RESUMO
Cancers in the bone produce a number of severe symptoms including pain that compromises patient functional status, quality of life, and survival. The source of this pain is multifaceted and includes factors secreted from tumor cells. Malignant cells release the neurotransmitter and cell-signaling molecule glutamate via the oxidative stress-related cystine/glutamate antiporter, system xC-, which reciprocally imports cystine for synthesis of glutathione and the cystine/cysteine redox cycle. Pharmacological inhibition of system xC- has shown success in reducing and delaying the onset of cancer pain-related behavior in mouse models. This investigation describes the development of a stable siRNA-induced knockdown of the functional trans-membrane system xC- subunit xCT ( SLC7A11) in the human breast cancer cell line MDA-MB-231. Clones were verified for xCT knockdown at the transcript, protein, and functional levels. RNAseq was performed on a representative clone to comprehensively examine the transcriptional cellular signature in response to xCT knockdown, identifying multiple differentially regulated factors relevant to cancer pain including nerve growth factor, interleukin-1, and colony-stimulating factor-1. Mice were inoculated intrafemorally and recordings of pain-related behaviors including weight bearing, mechanical withdrawal, and limb use were performed. Animals implanted with xCT knockdown cancer cells displayed a delay until the onset of nociceptive behaviors relative to control cells. These results add to the body of evidence suggesting that a reduction in glutamate release from cancers in bone by inhibition of the system xC- transporter may decrease the severe and intractable pain associated with bone metastases.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Neoplasias da Mama/complicações , Dor do Câncer/etiologia , Dor do Câncer/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Isótopos de Carbono/farmacocinética , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Cistina/farmacocinética , Modelos Animais de Doenças , Feminino , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Humanos , Interleucina-1/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Aggressive breast cancer subtypes utilize system xc-, a membrane antiporter, to import cystine for glutathione synthesis and maintenance of redox homeostasis, in turn releasing glutamate as a metabolic pro-nociceptive by-product. Metastatic breast cancers establish themselves at distal sites including bone, where changes in extracellular glutamate levels contribute to cancer-induced bone pain. We previously established that stearically blocking system xc- activity with sulfasalazine delays the onset of nociceptive behaviours and that xCT, the functional antiporter subunit, is positively regulated by signal transducer and activator of transcription 3 (STAT3). In the current investigation, a murine xenograft cancer-induced bone pain model was applied to examine whether pharmacological inhibition of phosphorylated STAT3 (pSTAT3) induces changes in nociception. A high glutamate-releasing, xCT/pSTAT3 over-expressing human breast cancer cell line was selected for injection into the distal epiphysis of the right femur of female nude mice. A 14-day regimen of intraperitoneal injections with either vehicle or the novel STAT3 inhibitor DR-1-55 commenced three weeks after initial intrafemoral bone injection. Nociceptive behaviours were temporally monitored by automated von Frey, dynamic weight bearing and open-field testing for the duration of the study, beginning at the baseline. Prior to sacrifice and at ethical end point, tumour-induced osteolytic lesions were radiographically assessed. Treatment with DR-1-55 significantly delayed the onset and severity of spontaneous and induced nociceptive behaviours, also decreasing human SLC7A11 ( xCT) mRNA levels in tumour-bearing limbs without altering osteolysis. In addition, two pro-inflammatory cytokines released by this cell line, interleukin 6 and interleukin 1ß, were also down-regulated at the mRNA level in response to DR-1-55 treatment in vivo, with lower human interleukin 6 levels detected in the host circulation. This study demonstrates that targeting pSTAT3 may be a viable therapeutic means to manage cancer-induced bone pain, alone or in combination with stearic system xc- blockers.
