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Spinal neuroinflammation plays a critical role in the genesis of neuropathic pain. Accumulating data suggest that abscisic acid (ABA), a phytohormone, regulates inflammatory processes in mammals. In this study, we found that reduction of the LANCL2 receptor protein but not the agonist ABA in the spinal cord is associated with the genesis of neuropathic pain. Systemic or intrathecal administration of ABA ameliorates the development and pre-existence of mechanical allodynia and heat hyperalgesia in animals with partial sciatic nerve ligation (pSNL). LANCL2 is expressed only in microglia in the spinal dorsal horn. Pre-emptive treatment with ABA attenuates activation of microglia and astrocytes, ERK activity, and TNFα protein abundance in the dorsal horn in rats with pSNL. These are accompanied by restoration of spinal LANCL2 protein abundance. Spinal knockdown of LANCL2 gene with siRNA recapitulates the behavioral and spinal molecular changes induced by pSNL. Activation of spinal toll-like receptor 4 (TLR4) with lipopolysaccharide leads to activation of microglia, and over production of TNFα, which are concurrently accompanied by suppression of protein levels of LANCL2 and peroxisome proliferator activated-receptor γ. These changes are ameliorated when ABA is added with LPS. The anti-inflammatory effects induced by ABA do not requires Gi protein activity. Our study reveals that the ABA/LANCL2 system is a powerful endogenous system regulating spinal neuroinflammation and nociceptive processing, suggesting the potential utility of ABA as the management of neuropathic pain.
Assuntos
Ácido Abscísico , Neuralgia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Animais , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Lipopolissacarídeos/farmacologia , Mamíferos , Proteínas de Membrana/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Ratos , Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Glioblastoma (GBM) is the most common and aggressive form of malignant glioma. The GBM tumor microenvironment (TME) is a complex ecosystem of heterogeneous cells and signaling factors. Glioma associated macrophages and microglia (GAMs) constitute a significant portion of the TME, suggesting that their functional attributes play a crucial role in cancer homeostasis. In GBM, an elevated GAM population is associated with poor prognosis and therapeutic resistance. Neoplastic cells recruit these myeloid populations through release of chemoattractant factors and dysregulate their induction of inflammatory programs. GAMs become protumoral advocates through production a variety of cytokines, inflammatory mediators, and growth factors that can drive cancer proliferation, invasion, immune evasion, and angiogenesis. Among these inflammatory factors, cyclooxygenase-2 (COX-2) and its downstream product, prostaglandin E2 (PGE2), are highly enriched in GBM and their overexpression is positively correlated with poor prognosis in patients. Both tumor cells and GAMs have the ability to signal through the COX-2 PGE2 axis and respond in an autocrine/paracrine manner. In the GBM TME, enhanced signaling through the COX-2/PGE2 axis leads to pleotropic effects that impact GAM dynamics and drive tumor progression.
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Patients receiving paclitaxel for cancer treatment often develop an acute pain syndrome (paclitaxel-associated acute pain syndrome, P-APS), which occurs immediately after paclitaxel treatment. Mechanisms underlying P-APS remain largely unknown. We recently reported that rodents receiving paclitaxel develop acute pain and activation of spinal microglial toll like receptor 4 (TLR4) by paclitaxel penetrating into the spinal cord is a critical event in the genesis of P-APS. Our current study dissected cellular and molecular mechanisms underlying the P-APS. We demonstrated that bath-perfusion of paclitaxel, at a concentration similar to that found in the cerebral spinal fluid in animals receiving i.v. paclitaxel (2 mg/kg), resulted in increased calcium activity in microglia instantly, and in astrocytes with 6 min delay. TLR4 activation in microglia by paclitaxel caused microglia to rapidly release interleukin-1ß (IL-1ß) but not tumor necrosis factor α, IL-6, or interferon-γ. IL-1ß release from microglia depended on capthepsin B. IL-1ß acted on astrocytes, leading to elevated calcium activity and suppressed glutamate uptake. IL-1ß also acted on neurons to increase presynaptic glutamate release and postsynaptic AMPA receptor activity in the spinal dorsal horn. Knockout of IL-1 receptors prevented the development of acute pain induced by paclitaxel in mice. Our study indicates that IL-1ß is a crucial molecule used by microglia to alter functions in astrocytes and neurons upon activation of TLR4 in the genesis of P-APS, and targeting the signaling pathways regulating the production and function of IL-1ß from microglia is a potential avenue for the development of analgesics for the treatment of P-APS.
