Ghrelin Protects against the Detrimental Consequences of Porphyromonas gingivalis-Induced Akt Inactivation through S-Nitrosylation on Salivary Mucin Synthesis

Disturbances in nitric oxide synthase isozyme system and the impairment in salivary mucin synthesis are well-recognized features associated with oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. In this study, using rat sublingual gland acinar cells, we report that P. gingivalis LPS-induced impairment in mucin synthesis and associated suppression in Akt kinase activity were accompanied by a decrease in constitutive nitric oxide synthase (cNOS) activity and an induction in inducible nitric oxide synthase (iNOS) expression. The LPS effect on Akt inactivation was manifested in the kinase S-nitrosylation and a decrease in its phosphorylation at Ser473. Further, we demonstrate that a peptide hormone, ghrelin, countered the LPS-induced impairment in mucin synthesis. This effect of ghrelin was reflected in the suppression of iNOS and the increase in Akt activation, associated with the loss in S-nitrosylation and the increase in phosphorylation, as well as cNOS activation through phosphorylation. Our findings suggest that induction in iNOS expression by P. gingivalis-LPS leads to Akt kinase inactivation through S-nitrosylation that detrimentally impacts cNOS activation through phosphorylation as well as mucin synthesis. We also show that the countering effect of ghrelin on P. gingivalis-induced impairment in mucin synthesis is associated with Akt activation through phosphorylation

Ghrelin Protection against Lipopolysaccharide-Induced Gastric Mucosal Cell Apoptosis Involves Constitutive Nitric Oxide Synthase-Mediated Caspase-3 S-Nitrosylation

Ghrelin, a peptide hormone produced mainly in the stomach, has emerged as an important modulator of the inflammatory responses that are of significance to the maintenance of gastric mucosal integrity. Here, we report on the role of ghrelin in controlling the apoptotic processes induced in gastric mucosal cells by H. pylori lipopolysaccharide (LPS). The countering effect of ghrelin on the LPS-induced mucosal cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (NOS-2). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME. Moreover, the effect of ghrelin on the LPS-induced changes in cNOS activity was reflected in the increased cNOS phosphorylation that was sensitive to SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by L-NAME. Therefore, ghrelin protection of gastric mucosal cells against H. pylori LPS-induced apoptosis involves Src/Akt-mediated up-regulation in cNOS activation that leads to the apoptotic signal inhibition through the NO-induced caspase-3 S-nitrosylation

Mechanism of Cytosolic Phospholipase A2 Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity

Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA2 activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA2 phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA2 activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis

<i>Helicobacter pylori</i> Induces Disturbances in Gastric Mucosal Akt Activation through Inducible Nitric Oxide Synthase-Dependent S-Nitrosylation: Effect of Ghrelin

Gastric mucosal inflammatory response to H. pylori and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in apoptosis and the disturbances in NO signaling pathways. Here, we report that H. pylori LPS-induced enhancement in the mucosal inducible nitric oxide synthase (iNOS) was associated with the suppression in Akt kinase activity and the impairment in constitutive nitric oxide synthase (cNOS) phosphorylation. Further, we demonstrate that the LPS effect on Akt inactivation, manifested in the kinase protein S-nitrosylation and a decrease in its phosphorylation at Ser473, was susceptible to suppression by iNOS inhibition. Moreover, the countering effect of hormone, ghrelin, on the LPS-induced changes in Akt activity was reflected in the loss in Akt S-nitrosylation and the increase in its phosphorylation at Ser473, as well as cNOS activation through phosphorylation. Our findings demonstrate that up-regulation in iNOS with H. pylori infection leads to Akt inactivation through S-nitrosylation that exerts the detrimental effect on the processes of cNOS activation through phosphorylation. We also report that ghrelin protection against H. pylori-induced disturbances is manifested in a marked increase in Akt activity and evoked by a decrease in the kinase S-nitrosylation and the increase in its phosphorylation at Ser473.</jats:p

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus

CD147 and MCT1 - potential partners in bladder cancer aggressiveness and cisplatin resistance

