RESUMEN
Impact of reactive oxygen species (ROS) in development of hyperalgesia has recently motivated scientists to focus on ROS as novel target of anti-hyperalgesic interventions. Studies have indicated the usefulness of ROS scavengers and exogenous antioxidants as anti-nociceptive agents in animal models of neuropathic and inflammatory hyperalgesia. In present study, we suggest the anti-hyperalgesic potential of the dietary antioxidant quercetin on chronic inflammatory hyperalgesia induced by Complete Freund's Adjuvant (CFA). Three doses of quercetin (25, 50 and 75 mg/kg body weight) for consecutive 7 days were used for the study. Thermal hyperalgesia was assessed by paw withdrawal latency (PWL) test and inflammation was checked in terms of changes in paw edema. The insight of molecular signaling during chronic hyperalgesia was analyzed by TNF-α-TNFR1-ERK1/2 pathway in relation to change in ROS level in DRG and spinal cord. CFA-induced hyperalgesia was confirmed by decreased PWL and increased c-Fos activity in dorsal horn of spinal cord, determined by immunohistochemical analysis. It was characterized with elevated level of ROS and TNF-α estimated by ELISA. The activation of ERK1/2 and NF-κB in DRG and spinal cord and over-expression of TNFR1 in DRG were analyzed by Western blotting. Up-regulation of Iba1 and GFAP indicates glial activation in spinal cord. Expression of GFAP and its co-localization with NF-κB were examined by immunofluorescence. All the molecular modulators of hyperalgesia were brought towards normal after quercetin treatment showing its anti-hyperalgesic activity, indicating that repeated quercetin treatment is able to alleviate chronic inflammatory hyperalgesia by attenuating TNF-α-TNFR1-ERK1/2 signaling pathway via modulation of ROS and by suppression of central sensitization via inhibition of spinal glial activation.
Asunto(s)
Astrocitos/efectos de los fármacos , Adyuvante de Freund/toxicidad , Hiperalgesia/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microglía/efectos de los fármacos , Quercetina/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/etiología , Hiperalgesia/patología , Inflamación , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Quercetina/farmacología , Distribución Aleatoria , Ratas , Receptores Tipo I de Factores de Necrosis Tumoral/biosíntesis , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Role of NADPH oxidase1 in the development of inflammatory pain has been demonstrated by gene knockout studies. Nevertheless, pharmacological inhibition of NOX1 is a requisite approach for therapeutic utility. Recently, we have reported the anti-nociceptive effect of newly identified NOX1 specific inhibitor ML171 (2-acetylphenothiazine). Inhibition of NOX1 resulted in attenuation of nociceptive sensitization during acute inflammatory pain via inhibition of ROS generation and its downstream ERK1/2 activation. However, glial activation accompanying inflammation is closely related to the initiation and maintenance of pain. Peripheral nociceptive inputs activate the primary afferents via release of various chemical mediators which are potentially capable of mediating signals from neuron to glia in DRG and subsequently in spinal cord dorsal horn. The subsequent interactions between neuron and glia contribute to pain hypersensitivity. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in a mouse model of formalin induced acute nociception. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in DRG and dorsal horn of the spinal cord of lumbar region (L3-L5) in a mouse model of formalin induced acute nociception. Intraperitoneal administration of ML171 decreased nociceptive behavioral responses, i.e. the flinch and lick counts, in formalin induced nociceptive mice. Immunofluorescence and Western blot analysis demonstrated decreased levels of nociceptive mediators like p-ERK1/2, p-NFκB p65, Iba1 and GFAP in DRG as well as in spinal cord dorsal horn; supporting anti-nociceptive potential of ML171. Further, co-localization studies showed the neuron-glia crosstalk in tissue dependent manner. ERK1/2 was found to be activated in glia and NFκB in neurons in DRG; whereas in case of spinal cord ERK1/2 was activated in neurons and NFκB in astrocytes. Decrease in nociceptive behavioral response and activation of nociceptive mediators after intraperitoneal administration of ML171 strongly advocate anti-nociceptive potential of ML171. This is the first report demonstrating modulation of ERK1/2-NFκB signaling pathway, glial activation and regulation of neuron-glia crosstalk by NADPH oxidase1 inhibition towards its anti-nociceptive action.
