RESUMO
Much is understood about the structure and gating properties of NMDA receptors (NMDAR), but the function of the carboxy-terminal splice variant of the NR1 subunit, NR1 C2 has never been identified. By studying the scaffolding protein Magi-2 in animal models of inflammatory pain, we discovered how NR1 C2 protein is specifically regulated. We found that Magi-2 deficiency resulted in decreased pain behavior and a concomitant reduction in NR1 C2 protein. Magi-2 contains WW domains, domains typically found in ubiquitin ligases. We identified an atypical WW-binding domain within NR1 C2 which conferred susceptibility to Nedd4-1 ubiquitin-ligase dependent degradation. We used lipidated peptidomimetics derived from the NR1 C2 sequence and found that NR1 C2 protein levels and pain behavior can be pharmacologically targeted. The function of NR1 C2 is to give lability to a pool of NMDAR, important for pain signaling.
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Chronic constriction injuries (CCI) of the sciatic nerve are widely used nerve entrapment animal models of neuropathic pain. Two common pain behaviors observed following CCI are thermal hyperalgesia and mechanical allodynia, measured by the Hargreaves and von Frey tests, respectively. While thermal hyperalgesia tends to recover by 30 days, mechanical allodynia can persist for many more months thereafter. Consequently, mechanical allodynia has been used extensively as a measure of 'chronic pain' focusing on the circuitry changes that occur within the spinal cord. Here, using the sciatic nerve cuff variant of CCI in mice, we propose that in contrast to these evoked measures of nociceptive hypersensitivity, dynamic weight bearing provides a more clinically relevant behavioral measure for ongoing pain during nerve injury. We found that the effect of sciatic nerve cuff on the ratio of weight bearing by the injured relative to uninjured hindlimbs more closely resembled that of thermal hyperalgesia, following a trend toward recovery by 30 days. We also found an increase in the percent of body weight bearing by the contralateral paw that is not seen in the previously tested behaviors. These results demonstrate that dynamic weight bearing is a reliable measure of non-evoked neuropathic pain and suggest that thermal hyperalgesia, rather than mechanical allodynia, provides a proxy measure for nerve entrapment-induced ongoing pain.
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The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP+/IB4- nociceptors in rodents and in CGRP+ dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dor Crônica/tratamento farmacológico , Endocitose/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Complexo 2 de Proteínas Adaptadoras/antagonistas & inibidores , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades alfa do Complexo de Proteínas Adaptadoras/antagonistas & inibidores , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Epiderme/inervação , Feminino , Gânglios Espinais/metabolismo , Humanos , Inflamação , Masculino , Camundongos , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Nociceptores/fisiologia , Peptídeos/administração & dosagem , Peptídeos/metabolismo , Peptídeos/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Voltage-dependent sodium (NaV) 1.8 channels regulate action potential generation in nociceptive neurons, identifying them as putative analgesic targets. Here, we show that NaV1.8 channel plasma membrane localization, retention, and stability occur through a direct interaction with the postsynaptic density-95/discs large/zonula occludens-1-and WW domain-containing scaffold protein called membrane-associated guanylate kinase with inverted orientation (Magi)-1. The neurophysiological roles of Magi-1 are largely unknown, but we found that dorsal root ganglion (DRG)-specific knockdown of Magi-1 attenuated thermal nociception and acute inflammatory pain and produced deficits in NaV1.8 protein expression. A competing cell-penetrating peptide mimetic derived from the NaV1.8 WW binding motif decreased sodium currents, reduced NaV1.8 protein expression, and produced hypoexcitability. Remarkably, a phosphorylated variant of the very same peptide caused an opposing increase in NaV1.8 surface expression and repetitive firing. Likewise, in vivo, the peptides produced diverging effects on nocifensive behavior. Additionally, we found that Magi-1 bound to sequence like a calcium-activated potassium channel sodium-activated (Slack) potassium channels, demonstrating macrocomplexing with NaV1.8 channels. Taken together, these findings emphasize Magi-1 as an essential scaffold for ion transport in DRG neurons and a central player in pain.-Pryce, K. D., Powell, R., Agwa, D., Evely, K. M., Sheehan, G. D., Nip, A., Tomasello, D. L., Gururaj, S., Bhattacharjee, A. Magi-1 scaffolds NaV1.8 and Slack KNa channels in dorsal root ganglion neurons regulating excitability and pain.
