvation-induced increase in intracellular ROS levels, mitochondrial fragmentation and cell death. In summary, these results suggest that CHAC1 degradation of GSH enhances cystine-starvation-induced necroptosis and ferroptosis through the activated GCN2-eIF2α-ATF4 pathway in TNBC cells. Our findings improve our understanding of the mechanism underlying cystine-starvation-induced TNBC cell death.

Abstract 4793: TRAF2 is required for the survival of ErbB2-transfored mammary tumor cells

Abstract TRAF2 regulates signaling pathways downstream of many members of the TNF receptor superfamily, such as TNFR1 and CD40. Recently, we found that TRAF2 also plays an important role in breast cancer cell survival under conditions of endoplasmic reticulum (ER) stress. It is known that TRAF2 knockout (KO) mice die within a week of birth and this lethality can be rescued by KO of TNFα or TNFR1. In order to determine the role of TRAF2 in mammary tumor development and progression, we crossed MMTV-ErbB2 mice (FVB) with TNF-/-/TRAF2-/+ mice (B6), and generated MMTV-ErbB2 (ErbB2), MMTV-ErbB2-TNF-/- (ErbB2/T-KO) and MMTV-ErbB2-TNF-/-/TRAF2-/- (ErbB2/T/T2-DKO) mice all in FVB/B6 mixed background. In 43 weeks, only 1 of 10 ErbB2/T/T2-DKO mice developed a mammary tumor, while 50% of ErbB2 and ErbB2/T-KO mice developed multiple and larger tumors in 30-32 weeks. Interestingly, the tumor derived from the ErbB2/T/T2-DKO mice did not show decreased cyclin D1 expression, nor the tumor cells cultured in vitro show decreased NF-κB activation in response to RANKL. In addition, ErbB2/T/T2-DKO cells formed mammospheres of similar appearance with that of ErbB2 cells in HEMA-coated plates. However, after dispersion of the mammospheres into single cells, ErbB2/T/T2-DKO cells formed dead cell aggregates, while ErbB2 and ErbB2/T-KO cells formed secondary mammospheres, suggesting that the self-renewal capacity of ErbB2/T/T2-DKO cells is impaired. Mechanistic studies reveal that ErbB2/T/T2-DKO cells are sensitive to death caused by ER stress but not by TRAIL or DNA damage, and that this sensitivity is significantly inhibited by knockdown of RIP1. Collectively, these findings suggest that TRAF2 plays a critical role in mammary tumor development and progression by suppressing the pro-death activity of RIP1, and that TRAF2 and RIP1 interaction could be an attractive target for development of new anti-breast cancer drugs. Citation Format: Hasem Habelhah, Yumei Fan, Laiqun Zhang. TRAF2 is required for the survival of ErbB2-transfored mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4793. doi:10.1158/1538-7445.AM2017-4793

RIP1-dependent reactive oxygen species production executes artesunate-induced cell death in renal carcinoma Caki cells.

Artesunate is a well-known anti-malarial drug originated from artemisinin as a Chinese herb and has been reported to have anti-cancer potential in many cancer cells. In the present study, we examined the efficacy of artesunate against the renal carcinoma Caki cells and explored its mechanism of cytotoxicity. A steep decline in cell viability within 18h was recorded upon artesunate exposure, but pretreatment of z-VAD-FMK had no effect on the loss of the cell viability by artesunate. On the other hand, necrostatin-1 pretreatment and knockdown of RIP-1 significantly reduced the cytotoxicity of artesunate against Caki cell. Moreover, the generation of mitochondrial ROS prompted by artesunate was found to be the principle mechanism of cell death. Pretreatment with necrostatin-1 or knockdown of RIP-1 inhibited the generation of ROS by artesunate, resulting in the protection of the cells from artesunate toxicity. Moreover, the similar results were observed in the case of other renal carcinoma cell lines (ACHN and A498). The results suggest that artesunate induces the generation of ROS and cell death in RIP1-dependent manner. Therefore, our data suggest that artesunate could induce RIP1-dependent cell death in human renal carcinoma.

