Activation Of Cell Survival Pathways Research Articles

Structure, Function and Inhibition of Poly(ADP-ribose)polymerase, Member 14 (PARP14).

Poly(ADP-ribose)polymerase, member 14 (PARP14, alternatively named ARTD8, BAL2, and COAST6) is an intracellular mono(ADP-ribosyl) transferase. PARP14 transfers a negatively charged ADP-ribose unit from a donor NAD+ molecule onto a target protein, post-translationally. PARP14's domain architecture consists of three macrodomains (Macro1, Macro2 and Macro3), a WWE domain and an ARTD (or catalytic domain). The Macro2 and Macro3 domains bind ADPribose (ADPr) with high affinity, whereas the WWE domain stabilizes the protein structure by binding to ADPr derivatives. The catalytic domain is involved in binding the NAD+ and catalyzing the mono- ADP-ribosylation reaction. PARP14 has been identified as a possible anti-cancer and antiinflammatory target. Acting as a transcriptional co-activator for STAT6, PARP14 acts to promote the over activation of the Th2 immune response, thus promoting the metabolic change to an anaerobic state (Warburg effect) and activation of cell survival pathways through JNK2 and the PGI/AMF complex. These changes are consistent with the metabolic sophistication observed in cancer, and the immune imbalance in inflammatory diseases. Current literature on selective and unselective PARP14 inhibitors are reviewed and discussed. Although there is no evidence that selective PARP inhibitors would be advantageous we have proposed some strategies for future design of selective PARP14 inhibitors.

Enhanced Chemotherapeutic Efficacy of Paclitaxel Nanoparticles Co-delivered with MicroRNA-7 by Inhibiting Paclitaxel-Induced EGFR/ERK pathway Activation for Ovarian Cancer Therapy

Chemotherapy-induced activation of cell survival pathways leads to drug resistance. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression in many biological pathways. Paclitaxel (PTX) is one of the first-line chemotherapy drugs for ovarian cancer, and it induces the activation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) pathway that leads to tumor cell proliferation, survival, invasion, and drug resistance. MicroRNA-7 (miR-7) has the ability to suppress the EGFR/ERK pathway. To sensitize chemotherapy, we developed monomethoxy(poly(ethylene glycol))-poly(d,l-lactide- co-glycolide)-poly(l-lysine) nanoparticles for the simultaneous co-delivery of PTX and miR-7. The resulting PTX/miR-7 nanoparticles (P/MNPs) protect miRNA from degradation, possess a sequential and controlled release of drugs, improve the transfection efficiency of miRNA, decrease the half-maximal inhibitory concentration of PTX, and increase the apoptosis of ovarian cancer cells. The chemotherapeutic efficacy of PTX is prominently enhanced in vitro and in vivo via the inhibition of PTX-induced EGFR/ERK pathway activation by miR-7. Our studies in P/MNPs reveal a novel paradigm for a dual-drug-delivery system of chemotherapeutics and gene therapy in treating cancers.

GMI-1271 Improves Efficacy and Safety of Chemotherapy in R/R and Newly Diagnosed Older Patients with AML: Results of a Phase 1/2 Study

