Receptor-interacting Protein Kinase 3 Expression Research Articles

The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death

Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was associated with reduced promoter activity of the RIPK1, RIPK3 and MLKL genes. Collectively, these results indicate that PCB-95 was associated with REST-induced necroptotic cell death by increasing RIPK1, RIPK3 and MLKL expression and reducing caspase-8 levels. In addition, since REST is involved in several neurological disorders, therapies that block REST-induced necroptosis could be a new strategy to revert the neurodetrimental effects associated to its overexpression.

Abstract 4462: ACTN4 stabilises RIPK1 to function as an oncogenic driver in melanoma

Abstract We have recently reported that receptor-interacting protein kinase 1 (RIPK1) is commonly up-regulated through cellular inhibitor apoptosis protein (cIAP)-mediated stabilization and functions as an oncogenic driver via activation of NF-κB in melanoma cells. Here we show that α-actinin-4 (ACTN4), an isoform of α-actinins of spectrin superfamily proteins that is emerging as an oncogenic regulator, is required for cIAP-mediated stabilization of RIPK1 and plays a pro-oncogenic role in melanoma. Similar to RIPK1, ACTN4 was found to be commonly increased in melanoma cells. While knockdown of ACTN4 inhibited melanoma cell proliferation that was associated with down-regulation of RIPK1 and reduction in the basal levels of NF-κB activation, overexpression of ACTN4 resulted in enhanced proliferation of melanocytes that was associated with elevation in RIPK1 expression and increased NF-κB activation. The inhibitory effect of ACTN4 knockdown on melanoma cell proliferation and activation of NF-κB was due to decreased expression of RIPK1, as it was abolished by overexpression of RIPK1. On the other hand, knockdown of RIPK1 diminished ACTN4 overexpression-triggered enhancement in proliferation in melanocytes. Strikingly, knockdown of ACTN4 attenuated the association between cIAPs and RIPK1 and reduced K63-linked ubiquitination of the protein, leading to RIPK1 protein degradation. Mechanistic studies showed that ACTN4 bound to RIPK1 through its N-terminus, whereas it was associated with cIAPs via its C-terminus, and that dimerization of ACTN4 in an anti-parallel manner is required for the interaction between cIAPs and RIPK1. Collectively, ACTN4 is necessary for stabilization of RIPK1 by cIAPs and functions as an oncogenic driver in melanoma. Citation Format: Lei Jin, Hessam Tabatabaeehatambakhsh, Chen Chen Jiang, Xu Guang Yan, Jia Yu Wang, Yuan Yuan Zhang, Hamed Yari, Chun Yan Wang, Ting La, Fu Xi Lei, Yu Chen Feng, Su Tang Guo, Xu Dong Zhang. ACTN4 stabilises RIPK1 to function as an oncogenic driver in melanoma [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 4462. doi:10.1158/1538-7445.AM2017-4462

Role of necroptosis in liver injury in septic rats

Objective To evaluate the role of necroptosis in liver injury in septic rats. Methods Twenty-four SPF healthy male adult Sprague-Dawley rats, weighing 200-220 g, aged 6-8 weeks, were divided into 3 groups (n=8 each) using a random number table: sham operation group (Sh group), sepsis group (Sep group) and specific necroptosis inhibitor necrostatin-1 group (N group). Sepsis was induced by cecal ligation and puncture in anesthetized rats in N and Sep groups.Necrostatin-1 1.0 mg/kg was intravenously injected at 1 h before operation in group N, while the equal volume of dimethyl sulfoxide was given instead in group Sep.Rats were sacrificed at 6 h after operation, and livers were removed for examination of the pathological changes (with a light microscope) and for determination of the level of reactive oxygen species (ROS) in liver tissues (by using chemiluminescence assay) and expression of receptor-interacting protein kinase-1 (RIPK1), RIPK3, mixed lineage kinase domain-like (MLKL) and high-mobility group box 1 protein (HMGB1) in liver tissues (using Western blot). Results Compared with Sh group, the ROS level in liver tissues was significantly increased, and the expression of RIPK1, RIPK3, MLKL and HMGB1 in liver tissues was up-regulated in Sep and N groups (P<0.05). Compared with Sep group, the ROS level in liver tissues was significantly decreased, and the expression of RIPK1, RIPK3, MLKL and HMGB1 in liver tissues was down-regulated in group N (P<0.05). The pathological changes of liver tissues were significantly attenuated in group N when compared with group Sep. Conclusion Necroptosis is involved in liver injury in septic rats. Key words: Sepsis; Liver; Necrosis