Dichotomy between Receptor-Interacting Protein 1– and Receptor-Interacting Protein 3–Mediated Necroptosis in Experimental Pancreatitis

Pancreatic acinar cell necrosis and inflammatory responses are two key pathologic processes in acute pancreatitis (AP), which determines the severity and outcome of the disease. Recent studies suggest that necroptosis, a programed form of necrosis, is involved in the pathogenesis of AP, but the underlying mechanisms remain unknown. We investigated the expression of necrosome components, including receptor-interacting protein (RIP) 1, RIP3, and mixed lineage kinase domain-like (MLKL), and the molecular mechanisms in pancreatitis-associated necroptosis. We found that RIP3 and phosphorylated MLKL expression was positively related to the degree of necrosis, whereas RIP1 expression was negatively related to the degree of necrosis. Pharmacologic inhibition of RIP1 kinase activity exerted no protection against caerulein/cholecystokinin-8-induced AP, but knockdown of RIP1 with siRNA increased acinar cell necrosis and inhibition of NF-κB activation. RIP1 inhibition led to enhanced RIP3 expression. RIP3 and MLKL inhibition decreased acinar cell necrosis, in which the inhibition of RIP3 reduced the phosphorylation level of MLKL. RIP3 inhibition had no effect on trypsinogen activation but partly inhibited inflammasome activation. Our study strongly suggests that the imbalance between RIP1and RIP3 shifts the cell death to necrosis, which unravels a new molecular pathogenesis of mechanism of AP and may provide insight into the development of novel therapeutic agent for other necrosis-related diseases.

Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43.

Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.

Basal autophagy is negatively regulated by RIP1

Basal autophagy is negatively regulated by RIP1

Smac mimetic and oleanolic acid synergize to induce cell death in human hepatocellular carcinoma cells

Chemotherapy resistance of hepatocellular carcinoma (HCC) is still a major unsolved problem highlighting the need to develop novel therapeutic strategies. Here, we identify a novel synergistic induction of cell death by the combination of the Smac mimetic BV6, which antagonizes Inhibitor of apoptosis (IAP) proteins, and the triterpenoid oleanolic acid (OA) in human HCC cells. Importantly, BV6 and OA also cooperate to suppress long-term clonogenic survival as well as tumor growth in a preclinical in vivo model of HCC underscoring the clinical relevance of our findings. In contrast, BV6/OA cotreatment does not exert cytotoxic effects against normal primary hepatocytes, pointing to some tumor selectivity. Mechanistic studies show that BV6/OA cotreatment leads to DNA fragmentation and caspase-3 cleavage, while supply of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) revealed a cell type-dependent requirement of caspases for BV6/OA-induced cell death. The receptor interacting protein (RIP)1 kinase Inhibitor Necrostatin-1 (Nec-1) or genetic knockdown of RIP1 fails to rescue BV6/OA-mediated cell death, indicating that BV6/OA cotreatment does not primarily engage necroptotic cell death. Notably, the addition of several reactive oxygen species (ROS) scavengers significantly decreases BV6/OA-triggered cell death, indicating that ROS production contributes to BV6/OA-induced cell death. In conclusion, cotreatment of Smac mimetic and OA represents a novel approach for the induction of cell death in HCC and implicates further studies.

RIP1 modulates death receptor mediated apoptosis and autophagy in macrophages

Macrophages are responsible for defending against diverse pathogens and play a crucial role in the innate immune system. Macrophage's lifespan is determined by homeostatic balance between survival and apoptosis. Here we report that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers both apoptosis and autophagy in human U937 cells. Inhibition of autophagy facilitates TRAIL-induced apoptosis, suggesting that autophagy of macrophages protects against TRAIL-induced apoptosis. TRAIL treatment influences the expression of death receptors, indicating that TRAIL-induced apoptosis and autophagy are mediated by death receptors. RIP1 ubiquitination and expression regulate apoptosis and autophagy. Furthermore, expression and bioactivity of the p43/41-caspase-8 variant are critical to TRAIL-induced autophagy and apoptosis. Knockdown of RIP1 suppresses autophagy in macrophage. These data demonstrate that RIP1 is essential for the regulation of death receptor mediated autophagy and apoptosis. The results in this study contribute to understanding the regulation of autophagy and apoptosis in macrophages, and shed lights on death receptor-targeted therapy for cancer, inflammation and autoimmune diseases.

Role of RIP1 in physiological enterocyte turnover in mouse small intestine via nonapoptotic death.