Background Binding of E-selectin (E-sel), an adhesion molecule expressed in the vasculature of the bone marrow, to sialyl Lex on the leukemic cell surface activates cell survival pathways and promotes chemotherapy resistance in AML. GMI-1271 is a novel E-selectin antagonist that disrupts activation of cell survival pathways, enhances chemotherapy response with improved survival in mouse xenograft and syngeneic models and protects from chemotherapy toxicity in vivo (Becker 2013; Winkler 2013, 2014, 2016). We added GMI-1271 to salvage with MEC for relapsed/refractory AML patients or to induction with 7+3 for older treatment naive (TN) AML patients. Methods A Phase (Ph) 1 trial escalated GMI-1271 (5-20 mg/kg) combined with MEC (mitoxantrone, etoposide, cytarabine) in pts with R/R AML, to evaluate safety, tolerability and anti-leukemic activity. Dose limiting toxicity (DLT) was defined as persistent cytopenias in the absence of disease or any Grade 3-5 non-hematologic toxicity beyond day 42. The recommended Ph 2 dose (RP2D) of GMI-1271 was 10 mg/kg. A Ph 2 study then enrolled 47 pts with R/R AML for GMI-1271 plus MEC chemotherapy. Responders could receive 1 cycle of consolidation with GMI-1271 plus MEC. We also enrolled a separate Ph 2 cohort of 25 pts ≥60 yrs with TN AML for GMI-1271 (10 mg/kg) plus cytarabine and idarubicin (7+3). Two cycles of induction were allowed. Responders could receive 3 cycles of consolidation with GMI-1271 plus intermediate dose cytarabine. Prior treatment of MDS was allowed; for R/R disease prior SCT was allowed. GMI-1271 was given 24 hrs prior, then every 12 hrs during and for 48 hrs post chemotherapy. E-sel ligand expression on leukemic blasts in blood and bone marrow was assessed by flow cytometry, for the percentage of blasts binding to E-sel-Fc chimera and HECA452 (antibody to sialyl Lex). Results 91 pts were enrolled (Ph 1 R/R=19; Ph 2 R/R=47, TN=25). Median age for R/R pts was 59 yrs (range 26-84) and 62% were males. Prior AML history included 32% primary refractory, 33% CR1 Median age for TN pts was 67 yrs (range, 60-79) 56% were males, and 32% had high-risk CG. 56% had secondary AML (sAML), 43% of sAML had prior hypomethylating therapy. GMI-1271 was well tolerated, adverse events were typical for AML induction in the TN population, and Grade 3/4 mucositis was not seen. 30 and 60 d mortality were 8 and 12%, respectively; two deaths were from sepsis before response assessment and one involved persistent disease. Median time to ANC 500 was 28d (range 21-63). 8 pts required two induction courses, and 4 (16%) proceeded to SCT by data cut-off. The CR/CRi rate was 68% (73% for de novo and 64% for sAML) and ORR was 80%. Median OS and DFS are not yet reached; median follow up is 6 mos. Median E-sel ligand expression (binding to HECA452) at baseline was 31% (range, 2-92%) of bone marrow blasts. Conclusion The addition of GMI-1271 to chemotherapy was well tolerated with high remission rate, low induction mortality, and low rate of mucositis suggesting improved tolerance of chemotherapy. In R/R AML, correlative studies showed blasts expressing the E-sel ligand were predictive of response. Initial survival outcomes are promising and updated survival outcomes will be presented at ASH. FDA granted Breakthrough Therapy Designation to GMI-1271 for treatment of adults with R/R AML and randomized trials are planned. Disclosures DeAngelo: Amgen: Consultancy, Research Funding; BMS: Consultancy; Immunogen: Honoraria, Research Funding; Celgene: Research Funding; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Honoraria, Research Funding; Incyte: Consultancy, Honoraria; Glycomimetics: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Takeda Pharmaceuticals U.S.A., Inc.: Honoraria; Shire: Honoraria; Pfizer Inc.: Consultancy, Honoraria, Research Funding. Jonas: Celgene: Membership on an entity9s Board of Directors or advisory committees; AbbVie, Celgene, Daiichi Sankyo, Pharmacyclics, Genentech/Roche, Glycomimetics, Esanex, Kalobios: Research Funding; Rigel: Consultancy. Liesveld: Seattle Genetics: Honoraria; Onconova: Honoraria. Advani: Pfizer: Consultancy; Takeda/ Millenium: Research Funding. O9Dwyer: Onkimmune Ltd: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Magnani: GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees. Thackray: GlycoMimetics, Inc.: Employment, Equity Ownership. Becker: GlycoMimetics, Inc.: Research Funding.

Thiamethoxam induced hepatotoxicity and pro-carcinogenicity in rabbits via motivation of oxidative stress, inflammation, and anti-apoptotic pathway.

