[Show abstract][Hide abstract] ABSTRACT: The overactivation of signaling pathways, such as the PI3K and MAPK, which are crucial to cell growth and survival, is a common feature in many cancer types. Though a number of advances have been made in the development of molecular agents targeting these pathways, their application as monotherapies has not significantly improved clinical outcome. A novel liposomal preparation was developed, co-loaded with NCL-240, a small-molecule inhibitor of the PI3K/mTOR pathway, along with cobimetinib, a MEK/ERK pathway inhibitor. This combination drug-loaded nanocarrier, (N + C)-LP, was able to significantly enhance the cytotoxicity of these drugs against colon carcinoma cells in vitro demonstrating a clear synergistic effect (combination index of 0.79). The (N + C)-LP was also able to induce cell cycle arrest of the cells, specifically in the G1 phase thereby preventing their progression to the S-phase, typical of the action of MEK inhibitors. Analyzing the apoptotic events, it was found that this effect on cell cycle regulation is followed by the induction of apoptosis. The quantified distribution of apoptotic events showed that the (N + C)-LP induced apoptosis significantly by over 3–4 fold (P < 0.001) compared to other treatment groups. The co-loaded liposomal preparation was also targeted to the transferrin receptor of cancer cells by modifying the surface of the liposome with transferrin. FACS analysis showed that transferrin-mediated targeting enhanced the association of liposomes to HCT 116 cells by almost 5-fold. This could potentially allow for cancer cell-specific effects in vivo thereby minimizing any non-specific interactions of the liposomes with non-cancerous cells. Taken together, this study clearly shows that the combined inhibition of the PI3K and MEK pathways correlates with a significant anti-proliferative effect, due to cell-cycle regulation leading to the induction of apoptosis.
Full-text · Article · Oct 2015 · Journal of Controlled Release
[Show abstract][Hide abstract] ABSTRACT: Receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3 ) are homologous serine-threonine kinases that were recognized for their roles in directing programmed necrotic cell death or necroptosis under a broad range of pathologic settings. Emerging evidence suggests new physiologic roles for RIPK1 and RIPK3 in mediating cell death of innate immune responses. Our review discusses current evidence on the mechanisms and the impact of RIPK1- and/or RIPK3-dependent cell death in responses to a variety of viral and bacterial pathogens. Furthermore, the discussion also summarizes emerging roles for RIPK1 and RIPK3 in other facets of host immunity, including the maintenance of epithelial barrier function and pro-inflammatory processes that may, in some cases, manifest independent of cell death. Finally, we briefly consider the therapeutic opportunities in targeting RIPK1- and RIPK3-dependent processes in infection and immunity.
No preview · Article · Sep 2015 · Current topics in microbiology and immunology
[Show abstract][Hide abstract] ABSTRACT: Necrosis is a primary form of cell death in a variety of human pathologies. The deleterious nature of necrosis, including its propensity to promote inflammation, and the relative lack of the cells displaying necrotic morphology under physiologic settings, such as during development, have contributed to the notion that necrosis represents a form of pathologic stress-induced nonspecific cell lysis. However, this notion has been challenged in recent years by the discovery of a highly regulated form of necrosis, termed regulated necrosis or necroptosis. Necroptosis is now recognized by the work of multiple labs, as an important, drug-targetable contributor to necrotic injury in many pathologies, including ischemia-reperfusion injuries (heart, brain, kidney, liver), brain trauma, eye diseases, and acute inflammatory conditions. In this review, we describe the methods to analyze cellular necroptosis and activity of its key mediator, RIP1 kinase.
No preview · Article · Jul 2014 · Methods in Enzymology
[Show abstract][Hide abstract] ABSTRACT: We previously reported encouraging in vitro and in vivo anti-cancer activity of N-((3-chloro-2-hydroxy-5-nitrophenyl)carbamothioyl)benzamide (termed PITENIN-1). In the current work, we describe the structure–activity relationship study of the PIT-1 series, based on the replacement of a central thiourea unit with 1,2,3-triazole, which leads to increased liver microsomal stability, drug likeness and toxicity towards cancer cells.