Asunto(s)
Sensibilización del Sistema Nervioso Central/efectos de los fármacos , Formaldehído/farmacología , NADPH Oxidasas/efectos de los fármacos , Nocicepción/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Masculino , Ratones , NADP/efectos de los fármacos , NADP/metabolismo , FN-kappa B/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Dolor/tratamiento farmacológico , Dolor/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The spread of metastatic cancer cell is the main cause of death worldwide. Cellular and molecular basis of the action of phytochemicals in the modulation of metastatic cancer highlights the importance of fruits and vegetables. Quercetin is a natural bioflavonoid present in fruits, vegetables, seeds, berries, and tea. The cancer-preventive activity of quercetin is well documented due to its anti-inflammatory, anti-proliferative and anti-angiogenic activities. However, poor water solubility and delivery, chemical instability, short half-life, and low-bioavailability of quercetin limit its clinical application in cancer chemoprevention. A better understanding of the molecular mechanism of controlled and regulated drug delivery is essential for the development of novel and effective therapies. To overcome the limitations of accessibility by quercetin, it can be delivered as nanoconjugated quercetin. Nanoconjugated quercetin has attracted much attention due to its controlled drug release, long retention in tumor, enhanced anticancer potential, and promising clinical application. The pharmacological effect of quercetin conjugated nanoparticles typically depends on drug carriers used such as liposomes, silver nanoparticles, silica nanoparticles, PLGA (Poly lactic-co-glycolic acid), PLA (poly(D,L-lactic acid)) nanoparticles, polymeric micelles, chitosan nanoparticles, etc. In this review, we described various delivery systems of nanoconjugated quercetin like liposomes, silver nanoparticles, PLGA (Poly lactic-co-glycolic acid), and polymeric micelles including DOX conjugated micelles, metal conjugated micelles, nucleic acid conjugated micelles, and antibody-conjugated micelles on in vitro and in vivo tumor models; as well as validated their potential as promising onco-therapeutic agents in light of recent updates.
Asunto(s)
Antineoplásicos/administración & dosificación , Nanopartículas , Neoplasias/tratamiento farmacológico , Quercetina/administración & dosificación , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Portadores de Fármacos , Humanos , Estructura Molecular , Quercetina/química , Quercetina/uso terapéutico , SolubilidadRESUMEN
Reactive oxygen species (ROS) have a key role in different etiologies of pain. At sub-cellular level, mitochondria and plasma membranes have been identified as endogenous sources of ROS required for pain generation. NADPH oxidase (NOX) is the main contributor of membrane associated ROS generation. Out of 7 isozymes, NOX1, NOX2 and NOX4 are reported to be associated with nociceptive sensitization. Therefore, it has been hypothesized that specific inhibition of the NOX isozymes could be putative strategy for treatment of pain. However, unavailability of specific inhibitors was the biggest obstacle to test this hypothesis. Here, we investigated anti-nociceptive potential of a newly identified specific NOX1 inhibitor ML171 in formalin induced inflammatory pain. ML171 administration decreased the paw lickings and flinching response in both phases of formalin test. Behavioral response was supported with decreased activation of c-Fos in spinal dorsal horn. The increased level of total NOX activity, ROS and pERK1/2 in dorsal root ganglion (DRG) and spinal dorsal horn of formalin induced nociception were reversed by ML171 administration. ML171 also inhibited the upregulated Tumor necrosis factor receptor 1 (TNFR1) expression in DRG, whereas did not show any effect in spinal dorsal horn which was unaltered after formalin insult. The study for the first time depicts anti-nociceptive potential of ML171 via regulation of ROS mediated ERK1/2 signaling by inhibition of NOX1 activity.