RIPK1 binds to vitamin D receptor and decreases vitamin D-induced growth suppression

Receptor interacting protein kinase 1 (RIPK1) is an enzyme acting downstream of tumor necrosis factor alpha to control cell survival and death. RIPK1 expression has been reported to cause drug resistance in cancer cells, but so far, no published studies have investigated the role of RIPK1 in vitamin D signaling. In the present study, we investigated whether RIPK1 plays any roles in 1,25-dihydroxyvitamin D3 (1,25D3)-induced growth suppression. In our studies, RIPK1 decreased the transcriptional activity of vitamin D receptor (VDR) in luciferase reporter assays independent of its kinase activity, suggesting a negative role of RIPK1 in 1,25D3 action. RIPK1 also formed a complex with VDR, and deletion analyses mapped the RIPK1 binding region to the C-terminal ligand-binding domain of the VDR. Subcellular fractionation analyses indicated that RIPK1 increased VDR retention in the cytoplasm, which may account for its inhibition of VDR transcriptional activity. Consistent with the reporter analyses, 1,25D3-induced growth suppression was more pronounced in RIPK1-null MEFs and RIPK1-knockdown ovarian cancer cells than in control cells. Our studies have defined RIPK1 as a VDR repressor, projecting RIPK1 depletion as a potential strategy to increase the potency of 1,25D3 and its analogs for cancer intervention.

Involvement of XBP1s in Blue Light-Induced A2E-Containing Retinal Pigment Epithelium Cell Death

Purpose: Retinal pigment epithelium (RPE) cell dysfunction is essential to the development of retinal degenerative disease. This study was designed to investigate how spliced X-box-binding protein 1 (XBP1s) regulates different modes of RPE cell death in vitro. Methods: Human ARPE19 cells were incubated with 25 μ<smlcap>M</smlcap> N-retinylidene-N-retinylethanolamine (A2E) and irradiated with blue light. Expressions of glucose-regulated protein 78 (GRP78) and XBP1s were detected by real-time quantitative PCR and Western blot. STF-083010 was used to suppress XBP1s expression. ARPE19 cell apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling and flow cytometry. Receptor-interacting protein kinase-3 (RIP3) was detected by Western blot. Changes in the morphology of ARPE19 cells were identified by transmission electron microscopy. Results: Blue light-induced A2E-containing ARPE19 cell damage caused a transient elevation of GRP78 and XBP1s, while RIP3 rose in the late stage. STF-083010 effectively inhibited XBP1s expression and brought about the aggravation of apoptosis together with an alleviation of RIP3 expression. Most of the dying cells exhibited apoptotic morphology. Conclusion: A2E, along with blue light, brought about apoptosis and necroptosis of ARPE19 cells, and XBP1s was transiently elevated. The suppression of XBP1s induced ARPE19 cell death by promoting apoptosis rather than necroptosis. XBP1s might play a role in the pathogenesis of retinal degenerative diseases.

