Modulating MAPK Signaling Research Articles

Sirt1 Protects against Oxidative Stress-Induced Apoptosis in Fibroblasts from Psoriatic Patients: A New Insight into the Pathogenetic Mechanisms of Psoriasis.

Psoriasis, a multisystem chronic disease characterized by abnormal keratinocyte proliferation, has an unclear pathogenesis where systemic inflammation and oxidative stress play mutual roles. Dermal fibroblasts, which are known to provide a crucial microenvironment for epidermal keratinocyte function, represented the selected experimental model in our study which aimed to clarify the potential role of SIRT1 in the pathogenetic mechanisms of the disease. We firstly detected the presence of oxidative stress (lipid peroxidation and total antioxidant capacity), significantly reduced SIRT1 expression level and activity, mitochondrial damage and apoptosis (caspase-3, -8 and -9 activities) in psoriatic fibroblasts. Upon SIRT1 activation, redox balance was re-established, mitochondrial function was restored and apoptosis was no longer evident. Furthermore, we examined p38, ERK and JNK activation, which was strongly altered in psoriatic fibroblasts, in response to SIRT1 activation and we measured caspase-3 activity in the presence of specific MAPK inhibitors demonstrating the key role of the SIRT1 pathway against apoptotic cell death via MAPK modulation. Our results clearly demonstrate the involvement of SIRT1 in the protective mechanisms related to fibroblast injury in psoriasis. SIRT1 activation exerts an active role in restoring both mitochondrial function and redox balance via modulation of MAPK signaling. Hence, SIRT1 can be proposed as a specific tool for the treatment of psoriasis.

P-Coumaric Acid Protects Human Lens Epithelial Cells against Oxidative Stress-Induced Apoptosis by MAPK Signaling.

To protect against oxidative stress-induced apoptosis in lens epithelial cells is a potential strategy in preventing cataract formation. The present study aimed at studying the protective effect and underlying mechanisms of p-coumaric acid (p-CA) on hydrogen peroxide- (H2O2-) induced apoptosis in human lens epithelial (HLE) cells (SRA 01–04). Cells were pretreated with p-CA at a concentration of 3, 10, and 30 μM before the treatment of H2O2 (275 μM). Results showed that pretreatment with p-CA significantly protected against H2O2-induced cell death in a dose-dependent manner, as well as downregulating the expressions of both cleaved caspase-3 and cleaved caspase-9 in HLE cells. Moreover, p-CA also greatly suppressed H2O2-induced intracellular ROS production and mitochondrial membrane potential loss and elevated the activities of T-SOD, CAT, and GSH-Px of H2O2-treated cells. As well, in vitro study showed that p-CA also suppressed H2O2-induced phosphorylation of p-38, ERK, and JNK in HLE cells. These findings demonstrate that p-CA suppresses H2O2-induced HLE cell apoptosis through modulating MAPK signaling pathways and suggest that p-CA has a potential therapeutic role in the prevention of cataract.

Copper Regulates Maturation and Expression of an MITF:Tryptase Axis in Mast Cells.

Copper has previously been implicated in the regulation of immune responses, but the impact of this metal on mast cells is poorly understood. In this article, we address this issue and show that copper starvation of mast cells causes increased granule maturation, as indicated by higher proteoglycan content, stronger metachromatic staining, and altered ultrastructure in comparison with nontreated cells, whereas copper overload has the opposite effects. In contrast, copper status did not impact storage of histamine in mast cells, nor did alterations in copper levels affect the ability of mast cells to degranulate in response to IgER cross-linking. A striking finding was decreased tryptase content in mast cells with copper overload, whereas copper starvation increased tryptase content. These effects were associated with corresponding shifts in tryptase mRNA levels, suggesting that copper affects tryptase gene regulation. Mechanistically, we found that alterations in copper status affected the expression of microphthalmia-associated transcription factor, a transcription factor critical for driving tryptase expression. We also found evidence supporting the concept that the effects on microphthalmia-associated transcription factor are dependent on copper-mediated modulation of MAPK signaling. Finally, we show that, in MEDNIK syndrome, a condition associated with low copper levels and a hyperallergenic skin phenotype, including pruritis and dermatitis, the number of tryptase-positive mast cells is increased. Taken together, our findings reveal a hitherto unrecognized role for copper in the regulation of mast cell gene expression and maturation.

Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor.

Brown adipose tissue with its constituent brown adipocytes is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. The molecular control of brown adipocyte differentiation and function has been extensively studied in mice, but relatively little is known about such regulatory mechanisms in humans, which in part is due to lack of human brown adipose tissue derived cell models. Here, we used retrovirus-mediated overexpression to stably integrate human telomerase reverse transcriptase (TERT) into stromal-vascular cell fractions from deep and superficial human neck adipose tissue biopsies from the same donor. The brown and white pre-adipocyte cell models (TERT-hBA and TERT-hWA, respectively) displayed a stable proliferation rate and differentiation until at least passage 20. Mature TERT-hBA adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature TERT-hWA adipocytes. TERT-hBA adipocytes were UCP1-positive and responded to β-adrenergic stimulation by activating the PKA-MKK3/6-p38 MAPK signaling module and increasing thermogenic gene expression and oxygen consumption. Mature TERT-hWA adipocytes underwent efficient rosiglitazone-induced ‘browning’, as demonstrated by strongly increased expression of UCP1 and other brown adipocyte-enriched genes. In summary, the TERT-hBA and TERT-hWA cell models represent useful tools to obtain a better understanding of the molecular control of human brown and white adipocyte differentiation and function as well as of browning of human white adipocytes.

Anti-encephalitogenic effects of cucumber leaf extract

Cucumber (Cucumis sativus) fruit has been used in cuisine worldwide, while its leaves are rich in immunomodulatory compounds. Cucumber leaf extract (CLE) was characterized by the predominance of triterpenoids cucurbitacins and significant levels of phenolics. Effects of CLE on CD4+ T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system were investigated in our study. CLE potently inhibited production of major pathogenic Th cytokines: interferon-gamma and interleukin-17, as well as of nitric oxide and reactive oxygen species in macrophages. Antigen-presenting activity of macrophages and dendritic cells was also affected by CLE. The effects of CLE were co-incident with modulation of NFκB and p38 MAPK signaling. Concentrations of CLE used in vitro did not show toxic effects on zebrafish embryos. Moreover, CLE inhibited generation of encephalitogenic cells in vivo. These results demonstrate that CLE deserve further investigation on its anti-encephalitogenic therapeutic properties.

All-Trans Retinoic Acid Ameliorates Arsenic-Induced Oxidative Stress and Apoptosis in the Rat Uterus by Modulating MAPK Signaling Proteins.

Exposure to arsenic leads to inhibition of the anti-oxidant defense mechanism of the body. Reactive oxygen species generated in response to arsenic causes reproductive failures in exposed females and also acts as an inducer of apoptosis. As a prospective remedial agent, all-trans retinoic acid (ATRA) was assessed for reversing arsenic-induced oxidative stress and apoptosis. Rats exposed to arsenic for 28 days were allowed to recover naturally or were treated simultaneously with ATRA for 28 days or up to 56 days. Production of H2 O2 was detected using 2',7'-dichlorfluorescein diacetate (DCFCA) by flow cytometry. Catalase, superoxide dismutase, glutathione, ALT, and AST were estimated by biochemical assays and Western blot analyses. Detection of apoptosis was performed using annexin V-FITC/propidium iodide. Expressions of p53, p21, cleaved caspase 3, JNK/pJNK, and ERK/pERK levels were estimated using Western blot analysis. Elemental arsenic deposition in the rat uterus and liver was estimated by atomic absorption spectrophotometry. Our results confirmed that ATRA ameliorated sodium arsenite-induced ROS generation, restored redox balance, and prevented apoptosis. Concomitant recovery was observed to be more prominent for ATRA-treated rats as compared to the rats that were allowed to recover naturally for 56 days. Tissue arsenic deposition was significantly reduced in the uterus upon continuous ATRA treatment. The results revealed that ATRA reversed arsenic-induced free radical generation, activated the anti-oxidant defence system, and subsequently repressed p53-dependent apoptosis through inhibition of the MAPK signaling components. J. Cell. Biochem. 118: 3796-3809, 2017. © 2017 Wiley Periodicals, Inc.