Preview · Article · Jul 2014 · Medicinal Chemistry Communication
[Show abstract][Hide abstract] ABSTRACT: Akt (protein kinase B) and mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of cell death and cognitive outcome after cerebral contusion in mice; however, a role for Akt/mTOR in concussive brain injury has not been well characterized. In a mouse closed head injury (CHI) concussion traumatic brain injury (TBI) model, phosphorylation of Akt (p-Akt), mTOR (p-mTOR), and S6RP (p-S6RP) was increased by 24 hours in cortical and hippocampal brain homogenates (P<0.05 versus sham for each), and p-S6RP was robustly induced in IBA-1+ microglia and glial fibrillary acidic protein-positive (GFAP+) astrocytes. Pretreatment with inhibitors of Akt or mTOR individually by the intracerebroventricular route reduced phosphorylation of their respective direct substrates FOXO1 (P<0.05) or S6RP (P<0.05) after CHI, confirming the activity of inhibitors. Rapamycin pretreatment significantly worsened hidden platform (P<0.01) and probe trial (P<0.05) performance in CHI mice. Intracerebroventricular administration of necrostatin-1 (Nec-1) before CHI increased hippocampal Akt and S6RP phosphorylation and improved place learning (probe trials, P<0.001 versus vehicle), whereas co-administration of rapamycin or Akt inhibitor with Nec-1 eliminated improved probe trial performance. These data suggest a beneficial role for Akt/mTOR signaling after concussion TBI independent of cell death that may contribute to improved outcome by Nec-1.Journal of Cerebral Blood Flow & Metabolism advance online publication, 18 June 2014; doi:10.1038/jcbfm.2014.113.
Preview · Article · Jun 2014 · Journal of Cerebral Blood Flow & Metabolism
[Show abstract][Hide abstract] ABSTRACT: Retinal ischemia-reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.
No preview · Article · Apr 2014 · Experimental Eye Research
[Show abstract][Hide abstract] ABSTRACT: Pathogen recognition by the innate immune system initiates the production of pro-inflammatory cytokines but can also lead to programmed host cell death. Necroptosis, a caspase-independent cell death pathway, can contribute to host defense against pathogens or cause damage to host tissues. Receptor-interacting Protein (RIP1) is a serine/threonine kinase that integrates inflammatory and necroptotic responses. To investigate mechanisms of RIP1-mediated activation of immune cells, we established a genetic screen based on RIP1-mediated necroptosis in wild-derived MOLF mice, which diverged from classical laboratory mice over a million years ago. When compared to C57BL/6, MOLF macrophages were resistant to RIP1-mediated necroptosis induced by TLRs. Using a forward genetic approach in a backcross panel of mice, we identified CYLD, a deubiquitinase known to act directly on RIP1 and promote necroptosis in TNFR signaling, as the gene conferring the trait. We demonstrate that CYLD is required for TLR-induced necroptosis and describe a novel mechanism by which CYLD is downregulated at the transcriptional level in MOLF macrophages to confer protection from necroptosis.
No preview · Article · Apr 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3β, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.
Full-text · Article · Feb 2014 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: Sirtuins can promote deacetylation of a wide range of substrates in diverse cellular compartments and regulate many cellular processes¹,². Recently Narayan et al., reported that SIRT2 was required for necroptosis based on their findings that SIRT2 inhibition, knock-down or knock-out prevented necroptosis. We sought to confirm and explore the role of SIRT2 in necroptosis and tested four different sources of the SIRT2 inhibitor AGK2, three independent siRNAs against SIRT2, and cells from two independently generated Sirt2−/− mouse strains, however we were unable to show that inhibiting or depleting SIRT2 protected cells from necroptosis. Furthermore, Sirt2−/− mice succumbed to TNF induced Systemic Inflammatory Response Syndrome (SIRS) more rapidly than wild type mice while Ripk3−/− mice were resistant. Our results therefore question the importance of SIRT2 in the necroptosis cell death pathway.
[Show abstract][Hide abstract] ABSTRACT: Retinal ischemia–reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.
No preview · Article · Jan 2014 · Experimental Eye Research
[Show abstract][Hide abstract] ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
[Show abstract][Hide abstract] ABSTRACT: Interferons (IFNs) are cytokines with powerful immunomodulatory and antiviral properties, but less is known about how they induce cell death. Here, we show that both type I (α/β) and type II (γ) IFNs induce precipitous receptor-interacting protein (RIP)1/RIP3 kinase-mediated necrosis when the adaptor protein Fas-associated death domain (FADD) is lost or disabled by phosphorylation, or when caspases (e.g., caspase 8) are inactivated. IFN-induced necrosis proceeds via progressive assembly of a RIP1-RIP3 "necrosome" complex that requires Jak1/STAT1-dependent transcription, but does not need the kinase activity of RIP1. Instead, IFNs transcriptionally activate the RNA-responsive protein kinase PKR, which then interacts with RIP1 to initiate necrosome formation and trigger necrosis. Although IFNs are powerful activators of necrosis when FADD is absent, these cytokines are likely not the dominant inducers of RIP kinase-driven embryonic lethality in FADD-deficient mice. We also identify phosphorylation on serine 191 as a mechanism that disables FADD and collaborates with caspase inactivation to allow IFN-activated necrosis. Collectively, these findings outline a mechanism of IFN-induced RIP kinase-dependent necrotic cell death and identify FADD and caspases as negative regulators of this process.