Thiamethoxam (TMX) is a non-mutagenic neonicotinoid insecticide that is widely used to combat different types of insects. The hepatotoxicity and carcinogenicity of TMX have been approved previously in mice but not in rats. However, the TMX-induced hepatotoxic and pro-carcinogenic effects on rabbits remain unclear. The present study elucidated the roles of oxidative stress, pro-inflammatory cytokines, and apoptosis-related genes in the hepatotoxic and carcinogenic effects of TMX on rabbits. Sixteen male rabbits were equally divided into two groups; eight rabbits orally treated with TMX at a dose of 250 mg/kg b.w for 90 successive days. Hepatotoxic effects of TMX were evidenced by attenuation of liver enzyme activities, elevation of bilirubin levels, and alterations in the hepatic architecture, including hepatocyte death by necrosis and apoptosis, lymphocyte infiltration and fibrosis. TMX induced oxidative stress, as evidenced by the significant increases in malondialdehyde levels and antioxidant enzyme (glutathione transferase and catalase) activities along with a decrease in glutathione levels. TMX also up-regulated the mRNA levels of interleukin-6 (1.6-fold) and B cell lymphoma-2 (1.8-fold) and down-regulated the mRNA level of the tumor necrosis factor-α (0.8-fold), indicating its effects on cell survival and proliferation through the inhibition of apoptosis. Interestingly, the elevated level of carcinoembryonic antigen and the appearance of ground glass-like hepatocytes suggested that TMX exerted a pro-carcinogenic effect. In conclusion, TMX exerts potentially hepatotoxic and pro-carcinogenic effects on rabbits by modulating oxidative/antioxidative status and pro-inflammatory cytokine production, inhibiting apoptosis and activating cell survival pathways.

The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways.

Bacterial effectors potently manipulate host signaling pathways. The marine bacterium Vibrio parahaemolyticus (V. para) delivers effectors into host cells through two type 3 secretion systems (T3SSs). T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate nonapoptotic cell death. To understand how the concerted action of T3SS1 effectors globally affects host cell signaling, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1+) to those in cells infected with V. para lacking T3SS1 (T3SS1-). Overall, the host transcriptional response to both T3SS1+ and T3SS1-V. para was rapid, robust, and temporally dynamic. T3SS1 rewired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors targeted host cells at the posttranslational level to cause cytotoxicity, V. para T3SS1 also precipitated a host transcriptional response that initially activated cell survival and repressed cell death networks. The increased expression of several key prosurvival transcripts mediated by T3SS1 depended on a host signaling pathway that is silenced posttranslationally later in infection. Together, our analysis reveals a complex interplay between the roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling.

Sex Differences of Human Cardiac Progenitor Cells in the Biological Response to TNF-α Treatment.

Adult cardiac progenitor cells (CPCs), isolated as cardiosphere-derived cells (CDCs), represent promising candidates for cardiac regenerative therapy. CDCs can be expanded in vitro manyfolds without losing their differentiation potential, reaching numbers that are appropriate for clinical applications. Since mechanisms of successful CDC survival and engraftment in the damaged myocardium are still critical and unresolved issues, we aimed at deciphering possible key factors capable of bolstering CDC function. In particular, the response and the phenotype of CDCs exposed to low concentrations of the multifunctional cytokine tumor necrosis factor α (TNF-α), known to be capable of activating cell survival pathways, have been investigated. Furthermore, differential biological responses of CDCs from male and female donors, in terms of cell cycle progression and cell spreading, have also been assessed. The results obtained indicate that (i) the intracellular signaling activated in our experimental conditions is most likely due to the prosurvival and proliferative signaling of TNF-α receptor 2 and that (ii) cells from female patients appear more responsive to TNF-α treatment in terms of cell cycle progression and migration ability. In conclusion, the present report highlights the hypothesis that TNF-stimulated CDCs isolated from females may represent a promising candidate for cardiac regenerative therapy applications.

Identification of initial leads directed at the calmodulin-binding region on the Src-SH2 domain that exhibit anti-proliferation activity against pancreatic cancer.

Cellular calmodulin binds to the SH2 domain of Src kinase, and upon Fas activation it recruits Src into the death-inducing signaling complex. This results in Src-ERK activation of cell survival pathway through which pancreatic cancer cells survive and proliferate. We had proposed that the inhibition of the interaction of calmodulin with Src-SH2 domain is an attractive strategy to inhibit the proliferation of pancreatic cancer. Thus we have performed screening of compound libraries by a combination of methods and identified some compounds (initial leads) that target the calmodulin-binding region on the SH2 domain and inhibit the proliferation of pancreatic cancer cells in in vitro assays. Most of these compounds also exhibited varying degrees of cytotoxicity when tested against immortalized breast epithelial cell line (MCF10A). These initial leads are likely candidates for development in targeted delivery of compounds to cancer cells without affecting normal cells.