RIPK1 Suppresses a TRAF2-Dependent Pathway to Liver Cancer

Receptor-interacting protein kinase 1 (RIPK1) represents an essential signaling node in cell death and inflammation. Ablation of Ripk1 in liver parenchymal cells (LPC) did not cause a spontaneous phenotype, but led to tumor necrosis factor (TNF)-dependent hepatocyte apoptosis and liver injury without affecting inducible nuclear factor κB (NF-κB) activation. Loss of Ripk1 induced the TNF-dependent proteasomal degradation of the E3-ligase, TNF receptor-associated factor 2 (TRAF2), in a kinase-independent manner, thereby activating caspase-8. Moreover, loss of both Ripk1 and Traf2 in LPC not only resulted in caspase-8 hyperactivation but also impaired NF-κB activation, promoting the spontaneous development of hepatocellular carcinoma. In line, low RIPK1 and TRAF2 expression in human HCCs was associated with an unfavorable prognosis, suggesting that RIPK1 collaborates with TRAF2 to inhibit murine and human hepatocarcinogenesis.

RIP1 upregulation promoted tumor progression by activating AKT/Bcl-2/BAX signaling and predicted poor postsurgical prognosis in HCC.

Although growing body of evidences have identified a critical role for receptor-interacting protein kinase 1 (RIP1) in mediating cell death signaling, its possible contribution to hepatocellular carcinoma (HCC) progression remains unclear. Here, we displayed that expression of RIP1 was significantly upregulated in HCC tissues than in adjacent liver tissues from 81.9% HCC patients (P<0.001) by immunohistochemistry (IHC) staining. Overexpression of RIP1 was found positively associated with HBV infection, advanced TNM staging, portal vein invasion, and intrahepatic metastases. Kaplan-Meier curve analysis indicated that patients with higher RIP1 expression in HCC tissues suffered from unfavorable postsurgical survival. Higher RIP1 expression in HCC tissues was also confirmed to be an independent poor prognostic predictor. Knockdown of RIP1 resulted in suppression of cell viability and proliferation and induced cell apoptosis in MHCC97h cells. Nevertheless, enforced expression of RIP1 promoted cell viability and proliferation of Huh7 cells and inhibited cell apoptosis. Mechanistic studies revealed that RIP1 exerted its function on HCC progression via activating AKT/Bcl-2/BAX signaling. In conclusion, our results provided the evidence of RIP1 overexpression in HCC, and RIP1 could be a novel predictive factor of unfavorable prognosis in HCC patients. Activation of AKT/Bcl-2/BAX signaling contributed to RIP1 promoting HCC progression.

Critical function of the necroptosis adaptor RIPK3 in protecting from intestinal tumorigenesis.

Necroptosis is a programmed form of non-apoptotic cell death that requires the kinase activity of the receptor interacting protein kinase 3 (RIPK3). Although in vitro data suggests that cancer cells lacking expression of RIPK3 are invasive, the physiological role of RIPK3 in a disease-relevant setting remains unknown. Here we provide evidence that RIPK3 has a critical role in suppressing colorectal cancer (CRC). RIPK3-deficient mice were highly susceptible to colitis-associated CRC and exhibited greater production of pro-inflammatory mediators and tumor promoting factors. Tumorigenesis in RIPK3-deficiency resulted from uncontrolled activation of NF-κB, STAT3, AKT and Wnt-β-catenin signaling pathways that enhanced the ability of intestinal epithelial cells (IECs) to aberrantly proliferate in the face of the sustained inflammatory microenvironment and promote CRC. We found that RIPK3 expression is reduced in tumors from patients with inflammatory bowel diseases, and further confirmed that expression of RIPK3 is downregulated in human CRC and correlated with cancer progression. Thus, our results reveal that the necroptosis adaptor RIPK3 has key anti-inflammatory and anti-tumoral functions in the intestine, and define RIPK3 as a novel colon tumor suppressor.

Receptor-interacting protein kinase3-mediated programmed cell necrosis in rats subjected to focal cerebral ischemia-reperfusion injury.