Preview · Article · Jul 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Many lipids present in cellular membranes are phosphorylated as part of signaling cascades and participate in the recruitment, localization, and activation of downstream protein effectors. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is one of the most important second messengers and is capable of interacting with a variety of proteins through specific PtdIns(3,4,5)P3 binding domains. Localization and activation of these effector proteins controls a myriad of cellular functions including cell survival, proliferation, cytoskeletal rearrangement, and gene expression. Aberrations in the production and metabolism of PtdIns(3,4,5)P3 have been implicated in many human diseases including cancer, diabetes, inflammation, and heart disease. This chapter provides an overview of the role of PtdIns(3,4,5)P3 in cellular regulation and the implications of PtdIns(3,4,5)P3 dysregulation in human diseases. Additionally, recent attempts at targeting PtdIns(3,4,5)P3 signaling via small molecule inhibitors are summarized.
No preview · Article · Jun 2013 · Advances in Experimental Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: Necroptosis is a novel form of regulated non-apoptotic cell death, which displays morphological features of necrosis. The kinase activity of receptor-interacting protein kinase 1 (RIP1) is a critical component in signaling for necroptosis. The development of assays to evaluate RIP1 kinase activity is important in the further development of existing and novel inhibitors of necroptosis. Here, we describe RIP1 protein expression and purification from mammalian and insect cells as well as two in vitro kinase assays to detect RIP1 kinase activity and inhibition.
No preview · Article · Jun 2013 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Receptor Interacting Protein 1 (RIP1) kinase is one of the key mediators of tumor necrosis factor alpha (TNF-α) signaling and is critical for activation of necroptotic cell death. We developed a method for expression of recombinant kinase, utilizing baculovirus co-infection of Cdc37, an Hsp90 co-chaperone, and RIP1-His, followed by a two-step purification scheme. After optimization, 1-3 mg of highly purified RIP1 kinase was typically obtained from a 1 L of Sf9 cells. The recombinant protein displayed kinase activity that was blocked by RIP1 inhibitors, necrostatins. The purified protein was used to develop a simple and robust thermal shift assay for further assessment of RIP1 inhibitors.
Full-text · Article · Mar 2013 · Protein Expression and Purification
[Show abstract][Hide abstract] ABSTRACT: Abstract We have developed and characterized micellar formulations of analogs to the recently developed inhibitor of the phosphatidylinositol-3-kinase (PI3K) pathway (N-[(2-hydroxy-5-nitrophenyl)amino]carbonothioyl-3,5-dimethylbenzamide (DM-PIT-1)) for their physicochemical, loading and cytotoxic properties. The first generation inhibitor DM-PIT-1 is a non-lipid, small molecule inhibitor of phosphatidylinositol-3,4,5-triphosphate/Pleckstrin homology (PIP3/PH) binding capable of inhibiting the growth of tumor cells both in vitro and in vivo. A second generation of improved and druggable analogs has been developed. All compounds were successfully loaded (>70%) in PEG2000-PE micelles of 16-20 nm in size with several analogs demonstrating favorable cytotoxic activity against A2780 ovarian carcinoma. These compounds were also successfully incorporated into polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles combined with surface-bound tumor necrosis factor related apoptosis inducing ligand (TRAIL). The resulting multifunctional combination micelles were able to significantly enhance cytotoxic activity in the TRAIL-resistant A2780 cell line. Additionally, analogs NCL-176 and NCL-240 were effective in inhibiting tumor growth in an in vivo subcutaneous tumor model of A2780. These results indicate the utility of delivering TRAIL and PI3K pathway inhibitors in a combined micellar preparation.
[Show abstract][Hide abstract] ABSTRACT: Necroptosis is a cellular mechanism that mediates necrotic cell death. The receptor-interacting serine/threonine protein kinase 1 (RIP1) is an essential upstream signaling molecule in tumor-necrosis-factor-α-induced necroptosis. Necrostatins, a series of small-molecule inhibitors, suppress necroptosis by specifically inhibiting RIP1 kinase activity. Both RIP1 structure and the mechanisms by which necrostatins inhibit RIP1 remain unknown. Here, we report the crystal structures of the RIP1 kinase domain individually bound to necrostatin-1 analog, necrostatin-3 analog, and necrostatin-4. Necrostatin, caged in a hydrophobic pocket between the N- and C-lobes of the kinase domain, stabilizes RIP1 in an inactive conformation through interactions with highly conserved amino acids in the activation loop and the surrounding structural elements. Structural comparison of RIP1 with the inhibitor-bound oncogenic kinase B-RAF reveals partially overlapping binding sites for necrostatin and for the anticancer compound PLX4032. Our study provides a structural basis for RIP1 inhibition by necrostatins and offers insights into potential structure-based drug design.