Phosphatase POPX2 Exhibits Dual Regulatory Functions in Cancer Metastasis.

Cancer metastasis is a complex mechanism involving multiple processes. Previously, our integrative proteome, transcriptome, and phosphoproteome study reported that the levels of serine/threonine phosphatase POPX2 were positively correlated with cancer cell motility through modulating MAPK signaling. Surprisingly, here we found that POPX2 knockdown cells induced more numerous and larger tumor nodules in lungs in longer term animal studies. Interestingly, our analysis of DNA microarray data from cancer patient samples that are available in public databases shows that low POPX2 expression is linked to distant metastasis and poor survival rate. These observations suggest that lower levels of POPX2 may favor tumor progression in later stages of metastasis. We hypothesize that POPX2 may do so by modulation of angiogenesis. Secretome analysis of POPX2-knockdown MDA-MB-231 cells using LC-MS/MS-based SILAC quantitative proteomics and cytokine array show that silencing of POPX2 leads to increased secretion of exosomes, which may, in turn, induce multiple pro-angiogenic cytokines. This study, combined with our previous findings, suggests that a single ubiquitously expressed phosphatase POPX2 influences cancer metastasis via modulating multiple biological processes including MAPK signaling and exosome cytokine secretion.

Targeting the metabolic pathway of human colon cancer overcomes resistance to TRAIL-induced apoptosis.

Colon cancer is a leading cause of cancer-related mortality for which targeted therapy is needed; however, trials using apoptosis-inducing ligand monotherapy to overcome resistance to apoptosis have not shown clinical responses. Since colon cancer cells selectively uptake and rapidly metabolize glucose, a property utilized for clinical staging, we investigated mechanisms to alter glucose metabolism in order to selectively target the cancer cells and to overcome evasion of apoptosis. We demonstrate TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) resistance in the majority of human colon cancers tested and utilize the glucose analog 2-deoxy-d-glucose to sensitize TRAIL-resistant gastrointestinal adenocarcinoma cells, and not normal gastrointestinal epithelial cells, to TRAIL-induced apoptosis through enhanced death receptor 5 expression, downstream modulation of MAPK signaling and subsequent miRNA expression modulation by increasing the expression of miR-494 via MEK activation. Further, established human colon cancer xenografts treated with this strategy experience anti-tumor responses. These findings in colon adenocarcinoma support further investigation of manipulation of cellular energetics to selectively overcome resistance to apoptosis and to impart tumor regressions in established colon cancer tumors.

MicroRNA-106a suppresses proliferation, migration, and invasion of bladder cancer cells by modulating MAPK signaling, cell cycle regulators, and Ets-1-mediated MMP-2 expression.

Despite the clinical significance of tumorigenesis, little is known about the cellular signaling networks of microRNAs (miRs). Here we report a new finding that mir‑106a regulates the proliferation, migration, and invasion of bladder cancer cells. Basal expression levels of mir‑106a were significantly lower in bladder cancer cells than in normal urothelial cells. Overexpression of mir‑106a suppressed the proliferation of bladder cancer cell line EJ. Transient transfection of mir‑106a into EJ cells led to downregulation of ERK phosphorylation and upregulation of p38 and JNK phosphorylation over their levels in the control. Flow cytometry analysis revealed that mir‑106a-transfected cells accumulated in the G1-phase of the cell cycle, and cyclinD1 and CDK6 were significantly downregulated. This G1-phase cell cycle arrest was due in part to the upregulation of p21CIP1/WAF1. In addition, mir‑106a overexpression blocked the wound-healing migration and invasion of EJ cells. Furthermore, mir‑106a transfection resulted in decreased expression of MMP-2 and diminished binding activity of transcription factor Ets-1 in EJ cells. Collectively, we report the novel mir‑106a-mediated molecular signaling networks that regulate the proliferation, migration, and invasion of bladder cancer cells, suggesting that mir‑106a may be a therapeutic target for treating advanced bladder tumors.