Assuntos
Antineoplásicos/efeitos adversos , Ácido Glutâmico/metabolismo , Interleucina-1beta/metabolismo , Microglia/metabolismo , Paclitaxel/efeitos adversos , Dor/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Animais , Cálcio/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Camundongos , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Dor/induzido quimicamente , Medição da Dor , RatosRESUMO
The small regulator of G protein signaling protein RGS10 is a key regulator of neuroinflammation and ovarian cancer cell survival; however, the mechanism for RGS10 function in these cells is unknown and has not been linked to specific G protein pathways. RGS10 is highly enriched in microglia, and loss of RGS10 expression in microglia amplifies production of the inflammatory cytokine tumor necrosis factor α (TNFα) and enhances microglia-induced neurotoxicity. RGS10 also regulates cell survival and chemoresistance of ovarian cancer cells. Cyclooxygenase-2 (COX-2)-mediated production of prostaglandins such as prostaglandin E2 (PGE2) is a key factor in both neuroinflammation and cancer chemoresistance, suggesting it may be involved in RGS10 function in both cell types, but a connection between RGS10 and COX-2 has not been reported. To address these questions, we completed a mechanistic study to characterize RGS10 regulation of TNFα and COX-2 and to determine if these effects are mediated through a G protein-dependent mechanism. Our data show for the first time that loss of RGS10 expression significantly elevates stimulated COX-2 expression and PGE2 production in microglia. Furthermore, the elevated inflammatory signaling resulting from RGS10 loss was not affected by Gαi inhibition, and a RGS10 mutant that is unable to bind activated G proteins was as effective as wild type in inhibiting TNFα expression. Similarly, suppression of RGS10 in ovarian cancer cells enhanced TNFα and COX-2 expression, and this effect did not require Gi activity. Together, our data strongly indicate that RGS10 inhibits COX-2 expression by a G protein-independent mechanism to regulate inflammatory signaling in microglia and ovarian cancer cells.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/fisiologia , Citocinas/metabolismo , Dinoprostona/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Camundongos , Microglia/metabolismo , Neoplasias Ovarianas/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Regulators of G protein signaling (RGS) are a family of proteins classically known to accelerate the intrinsic GTPase activity of G proteins, which results in accelerated inactivation of heterotrimeric G proteins and inhibition of G protein coupled receptor signaling. RGS proteins play major roles in essential cellular processes, and dysregulation of RGS protein expression is implicated in multiple diseases, including cancer, cardiovascular and neurodegenerative diseases. The expression of RGS proteins is highly dynamic and is regulated by epigenetic, transcriptional and post-translational mechanisms. This review summarizes studies that report dysregulation of RGS protein expression in disease states, and presents examples of drugs that regulate RGS protein expression. Additionally, this review discusses, in detail, the transcriptional and post-transcriptional mechanisms regulating RGS protein expression, and further assesses the therapeutic potential of targeting these mechanisms. Understanding the molecular mechanisms controlling the expression of RGS proteins is essential for the development of therapeutics that indirectly modulate G protein signaling by regulating expression of RGS proteins.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Drogas em Investigação/uso terapêutico , Epigênese Genética , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Processamento de Proteína Pós-Traducional , Proteínas RGS/genética , Animais , Azacitidina/uso terapêutico , Benzodiazepinas/uso terapêutico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Olanzapina , Proteínas RGS/agonistas , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/metabolismo , Transdução de Sinais , VorinostatRESUMO
RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.