Enterocyte shedding in the small intestine is often referred as an example of programmed cell death. However, little is known about the underlying mechanisms, although both apoptotic and nonapoptotic cell death have been suggested to play an important role. Here, we show by electron microscope that the majority of cells dying in the mouse small intestine do not display apoptotic characteristics. Chemical biological approach in vivo and in an organ culture showed that necrostatin-1 (Nec-1), an inhibitor of receptor-interacting protein 1 (RIP1, also called RIPK1), inhibited the shedding/nonapoptotic death of enterocyte, resulting in suppression of physiological enterocyte turnover. Moreover, RIP1 knockdown in vivo and RIP1 haploinsufficiency significantly suppressed physiological enterocyte turnover. Unlike Nec-1-sensitive (RIP1-dependent) cell death, so called necroptosis, which is also dependent on RIP3, physiological enterocyte turnover in RIP3-deficient mice was executed normally and still inhibited by Nec-1. As inhibition of the shedding/nonapoptotic death of enterocyte by Nec-1 resulted in suppression of crypt cell proliferation, the shedding process plays a dominant role over cell proliferation in maintaining homeostasis of enterocyte turnover. These results indicate that RIP1 plays a major role in physiological enterocyte turnover through a RIP3-independent nonapoptotic death mechanism in the mouse small intestine.

Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly

AbstractApoptosis resistance contributes to poor outcome in pediatric acute lymphoblastic leukemia (ALL). Here, we identify a novel synergistic combination of Smac mimetic BV6 and glucocorticoids (GCs) (ie, dexamethasone, prednisolone) to trigger apoptosis in ALL cells. BV6 and GCs similarly cooperate to induce apoptosis in patient-derived leukemia samples, underlining the clinical relevance. Importantly, BV6/dexamethasone cotreatment is significantly more effective than monotherapy to delay leukemia growth in a patient-derived xenograft model of pediatric ALL without causing additional side effects. In contrast, BV6 does not increase cytotoxicity of dexamethasone against nonmalignant peripheral blood lymphocytes, mesenchymal stromal cells, and CD34-positive hematopoietic cells. We identify a novel mechanism by showing that BV6 and dexamethasone cooperate to deplete cIAP1, cIAP2, and XIAP, thereby promoting assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing complex. This complex is critical and is required for BV6/dexamethasone-induced cell death, because RIP1 knockdown reduces caspase activation, reactive oxygen species production, and cell death. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, because blocking antibodies for TNFα, tumor necrosis factor-related apoptosis-inducing ligand, or CD95 ligand or knockdown of death receptors fail to rescue BV6/dexamethasone-induced cell death. This is the first report showing that BV6 sensitizes for GC-triggered cell death by promoting ripoptosome formation with important implications for apoptosis-targeted therapies of ALL.

Effects of silencing RIP1 with siRNA on the biological behavior of the LoVo human colon cancer cell line.

The present study aimed to investigate the effects of silencing RIP1 by small interfering RNA (siRNA) on the biological behavior of the LoVo human colorectal carcinoma cell line and to provide evidence for the feasibility of colorectal cancer gene therapy. LoVo cells were divided into the RIP1 siRNA group, the blank control group and the negative control group. Chemically synthesized siRNA targeting RIP1 (RIP1 siRNA) was transfected into LoVo cells. Following transfection of the RIP1-targeted siRNA into the LoVo cells, the expression of the RIP1 gene was effectively inhibited. The results demonstrated that RIP1 effectively regulated the malignant biological behavior of the LoVo colon cancer cell line. Furthermore, the proliferation, motility and invasiveness of LoVo cells were inhibited by siRNA knockdown of RIP1. The results revealed that the RIP1 gene has an important role in the regulation of proliferation and apoptosis in colorectal carcinoma cells.

Necroptosis mediates TNF-induced toxicity of hippocampal neurons.