Assuntos
Osso e Ossos/patologia , Dor do Câncer/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Nociceptividade/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/complicações , Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/complicações , Dor do Câncer/etiologia , Dor do Câncer/terapia , Carcinoma/complicações , Linhagem Celular Tumoral/patologia , Citocinas/sangue , Citocinas/genética , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Masculino , Camundongos , Camundongos Nus , Nociceptividade/efeitos dos fármacos , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Suporte de Carga/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer cells release the signalling molecule glutamate via the system xC- antiporter, which is upregulated to exchange extracellular cystine for intracellular glutamate to protect against oxidative stress. Here, we demonstrate that this antiporter is functionally influenced by the actions of the neurotrophin nerve growth factor on its cognate receptor tyrosine kinase, TrkA, and that inhibiting this complex may reduce cancer-induced bone pain via its downstream actions on xCT, the functional subunit of system xC-. We have characterized the effects of the selective TrkA inhibitor AG879 on system xC- activity in murine 4T1 and human MDA-MB-231 mammary carcinoma cells, as well as its effects on nociception in our validated immunocompetent mouse model of cancer-induced bone pain, in which BALB/c mice are intrafemorally inoculated with 4T1 murine carcinoma cells. AG879 decreased functional system xC- activity, as measured by cystine uptake and glutamate release, and inhibited nociceptive and physiologically relevant responses in tumour-bearing animals. Cumulatively, these data suggest that the activation of TrkA by nerve growth factor may have functional implications on system xC--mediated cancer pain. System xC--mediated TrkA activation therefore presents a promising target for therapeutic intervention in cancer pain treatment.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Neoplasias Ósseas/complicações , Dor do Câncer/etiologia , Dor do Câncer/metabolismo , Ácido Glutâmico/metabolismo , Receptor trkA/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Comportamento Animal , Contagem de Células , Linhagem Celular Tumoral , Feminino , Fêmur/patologia , Humanos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Fator de Crescimento Neural/farmacologia , Nociceptividade/efeitos dos fármacos , Osteólise/metabolismo , Osteólise/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor trkA/metabolismo , Tirfostinas/farmacologiaAssuntos
Neoplasias Ósseas/metabolismo , Dor do Câncer/metabolismo , Ácido Glutâmico/metabolismo , Sistema Nervoso Periférico/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Comportamento Animal , Neoplasias Ósseas/fisiopatologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Transplante de Neoplasias , Neurônios/metabolismo , Nociceptividade , Manejo da Dor , Medição da Dor , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sulfassalazina/farmacologiaRESUMO
System Xc- is a cystine/glutamate antiporter that contributes to the maintenance of cellular redox balance. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. Transcription factors regulating antioxidant defense mechanisms including system Xc- are of therapeutic interest, especially given that aggressive breast cancer cells exhibit increased system Xc- function. This investigation provides evidence that xCT expression is regulated by STAT3 and/or STAT5A, functionally affecting the antiporter in human breast cancer cells. Computationally analyzing two kilobase pairs of the xCT promoter/5' flanking region identified a distal gamma-activated site (GAS) motif, with truncations significantly increasing luciferase reporter activity. Similar transcriptional increases were obtained after treating cells transiently transfected with the full-length xCT promoter construct with STAT3/5 pharmacological inhibitors. Knock-down of STAT3 or STAT5A with siRNAs produced similar results. However, GAS site mutation significantly reduced xCT transcriptional activity, suggesting that STATs may interact with other transcription factors at more proximal promoter sites. STAT3 and STAT5A were bound to the xCT promoter in MDA-MB-231 cells, and binding was disrupted by pre-treatment with STAT inhibitors. Pharmacologically suppressing STAT3/5 activation significantly increased xCT mRNA and protein levels, as well as cystine uptake, glutamate release, and total levels of intracellular glutathione. Our data suggest that STAT proteins negatively regulate basal xCT expression. Blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism to protect breast cancer cells from stress, including reactive oxygen species, by up-regulating xCT expression and the function of system Xc-. We propose that targeting system Xc- together with STAT3/5 inhibitors may heighten therapeutic anti-cancer effects.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Cistina/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Humanos , Oxirredução , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional/genética , Regulação para Cima/genéticaRESUMO