Assuntos
Inativação Gênica , Histona Desacetilases/metabolismo , Microglia/metabolismo , Proteínas RGS/genética , Transcrição Gênica , Acetilação/efeitos dos fármacos , Animais , Azacitidina/farmacologia , Linhagem Celular , Quimiocina CXCL2/metabolismo , Modelos Animais de Doenças , Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Lisofosfolipídeos/farmacologia , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Proteínas RGS/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Emerging studies have shown that pharmacological activation of adenosine monophosphate-activated protein kinase (AMPK) produces potent analgesic effects in different animal pain models. Currently, the spinal molecular and synaptic mechanism by which AMPK regulates the pain signaling system remains unclear. To address this issue, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice. We demonstrated that AMPKα1 is imperative for maintaining normal nociception, and mice deficient for AMPKα1 exhibit mechanical allodynia. This is concomitantly associated with increased glutamatergic synaptic activities in neurons located in the superficial spinal dorsal horn, which results from the increased glutamate release from presynaptic terminals and function of ligand-gated glutamate receptors at the postsynaptic neurons. Additionally, AMPKα1 knockout mice have increased activities of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinases (p38), as well as elevated levels of interleukin-1ß (IL-1ß), reactive oxygen species (ROS), and heme oxygenase 1 (HO-1) in the spinal dorsal horn. Systemic administration of a non-specific ROS scavenger (phenyl-N-tert-butylnitrone, PBN) or a HO-1 activator (Cobalt protoporphyrin IX, CoPP) attenuated allodynia in AMPKα1 knockout mice. Bath-perfusion of the ROS scavenger or HO-1 activator effectively attenuated the increased ROS levels and glutamatergic synaptic activities in the spinal dorsal horn. Our findings suggest that ROS are the key down-stream signaling molecules mediating the behavioral hypersensitivity in AMPKα1 knockout mice. Thus, targeting AMPKα1 may represent an effective approach for the treatment of pathological pain conditions associated with neuroinflammation at the spinal dorsal horn.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácido Glutâmico/fisiologia , Hiperalgesia/metabolismo , Nociceptividade/fisiologia , Terminações Pré-Sinápticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Óxidos N-Cíclicos/farmacologia , Encefalite/metabolismo , Potenciais Pós-Sinápticos Excitadores , Feminino , Sequestradores de Radicais Livres/farmacologia , Heme Oxigenase-1/metabolismo , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos KnockoutRESUMO
More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Ovário/efeitos dos fármacos , Proteínas RGS/genética , Animais , Antineoplásicos/uso terapêutico , Epigênese Genética , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ovário/metabolismo , Ovário/patologia , Proteínas RGS/análise , Proteínas RGS/metabolismoRESUMO
Nucleoside analogs are used as chemotherapeutic options for the treatment of platinum-resistant ovarian cancers. Human concentrative nucleoside transporter 1 (hCNT1) is implicated in sensitizing solid tumors to nucleoside analogs although its role in determining drug efficacy in ovarian cancers remains unclear. Here we examined the functional expression of hCNT1 and compared its contributions toward gemcitabine efficacy in histological subtypes of ovarian cancer. Radioactivity analysis identified hCNT1-mediated (3)H-gemcitabine transport in ovarian cancer cells to be significantly reduced compared with that of normal ovarian surface epithelial cells. Biochemical and immunocytochemical analysis identified that unlike normal ovarian cells which expressed high levels of hCNT1 at the apical cell surface, the transporter was either diminished in expression and/or mislocalized in cell lines of various subtypes of ovarian cancer. Retroviral expression of hCNT1 selectively rescued gemcitabine transport in cell lines representing serous, teratocarcinoma, and endometrioid subtypes, but not clear cell carcinoma (CCC). In addition, exogenous hCNT1 predominantly accumulated in intracytoplasmic vesicles in CCC suggesting defective cellular trafficking of hCNT1 as a contributing factor to transport deficiency. Despite diminution of hCNT1 transport in the majority of ovarian cancers and apparent trafficking defects with CCC, the chemotherapeutic efficacy of gemcitabine was broadly enhanced in all subtypes when delivered via engineered nanoparticles (NPs). Additionally, by bypassing the transport requirement, the delivery of a gemcitabine-cisplatin combination in NP formulation increased their synergistic interactions. These findings uncover hCNT1 as a putative determinant for nucleoside analog chemoresistance in ovarian cancer and may help rationalize drug selection and delivery strategies for various histological subtypes of ovarian cancer.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Proteínas de Membrana Transportadoras/metabolismo , Antimetabólitos Antineoplásicos/química , Transporte Biológico , Linhagem Celular Tumoral , Cisplatino/química , Cisplatino/farmacologia , Desoxicitidina/química , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Feminino , Humanos , Concentração Inibidora 50 , Nanocápsulas/química , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , GencitabinaRESUMO
The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.
Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Lisofosfolipídeos/farmacologia , MAP Quinase Quinase Quinase 3/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingosina/análogos & derivados , Linhagem Celular Transformada , Relação Dose-Resposta a Droga , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoxazóis/farmacologia , MAP Quinase Quinase Quinase 3/genética , Propionatos/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Esfingosina/farmacologiaRESUMO
RGS10 is an important regulator of cell survival and chemoresistance in ovarian cancer. We recently showed that RGS10 transcript expression is suppressed during acquired chemoresistance in ovarian cancer. The suppression of RGS10 is due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to silencing of tumor suppressor genes during cancer progression. Here, we fully investigate the molecular mechanisms of epigenetic silencing of RGS10 expression in chemoresistant A2780-AD ovarian cancer cells. We identify two important epigenetic regulators, HDAC1 and DNMT1, that exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down also decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Neoplasias Ovarianas/metabolismo , Proteínas RGS/metabolismo , Imunoprecipitação da Cromatina , DNA (Citosina-5-)-Metiltransferase 1 , Primers do DNA/genética , Feminino , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.
Assuntos
Metilação de DNA/genética , Histonas/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteínas RGS/genética , Acetilação , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
BACKGROUND: A critical therapeutic challenge in epithelial ovarian carcinoma is the development of chemoresistance among tumor cells following exposure to first line chemotherapeutics. The molecular and genetic changes that drive the development of chemoresistance are unknown, and this lack of mechanistic insight is a major obstacle in preventing and predicting the occurrence of refractory disease. We have recently shown that Regulators of G-protein Signaling (RGS) proteins negatively regulate signaling by lysophosphatidic acid (LPA), a growth factor elevated in malignant ascites fluid that triggers oncogenic growth and survival signaling in ovarian cancer cells. The goal of this study was to determine the role of RGS protein expression in ovarian cancer chemoresistance. RESULTS: In this study, we find that RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant to chemotherapy compared with parental, chemo-sensitive cells in gene expression datasets of multiple models of chemoresistance. Further, exposure of SKOV-3 cells to cytotoxic chemotherapy causes acute, persistent downregulation of RGS10 and RGS17 transcript expression. Direct inhibition of RGS10 or RGS17 expression using siRNA knock-down significantly reduces chemotherapy-induced cell toxicity. The effects of cisplatin, vincristine, and docetaxel are inhibited following RGS10 and RGS17 knock-down in cell viability assays and phosphatidyl serine externalization assays in SKOV-3 cells and MDR-HeyA8 cells. We further show that AKT activation is higher following RGS10 knock-down and RGS 10 and RGS17 overexpression blocked LPA mediated activation of AKT, suggesting that RGS proteins may blunt AKT survival pathways. CONCLUSIONS: Taken together, our data suggest that chemotherapy exposure triggers loss of RGS10 and RGS17 expression in ovarian cancer cells, and that loss of expression contributes to the development of chemoresistance, possibly through amplification of endogenous AKT signals. Our results establish RGS10 and RGS17 as novel regulators of cell survival and chemoresistance in ovarian cancer cells and suggest that their reduced expression may be diagnostic of chemoresistance.
Assuntos
Neoplasias Ovarianas/metabolismo , Proteínas RGS/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Biologia Computacional , Docetaxel , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Lisofosfolipídeos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase , Proteínas RGS/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Taxoides/farmacologia , Vincristina/farmacologiaRESUMO
The regulator of G-protein signaling (RGS) family is a diverse group of multifunctional proteins that regulate cellular signaling events downstream of G-protein coupled receptors (GPCRs). In recent years, GPCRs have been linked to the initiation and progression of multiple cancers; thus, regulators of GPCR signaling are also likely to be important to the pathophysiology of cancer. This review highlights recent studies detailing changes in RGS transcript expression during oncogenesis, single nucleotide polymorphisms in RGS proteins linked to lung and bladder cancers, and specific roles for RGS proteins in multiple cancer types.