Substance P induces cardioprotection in ischemia-reperfusion via activation of AKT.

Accumulating evidence indicates that substance P is cardioprotective following ischemia-reperfusion primarily due to its potent coronary vasodilator actions. However, an anti-apoptotic effect of substance P has been observed in tenocytes following ischemia, which involved activation of the AKT pathway. This suggests the possibility that substance P also provides cardioprotection via direct actions to activate AKT in myocardial cells. The purpose of this study was to test the hypothesis that substance P attenuates ischemia-related cell death via direct effects on myocardial cells by activating cell survival pathways. Seven-week-old male Sprague-Dawley rats, anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg), were used. The ability of substance P to prevent cellular damage was assessed following ischemia-reperfusion in an isolated heart preparation and in short-term hypoxia without reperfusion using a left ventricular tissue slice culture preparation. In addition, the NK-1 receptor and AKT involvement was assessed using the NK-1 receptor antagonist L732138 and the AKT inhibitor LY294002. The results indicate that substance P reduced the ischemia-related release of lactate dehydrogenase in both preparations and the degree of apoptosis and necrosis in the hypoxic left ventricular slices, indicating its ability to attenuate cell damage; and induced AKT phosphorylation, with both the AKT inhibitor and NK-1 receptor antagonist preventing the increased phosphorylation of AKT and the ability of substance P to attenuate hypoxic cellular damage. It is concluded that substance P reduces ischemia/hypoxia-induced myocardial cell death by acting directly on cardiac cells to initiate cell survival pathways via the NK-1 receptor and AKT.

Growth arrest–specific protein 6 protects against renal ischemia–reperfusion injury

BackgroundRenal injury caused by ischemia–reperfusion (I/R) often occurs after shock or transplantation. Growth arrest–specific protein 6 (Gas6) is a secreted protein that binds to the TAM—Tyro3, Axl, Mer—family tyrosine kinase receptors, which modulate the inflammatory response and activate cell survival pathways. We hypothesized that Gas6 could have a protective role in attenuating the severity of renal injury after I/R. Materials and methodsAdult mice were subjected to 45 min of bilateral renal ischemia. Recombinant mouse Gas6 (rmGas6, 5 μg per mouse) or normal saline (vehicle) was administered intraperitoneally 1 h before ischemia and all subjects were sacrificed at 23 h after I/R for blood and tissue analysis. The expression of protein and messenger RNA (mRNA) was assessed by Western blotting and quantitative polymerase chain reaction, respectively. ResultsTreatment with rmGas6 significantly decreased serum levels of creatinine and blood urea nitrogen by 29% and 27%, respectively, improved the renal histologic injury index, and reduced the apoptosis in the kidneys, compared with the vehicle. Renal mRNA levels of interleukin 1β, interleukin 6, tumor necrosis factor α, keratinocyte-derived chemokine and macrophage inflammatory protein 2 were decreased significantly by 99%, 60%, 53%, 58%, and 43%, with rmGas6 treatment, respectively. After I/R, renal I-kappa-B α levels were reduced by 40%, whereas they returned to sham levels with rmGas6 treatment. The mRNA levels of inducible nitric oxide synthase and cyclooxygenase 2 were reduced by 79% and 70%, respectively, whereas the expression of cyclin D1 was increased by 2.1-fold in the rmGas6-treated group, compared with the vehicle. ConclusionsGas6 suppresses the nuclear factor κB pathway and promotes cell proliferation, leading to the reduction of inflammation and protection of renal injury induced by I/R.