Asunto(s)
Sistema de Señalización de MAP Quinasas/efectos de los fármacos , NADPH Oxidasa 1/efectos de los fármacos , Sulfonamidas/farmacología , Tiofenos/farmacología , Animales , Formaldehído/farmacología , Masculino , Ratones , NADPH Oxidasas/metabolismo , Proteínas Proto-Oncogénicas c-fos , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Flavonoids have been chronicles of the history of a long way journey in the cure of physiological or pathophysiological conditions in various diseases including cancer. Our previous findings suggest the extensive mechanism of quercetin (QUE) mediated regression of cell survival, cell proliferation, oxidative stress, inflammation, and angiogenesis via modulating PI3K and PKC signaling in lymphoma as well as hepatocellular carcinoma. PI3K-PKC pathway is a key monitor of mammalian cells regulated by its different isoenzymes, which may exert similar or opposite cellular effects by differential coupling of signaling pathways. Put forward the invention of selective inhibitors against various isoenzymes is beneficial to reduce the burden of inclusive deleterious effects of drug for normal physiological process. Therefore, we hypothesized the improved anticancer efficacy of QUE in combination with isoenzyme inhibitors-rottlerin (ROT-PKCδ inhibitor), G0 6983 (PKCα inhibitor), and PI-103 (p110α-class I PI3K inhibitor) in MCF-7 and RAW 264.7 cells. QUE significantly improves the cytotoxicity of ROT + G0 6983 ranged 30-55% and PI-103 ranged 24-63% after 24-48 h against MCF-7 cells. Additionally in the presence of QUE, the improved cytotoxicity of ROT + G0 6983 is observed to range 69-75% and PI-103 ranged 45-88% after 24-48 h in RAW 264.7 cells. This increment in cell deaths are positively correlated with enhanced morphological alteration observed in MCF-7 cells. Further, QUE significantly increases the attenuation of PKCα level approximately by 50% in combination with PI-103. Overall results of the current study suggested that QUE improves the synergistic anticancer efficacy in combination with PI-103, ROT, and G0 6983 in MCF-7 and RAW 264.7 cells.
Asunto(s)
Acetofenonas/farmacología , Antineoplásicos/farmacología , Benzopiranos/farmacología , Furanos/farmacología , Indoles/farmacología , Maleimidas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quercetina/farmacología , Acetofenonas/química , Animales , Apoptosis/efectos de los fármacos , Benzopiranos/química , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Furanos/química , Humanos , Indoles/química , Células MCF-7 , Maleimidas/química , Ratones , Proteína Quinasa C-alfa/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/química , Pirimidinas/química , Quercetina/química , Células RAW 264.7RESUMEN
Continuous accumulation of ROS maintains oxidative stress in tumor microenvironment and is implicated in hyperactivation of signaling pathways that promote cell proliferation, survival, and metabolic adaptations to cancer. Antioxidants are supposed to interfere with tumor microenvironment. Plant-derived flavonoids and curcuminoids are considered as promising therapeutic molecules due to least side effects. Anticarcinogenic activity of various herbal antioxidants is widely reported. However, supports from preclinical studies and clinical trials are insufficient. Critical insight in signaling pathways leading to cancer progression and regulation of specific targets by herbal antioxidants in preclinical studies is a prerequisite to overcome the lacuna in its successful translation into therapeutical medicine. Most of the studies are limited to in vitro tests, revealing the action at cellular level. However, the natural antioxidants need validation at the level of cancer regression and better survival of laboratory animals. This review will focus on various signaling pathways leading to cancer progression and their interception by herbal antioxidants with a greater emphasis on the antioxidants being most widely validated in vitro and in animal models. These drugs are at the preliminary stage of testing as supplementary or alternative drugs in patients. The review will provide insight to overcome the failures in clinical practice and scope for development of novel anticancer drugs.
Asunto(s)
Anticarcinógenos/farmacología , Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Neoplasias/patología , Control de Calidad , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Warburg effect is characterized by the upregulation of HIF-1 and c-Myc regulated LDH-A, even aerobically owing to hypoxic environment and alterations in oncogenes or tumor suppressor genes in cancer. Reduced antioxidant defence system in transformed cells favors higher ROS production, which plays a significant role in carcinogenesis and acts as an important regulator of NF-κB. In addition, various proinflammatory cytokines play active roles in maintenance and progression of cancer. OBJECTIVE: In continuation with our previous studies illustrating the long-term effect of curcumin using a liver tissue, present study was aimed to elucidate the anti-cancer effect of curcumin due to its long-term effect in the regulation of glycolytic metabolism, NF-κB activation, expression of proinflammatory cytokines in Dalton's lymphoma ascites cells in vivo. METHOD: Spectrophotometric assays, RT-PCR and EMSA were performed to address the problems. RESULTS: Results revealed that curcumin-induced activation of antioxidant enzymes, Nrf2 and downstream signaling gene NQO1. Reduction of oxidative stress, down-regulation of NADPH: Oxidase, decline in ROS and H2O2 levels were also observed. Activation of NF-κB, expression of COX2, HIF-1α and cMyc, as well as expression and activity of LDH-A were significantly reduced by curcumin. Besides, expression of proinflammatory cytokines was significantly down-regulated via reducing binding of nuclear protein with AP-1, NF-IL6, ETS and NF-κB binding elements of IL-1α, IL-1ß, TNF-α and IL-6 promoters, respectively. CONCLUSION: Curcumin downregulates glycolytic metabolism via modulation of stress-activated genes and reduces oxidative stress by enhancing antioxidant defence system, which inhibits activation of NF-κB signaling and expression of proinflammatory cytokines in Dalton's lymphoma ascites cells in vivo.