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Neoplasias/metabolismo , Proteínas RGS , Animais , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Proteínas RGS/biossíntese , Proteínas RGS/genética , Proteínas RGS/fisiologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de SinaisRESUMO
BACKGROUND/AIMS: Lysophosphatidic acid (LPA) is an autocrine growth signal critical to the initiation and progression of ovarian cancer. In the current study, we investigated the receptors and signaling cascades responsible for mediating LPA-stimulated cell growth in SKOV-3 and Caov-3 ovarian cancer cell lines. METHODS: Pharmacological inhibitors of distinct LPA and epidermal growth factor receptors, G proteins and kinases were tested for their effect on LPA-stimulated cell growth, MAP kinase activation and Akt activation in SKOV-3 and Caov-3 cells. RESULTS: Distinct agonist pharmacological profiles were observed. Saturated and unsaturated LPA species were equally potent in Caov-3 cells, while saturated LPA was less potent than unsaturated LPA in SKOV-3 cells. Further, the LPA1/LPA3 receptor antagonist Ki16425 was more potent in SKOV-3 cells. The effect of LPA on cell growth in both cell lines was dependent on phosphatidylinositol-3 kinases and MAP kinases. However, LPA-stimulated SKOV-3 cell growth required Gi G proteins, while Caov-3 cell growth was dependent on the Rho effector p160 Rho kinase. Finally, we demonstrated that regulator of G protein signaling proteins significantly regulated Gi-dependent LPA-stimulated cell growth in SKOV-3 cells. CONCLUSIONS: LPA-stimulated cell growth is mediated by distinct but overlapping receptors and signaling pathways in these two model ovarian cancer cell lines.
Assuntos
Proliferação de Células/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Humanos , Isoxazóis/farmacologia , Fosforilação/efeitos dos fármacos , Propionatos/farmacologia , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Regulator of G-protein signalling (RGS)(2) proteins critically regulate signalling cascades initiated by G-protein coupled receptors (GPCRs) by accelerating the deactivation of heterotrimeric G-proteins. Lysophosphatidic acid (LPA) is the predominant growth factor that drives the progression of ovarian cancer by activating specific GPCRs and G-proteins expressed in ovarian cancer cells. We have recently reported that RGS proteins endogenously expressed in SKOV-3 ovarian cancer cells dramatically attenuate LPA stimulated cell signalling. The goal of this study was twofold: first, to identify candidate RGS proteins expressed in SKOV-3 cells that may account for the reported negative regulation of G-protein signalling, and second, to determine if these RGS protein transcripts are differentially expressed among commonly utilized ovarian cancer cell lines and non-cancerous ovarian cell lines. Reverse transcriptase-PCR was performed to determine transcript expression of 22 major RGS subtypes in RNA isolated from SKOV-3, OVCAR-3 and Caov-3 ovarian cancer cell lines and non-cancerous immortalized ovarian surface epithelial (IOSE) cells. Fifteen RGS transcripts were detected in SKOV-3 cell lines. To compare the relative expression levels in these cell lines, quantitative real time RT-PCR was performed on select transcripts. RGS19/GAIP was expressed at similar levels in all four cell lines, while RGS2 transcript was detected at levels slightly lower in ovarian cancer cells as compared to IOSE cells. RGS4 and RGS6 transcripts were expressed at dramatically different levels in ovarian cancer cell lines as compared to IOSE cells. RGS4 transcript was detected in IOSE at levels several thousand fold higher than its expression level in ovarian cancer cells lines, while RGS6 transcript was expressed fivefold higher in SKOV-3 cells as compared to IOSE cells, and over a thousand fold higher in OVCAR-3 and Caov-3 cells as compared to IOSE cells. Functional studies of RGS 2, 6, and 19/GAIP were performed by measuring their effects on LPA stimulated production of inositol phosphates. In COS-7 cells expressing individual exogenous LPA receptors, RGS2 and RSG19/GAIP attenuated signalling initiated by LPA1, LPA2, or LPA3, while RGS6 only inhibited signalling initiated by LPA2 receptors. In SKOV-3 ovarian cancer cells, RGS2 but not RGS6 or RGS19/GAIP, inhibited LPA stimulated inositol phosphate production. In contrast, in CAOV-3 cells RGS19/GAIP strongly attenuated LPA signalling. Thus, multiple RGS proteins are expressed at significantly different levels in cells derived from cancerous and normal ovarian cells and at least two candidate RGS transcripts have been identified to account for the reported regulation of LPA signalling pathways in ovarian cancer cells.