Coupling of β1-adrenergic receptor to type 5 adenylyl cyclase and its physiological relevance in cardiac myocytes

Myocardial β-adrenergic receptor (β-AR) β1- and β2-subtypes are highly homologous, but play opposite roles in cardiac apoptosis and heart failure, as do cardiac adenylyl cyclase (AC) subtypes 5 (AC5) and 6 (AC6): β1-AR and AC5 promote cardiac remodeling, while β2-AR and AC6 activate cell survival pathways. However, the mechanisms involved remain poorly understood. We hypothesized that AC5 is coupled preferentially to β1-AR rather than β2-AR, and we examined this idea by means of pharmacological and genetic approaches. We found that selective inhibition of AC5 with 2′5′-dideoxyadenosine significantly suppressed cAMP accumulation and cardiac apoptosis induced by selective β1-AR stimulation, but had no effect on cAMP accumulation and cardiac apoptosis in response to selective β2-AR stimulation. The results of selective stimulation of β1-AR and β2-AR in neonatal cardiac myocytes prepared from wild-type and AC5-knockout mice were also consistent with the idea that β1-AR selectively couples with AC5. We believe these results are helpful for understanding the mechanisms underlying the different roles of AR subtypes in healthy and diseased hearts.

Ghrelin's Effects on Proinflammatory Cytokine Mediated Apoptosis and Their Impact on β-Cell Functionality.

Ghrelin is a peptidic hormone, which stimulates cell proliferation and inhibits apoptosis in several tissues, including pancreas. In preclinical stage of type 1 diabetes, proinflammatory cytokines generate a destructive environment for β-cells known as insulitis, which results in loss of β-cell mass and impaired insulin secretion, leading to diabetes. Our aim was to demonstrate that ghrelin could preserve β-cell viability, turnover rate, and insulin secretion acting as a counter balance of cytokines. In the present work we reproduced proinflammatory milieu found in insulitis stage by treating murine cell line INS-1E and rat islets with a cytokine cocktail including IL-1β, IFNγ, and TNFα and/or ghrelin. Several proteins involved in survival pathways (ERK 1/2 and Akt/PKB) and apoptosis (caspases and Bcl-2 protein family and endoplasmic reticulum stress markers) as well as insulin secretion were analyzed. Our results show that ghrelin alone has no remarkable effects on β-cells in basal conditions, but interestingly it activates cell survival pathways, downregulates apoptotic mediators and endoplasmic reticulum stress, and restores insulin secretion in response to glucose when beta-cells are cytokine-exposed. These data suggest a potential role of ghrelin in preventing or slowing down the transition from a preclinical to clinically established diabetes by ameliorating the effects of insulitis on β-cells.

Host-microbe interactions via membrane transport systems.

Living organisms take in essential molecules and get rid of wastes effectively through the selective transport of materials. Especially in the digestive tract, advanced transport systems are indispensable for the absorption of nutrients and elimination of waste products. These transport pathways control physiological functions by modulating the ionic environment inside and outside the cells. Moreover, recent studies have shown the importance of the expression of trafficking-related molecules and the population of gut microbiota. We found that the molecules secreted from microorganisms are imported into the cells via transporters or endocytosis and that they activate cell survival pathways of intestinal epithelial cells. These findings indicate that the interactions between the gut microbiota and host cells are mediated, at least partly, by the membrane transport systems. In addition, it is well known that the breakdown of transport systems induces various diseases. This review highlights the significance of the transport systems as the pathogenic molecules and therapeutic targets in gastrointestinal disorders. For example, abnormal expression of the genes encoding membrane transport-related molecules is frequently involved in digestive diseases, such as colorectal cancer and inflammatory bowel disease. We herein review the significance of these molecules as pathogenic and therapeutic targets for digestive diseases.

Thrombopoietin receptor agonists protect human cardiac myocytes from injury by activation of cell survival pathways.