BACKGROUND: Liver kinase 1 (LKB1) is an important multi-tasking protein linked with metabolic signaling, also controlling polarity and cytoskeletal rearrangements in diverse cell types including cancer cells. Prolactin (PRL) and Signal transducer and activator of transcription (STAT) proteins have been associated with breast cancer progression. The current investigation examines the effect of PRL and STAT-mediated signaling on the transcriptional regulation of LKB1 expression in human breast cancer cells. METHODS: MDA-MB-231, MCF-7, and T47D human breast cancer cells, and CHO-K1 cells transiently expressing the PRL receptor (long form), were treated with 100 ng/ml of PRL for 24 hours. A LKB1 promoter-luciferase construct and its truncations were used to assess transcriptional changes in response to specific siRNAs or inhibitors targeting Janus activated kinase 2 (JAK2), STAT3, and STAT5A. Real-time PCR and Western blotting were applied to quantify changes in mRNA and protein levels. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays were used to examine STAT3 and STAT5A binding to the LKB1 promoter. RESULTS: Consistent with increases in mRNA, the LKB1 promoter was up-regulated by PRL in MDA-MB-231 cells, a response that was lost upon distal promoter truncation. A putative GAS element that could provide a STAT binding site mapped to this region, and its mutation decreased PRL-responsiveness. PRL-mediated increases in promoter activity required signaling through STAT3 and STAT5A, also involving JAK2. Both STATs imparted basally repressive effects in MDA-MB-231 cells. PRL increased in vivo binding of STAT3, and more definitively, STAT5A, to the LKB1 promoter region containing the GAS site. In T47D cells, PRL down-regulated LKB1 transcriptional activity, an effect that was reversed upon culture in phenol red-free media. Interleukin 6, a cytokine activating STAT signaling in diverse cell types, also increased LKB1 mRNA levels and promoter activity in MDA-MB-231 cells. CONCLUSIONS: LKB1 is differentially regulated by PRL at the level of transcription in representative human breast cancer cells. Its promoter is targeted by STAT proteins, and the cellular estrogen receptor status may affect PRL-responsiveness. The hormonal and possibly cytokine-mediated control of LKB1 expression is particularly relevant in aggressive breast cancer cells, potentially promoting survival under energetically unfavorable conditions.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética , Quinases Proteína-Quinases Ativadas por AMP , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Prolactina/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
Neurotrophic factors were first identified to promote the growth, survival or differentiation of neurons and have also been associated with the early stages of ovarian folliculogenesis. More recently, their effects on the final stage of follicular development, including oocyte maturation and early embryonic development, have been reported. Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which are expressed in numerous peripheral tissues outside of the CNS, most notably the ovary, are now known to stimulate oocyte maturation in various species, also enhancing developmental competence. The mechanisms that underlie their actions in antral follicles, as well as the targets ultimately controlled by these factors, are beginning to emerge. GDNF, BDNF and NGF, alone or in combination, could be added to the media currently utilized for in vitro oocyte maturation, thereby potentially increasing the production and/or quality of early embryos.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Fator de Crescimento Neural/fisiologia , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Humanos , Técnicas de Maturação in Vitro de Oócitos/métodos , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismoRESUMO
BACKGROUND: Liver kinase 1 (LKB1) is emerging as a multifunctional protein, acting as a key metabolic enzyme, regulator of cell polarity, and transcription factor. Altered LKB1 expression has been linked with various cancers and may be a potential prognostic marker. While the functional role of LKB1 continues to undergo intensive investigation, the molecular mechanisms that regulate its expression remain to be defined more clearly. Recent reports have established a possible link between estrogen receptor alpha (ERα) signaling and LKB1 in MCF-7 human breast cancer cells. The current study aimed to investigate whether LKB1 is transcriptionally regulated by ERα in MCF-7 cells. METHODS: siRNA transfections were used to transiently knock down LKB1 and ERα. LKB1 and ERα mRNA and protein levels were evaluated by real-time PCR and Western blotting, respectively. An approximately 3 kilobase pair human LKB1 promoter construct and various truncations were generated, transfected into MCF-7 cells, and luciferase reporter assays were performed. Cells were also treated with various doses of 17-ß-estradiol (E2) to evaluate the effect on LKB1 and ERα mRNA levels. RESULTS: LKB1 mRNA and protein levels