Assuntos
Neoplasias Ovarianas/metabolismo , Proteínas RGS/metabolismo , Transdução de Sinais , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fosfatos de Inositol/metabolismo , Neoplasias Ovarianas/genética , Proteínas RGS/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. RESULTS: Our results demonstrate that Lysophosphatidic Acid (LPA) and Sphingosine-1-phosphate (S1P) receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to G i/o G-proteins that inhibit adenylyl cyclase and to G q-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via G i/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR)- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. CONCLUSION: Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.
Assuntos
Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Células Neuroepiteliais/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Proliferação de Células , Células-Tronco Embrionárias/citologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Microscopia de Vídeo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/fisiologia , RNA Mensageiro/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/fisiologia , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismoRESUMO
Phospholipase A(2) (PLA(2)) are esterases that cleave glycerophospholipids to release fatty acids and lysophospholipids. Several studies demonstrate that PLA(2) regulate growth and signaling in several cell types. However, few of these studies have focused on Ca2+-independent phospholipase A(2) (iPLA(2) or Group VI PLA(2)). This class of PLA(2) was originally suggested to mediate phospholipid remodeling in several cell types including macrophages. As such, it was labeled as a housekeeping protein and thought not to play as significant of roles in cell growth as its older counterparts cytosolic PLA(2) (cPLA(2) or Group IV PLA(2)) and secretory PLA(2) (sPLA(2) or Groups I-III, V and IX-XIV PLA(2)). However, several recent studies demonstrate that iPLA(2) mediate cell growth, and do so by participating in signal transduction pathways that include epidermal growth factor receptors (EGFR), mitogen activated protein kinases (MAPK), mdm2, and even the tumor suppressor protein p53 and the cell cycle regulator p21. The exact mechanism by which iPLA(2) mediates these pathways are not known, but likely involve the generation of lipid signals such as arachidonic acid, lysophosphatidic acid (LPA) and lysophosphocholines (LPC). This review discusses the role of iPLA(2) in cell growth with special emphasis placed on their role in cell signaling. The putative lipid signals involved are also discussed.
Assuntos
Proliferação de Células , Fosfolipases A2 do Grupo VI/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Fosfolipases A2 do Grupo VI/química , Fosfolipases A2 do Grupo VI/classificação , Humanos , Lipídeos/química , Lipídeos/fisiologiaRESUMO
Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G(i) subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G(i/o) signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G(i) proteins as compared to RGSwt G(i). We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Galpha(i2) RGSi as compared to cells expressing Galpha(i2) RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/farmacologia , Neoplasias Ovarianas/patologia , Proteínas RGS/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Feminino , Humanos , Mutagênese , Proteínas Mutantes/metabolismo , Neoplasias Ovarianas/enzimologia , Toxina Pertussis/farmacologia , Isoformas de Proteínas/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
The human P2Y12 receptor (P2Y12-R) is a member of the G protein coupled P2Y receptor family, which is intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y12-R, Gbeta1gamma2, and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y12-R and Galphai2beta1gamma2 was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was markedly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 = 80 nM) examined, whereas ADP, the cognate agonist of the P2Y12-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action of 2MeSADP; therefore, ATP is a relatively low-affinity antagonist of the P2Y12-R. The G protein selectivity of the P2Y12-R was examined by reconstitution with various G protein alpha-subunits in heterotrimeric form with Gbeta1gamma2. The most robust coupling of the P2Y12-R was to Galphai2, but effective coupling also occurred to Galphai1 and Galphai3. In contrast, little or no coupling occurred to Galphao or Galphaq. These results illustrate that the signaling properties of the P2Y12-R can be studied as a purified protein under conditions that circumvent the complications that occur in vivo because of nucleotide metabolism and interconversion as well as nucleotide release.