Thrombopoietin confers immediate protection against injury caused by ischemia/reperfusion in the rat heart. Eltrombopag is a small molecule agonist of the thrombopoietin receptor, the physiologic target of thrombopoietin. However, the ability of eltrombopag and thrombopoietin to protect human cardiac myocytes against injury and the mechanisms underlying myocyte protection are not known. Human cardiac myocytes (n = 6-10/group) were treated with eltrombopag (0.1-30.0 µM) or thrombopoietin (0.1-30.0 ng/ml) and then subjected to 5 hours of hypoxia (95% N2/5% CO2) and 16 hours of reoxygenation to determine their ability to confer resistance to myocardial injury. The thrombopoietin receptor c-Mpl was detected in unstimulated human cardiac myocytes by Western blotting. Eltrombopag and thrombopoietin confer immediate protection to human cardiac myocytes against injury from hypoxia/reoxygenation by decreasing necrotic and apoptotic cell death in a concentration-dependent manner, with an optimal concentration of 3 µM for eltrombopag and 1.0 ng/ml for thrombopoietin. The extent of protection conferred with eltrombopag is equivalent to that of thrombopoietin. Eltrombopag and thrombopoietin activate multiple prosurvival pathways; inhibition of Janus kinase-2, proto-oncogene tyrosine-protein kinase, protein kinase B/phosphatidylinositol-3 kinase, p44/42 mitogen-activated protein kinase (MAPK), and p38 MAPK abolished cardiac myocyte protection by eltrombopag and thrombopoietin. Eltrombopag and thrombopoietin may represent important and potent agents for immediately and substantially increasing protection of human cardiac myocytes, and may offer a long-lasting benefit through activation of prosurvival pathways during ischemia.

Anti-apoptotic effect of San Huang Shel Shin Tang cyclodextrin complex (SHSSTc) on CCl4 -induced hepatotoxicity in rats.

The metabolic loading is heavier in liver especially when injured or inflammation. San Huang Shel Shin Tang (SHSST) was an old traditional herbal decoction, which composed with Rheum officinale Baill, Scutellaria baicalnsis Geprgi and Coptis chinensis Franch (1:1:2 in weight), can provide a liver protection effects. We used a beta-cyclodextrin (β-CD) drug modification method in reduce of the necessary dose of the SHSST. As the results, the FAS-FADD expressions leaded apoptosis in CCl4 intraperitoneal (IP) injection induced acute liver injury in rats. Silymarin, baicalein, SHSST, and SHSST β-CD complex (SHSSTc) pretreatments protected liver through the decreasing of the expressions of FAS-FADD and downstream caspase-3 and caspase-8. Particularly, SHSSTc (30 mg/kg day) treatment enhanced cell survival pathway activation through the PI3K, Akt and Bad phosphorylation. Compared with SHSST as well as silymarin and baicalein, SHSSTc provided a magnificent liver protection effect, especially in survival pathway activation/TUNEL-apoptotic cell reduction/serum cholesterol level suppression. All these data suggested that β-CD complex modified the SHSST and promoted the bioavailability and liver protection effects. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 663-670, 2016.

Abstract 172: Thrombopoietin Receptor Agonists Protect Human Cardiac Myocytes from Injury by Activation of Cell Survival Pathways

Thrombopoietin confers immediate protection against injury caused by ischemia/reperfusion in the rat heart at a dose that does not increase platelet levels. Eltrombopag is a small molecule agonist of the thrombopoietin receptor; the physiological target of thrombopoietin. Administration of thrombopoietin and eltrombopag result in a dose- and time-dependent increase in platelet counts in patients with thrombocytopenia. However, the ability of eltrombopag and thrombopoietin to immediately protect human cardiac myocytes against injury and the mechanisms underlying myocyte protection are not known. Human cardiac myocytes (7500 cells, n=10/group) were treated with eltrombopag (0.1- 30.0 μM) or thrombopoietin ( 0.1 - 30.0 ng/ml) and then subjected to 5 hours of hypoxia (95% N 2 /5%CO 2 ) and 16 hours of reoxygenation to determine their ability to confer resistance to necrotic and apoptotic myocardial injury . The thrombopoietin receptor (c-Mpl) was detected in unstimulated human cardiac myocytes by western blotting. Eltrombopag and thrombopoietin confer immediate protection to human cardiac myocytes against injury from hypoxia/reoxygenation by decreasing necrotic and apoptotic cell death in a concentration-dependent manner with an optimal concentration of 3 μM for eltrombopag and 1.0 ng/ml for thrombopoietin. The extent of protection conferred to cardiac myocytes with eltrombopag is equivalent to that of thrombopoietin. Eltrombopag and thrombopoietin activate multiple pro-survival pathways; inhibition of JAK-2 (AG-490, 10 μM), p38 MAPK (SB203580, 10 μM), p44/42 MAPK (PD98059, 10 μM), Akt/PI 3 kinase (Wortmannin, 100 nM), and src kinase (PP1, 20 μM) prior to and during hypoxia abolished cardiac myocyte protection by eltrombopag and thrombopoietin. These inhibitors had no effect on hypoxia/reoxygenation injury in myocytes when used alone. Eltrombopag and thrombopoietin may represent important and potent agents for immediately and substantially increasing protection of human cardiac myocytes, and may offer long-lasting benefit through activation of pro-survival pathways during ischemia.