Cucurbitacin E Potently Modulates the Activity of Encephalitogenic Cells.

Cucurbitacin E (CucE) is a highly oxidized steroid consisting of a tetracyclic triterpene. It is a member of a Cucurbitacin family of biomolecules that are predominantly found in Cucurbitaceae plants. CucE has already been identified as a potent anti-inflammatory compound. Here, its effects on CD4(+) T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system, were investigated. Production of major pathogenic Th cell cytokines: interferon-gamma and interleukin-17 were inhibited under the influence of CucE. The effects of CucE on CD4(+) T cells were mediated through the modulation of aryl hydrocarbon receptor, STAT3, NFκB, p38 MAPK, and miR-146 signaling. Further, production of nitric oxide and reactive oxygen species, as well as phagocytic ability, were inhibited in macrophages treated with CucE. These results imply that CucE possesses powerful antiencephalitogenic activity.

BMP-6 modulates somatostatin effects on luteinizing hormone production by gonadrotrope cells

The effects of somatostatin analogs and roles of BMP-6 in the regulation of luteinizing hormone (LH) secretion were investigated using mouse gonadotrope LβT2 cells. LH mRNA expression and LH secretion induced by GnRH were suppressed by treatments with somatostatin analogs, including octreotide and pasireotide, in LβT2 cells. Of note, the inhibitory effects of somatostatin analogs on LH secretion were enhanced by the action of BMP-6. BMP-6 increased the expression levels of somatostatin receptor (SSTR)5, suggesting that BMP-6 upregulates SSTR activity that leads to reduction of GnRH-induced LH secretion. In addition, GnRH-induced phosphorylation of MAPKs including ERK, but not P38 or SAPK, was suppressed by pasireotide in the presence of BMP-6. Given that each inhibitor of ERK, JNK or P38 signaling suppressed GnRH-induced LH transcription, MAPKs are individually involved in the induction of LH production by LβT2 cells. Somatostatin analogs also impaired BMP-6-induced Smad1/5/8 phosphorylation by suppressing BMPRs and augmenting Smad6/7 expression. Collectively, the results indicate that somatostatin analogs have dual effects on the modulation of GnRH-induced MAPK signaling and BMP activity. The pituitary BMP system may play a regulatory role in GnRH-induced LH secretion by tuning the responsiveness to somatostatin analogs in gonadotrope cells.

In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment.

An in silico pathway analysis was performed in order to improve current knowledge on the molecular drivers of cervical cancer and detect potential targets for treatment. Three publicly available Affymetrix gene expression data-sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total of 9 cervical cancer cell lines (CCCLs), 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples (CCSs). Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. To select cancer cell-specific genes the CCSs were compared to the CCCLs. Validated genes were submitted to a gene set enrichment analysis (GSEA) and Expression2Kinases (E2K). In the CCSs a total of 1,547 probe sets were identified that were overexpressed (FDR < 0.1). Comparing to CCCLs 560 probe sets (481 unique genes) had a cancer cell-specific expression profile, and 315 of these genes (65%) were validated. GSEA identified 5 cancer hallmarks enriched in CCSs (P < 0.01 and FDR < 0.25) showing that deregulation of the cell cycle is a major component of cervical cancer biology. E2K identified a protein-protein interaction (PPI) network of 162 nodes (including 20 drugable kinases) and 1626 edges. This PPI-network consists of 5 signaling modules associated with MYC signaling (Module 1), cell cycle deregulation (Module 2), TGFβ-signaling (Module 3), MAPK signaling (Module 4) and chromatin modeling (Module 5). Potential targets for treatment which could be identified were CDK1, CDK2, ABL1, ATM, AKT1, MAPK1, MAPK3 among others. The present study identified important driver pathways in cervical carcinogenesis which should be assessed for their potential therapeutic drugability.