Asunto(s)
Antineoplásicos/farmacología , Ascitis/metabolismo , Curcumina/farmacología , Citocinas/metabolismo , Glucosa/metabolismo , Inflamación/metabolismo , Linfoma/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Antineoplásicos/química , Ascitis/patología , Curcumina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glucólisis/efectos de los fármacos , Linfoma/metabolismo , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos DBA , Estructura Molecular , Factor 2 Relacionado con NF-E2/genética , Relación Estructura-ActividadRESUMEN
INTRODUCTION: Pain is an unpleasant sensation triggered by noxious stimulation. It is one of the most prevalent conditions, limiting productivity and diminishing quality of life. Non steroidal anti inflammatory drugs (NSAIDs) are widely used as pain relievers in present day practice as pain is mostly initiated due to inflammation. However, due to potentially serious side effects, long term use of these antihyperalgesic drugs raises concern. Therefore there is a demand to search novel medicines with least side effects. Herbal products have been used for centuries to reduce pain and inflammation, and phytochemicals are known to cause fewer side effects. However, identification of active phytochemicals of herbal medicines and clear understanding of the molecular mechanism of their action is needed for clinical acceptance. MATERIALS AND METHODS: In this review, we have briefly discussed the cellular and molecular changes during hyperalgesia via inflammatory mediators and neuro-modulatory action involved therein. The review includes 54 recently reported phytochemicals with antihyperalgesic action, as per the literature available with PubMed, Google Scholar and Scopus. CONCLUSION: Compounds of high interest as potential antihyperalgesic agents are: curcumin, resveratrol, capsaicin, quercetin, eugenol, naringenin and epigallocatechin gallate (EGCG). Current knowledge about molecular targets of pain and their regulation by these phytochemicals is elaborated and the scope of further research is discussed.
Asunto(s)
Analgésicos/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Dolor/tratamiento farmacológico , Fitoquímicos/uso terapéutico , Analgésicos/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Citocinas/metabolismo , Humanos , Óxido Nítrico Sintasa/metabolismo , Nociceptores/metabolismo , Dolor/metabolismo , Dolor/fisiopatología , Fitoquímicos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
Concomitant generation of reactive oxygen species during tissue inflammation has been recognised as a major factor for the development and the maintenance of hyperalgesia, out of which H2O2 is the major player. However, molecular mechanism of H2O2 induced hyperalgesia is still obscure. The aim of present study is to analyse the mechanism of H2O2-induced hyperalgesia in rats. Intraplantar injection of H2O2 (5, 10 and 20 µmoles/paw) induced a significant thermal hyperalgesia in the hind paw, confirmed by increased c-Fos activity in dorsal horn of spinal cord. Onset of hyperalgesia was prior to development of oxidative stress and inflammation. Rapid increase in phosphorylation of extracellular signal regulated kinase (ERK) was observed in neurons of dorsal root ganglia after 20 min of H2O2 (10 µmoles/paw) administration, which gradually returned towards normal level within 24 h, following the pattern of thermal hyperalgesia. The expression of TNFR1 followed the same pattern and colocalised with pERK. ERK phosphorylation was observed in NF-200-positive and -negative neurons, indicating the involvement of ERK in C-fibres as well as in A-fibres. Intrathecal preadministration of Src family kinases (SFKs) inhibitor (PP1) and MEK inhibitor (PD98059) prevented H2O2 induced augmentation of ERK phosphorylation and thermal hyperalgesia. Pretreatment of protein tyrosine phosphatases (PTPs) inhibitor (sodium orthovanadate) also diminished hyperalgesia, although it further increased ERK phosphorylation. Combination of orthovanadate with PP1 or PD98059 did not exhibit synergistic antihyperalgesic effect. The results demonstrate SFKs-mediated ERK activation and increased TNFR1 expression in nociceptive neurons during H2O2 induced hyperalgesia. However, the role of PTPs in hyperalgesic behaviour needs further molecular analysis.