were significantly lower in ERα-positive MCF-7 compared to ERα-negative MDA-MB-231 breast cancer cells, suggesting that ERα may act as a repressor. siRNA-mediated knock-down of ERα in MCF-7 cells significantly increased LKB1 promoter activity and expression at the mRNA and protein levels, and computational analysis revealed the presence of several putative estrogen response element (ERE) DNA binding sites in the LKB1 promoter region. In addition, treatment with E2 led to an increase in LKB1 expression, concomitant with decreased expression of ERα in MCF-7 cells. The E2-mediated increase was abrogated by pretreatment with actinomycin D, supporting that the observed changes in LKB1 levels were transcriptionally regulated. CONCLUSIONS: ERα repressively modulates the expression of LKB1 at the transcriptional level. Targeting the expression of LKB1 by modulating ERα signaling may provide a potential approach to further evaluate its function in ERα-positive breast cancers.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Mammary carcinomas have been associated with a high-fat diet, and the rate of breast cancer in overweight post-menopausal women is up to 50% higher than in their normal-weight counterparts. Epidemiological studies suggest that prolactin (PRL) plays a role in the progression of breast cancer. The current study examined breast cancer as a metabolic disease in the context of altered fatty acid catabolism by examining the effect of PRL on carnitine palmitoyl transferase 1 (CPT1), an enzyme that shuttles long-chain fatty acids into the mitochondrial matrix for ß-oxidation. The effect of PRL on the adenosine 5'-monophosphate-activated protein kinase (AMPK) energy sensing pathway was also investigated. METHODS: MCF-7 and MDA-MB-231 breast cancer cells and 184B5 normal breast epithelial cells treated with 100 ng/ml of PRL for 24 hr were used as in vitro models. Real-time PCR was employed to quantify changes in mRNA levels and Western blotting was carried out to evaluate changes at the protein level. A non-radioactive CPT1 enzyme activity assay was established and siRNA transfections were performed to transiently knock down specific targets in the AMPK pathway. RESULTS: PRL stimulation increased the expression of CPT1A (liver isoform) at the mRNA and protein levels in both breast cancer cell lines, but not in 184B5 cells. In response to PRL, a 20% increase in CPT1 enzyme activity was observed in MDA-MB-231 cells. PRL treatment resulted in increased phosphorylation of the α catalytic subunit of AMPK at Thr172, as well as phosphorylation of acetyl-CoA carboxylase (ACC) at Ser79. A siRNA against liver kinase B1 (LKB1) reversed these effects in breast cancer cells. PRL partially restored CPT1 activity in breast cancer cells in which CPT1A, LKB1, or AMPKα-1 were knocked down. CONCLUSIONS: PRL enhances fatty acid ß-oxidation by stimulating CPT1 expression and/or activity in MCF-7 and MDA-MB-231 breast cancer cells. These PRL-mediated effects are partially dependent on the LKB1-AMPK pathway, although the regulation of CPT1 is also likely to be influenced by other mechanisms. Ultimately, increased CPT1 enzyme activity may contribute to fueling the high energy demands of cancer cells. Targeting metabolic pathways that are governed by PRL, which has already been implicated in the progression of breast cancer, may be of therapeutic benefit.
Assuntos
Neoplasias da Mama/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Prolactina/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Carnitina O-Palmitoiltransferase/biossíntese , Carnitina O-Palmitoiltransferase/genética , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Oxirredução , Fosforilação/efeitos dos fármacos , Prolactina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Chronic pain occurs with greater frequency in women, with a parallel sexually dimorphic trend reported in sufferers of many autoimmune diseases. There is a need to continue examining neuro-immune-endocrine crosstalk in the context of sexual dimorphisms in chronic pain. Several phenomena in particular need to be further explored. In patients, autoantibodies to neural antigens have been associated with sensory pathway hyper-excitability, and the role of self-antigens released by damaged nerves remains to be defined. In addition, specific immune cells release pro-nociceptive cytokines that directly influence neural firing, while T lymphocytes activated by specific antigens secrete factors that either support nerve repair or exacerbate the damage. Modulating specific immune cell populations could therefore be a means to promote nerve recovery, with sex-specific outcomes. Understanding biological sex differences that maintain, or fail to maintain, neuroimmune homeostasis may inform the selection of sex-specific treatment regimens, improving chronic pain management by rebalancing neuroimmune feedback. Given the significance of interactions between nerves and immune cells in the generation and maintenance of neuropathic pain, this review focuses on sex differences and possible links with persistent autoimmune activity using sciatica as an example.