In the current study, the activation of tumor necrosis factor-α receptor 1 (TNFR1) and receptor-interacting protein kinase 3 (RIP3) were investigated following cerebral ischemia-reperfusion injury (CIRI). Healthy SD rats were randomly divided into 3 groups: Sham operation group, model group and inhibitor group. The model group and inhibitor group were further divided into 4 subgroups of 6, 12, 24 and 72 h following CIRI. Using right middle cerebral artery embolization, the CIRI model was generated. To confirm that the CIRI model was established, neurological scores, TTC staining and brain water content measurements were conducted. Immunohistochemistry and western blotting were conducted to investigate the expression of TNFR1 and RIP3 in the cerebral cortex. It was observed that nerve cell necrosis occurred following 6 h of CIRI. The appearance of necrotic cells was gradually increased with increasing CIRI duration. TNFR1 and RIP3 were positively expressed following 6 h of CIRI. With increasing durations of CIRI, the protein expression levels of TNFR1 and RIP3 were significantly increased. Pre-administration with Z-VAD-FMK (zVAD) significantly increased the protein level of RIP3, however, had no effect on the levels of TNFR1, and was accompanied by a reduction in necrosis. In conclusion, RIP3-mediated cell necrosis was enhanced by caspase blockade zVAD and the function of zVAD was independent of TNFR1 signaling following IR.

Hypothermic machine perfusion increases A20 expression which protects renal cells against ischemia/reperfusion injury by suppressing inflammation, apoptosis and necroptosis.

There is an urgent need to improve the quality of donor organs obtained after cardiac death. In the present study, we examined the potential mechanisms through which A20 protects renal cells against ischemia/reperfusion injury (IRI) following either hypothermic machine perfusion (HMP) or static cold storage (CS) of the kidneys in a rabbit model. The expression of markers of apoptosis, necroptosis and inflammation in frozen kidney tissues were detected by western blot analysis, RT-qPCR and ELISA. Compared with the CS group, A20 expression was significantly higher in the tissue from the HMP group (P<0.01). By contrast, the expression of nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) was significantly lower in HMP group (P<0.01), whereas IκBα expression was significantly higher (P<0.01). The expression of apoptosis signal-regulating kinase 1 (ASK1), phosphorylated (p-)c-Jun N-terminal kinase (JNK) and activated caspase-3 in the HMP group was significantly downregulated compared with that in the CS group (all P<0.01). In addition, A20 inhibited receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis in the kidney. RIPK3 expression in the HMP group was significantly lower than that in the CS group (P<0.01), although the levels in both groups were higher than those in the sham group (P<0.01). Based on these findings, we propose a novel mechanism underlying the anti-apoptotic effect of A20 in renal cells in which A20 binds to ASK1 and promotes the degradation of ASK1 leading to the suppression of JNK activation and eventually, to the blockade of apoptosis. Thus, HMP reduces inflammation, apoptosis and necroptosis by upregulating the expression of A20; this mechanism may be responsible for protecting the kidney against IRI.

Necroptosis in Niemann-Pick disease, type C1: a potential therapeutic target.

Niemann–Pick disease, type C1 (NPC1) is a neurodegenerative, lysosomal storage disorder due to mutation of the NPC1 gene. The NPC1 phenotype is characterized by progressive neuronal dysfunction, including cerebellar ataxia and dementia. There is histological evidence of neuroinflammation and progressive neuronal loss, with cerebellar Purkinje cells particularly vulnerable to loss of NPC1 function. Necroptosis was evaluated as a mechanism of neuronal loss. Receptor-interacting protein kinase 1 (RIP1) and RIP3 are key components of the necrosomal complex that regulates necroptotic cell death. We report increased expression of RIP1 and RIP3 in NPC1 fibroblasts, NPC1 iPS cell-derived neuronal precursors, and in cerebellar tissue from both NPC1 mice and patients. Our data suggest a positive correlation between NPC1 neurological disease severity and assembly of the necrosome complex. Furthermore, we demonstrate that pharmacological inhibition of RIP1 decreases cell death both in vitro and in vivo. Treatment of Npc1-mutant mice with necrostatin-1, an allosteric inhibitor of RIP1, significantly delayed cerebellar Purkinje cell loss, progression of neurological symptoms, and death. Collectively, our data identified necroptosis as a key component of the molecular network that contributes to neuronal loss in NPC1 and establish that inhibition of necroptosis is a potential therapeutic intervention.

CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3.

Receptor-interacting protein kinase 3 (RIPK3) functions as a key regulator of necroptosis. Here, we report that the RIPK3 expression level is negatively regulated by CHIP (carboxyl terminus of Hsp70-interacting protein; also known as STUB1) E3 ligase-mediated ubiquitylation. Chip(-/-) mouse embryonic fibroblasts and CHIP-depleted L929 and HT-29 cells exhibited higher levels of RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNF (also known as TNFα). These phenomena are due to the CHIP-mediated ubiquitylation of RIPK3, which leads to its lysosomal degradation. Interestingly, RIPK1 expression is also negatively regulated by CHIP-mediated ubiquitylation, validating the major role of CHIP in necrosome formation and sensitivity to TNF-mediated necroptosis. Chip(-/-) mice (C57BL/6) exhibit inflammation in the thymus and massive cell death and disintegration in the small intestinal tract, and die within a few weeks after birth. These phenotypes are rescued by crossing with Ripk3(-/-) mice. These results imply that CHIP is a bona fide negative regulator of the RIPK1-RIPK3 necrosome formation leading to desensitization of TNF-mediated necroptosis.

Serum levels of RIPK3 and troponin I as potential biomarkers for predicting impaired left ventricular function in patients with myocardial infarction with ST segment elevation and normal troponin I levels prior percutaneous coronary intervention.

The current study examined the serum levels of receptor-interacting protein kinase 3 (RIPK3) in 51 patients with New York Heart Association (NYHA) class III-IV heart failure, 53 patients with myocardial infarction with ST elevation (STEMI), and 19 healthy subjects serving as a control group. An enzyme-linked immunoadsorbent assay (ELISA) was used to measure the levels of RIPK3 expression in serum. The area under the receiver operating characteristic curve (AUC) was then used to evaluate the predictive performance of RIPK3 and troponin I in patients with STEMI. In patients with normal levels of troponin I prior to percutaneous coronary intervention (PCI), serum levels of RIPK3 and troponin I after PCI were sufficient to differentiate patients with a preserved left ventricular ejection fraction (LVEF) from those with impaired left ventricular function after PCI (AUC = 0.780 (95% CI: 0.565-0.995, p = 0.043) with a sensitivity of 76.9% and a specificity of 71.4% vs. AUC = 0.735 (95% CI: 0.530-0.941, p = 0.038) with a sensitivity of 88.2% and a specificity of 63.6% at the optimal cutoff values, respectively). Moreover, elevated levels of troponin I after PCI were associated with an increased risk of an LVEF < 50% prior to discharge (odds ratio, 1.014; 95 % CI, 1.001 to 1.027; p = 0.03), while elevated levels of RIPK3 were not associated with such a risk. The current findings suggest that in patients with normal levels of troponin I prior to PCI, serum levels of RIPK3 and troponin I can serve as a potential marker to identify patients with a decreased LVEF, thus possibly allowing an early shift to more intensive therapy.

Absence of RIPK3 predicts necroptosis resistance in malignant melanoma.

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.

Abstract 56: Receptor-Interacting protein kinase 1 functions as an oncogenic regulator in human melanoma