Asunto(s)
Hiperalgesia/enzimología , Sistema de Señalización de MAP Quinasas , Neuronas/enzimología , Familia-src Quinasas/fisiología , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ganglios Espinales/patología , Miembro Posterior , Calor , Peróxido de Hidrógeno , Hiperalgesia/inducido químicamente , Peroxidación de Lípido , Masculino , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Sensación Térmica , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVES: Impact of ROS in development of hyperalgesia has recently motivated scientists to focus on ROS as novel target of anti-hyperalgesic interventions. However, role of ROS in molecular signaling of hyperalgesia is still poorly understood. The present study is aimed to analyze the effect of dietary antioxidant resveratrol on antioxidant defense system, ROS level and TNFR1-ERK signaling pathway during early and late phase of inflammatory hyperalgesia. METHODS AND MATERIALS: Hyperalgesia was assessed by paw withdrawal latency test in complete Freund's adjuvant-induced hyperalgesic rats. Activities of antioxidant enzymes were measured by in-gel assays, ROS level was measured by DCFH2DA, and expression of pERK, ERK and TNFR1 was estimated by Western blotting. RESULTS: Anti-hyperalgesic effect of resveratrol was observed by paw withdrawal latency test. ROS level was increased in paw skin as well as spinal cord during early phase which was further increased in paw skin, but remained constant in spinal cord up to late phase. Resveratrol differentially regulated the activities of SOD, catalase and GPx in paw skin as well as spinal cord of hyperalgesic rats in both phases. Activities were normalized back showing anti-hyperalgesic effect of resveratrol. Upregulated ERK signaling was modulated by resveratrol, whereas TNFR1 level remained unchanged. CONCLUSION: Overall results suggest that resveratrol alleviates inflammatory hyperalgesia by downregulation of ERK activation, modulation of ROS and differential regulation of antioxidant enzymes during early and late phases.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Antioxidantes/farmacología , Hiperalgesia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Estilbenos/uso terapéutico , Animales , Antioxidantes/metabolismo , Adyuvante de Freund , Dimensión del Dolor , Ratas , Receptores Tipo I de Factores de Necrosis Tumoral/genética , ResveratrolRESUMEN
AKT signaling is important to maintaining normal physiology. Hyperactivation of AKT signaling is frequent in cancer, which maintains a high oxidative state in a tumor microenvironment that is needed for tumor adaptation. Therefore, antioxidants are proposed to exhibit anticancer properties by interfering with the tumor microenvironment. Quercetin is an ubiquitous bioactive antioxidant rich in vegetables and beverages. The present study aimed to analyze cancer preventive property of quercetin in ascite cells of Dalton's lymphoma-bearing mice. Protein level was determined by Western blotting. Nitric oxide (NO) level was estimated spectrophotometrically using Griess reagent. Results show downregulation in phosphorylation of AKT and PDK1 by quercetin, which was consistent with decreased phosphorylation of downstream survival factors such as BAD, GSK-3ß, mTOR, and IkBα. Further, quercetin attenuated the levels of angiogenic factor VEGF-A and inflammatory enzymes COX-2 and iNOS as well as NO levels, whereas it increased the levels of phosphatase PTEN. Overall results suggest that quercetin modulates AKT signaling leading to attenuation of cell survival, inflammation, and angiogenesis in lymphoma-bearing mice.
Asunto(s)
Moduladores de la Angiogénesis/farmacología , Inflamación/tratamiento farmacológico , Linfoma de Células T/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/farmacología , Animales , Antioxidantes/farmacología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Masculino , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Aberrant activation of PI3K-AKT signaling in many pathological conditions including cancer has attracted much of interest for drug targeting. Various isoforms are known from three classes of PI3K. Targeting selective isoform is advantageous to overcome the global deleterious effects of drug. PI-103 is a specific inhibitor of p110α of class I PI3K. The present study is aimed to analyze anti-carcinogenic activity of PI-103 in Dalton's lymphoma ascite (DLA) cells. Result shows regression in cell proliferation and increased apoptosis in terms of increased Annexin V binding, nuclear fragmentation and active caspase 3 level. It is correlated with attenuation of PI3K-AKT signaling by PI-103 via downregulation of the level of p110α, phospho-p85α, phospho- AKT, and PKCα in DLA cells as well as in H2O2 induced DLA cells. Additionally, ROS accumulation is declined in H2O2 induced DLA cells. Overall result suggests that PI-103 attenuates PI3K-AKT signaling via induction of apoptosis in murine T-cell lymphoma.