Assuntos
Autoimunidade , Dor Crônica/imunologia , Neuroimunomodulação , Ciática/imunologia , Caracteres Sexuais , Animais , Dor Crônica/genética , Hormônios Esteroides Gonadais/fisiologia , Humanos , Ciática/genética , Cromossomos Sexuais/genéticaRESUMO
Chronic neuropathic pain (NP) is a growing clinical problem for which effective treatments, aside from non-steroidal anti-inflammatory drugs and opioids, are lacking. Cannabinoids are emerging as potentially promising agents to manage neuroimmune effects associated with nociception. In particular, Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and their combination are being considered as therapeutic alternatives for treatment of NP. This study aimed to examine whether sex affects long-term outcomes on persistent mechanical hypersensitivity 7 weeks after ceasing cannabinoid administration. Clinically relevant low doses of THC, CBD, and a 1:1 combination of THC:CBD extracts, in medium chain triglyceride (MCT) oil, were orally gavaged for 14 consecutive days to age-matched groups of male and female sexually mature Sprague Dawley rats. Treatments commenced one day after surgically inducing a pro-nociceptive state using a peripheral sciatic nerve cuff. The analgesic efficacy of each phytocannabinoid was assessed relative to MCT oil using hind paw mechanical behavioural testing once a week for 9 weeks. In vivo intracellular electrophysiology was recorded at endpoint to characterize soma threshold changes in primary afferent sensory neurons within dorsal root ganglia (DRG) innervated by the affected sciatic nerve. The thymus, spleen, and DRG were collected post-sacrifice and analyzed for long-term effects on markers associated with T lymphocytes at the RNA level using qPCR. Administration of cannabinoids, particularly the 1:1 combination of THC, elicited a sustained mechanical anti-hypersensitive effect in males with persistent peripheral NP, which corresponded to beneficial changes in myelinated Aß mechanoreceptive fibers. Specific immune cell markers associated with T cell differentiation and pro-inflammatory cytokines, previously implicated in repair processes, were differentially up-regulated by cannabinoids in males treated with cannabinoids, but not in females, warranting further investigation into sexual dimorphisms that may underlie treatment outcomes.
Assuntos
Analgésicos/administração & dosagem , Analgésicos/farmacologia , Canabidiol/efeitos adversos , Canabidiol/farmacologia , Dronabinol/administração & dosagem , Dronabinol/farmacologia , Óleos/química , Administração Oral , Analgésicos/química , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Canabidiol/química , Dronabinol/química , Composição de Medicamentos , Interações Medicamentosas , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND PURPOSE: Chronic neuropathic pain (NEP) is associated with growing therapeutic cannabis use. To promote quality of life without psychotropic effects, cannabinoids other than Δ9-tetrahydrocannabidiol, including cannabidiol and its precursor cannabidiolic acid (CBDA), are being evaluated. Due to its instability, CBDA has been understudied, particularly as an anti-nociceptive agent. Adding a methyl ester group (CBDA-ME) significantly enhances its stability, facilitating analyses of its analgesic effects in vivo. This study examines early treatment efficacy of CBDA-ME in a rat model of peripherally induced NEP and evaluates sex as a biological variable. EXPERIMENTAL APPROACH: After 14 consecutive days of intraperitoneal CBDA-ME administration at 0.01, 0.1 and 1 µg·kg-1 , commencing 1 day after surgically implanting a sciatic nerve-constricting cuff to induce NEP, the anti-nociceptive efficacy of this cannabinoid was assessed in male and female Sprague-Dawley rats relative to vehicle-treated counterparts. In females, 2 and 4 µg·kg-1 daily doses of CBDA-ME were also evaluated. Behavioural tests were performed for hind paw mechanical and thermal withdrawal thresholds once a week for 8 weeks. At endpoint, in vivo electrophysiological recordings were obtained to characterize soma threshold changes in primary sensory neurons. KEY RESULTS: In males, CBDA-ME elicited a significant concentration-dependent chronic anti-hyperalgesic effect, also influencing both nociceptive and non-nociceptive mechanoreceptors, which were not observed in females at any of the concentrations tested. CONCLUSION AND IMPLICATIONS: Initiating treatment of a peripheral nerve injury with CBDA-ME at an early stage post-surgery provides anti-nociception in males, warranting further investigation into potential sexual dimorphisms underlying this response.