Abstract Although many studies in recent years have uncovered an important role of receptor-interacting protein kinase 1 (RIP1) in mediating cell survival and death signaling, its potential part in the pathogenesis of cancer remains less understood. Here we report that RIP1 functions as an oncogenic regulator in human melanoma. While the expression of RIP1 was commonly upregulated in melanoma, knockdown of RIP1 inhibited melanoma cell proliferation in vitro, and retarded melanoma growth in a xenograft model. Conversely, despite induction of apoptosis in a small proportion of melanoma cells, overexpression of RIP1 enhanced proliferation in the remaining cells. The promoting effect of RIP1 on melanoma cell proliferation was mediated by activation of NF-κB, as blockade of NF-κB activation eliminated RIP1 overexpression-triggered increase in cell proliferation, whereas hyperactivation of NF-κB abolished inhibition of cell proliferation caused by RIP1 knockdown. In support, RIP1 knockdown led to reduction, whereas its overexpression caused an increase, in NF-κB activation. Strikingly, ectopic expression of RIP1 enhanced melanocyte proliferation and triggered anchorage-independent growth of the cells similarly in a NF-κB-dependent manner. While upregulation of RIP1 was associated with DNA copy number gain in a subset of melanomas, constitutive ubiquitination and subsequent stabilization of the RIP1 protein driven by TNFα autocrine appeared to be another mechanism commonly responsible for upregulation of RIP1 in melanoma cells. Collectively, these results identify RIP1 as an oncogenic regulator in melanoma, and points to the possibility of targeting the NF-κB activating mechanism of RIP1 as a novel approach in the treatment of the disease. Citation Format: Lei Jin, Xiao Ying Liu, Fritz Lai, Xu Guang Yan, Chen Chen Jiang, Su Tang Guo, Chun Yan Wang, Amanda Croft, Hsin-Yi Tseng, James S. Wilmott, Richard A. Scolyer, Xu Dong Zhang. Receptor-Interacting protein kinase 1 functions as an oncogenic regulator in human melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 56. doi:10.1158/1538-7445.AM2015-56

Protective effect of 2-deoxy-D-glucose on the cytotoxicity of cyclosporin A in vitro.

The present study aimed to investigate the mechanism underlying the protective effect of 2-deoxy-D-glucose (2-DG) on the cytotoxicity of cyclosporin A (CsA) in vitro using NRK-52E cells. Staining with Hoechst 33342/propidium iodide prior to flow cytometric analysis was performed to assess the rate of cellular apoptosis and necrosis induced by CsA. The expression levels of lactate dehydrogenase (LDH), caspase 3, receptor-interacting protein kinase 3 (RIP3), reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) were detected using colorimetry, ELISA, western blotting or flow cytometric analysis to determine the protective effects of 2-DG on CsA-induced cell death. The results demonstrated that 2-DG inhibited the release of LDH, the activation of caspase 3 and the generation of ROS induced by CsA, but had no effect on the expression of RIP3. Treatment with 2-DG increased the expression of GSH and decreased the expression of MDA in dose-dependent manner, and reduced the rate of the cellular apoptosis and necrosis induced by CsA. Therefore, 2-DG inhibited CsA-induced cellular apoptosis and necrosis, possibly by reducing the production of ROS. Inhibiting the activation of caspase 3 is one of the protective mechanisms of 2-DG, however, the expression of RIP3 remained unaltered following treatment with 2-DG. Whether 2-DG inhibits the CsA-induced necrosis and apoptosis by inhibiting the RIP3 signaling pathway remains to be elucidated.

Receptor-interacting protein kinase 3 is apredictor of survival and plays atumor suppressive role in colorectal cancer.

Receptor-interacting protein kinase 3 (RIP3) is amember of the RIP Ser/Thr kinase family, plays an important role in regulating cell survival, cell apoptosis and cell necrosis. However, the role of RIP3 in the carcinogenesis of colorectal cancer is still poorly understood.We used quantitative PCR and Western blot analysis to examine RIP3 expression in primary colorectal cancer and paired normal colorectal mucosa. RIP3 clinicopathological significance was assessed by immunohistochemical staining in 112 cases of primary colorectal cancer paired with noncancerous tissues. The biological function of RIP3 overexpression was measured by CCK8 assay and plate colony formation assay. Dual staining with fluorescent Annexin Vand propidium iodide (PI) was used to discriminate apoptotic or necrotic cell death.RIP3 expression was significantly lower in colorectal cancer and associated with Tstage, Mstage and AJCC stage. Cox proportional hazard models showed that RIP3 expression was an independent prognostic factor for overall survival and disease-free survival in patients with colorectal cancer. Overexpression of RIP3 significantly suppressed the proliferation of colorectal cancer cells in vitro.Our results suggest that RIP3 may function as anovel prognostic indicator after surgery and play asuppressive role in the colorectal carcinogenesis.