Asunto(s)
Apoptosis/efectos de los fármacos , Furanos/farmacología , Linfoma de Células T/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Linfoma de Células T/genética , Linfoma de Células T/patología , Masculino , Ratones , Fosforilación/efectos de los fármacos , Proteína Quinasa C-alfa/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Phosphatidylinositol 3 kinase-protein kinase B (PI3K-AKT) pathway has been considered as major drug target site due to its frequent activation in cancer. AKT regulates the activity of various targets to promote tumorigenesis and metastasis. Accumulation of reactive oxygen species (ROS) has been linked to oxidative stress and regulation of signaling pathways for metabolic adaptation of tumor microenvironment. Hydrogen peroxide (H2O2) in this context is used as ROS source for oxidative stress preconditioning. Antioxidants are commonly considered to be beneficial to reduce detrimental effects of ROS and are recommended as dietary supplements. Quercetin, a ubiquitous bioactive flavonoid is a dietary component which has attracted much of interest due to its potential health-promoting effects. Present study is aimed to analyze PI3K-AKT signaling pathway in H2O2 exposed Dalton's lymphoma ascite (DLA) cells. Further, regulation of PI3K-AKT pathway by quercetin as well as PI-103, an inhibitor of PI3K was analyzed. Exposure of H2O2 (1mM H2O2 for 30min) to DLA cells caused ROS accumulation and resulted in increased phosphorylation of PI3K and downstream proteins PDK1 and AKT (Ser-473 and Thr-308), cell survival factors BAD and ERK1/2, as well as TNFR1. However, level of tumor suppressor PTEN was declined. Both PI-103 & quercetin suppressed the enhanced level of ROS and significantly down-regulated phosphorylation of AKT, PDK1, BAD and level of TNFR1 as well as increased the level of PTEN in H2O2 induced lymphoma cells. The overall result suggests that quercetin and PI3K inhibitor PI-103 attenuate PI3K-AKT pathway in a similar mechanism.
Asunto(s)
Furanos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Linfoma de Células T/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Quercetina/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Humanos , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Tamoxifen (Tmx) embedded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-Tmx) is prepared to evaluate its better DNA cleavage potential, cytotoxicity using Dalton's lymphoma ascite (DLA) cells and MDA-MB231 breast cancer cells. PLGA-Tmx nanoparticles are prepared through emulsified nanoprecipitation technique with varying dimension of 17-30nm by changing the concentrations of polymer, emulsifier and drug. Nanoparticles dimension are measured through electron and atomic force microscopy. Interactions between tamoxifen and PLGA are verified through spectroscopic and calorimetric methods. PLGA-Tmx shows excellent DNA cleavage potential as compared to pure Tmx raising better bioavailability. In vitro cytotoxicity studies indicate that PLGA-Tmx reduces DLA cells viability up to â¼38% against â¼15% in pure Tmx. Hoechst stain is used to detect apoptotic DLA cells through fluorescence imaging of nuclear fragmentation and condensation exhibiting significant increase of apoptosis (70%) in PLGA-Tmx vis-à-vis pure drug (58%). Enhanced DNA cleavage potential, nuclear fragmentation and condensation in apoptotic cells confirm greater bioavailability of PLGA-Tmx as compared to pure Tmx in terms of receptor mediated endocytosis. Hence, the sustained release kinetics of PLGA-Tmx nanoparticles shows much better anticancer efficacy through enhanced DNA cleavage potential and nuclear fragmentation and, thereby, reveal a novel vehicle for the treatment of cancer.