Plant MAPK cascades: Just rapid signaling modules?

Abscisic acid (ABA) is a major phytohormone mediating important stress-related processes. We recently unveiled an ABA-activated MAPK signaling module constituted of MAP3K17/18-MKK3-MPK1/2/7/14. Unlike classical rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests that MAPKs can regulate both early and long-term plant stress responses.

Interaction of cCMP with the cGK, cAK and MAPK Kinases in Murine Tissues.

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-32P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.

Intracellular Calcium Levels Determine Differential Modulation of Allosteric Interactions within G Protein-Coupled Receptor Heteromers

The pharmacological significance of the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer is well established and it is being considered as an important target for the treatment of Parkinson’s disease and other neuropsychiatric disorders. However, the physiological factors that control its distinctive biochemical properties are still unknown. We demonstrate that different intracellular Ca2+ levels exert a differential modulation of A2AR-D2R heteromer-mediated adenylyl-cyclase and MAPK signaling in striatal cells. This depends on the ability of low and high Ca2+ levels to promote a selective interaction of the heteromer with the neuronal Ca2+-binding proteins NCS-1 and calneuron-1, respectively. These Ca2+-binding proteins differentially modulate allosteric interactions within the A2AR-D2R heteromer, which constitutes a unique cellular device that integrates extracellular (adenosine and dopamine) and intracellular (Ca+2) signals to produce a specific functional response.

Altered CD31 expression and activity in helper T cells of acute coronary syndrome patients.

In acute coronary syndrome (ACS), T cell abnormalities are associated to a worse outcome. Loss of inhibitory activity of CD31, an Ig-like adhesion molecule, on peripheral leukocytes has been found to enhance atherosclerosis in experimental models. In this study, we examined the expression of CD31 on T cells, and its role on TCR signaling in 35 patients with non-ST elevation ACS, in 35 patients with stable angina (SA), and in 35 controls. Furthermore, 10 ACS and 10 SA patients were re-analyzed at 1-year follow-up. Flow-cytometry analysis showed that in ACS patients, CD31 expression was reduced on total CD4(+) and CD4(+)CD28(null) (P < 0.001, ACS vs. SA), on naïve (P < 0.001, ACS vs. SA) and on central-memory and effector-memory CD4(+) T cells (P < 0.05, ACS vs. SA and controls). The immunomodulatory effect of CD31 on TCR signaling of CD4(+) and CD4(+)CD28(null) T cells, was lower in ACS than SA patients (P < 0.05, for both comparisons). At 1-year follow-up, CD31 expression and function increased in ACS becoming similar to that found in SA. CD31 recruitment in the immunological synapse was lower in ACS than controls (P = 0.012). Moreover, CD31 modulated MAPK signaling and reduced the expression of T bet and Rorγ-t, necessary for Th1 and Th17 differentiation. Finally, we studied TCR signaling in CD31(+) naïve and primed T cell subsets observing a different pattern of protein phosphorylation. A CD31-mediated regulatory pathway is enhanced in SA and temporarily downregulated in ACS. As CD31 modulates both T cell activation, by increasing the threshold for TCR stimulation, and T cell differentiation, it might represent a novel molecular target to treat T cell abnormalities in ACS.