"Article first published online: 27 SEP 2014"The relapsing and progressive nature of bladder tumors, and the heterogeneity in the response to cisplatin-containing regimens, are the major concerns in the care of urothelial bladder carcinoma (UBC) patients. The metabolic adaptations that alter the tumor microenvironment and thus contribute to chemoresistance have been poorly explored in UBC setting. We found significant associations between the immunoexpressions of the microenvironment-related molecules CD147, monocarboxylate transporters (MCTs) 1 and 4, CD44 and CAIX in tumor tissue sections from 114 UBC patients. The presence of MCT1 and/or MCT4 expressions was significantly associated with unfavorable clinicopathological parameters. The incidence of CD147 positive staining significantly increased with advancing stage, grade and type of lesion, and occurrence of lymphovascular invasion. Similar associations were observed when considering the concurrent expression of CD147 and MCT1. This expression profile lowered significantly the 5-year disease-free and overall survival rates. Moreover, when selecting patients who received platinum-based chemotherapy, the prognosis was significantly worse for those with MCT1 and CD147 positive tumors. CD147 specific silencing by small interfering RNAs (siRNAs) in UBC cells was accompanied by a decrease in MCT1 and MCT4 expressions and, importantly, an increase in chemosensitivity to cisplatin. Our results provide novel insights for the involvement of CD147 and MCTs in bladder cancer progression and resistance to cisplatin-based chemotherapy. We consider that the possible cooperative role of CD147 and MCT1 in determining cisplatin resistance should be further explored as a potential theranostics biomarker.Grant sponsor: Life and Health Sciences Research Institute (ICVS); Grant sponsor: Portuguese Science and Technology Foundation (FCT); Grant number: SFRH/BD/51997/201

IGF binding protein‐6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here, we report that hypoxia induced the expression of insulin‐like growth factor binding protein‐6 (IGFBP‐6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a hypoxia‐inducible factor (HIF)‐mediated mechanism. Addition of human IGFBP‐6 to cultured human VECs inhibited angiogenesis in vitro . An IGFBP‐6 mutant with at least 10,000‐fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP‐6 is IGF‐independent. The functional relationship between IGFBP‐6 and vascular endothelial growth factor (VEGF), a major hypoxia‐inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro , co‐incubation with IGFBP‐6 abolished VEGF‐stimulated angiogenesis. The in vivo role of IGFBP‐6 in angiogenesis was tested in flk1 :GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP‐6 mRNA reduced the number of embryonic inter‐segmental blood vessels by ∼40%. This anti‐angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP‐6b had similar effects. To determine the anti‐angiogenic effect of IGFBP‐6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP‐6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP‐6‐transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP‐6 in VECs is up‐regulated by hypoxia and IGFBP‐6 inhibits angiogenesis in vitro and in vivo .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90159/1/26201_ftp.pd

GPER mediates the angiocrine actions induced by IGF1 through the HIF-1α/VEGF pathway in the breast tumor microenvironment

The G protein estrogen receptor GPER/GPR30 mediates estrogen action in breast cancer cells as well as in breast cancer-associated fibroblasts (CAFs), which are key components of microenvironment driving tumor progression. GPER is a transcriptional target of hypoxia inducible factor 1 alpha (HIF-1α) and activates VEGF expression and angiogenesis in hypoxic breast tumor microenvironment. Furthermore, IGF1/IGF1R signaling, which has angiogenic effects, has been shown to activate GPER in breast cancer cells. We analyzed gene expression data from published studies representing almost 5000 breast cancer patients to investigate whether GPER and IGF1 signaling establish an angiocrine gene signature in breast cancer patients. Next, we used GPER-positive but estrogen receptor (ER)-negative primary CAF cells derived from patient breast tumours and SKBR3 breast cancer cells to investigate the role of GPER in the regulation of VEGF expression and angiogenesis triggered by IGF1. We performed gene expression and promoter studies, western blotting and immunofluorescence analysis, gene silencing strategies and endothelial tube formation assays to evaluate the involvement of the HIF-1α/GPER/VEGF signaling in the biological responses to IGF1. We first determined that GPER is co-expressed with IGF1R and with the vessel marker CD34 in human breast tumors (n = 4972). Next, we determined that IGF1/IGF1R signaling engages the ERK1/2 and AKT transduction pathways to induce the expression of HIF-1α and its targets GPER and VEGF. We found that a functional cooperation between HIF-1α and GPER is essential for the transcriptional activation of VEGF induced by IGF1. Finally, using conditioned medium from CAFs and SKBR3 cells stimulated with IGF1, we established that HIF-1α and GPER are both required for VEGF-induced human vascular endothelial cell tube formation. These findings shed new light on the essential role played by GPER in IGF1/IGF1R signaling that induces breast tumor angiogenesis. Targeting the multifaceted interactions between cancer cells and tumor microenvironment involving both GPCRs and growth factor receptors has potential in future combination anticancer therapies

Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer

BACKGROUND. Monocarboxylate transporters (MCTs) are transmembrane proteins involved in the transport of monocarboxylates across the plasma membrane, which appear to play an important role in solid tumours, however the role of MCTs in prostate cancer is largely unknown.The aim of the present work was to evaluate the clinico-pathological value of monocarboxylate transporters (MCTs) expression, namely MCT1, MCT2 and MCT4, together with CD147 and gp70 as MCT1/4 and MCT2 chaperones, respectively, in prostate carcinoma. METHODS. Prostate tissues were obtained from 171 patients, who performed radical prostatectomy and 14 patients who performed cystoprostatectomy. Samples and clinico-pathological data were retrieved and organized into tissue microarray (TMAs) blocks. Protein expression was evaluated by immunohistochemistry in neoplastic (n= 171), adjacent non-neoplastic tissues (n= 135), PIN lesions (n=40) and normal prostatic tissue (n=14). Protein expression was correlated with patients' clinicopathologic characteristics. RESULTS. In the present study, a significant increase of MCT2 and MCT4 expression in the cytoplasm of tumour cells and a significant decrease in both MCT1 and CD147 expression in prostate tumour cells was observed when compared to normal tissue. All MCT isoforms and CD147 were expressed in PIN lesions. Importantly, for MCT2 and MCT4 the expression levels in PIN lesions were between normal and tumour tissue, which might indicate a role for these MCTs in the malignant transformation. Associations were found between MCT1, MCT4 and CD147 expressions and poor prognosis markers; importantly MCT4 and CD147 overexpression correlated with higher PSA levels, Gleason score and pT stage, as well as with perineural invasion and biochemical recurrence. CONCLUSIONS. Our data provides novel evidence for the involvement of MCTs in prostate cancer. According to our results, we consider that MCT2 should be further explored as tumour marker and both MCT4 and CD147 as markers of poor prognosis in prostate cancer.NPG, CP and VMG received fellowships from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009, SFRH/BPD/69479/ 2010 and SFRH/BI/33503/2008, respectively. This work was supported by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of Programa Operacional Temático Factores de Competitividade” (COMPETE) of Quadro Comunitário de Apoio III and co-financed by Fundo Comunitário Europeu FEDER

Role of monocarboxylate transporters in human cancers : state of the art

Monocarboxylate transporters (MCTs) belong to the SLC16 gene family, presently composed by 14 members. MCT1-MCT4 are proton symporters, which mediate the transmembrane transport of pyruvate, lactate and ketone bodies. The role of MCTs in cell homeostasis has been characterized in detail in normal tissues, however, their role in cancer is still far from understood. Most solid tumors are known to rely on glycolysis for energy production and this activity leads to production of important amounts of lactate, which are exported into the extracellular milieu, contributing to the acidic microenvironment. In this context, MCTs will play a dual role in the maintenance of the hyper-glycolytic acidresistant phenotype of cancer, allowing the maintenance of the high glycolytic rates by performing lactate efflux, and pH regulation by the co-transport of protons. Thus, they constitute attractive targets for cancer therapy, which have been little explored. Here we review the literature on the role of MCTs in solid tumors in different locations, such as colon, central nervous system, breast, lung, gynecologic tract, prostate, stomach, however, there are many conflicting results and in most cases there are no functional studies showing the dependence of the tumors on MCT expression and activity. Additional studies on MCT expression in other tumor types, confirmation of the results already published as well as additional functional studies are needed to deeply understand the role of MCTs in cancer maintenance and aggressiveness