Asunto(s)
Preparaciones de Acción Retardada/farmacología , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ascitis/patología , Disponibilidad Biológica , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , División del ADN/efectos de los fármacos , Liberación de Fármacos , Humanos , Cinética , Linfoma/patología , Ratones , Nanopartículas/ultraestructura , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Alcohol Polivinílico/química , Espectroscopía Infrarroja por Transformada de Fourier , Tamoxifeno/farmacologíaRESUMEN
Sensitization of nociceptive neurons by inflammatory mediators leads to hypersensitivity for normal painful stimuli which is termed hyperalgesia. Oxidative stress is an essential factor in pathological pain; therefore, antioxidants qualify as potential anti-hyperalgesic agents. The present study examines the efficacy of the natural antioxidant resveratrol in complete Freund's adjuvant (CFA) induced hyperalgesic rats. Thermal hyperalgesia was measured at different time points by paw withdrawal latency test and confirmed by c-Fos expression in spinal dorsal horn. The impact of resveratrol treatment on inflammatory mediators at peripheral (paw skin) and central (spinal cord) sites was determined during early (6 h) as well as late phase (48 h) of hyperalgesia. Intraplanter injection of CFA increased the level of cytokines IL-1ß, TNF-α and IL-6 as well as inflammatory enzymes COX-2 and iNOS in paw skin in both phases. In case of spinal cord, the level of COX-2 was found to be elevated in both phases, whereas iNOS could not be detected. The cytokines were found to be elevated only in late phase in spinal cord. Administration of resveratrol (20 mg/kg) shifted the level of all inflammatory mediators towards normal, except cytokines in paw skin. The present study suggests that the anti-nociceptive effect of resveratrol is implicated at both peripheral and central sites in a tissue specific manner. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Analgésicos/farmacología , Hiperalgesia/tratamiento farmacológico , Mediadores de Inflamación/análisis , Estilbenos/farmacología , Animales , Ciclooxigenasa 2/análisis , Ciclooxigenasa 2/fisiología , Citocinas/análisis , Masculino , Óxido Nítrico Sintasa de Tipo II/fisiología , Ratas , Resveratrol , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunologíaRESUMEN
BACKGROUND: Protein kinase C regulates various cellular processes including cell proliferation, cell adhesion, apoptosis, angiogenesis, invasion, and metastasis. Activation of different PKC isozymes results in distinct cellular responses. Novel PKCs are mainly involved in apoptotic process. Atypical PKC subfamily plays a critical role in cell proliferation and apoptosis, cell differentiation and motility. However, Atypical PKCs show contradictory regulation in different tissues or cancer cells. The mechanism of diversified effects is not well explored. Antioxidant ellagic acid shows hepatoprotective, anti-carcinogenic and anti-mutagenic properties. Present study is focused to analyze the effect of ellagic acid on novel and atypical isozymes of PKC in regulation of PKC-mediated apoptosis in liver of lymphoma bearing mice. Implication of ellagic acid treatment to DL mice was analyzed on caspase-3 mediated apoptosis via PKCδ induced activation; and on maintenance of adequate supply of energy during cancer growth. METHODS: 15-20 weeks old adult DL mice were divided into four groups (n=6). Group 2, 3, 4 were treated with different doses of ellagic acid (40 mg/kg, 60 mg/kg and 80 mg/kg bw). The mice were sacrificed after 19 days of treatment and liver was used for study. The effect of ellagic acid was determined on expression of novel and atypical PKC isozymes. Apoptotic potentiality of ellagic acid was checked on activities of caspase-3 and PKCδ in terms of their catalytic fragments. Aerobic glycolysis was monitored by LDH activity, especially activity of LDH A. RESULTS: Ellagic acid treatment caused up regulation of expression of almost all novel and atypical PKC isozymes. Activities of PKCδ and caspase-3 were enhanced by ellagic acid, however activities of total LDH and LDH-A were inhibited. CONCLUSION: The results show that ellagic acid promotes apoptosis in lymphoma bearing mice via novel and atypical PKCs which involves PKCδ induced caspase-3 activation; and inhibition of glycolytic pathway.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Ácido Elágico/farmacología , Linfoma/metabolismo , Extractos Vegetales/farmacología , Proteína Quinasa C/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ácido Elágico/uso terapéutico , Activadores de Enzimas/farmacología , Activadores de Enzimas/uso terapéutico , Glucólisis/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Linfoma/tratamiento farmacológico , Ratones , Fitoterapia , Extractos Vegetales/uso terapéutico , Transducción de Señal/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Protein kinase C (PKC) is a key regulator of cell growth and differentiation in mammalian cells and hyperactivation of PKC is believed to play an important role in tumor progression. PKC is downstream to signaling protein of phosphatidylinositol 3-Kinase (PI3K), a known up-regulator of cell proliferation and survival. Accumulation of reactive oxygen species (ROS) triggers oxidative stress in the tumor microenvironment, leading to the hyperactivation of various oxidative stress-stimulated signaling molecules. Quercetin (QUE) is a naturally occurring dietary flavonoid having antioxidant properties. QUE is reported to show antitumor activity both in vitro and in vivo; however, the molecular mechanism is yet to be thoroughly explored. HepG2 cells display cellular functions similar to the normal hepatocytes with high degree of morphological and functional differentiation, therefore HepG2 cell line is chosen as the suitable model for drug targeting. Present study is aimed to establish the signaling pathway involved in the anticarcinogenic action of QUE in HepG2 cell line. HepG2 cells were treated with different doses of QUE. Protein level and gene expression were analysed by Western blotting and RT-PCR, respectively. PKC activity was measured by non-radioactive-tagged phosphorylation. Results showed downregulation of expression of PI3K, PKC, COX-2 and ROS caused by QUE. Additionally, QUE enhanced the expression of p53 and BAX in HepG2 cells. Overall, results of the current study suggested that QUE elicited anticarcinogenic action by upregulation of p53 and BAX in HepG2 cells via downregulation of ROS, PKC, PI3K and COX-2, confirming our earlier report on the animal model.