Assuntos
Canabinoides , Neuralgia , Animais , Dronabinol , Ésteres , Feminino , Masculino , Neuralgia/tratamento farmacológico , Qualidade de Vida , Ratos , Ratos Sprague-DawleyRESUMO
INTRODUCTION: Pain is a common and debilitating comorbidity of metastatic breast cancer. The hippocampus has been implicated in nociceptive processing, particularly relating to the subjective aspect of pain. Here, a syngeneic mouse model was used to characterize the effects of peripheral tumors on hippocampal microglial activation in relation to cancer-induced pain (CIP). MATERIALS AND METHODS: Mice were systemically treated with the colony-stimulating factor 1 receptor inhibitor Pexidartinib prior to intrafemoral (IF) or subcutaneous 4T1 carcinoma cell inoculation. Spontaneous and evoked nociceptive responses were quantitated throughout tumor development, and contralateral hippocampi were collected via endpoint microdissection for RNA analysis. Additionally, IF tumor-bearing animals were sacrificed on days 5, 10, 15, and 20 post 4T1 cell inoculation, and brain sections were immunofluorescently stained for Iba1, a marker of activated microglia. RESULTS: Ablation of these neuroimmune cells with the CSF1R inhibitor Pexidartinib delayed the onset and severity of cancer-induced nociceptive behaviors in IF tumor-bearing animals, adding to the body of literature that demonstrates microglial contribution to the development and maintenance of CIP. Furthermore, in untreated IF tumor-bearing mice, nociceptive behaviors appeared to progress in parallel with microglial activation in hippocampal regions. Immunofluorescent Iba1+ microglia increased in the dentate gyrus and cornu ammonis 1 hippocampal regions in IF tumor-bearing animals over time, which was confirmed at the mRNA level using relevant microglial markers. CONCLUSION: This is the first experimental evidence to demonstrate the effects of peripheral tumor-induced nociception on hippocampal microglial activation. The increase in hippocampal microglia observed in the present study may reflect the emotional and cognitive deficits reported by patients with CIP.
RESUMO
BACKGROUND: Chronic pain is a major symptom that develops in cancer patients, most commonly emerging during advanced stages of the disease. The nature of cancer-induced pain is complex, and the efficacy of current therapeutic interventions is restricted by the dose-limiting sideeffects that accompany common centrally targeted analgesics. METHODS: This review focuses on how up-regulated glutamate production and export by the tumour converge at peripheral afferent nerve terminals to transmit nociceptive signals through the transient receptor cation channel, TRPV1, thereby initiating central sensitization in response to peripheral disease-mediated stimuli. RESULTS: Cancer cells undergo numerous metabolic changes that include increased glutamine catabolism and over-expression of enzymes involved in glutaminolysis, including glutaminase. This mitochondrial enzyme mediates glutaminolysis, producing large pools of intracellular glutamate. Upregulation of the plasma membrane cystine/glutamate antiporter, system xc -, promotes aberrant glutamate release from cancer cells. Increased levels of extracellular glutamate have been associated with the progression of cancer-induced pain and we discuss how this can be mediated by activation of TRPV1. CONCLUSION: With a growing population of patients receiving inadequate treatment for intractable pain, new targets need to be considered to better address this largely unmet clinical need for improving their quality of life. A better understanding of the mechanisms that underlie the unique qualities of cancer pain will help to identify novel targets that are able to limit the initiation of pain from a peripheral source-the tumour.
Assuntos
Dor do Câncer/metabolismo , Ácido Glutâmico/metabolismo , Vias Aferentes/metabolismo , Animais , Humanos , Canais de Cátion TRPV/metabolismoRESUMO
STAT3 and STAT5 mediate diverse cellular processes, transcriptionally regulating gene expression and interacting with cytoplasmic proteins. Their canonical activity is stimulated by cytokines/growth factors through JAK-STAT signaling. As targets of oncogenes with intrinsic tyrosine kinase activity, STAT3 and STAT5 become constitutively active in hematologic neoplasms and solid tumors, promoting cell proliferation and survival and modulating redox homeostasis. This review summarizes reactive oxygen species (ROS)-regulated STAT activation and how STATs influence ROS production. ROS-induced effects on post-translational modifications are presented, and STAT3/5-mediated regulation of xCT, a redox-sensitive target up-regulated in numerous cancers, is discussed with regard to transcriptional cross-talk.
Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Proliferação de Células , Sobrevivência Celular , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredução , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This "ying-yang" effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient's breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxirredução , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc(-), an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc(-), is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc(-). We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.
Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Sistema y+ de Transporte de Aminoácidos/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Células MCF-7 , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Evidence supporting that deleted in azoospermia-like (DAZL) plays a key role during gametogenesis and meiosis continues to emerge. Our study aimed to determine whether overexpression of DAZL using a lentiviral approach in a somatic stem cell to germ cell in vitro differentiation culture could enhance the formation of primordial germ cell-like cells (PLCs) and oocyte-like cells (OLCs). Introduction of DAZL at the beginning of induced differentiation significantly increased the formation of Fragilis-positive PLCs, which was independent of mitotic proliferation. In addition, mRNA levels of the germ cell markers Oct4, Stella, and Vasa were also higher in the DAZL-transduced group and suppressed when DAZL was knocked down using small interference RNA. At later stages of differentiation, the expression of several genes associated with meiosis, including Scp3, Dmc1, Rec8, and Stra8, was determined to be significantly higher when DAZL was overexpressed, which was abrogated by its knockdown. Exogenous introduction of DAZL also increased the protein levels of SCP3 and VASA, which again was reversed by its knockdown. Although not a common phenomenon in the in vitro differentiation system, the percentage of SCP3-positive cells displaying meiotic chromosome patterns in the DAZL-transduced group was higher than in the control, as was the overall percentage of OLCs that were generated. The introduction of factors such as DAZL into a stem cell-to-germ cell differentiation culture may provide an opportunity to better understand the key genes and their interactions during gametogenesis, also providing a means to enhance the generation of germ cells in vitro.
Assuntos
Células Germinativas/fisiologia , Meiose , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/fisiologia , Animais , Antígenos de Diferenciação/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Forma Celular , Células Cultivadas , Replicação do DNA , Expressão Gênica , Técnicas de Silenciamento de Genes , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Pele/citologia , Células-Tronco/fisiologia , Sus scrofaRESUMO
BACKGROUND: The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth. METHODS AND FINDINGS: Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1ß1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKß1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1ß1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKß1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism. CONCLUSIONS: Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/radioterapia , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Radiação Ionizante , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Feminino , Humanos , Modelos Biológicos , Fosforilação/efeitos da radiação , Ligação Proteica/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Frações Subcelulares/metabolismo , Frações Subcelulares/efeitos da radiaçãoRESUMO
PURPOSE: To analyze the (i) expression of AMPK in a variety of epithelial cancer cells, (ii) regulation of AMPK subunit expression by ionizing radiation (IR) and (iii) impact of AMPK on signaling pathways regulating cell cycle and survival. METHODS AND MATERIALS: Human lung, prostate, and breast normal and cancer cells were treated with 0 or 8 Gy IR and mRNA and protein levels of AMPK were evaluated by RT-PCR and immunoblotting 24 or 48 h later. Untreated and radiated wild type (WT) and AMPKα(-/-) mouse embryonic fibroblasts (MEFs) were analyzed by immunoblotting using total- and phosphorylation-specific antibodies. Histone H2Ax was examined by fluorescence microscopy. The cell cycle and survival of WT and AMPKα(-/-) MEFs was also evaluated following 8 Gy by IR. RESULTS: AMPK subunits were found widely expressed in normal and cancer epithelial cells. IR increased subunit protein levels and stimulated gene transcription in cancer cells. AMPKα(-/-)-MEFs showed enhanced basal total levels of ATM and phosphorylation of its substrates histone H2Ax, but inhibited response of these markers and of checkpoint kinase Chk2 phosphorylation to IR. AMPKα(-/-)-MEFs showed increased basal levels of p53 and cyclin-dependent kinase inhibitors p21(cip1), but lack of response of both genes to IR. These cells had increased basal levels and activation of the Akt-mTOR-p70(S6K)/4-EBP1 signalling pathway. IR increased Akt, p70(S6K) and 4-EBP1 phosphorylation in WT-MEFs, but this was reduced in AMPKα(-/-)-MEFs. AMPKα(-/-)-MEFs failed to arrest at the G2-M checkpoint after IR and showed a trend for radio-resistance in proliferation assays. CONCLUSIONS: AMPK is widely expressed in human normal and cancer epithelial cells and its gene transcription, protein levels, and enzymatic activity is stimulated by IR. Work with AMPKα knockout cells suggests that AMPK (i) may mediate a suppressive regulation on basal expression and activity of ATM and its downstream effector pathways Chk2/p53-p21(cip1) and Akt-mTOR, (ii) facilitates the normal response of these pathways to IR and, (iii) mediates the IR-induced G2-M checkpoint.