Chronic Treatment with a Water-Soluble Extract from the Culture Medium of Ganoderma lucidum Mycelia Prevents Apoptosis and Necroptosis in Hypoxia/Ischemia-Induced Injury of Type 2 Diabetic Mouse Brain.

Type 2 diabetes mellitus has been known to increase systemic oxidative stress by chronic hyperglycemia and visceral obesity and aggravate cerebral ischemic injury. On the basis of our previous study regarding a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (designed as MAK), which exerts antioxidative and neuroprotective effects, the present study was conducted to evaluate the preventive effects of MAK on apoptosis and necroptosis (a programmed necrosis) induced by hypoxia/ischemia (H/I) in type 2 diabetic KKAy mice. H/I was induced by a combination of unilateral common carotid artery ligation with hypoxia (8% O2 for 20 min) and subsequent reoxygenation. Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation. Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells. Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis. These results suggest that MAK confers resistance to apoptotic and necroptotic cell death and relieves H/I-induced cerebral ischemic injury in type 2 diabetic mice.

Down-Regulating Receptor Interacting Protein Kinase 1 (RIP1) Promotes Oxaliplatin-Induced Tca8113 Cell Apoptosis.

BackgroundOxaliplatin is a crucial chemotherapy drug that plays an important role in colorectal cancer and oral cancer treatment. However, the molecular mechanism of oxaliplatin in killing tongue squamous cell cancer cells is still unknown. This paper investigates the mechanism of by which oxaliplatin regulates tongue squamous cell carcinoma Tca8113 cell survival and death.Material/MethodsTca8113 was treated with 1 μmol/L oxaliplatin for 24 h. Tca8113 cell proliferation and apoptosis were determined by MTT method and flow cytometry, respectively. Western blot was applied to detect receptor-interacting protein kinase 1 (RIP1) level. Tca8113 was transfected with siRNA RIP1 and then treated with 1 μmol/L oxaliplatin, and the cell apoptosis was detected.ResultsWe found that 1 μmol/L oxaliplatin could inhibit Tca8113 cell growth (cell survival rate was 19.3%), reduce mitochondrial membrane potential (reduce 82.3%) and phosphatidylserine eversion (positive rate was 62.7%), and activate caspase-3 (increased 2.6 times). We also found that 1 μmol/L oxaliplatin treatment could increase RIP1 expression in Tca8113 cells. Cell apoptosis rate increased after siRNA RIP1 and 1 μmol/L oxaliplatin treatment (apoptosis rate was 90.2%).ConclusionsDown-regulating RIP1 promotes oxaliplatin induced Tca8113 cells apoptosis.

The role of the c-Jun N-terminal kinases 1/2 and receptor-interacting protein kinase 3 in furosemide-induced liver injury

1. The mechanisms of furosemide (FS) hepatotoxicity were explored in mice. Specifically, C57Bl/6 J mice were treated with 500 mg FS/kg bodyweight, and c-Jun N-terminal kinase (JNK) activation and receptor-interacting protein kinase 3 (RIP3) expression were measured by western blotting. Co-treatment with FS and the JNK inhibitor SP600125 was also performed, and FS-induced liver injury was compared in wild-type and RIP3 knockout (KO) mice.2. JNK phosphorylation and RIP3 expression were increased in livers from the FS-treated mice as early as 6 h after treatment and persisted until at least 24 h. JNK phosphorylation was also observed in primary mouse hepatocytes and human HepaRG cells treated with FS.3. Phosphorylated JNK translocated into mitochondria in livers, but no evidence of mitochondrial damage was observed.4. SP600125-treated mice, SP600125 co-treated primary mouse hepatocytes and RIP3 KO mice were not protected against FS hepatotoxicity. These data show that, although JNK activation and RIP3 expression are induced by FS, neither contributes to the liver injury.