Asunto(s)
Anticarcinógenos/farmacología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteína Quinasa C/metabolismo , Quercetina/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Ciclooxigenasa 2/genética , Regulación hacia Abajo , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Fosfatidilinositol 3-Quinasa/genética , Proteína Quinasa C/genética , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba , Proteína X Asociada a bcl-2/genéticaRESUMEN
Developmental stage-specific enhancer-promoter-insulator interactions regulate the chromatin configuration necessary for transcription at various loci and additionally for VDJ recombination at antigen receptor loci that encode immunoglobulins and T-cell receptors. To investigate these regulatory interactions, we analyzed the epigenetic landscape of the murine T-cell receptor ß (TCRß) locus in the presence and absence of an ectopic CTCF-dependent enhancer-blocking insulator, H19-ICR, in genetically manipulated mice. Our analysis demonstrated the ability of the H19-ICR insulator to restrict several aspects of enhancer-based chromatin alterations that are observed during activation of the TCRß locus for transcription and recombination. The H19-ICR insulator abrogated enhancer-promoter contact-dependent chromatin alterations and additionally prevented Eß-mediated histone modifications that have been suggested to be independent of enhancer-promoter interaction. Observed enhancer-promoter-insulator interactions, in conjunction with the chromatin structure of the Eß-regulated domain at the nucleosomal level, provide useful insights regarding the activity of the regulatory elements in addition to supporting the accessibility hypothesis of VDJ recombination. Analysis of H19-ICR in the heterologous context of the developmentally regulated TCRß locus suggests that different mechanisms proposed for CTCF-dependent insulator action might be manifested simultaneously or selectively depending on the genomic context and the nature of enhancer activity being curtailed.
Asunto(s)
Cromatina/genética , Elementos de Facilitación Genéticos , Proteínas Represoras/fisiología , Animales , Factor de Unión a CCCTC , Células Cultivadas , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Histonas , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Mutagénesis Insercional , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/genética , Timocitos , Activación TranscripcionalRESUMEN
Cancer cells are characterized by increased production of reactive oxygen species (ROS) and an altered redox environment as compared to normal cells. Continuous accumulation of ROS triggers oxidative stress leading to hyper-activation of signaling pathways that promote cell proliferation, survival, and metabolic adaptation to the tumor microenvironment. Therefore, antioxidants are proposed to contribute to cancer prevention. Protein kinase C (PKC) is a crucial regulator of diverse cellular processes and contributes to cancer progression. The activation of PKC is partially dependent on ROS signaling. In the present study, cancer preventive activity of natural flavonoid quercetin is analyzed in ascite cells of Dalton's lymphoma-bearing mice. The total ROS level and activity of PKC were downregulated after quercetin treatment in lymphoma-bearing mice. Quercetin modulates the expression of almost all isozymes of classical, novel, and atypical PKC as well as downregulates the level and expression of PKCα. Further, quercetin improves apoptotic potential, as observed by the levels of caspase 3, caspase 9, PARP, PKCδ, and nuclear condensation. Additionally, quercetin reduces cell survival and promotes death receptor-mediated apoptosis via differential localization of the TNFR1 level in ascite cells. The overall result suggests the cancer preventive activity of quercetin via the induction of apoptosis and modulates PKC signaling with the reduction of oxidative stress in ascite cells of lymphoma-bearing mice.