Abstract 2335: Thymoquinone induces the MUC4 mRNA-destabilizing activity of Tristetraprolin

Abstract Tristetraprolin (TTP) is an AU-rich element binding protein that destabilizes ARE-containing mRNAs. We previously reported that TTP suppresses several target genes such as those encoding proto-oncogenes and cytokines in various cancer cells. However, the signaling pathway to induce TTP expression is not well known. In this study, we investigate that Tymoquinone (TQ) which is an active ingredient derived from the medicinal plant Nigella satia induces the TTP mRNA and protein expression in AGS and MDA-MB231. TQ treatment destabilized a luciferase mRNA containing cIAP2, VEGF and MUC4 ARE. Transfected cells were treated with various kinds of MAPK inhibitors, treatment with Sorafenib (RAF inhibitor) and U0126 (MEK-1/2) reduced the TTP induction activity of TQ in a dose-dependent manner. We confirmed that co-treatment with sorafenib/U0126 and TQ also reduced TTP expression. Following TQ downregulated the expression of MUC4 in previous study, we next examined whether the changes of TTP expression affects the expression of MUC4. Overexpression of TTP decreased the stability of MUC4 mRNA and expression level of MUC4. On the contrary, inhibition of TTP using siRNA increased the MUC4 expression. MUC4 mRNA contains two AREs within the 3′UTR and TTP destabilized a luciferase mRNA containing MUC4 mRNA 3′UTR. Analyses of point mutants of MUC4 mRNA 3′UTR demonstrated that ARE2 were mostly responsible for the TTP mediated destabilization of MUC4 mRNA. Our data suggest that TQ enhances the mRNA and protein level of TTP via the modulation of the Raf-MEK MAPK signaling and suppressed cancer cell growth and promotion through destabilizing MUC4 mRNA as TTP novel target gene. Citation Format: Hyun Hee Lee, Se-Ra Lee, Sun-Hee Leem. Thymoquinone induces the MUC4 mRNA-destabilizing activity of Tristetraprolin. [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 2335. doi:10.1158/1538-7445.AM2014-2335

Complex Formation between S100B Protein and the p90 Ribosomal S6 Kinase (RSK) in Malignant Melanoma Is Calcium-dependent and Inhibits Extracellular Signal-regulated Kinase (ERK)-mediated Phosphorylation of RSK

S100B is a prognostic marker for malignant melanoma. Increasing S100B levels are predictive of advancing disease stage, increased recurrence, and low overall survival in malignant melanoma patients. Using S100B overexpression and shRNA(S100B) knockdown studies in melanoma cell lines, elevated S100B was found to enhance cell viability and modulate MAPK signaling by binding directly to the p90 ribosomal S6 kinase (RSK). S100B-RSK complex formation was shown to be Ca(2+)-dependent and to block ERK-dependent phosphorylation of RSK, at Thr-573, in its C-terminal kinase domain. Additionally, the overexpression of S100B sequesters RSK into the cytosol and prevents it from acting on nuclear targets. Thus, elevated S100B contributes to abnormal ERK/RSK signaling and increased cell survival in malignant melanoma.

Capsaicin induces apoptosis and modulates MAPK signaling in human gastric cancer cells

Capsaicin is known to have tumor suppressive effects. However, the molecular mechanisms and targets of capsaicin involved in exerting anticancer activity are complex and remain to be clarified. The aim of the current study was to investigate the effects of capsaicin on human gastric cancer cells (AGS cells) and demonstrate that capsaicin induced apoptosis in AGS cells. Results of the MTT assay and flow cytometry revealed that capsaicin potentially inhibited the proliferation of AGS cells and induced apoptosis in vitro in a dose-dependent manner. Cleaved caspase-3 was increased and Bcl-2 was reduced by treatment with capsaicin in AGS cells. Capsaicin treatment decreased the expression of phosphorylated ERK 1/2, p38 MAPK or JNK in AGS cells. The results of this study suggest that capsaicin may serve as an anti-tumorigenic agent in human gastric cancer.

Regulation Of Erythro-Megakaryocytic Lineage Bifurcation By The Gfi1b Gene Target Rgs18

The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis.Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively.Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures:No relevant conflicts of interest to declare.