Abstract 232: Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols

Abstract Some cancer-preventive agents such as selenium and green tea polyphenols effectively prevent cancer in some cases, but fail in other cases. It could be possible that the cancer-preventive actions of these agents may be altered by the interaction of cancer cells with the extracellular matrix (ECM) components, thus making this an important issue to study. Others have previously cloned a family of human prostate cancer cell lines with a common lineage that show the progression of characteristics from low to high degrees of malignancy and mimic different stages of tumor progression seen in human prostate cancer. For this study, we used these prostate cancer cell lines with increasing tumorigenicity and invasive ability: WPE1-NA22 showing the lowest, WPE1-NB14 and WPE1-NB11 showing intermediate, and WPE1-NB26 showing the greatest. The low tumorigenic cell line, WPE1-NA22 resembles noninvasive prostatic intra-epithelial neoplasia and represents a good target for chemoprevention in humans. Initially, we grew these cells on the surface of ECM components, laminin, collagen I, and fibronectin and determined the tumor cell sensitivity to methylseleninic acid (MSA) and epigallocatechin-3-gallate (EGCG). Poly-L-lysine was used as a control substratum. Cell growth was determined by sulforhodamine B staining assay. Then we determined whether these ECM proteins could enhance tumor cell resistance to MSA and EGCG. ECM components, but not polylysine, decreased tumor cell sensitivity to these agents. The effect of ECM components was prevented by β1 integrin-blocking antibodies suggesting that β1 integrin plays a role in mediating intracellular signaling conferring resistance against MSA and EGCG. Previously, we have shown an increase in expression of protein kinase Cε (PKCε) in advanced prostate tumor cells WPE1-NB26. A decrease in expression of PKCε by siRNA approach decreased the tumor cell resistance caused by ECM components. Furthermore, an inhibition of cell survival enzymes phosphoinositide-3-kinase (PI3K) with its specific inhibitor LY294002 (50 μM) as well as Akt with its specific inhibitor MK-2206 (1 μM) decreased the effect of ECM components on tumor cell survival against MSA and EGCG. WPE1-NB14, a low tumorigenic cell line having low PKCε and Akt activities, responded less to ECM components and showed higher sensitivity to MSA and EGCG. Conceivably, ECM components, laminin, collagen, and fibronectin induced β1 integrin signaling to activate cell survival pathways involving PKCε and PI3K-Akt, thereby decreasing prostate tumor cell sensitivity to MSA and EGCG. Thus, ECM may be one of the limiting factors for the action of cancer-preventive agents. Citation Format: Rayudu Gopalakrishna, Tiffany Fan, Ronald Deng, David Rayudu, Zachary W. Chen, William S. Tzeng, Usha Gundimeda. Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 232. doi:10.1158/1538-7445.AM2014-232

Pyridoxamine Reverts Methylglyoxal‐induced Impairment of Survival Pathways During Heart Ischemia

Increased levels of advanced glycation end-products (AGE) and their precursors, such as methylglyoxal (MG), in patients with diabetes may account for impaired response to heart ischemia. Pyridoxamine is a derivate of vitamin B6, which has been shown to reduce AGE formation. Our goal was to assess the role of pyridoxamine in protecting from MG-induced impaired heart response to ischemia. Wistar rats were subjected to MG administration (WM), MG plus pyridoxamine (WMPyr), or no treatment (W). Half of the hearts from each group were submitted to ischemia and the other half were perfused as control. The levels of CEL, Bcl-2, Bax, and total and phosphorylated forms of JNK and Akt were determined. Methylglyoxal led to higher levels of AGE and AGE receptor (RAGE) than in the W group. During ischemia, MG caused an impairment of survival pathways and Bcl-2/Bax ratio, a marker of apoptosis. Pyridoxamine treatment decreased glycation and restored the activation of JNK and Akt during ischemia. These events were followed by levels of Bcl-2/Bax ratio similar to W group. Methylglyoxal-induced AGE accumulation impairs the activation of cell survival pathways during ischemia. Pyridoxamine-induced decrease of glycation inhibited the effects of MG accumulation in the heart, suggesting that it can be of added value to usual diabetic therapy.