Tumor necrosis factor-α (TNF-α) is a critical proinflammatory cytokine regulating neuroinflammation. Elevated levels of TNF-α have been associated with various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the signaling events that lead to TNF-α-initiated neurotoxicity are still unclear. Here, we report that RIP3-mediated necroptosis, a form of regulated necrosis, is activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. Furthermore, we characterized the molecular mechanism of TNF-α-induced neurotoxicity in HT-22 hippocampal neuronal cells. HT-22 cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. The cell death is suppressed by knockdown of CYLD or RIP1 or RIP3 or MLKL, suggesting that this necrosis is necroptosis and mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. TNF-α-induced necroptosis of HT-22 cells is largely independent of both ROS accumulation and calcium influx although these events have been shown to be critical for necroptosis in certain cell lines. Taken together, these data not only provide the first in vivo evidence for a role of RIP3 in TNF-α-induced toxicity of hippocampal neurons, but also demonstrate that TNF-α promotes CYLD-RIP1-RIP3-MLKL-mediated necroptosis of hippocampal neurons largely bypassing ROS accumulation and calcium influx.

A Small Molecule, Shikonin, Dually Functions As a Proteasome Inhibitor and a Necroptosis Inducer In Multiple Myeloma Cells

BackgroundDespite of recent advances in therapeutic strategy for multiple myeloma (MM), MM still remains incurable and novel therapeutic approach is urgently needed. We have previously demonstrated that a natural small molecule, shikonin (SHK), induced both apoptosis and necroptosis (programmed necrosis) in MM cells. In this study, we attempted to elucidate biological mechanisms of SHK in inducing apoptosis and necroptosis. MethodsSix MM cell lines, KMS-12-PE, RPMI 8226, U266, KMM1, KMS-11 and a bortezomib-resistant MM cell line, KMS-11/BTZ (obtained from Kyowa Hakko Kirin Co. Ltd.), were utilized. Inhibitors of pan-caspase and necroptosis, ZVAD-fmk and Nec-1 (necrostatin-1), were employed to distinguish apoptosis and necroptosis, respectively. Cell death was analyzed using the trypan blue dye exclusion method (WST-8 assay) and flow cytometry analysis using AnnexinV/PI staining. Morphological examinations of cells were performed with May Giemza staining. Caspases, RIP1, ubiquitinated proteins, and heat shock proteins were analyzed with western blot. Knockdown of RIP1, an essential molecule for necroptosis, was performed using siRNA. ER stress was assessed by detecting activated XBP-1, which was analyzed by digestion of PCR products with ApaLI. Because the ApaLI site in XBP-1 mRNA is spliced out upon activation, the activated XBP-1 shows one large band after ApaLI digestion, while inactivated XBP-1 shows two ApaI-digested bands. Thapsigargin, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, was used as an ER stress inducer. ResultsBy screening natural compounds libraries (provided by Institute of Natural Medicine, Toyama University, Japan), we found that SHK, a natural compound derived from the root of Lithospermum erythrorhizon, induced cell death in MM cells. Apoptosis was induced at a relatively low concentration (2.5∼5 µM) and was inhibited by a caspase-inhibitor, while necroptosis was promptly induced at higher concentrations (10∼ 20 µM) within 5 hours and was completely inhibited by Nec-1. Morphological analysis showed that SHK at low concentrations induced typical apoptotic changes, such as fragmented nucleus, while SHK at higher concentrations induced necrotic morphology, such as translucent cytoplasm and swelling of cell membranes. By contrast, SHK did not induce apoptosis or necrptosis in peripheral blood mononuclear cells from healthy donors at low concentrations. SHK activated caspase-8 and -3 at low concentrations but did not at higher concentrations. RIP1, an essential molecule for necroptosis, was cleaved after treatment with SHK at low concentrations, which leads to the inhibition of necroptosis, while it was not cleaved and remained active at higher concentrations, suggesting that SHK dynamically regulates the cleavage of RIP-1.At low concentrations, shikonin induced an accumulation of ubiquitinated proteins and activated XBP-1, suggesting SHK may have a property of proteasome inhibitor eventually inducing endoplasmic reticulum stress. Finally, SHK at low concentration killed bortezomib resistant cells with lower IC50 comparing to that of the parental cells (0.91 vs 1.56 µM, respectively). ConclusionsWe here report, for the first time, that SHK induces apoptosis and necroptosis in MM cells at low and high concentrations, respectively, by regulating proteasome function and RIP-1 cleavage. Given the fact that SHK efficiently induces cell death in bortezomib-resistant cell line, SHK may act as a novel proteasome inhibitor for bortezomib-resistant myeloma cells. Moreover, SHK at higher concentrations, which induces nectoptosis, should be an attractive future therapeutic option potentially to eradicate MM cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.