Erythropoietin Activates Cell Survival Pathways in Breast Cancer Stem–like Cells to Protect Them from Chemotherapy

Recombinant erythropoietin (EPO) analogs [erythropoiesis-stimulating agents (ESA)] are clinically used to treat anemia in patients with cancer receiving chemotherapy. After clinical trials reporting increased adverse events and/or reduced survival in ESA-treated patients, concerns have been raised about the potential role of ESAs in promoting tumor progression, possibly through tumor cell stimulation. However, evidence is lacking on the ability of EPO to directly affect cancer stem-like cells, which are thought to be responsible for tumor progression and relapse. We found that breast cancer stem-like cells (BCSC) isolated from patient tumors express the EPO receptor and respond to EPO treatment with increased proliferation and self-renewal. Importantly, EPO stimulation increased BCSC resistance to chemotherapeutic agents and activated cellular pathways responsible for survival and drug resistance. Specifically, the Akt and ERK pathways were activated in BCSC at early time points following EPO treatment, whereas Bcl-xL levels increased at later times. In vivo, EPO administration counteracted the effects of chemotherapeutic agents on BCSC-derived orthotopic tumor xenografts and promoted metastatic progression both in the presence and in the absence of chemotherapy treatment. Altogether, these results indicate that EPO acts directly on BCSC by activating specific survival pathways, resulting in BCSC protection from chemotherapy and enhanced tumor progression.

Traffic Control of Bacteria-Derived Molecules: A New System of Host-Bacterial Crosstalk

Virulent microorganisms, such as pathogenic bacteria and viruses, are recognized by pattern recognition receptors (PRRs), including toll-like receptors (TLRs) and nucleotide-binding oligomerization-domain proteins (NODs), and induce inflammatory responses in mammalian hosts. Conversely, commensal bacteria and probiotics, which symbiotically confer health benefits on the host organisms, can lodge in the host intestinal tract without inducing intestinal inflammation. Recent advances in investigations concerning host-microbial interactions have shown that some effector molecules secreted from beneficial bacteria activate cell survival pathways, such as those mediated by p38 MAPK and Akt, and bring health benefits to mammalian hosts. It is noteworthy that such bacteria-derived molecules are taken into the intestinal epithelia through a transport or endocytosis system, thereafter exhibiting their beneficial effects. Understanding this traffic control process can aid in the comprehension of host and microbe interactions and may provide new insight to clarify the pathogenesis of intestinal disorders. This paper highlights the intestinal trafficking systems of bacteria-derived molecules that affect the bacterial functions and modulate epithelial signaling cascades. The latter mechanism may contribute to the maintenance of intestinal homeostasis by improving the host damage induced by virulence factors and various disease states.

Molecular Pathogenesis of Mantle Cell Lymphoma: New Perspectives and Challenges With Clinical Implications

Mantle cell lymphoma (MCL) is a B-cell neoplasia genetically characterized by the t(11;14)(q13;q32) translocation leading to the overexpression of its target gene CCND1. The aggressive clinical behavior of this tumor has been considered to be influenced by its genetic and molecular pathogenesis that integrates an accumulation of many chromosomal aberrations associated with frequent alterations in cell cycle and DNA damage response mechanisms and activation of cell survival pathways. Recent studies aimed to define new chromosomal regions, target genes, and signaling pathways that may contribute to the pathogenesis of this tumor. A subset of patients presenting with a leukemic and non-nodal disease and following a more indolent clinical evolution seem to have some differences in their chromosomal and genomic profiles compared to patients with conventional MCL. The new studies are opening new perspectives on the pathogenesis of this lymphoma that may influence our clinical practice in the diagnosis and management of patients.