Assuntos
Gânglios Espinais/citologia , Guanilato Quinases/fisiologia , Proteínas de Membrana/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.8/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Nociceptividade/fisiologia , Canais de Potássio Ativados por Sódio/fisiologia , Células Receptoras Sensoriais/fisiologia , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Células Cultivadas , Feminino , Guanilato Quinases/antagonistas & inibidores , Guanilato Quinases/genética , Injeções , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Domínios PDZ , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Nós Neurofibrosos/metabolismo , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Nervos EspinhaisRESUMO
A newly designed organoselenium compound, methyl substituted umbelliferone selenocyanate (MUS), was synthesized as a primary hit against the myelotoxic activity of carboplatin. MUS was administered at 6 mg/kg b.wt, p.o. in concomitant and pretreatment schedules with carboplatin (12 mg/kg b.wt, i.p. for 10 days) in female Swiss albino mouse. MUS treatment reduced (P < 0.001) the percentage of chromosomal aberrations, micronuclei formation, DNA damage and apoptosis in murine bone marrow cells and also enhanced (P < 0.001) the bone marrow cell proliferation of the carboplatin-treated mice. These activities cumulatively restored the viable bone marrow cell count towards normalcy. Myeloprotection by MUS was achieved, in part, due to a significant reduction in the ROS/RNS formation and restoration of glutathione redox pool. Additionally, MUS synergistically enhanced the cytotoxicity of carboplatin against two human cancer cell lines (MCF-7 and Colo-205). Furthermore, MUS can effectively potentiate the antitumour activity of carboplatin against two murine cancers (Dalton's Lymphoma and Sarcoma-180) in vivo. These preclinical findings clearly indicate that MUS can improve the therapeutic index of carboplatin and ensures more effective therapeutic strategy against cancer for clinical development.
Assuntos
Antineoplásicos/farmacologia , Cumarínicos/química , Desenho de Fármacos , Linfoma/tratamento farmacológico , Mielopoese/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Sarcoma Experimental/tratamento farmacológico , Animais , Antineoplásicos/química , Antioxidantes/química , Antioxidantes/farmacologia , Apoptose , Proliferação de Células , Quimioterapia Adjuvante , Aberrações Cromossômicas , Dano ao DNA/efeitos dos fármacos , Feminino , Peroxidação de Lipídeos , Linfoma/patologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Sarcoma Experimental/patologia , Células Tumorais CultivadasRESUMO
The sodium-activated potassium (KNa) channel Kcnt1 (Slack) is abundantly expressed in nociceptor (pain-sensing) neurons of the dorsal root ganglion (DRG), where they transmit the large outward conductance IKNa and arbitrate membrane excitability. Slack channel expression at the DRG membrane is necessary for their characteristic firing accommodation during maintained stimulation, and reduced membrane channel density causes hyperexcitability. We have previously shown that in a pro-inflammatory state, a decrease in membrane channel expression leading to reduced Slack-mediated IKNa expression underlies DRG neuronal sensitization. An important component of the inflammatory milieu, PKA internalizes Slack channels from the DRG membrane, reduces IKNa, and produces DRG neuronal hyperexcitability when activated in cultured primary DRG neurons. Here, we show that this PKA-induced retrograde trafficking of Slack channels also occurs in intact spinal cord slices and that it is carried out by adaptor protein-2 (AP-2) via clathrin-mediated endocytosis. We provide mass spectrometric and biochemical evidence of an association of native neuronal AP-2 adaptor proteins with Slack channels, facilitated by a dileucine motif housed in the cytoplasmic Slack C terminus that binds AP-2. By creating a competitive peptide blocker of AP-2-Slack binding, we demonstrated that this interaction is essential for clathrin recruitment to the DRG membrane, Slack channel endocytosis, and DRG neuronal hyperexcitability after PKA activation. Together, these findings uncover AP-2 and clathrin as players in Slack channel regulation. Given the significant role of Slack in nociceptive neuronal excitability, the AP-2 clathrin-mediated endocytosis trafficking mechanism may enable targeting of peripheral and possibly, central neuronal sensitization.
Assuntos
Membrana Celular/metabolismo , Clatrina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Fator de Transcrição AP-2/metabolismo , Animais , Células Cultivadas , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Neurônios/citologia , Canais de Potássio Ativados por Sódio , Ratos , Ratos Sprague-DawleyRESUMO
Early infantile epileptic encephalopathies (EOEE) are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl-]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a "change-of-function" KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient.
Assuntos
Epilepsia/metabolismo , Mutação de Sentido Incorreto , Canais de Potássio/genética , Potenciais de Ação , Animais , Células CHO , Células Cultivadas , Pré-Escolar , Cricetinae , Cricetulus , Epilepsia/genética , Epilepsia/fisiopatologia , Feminino , Heterozigoto , Humanos , Masculino , Fenótipo , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , XenopusRESUMO
The Slick (Kcnt2) sodium-activated potassium (KNa) channel is a rapidly gating and weakly voltage-dependent and sodium-dependent potassium channel with no clearly defined physiological function. Within the dorsal root ganglia (DRGs), we show Slick channels are exclusively expressed in small-sized and medium-sized calcitonin gene-related peptide (CGRP)-containing DRG neurons, and a pool of channels are localized to large dense-core vesicles (LDCV)-containing CGRP. We stimulated DRG neurons for CGRP release and found Slick channels contained within CGRP-positive LDCV translocated to the neuronal membrane. Behavioral studies in Slick knockout (KO) mice indicated increased basal heat detection and exacerbated thermal hyperalgesia compared with wild-type littermate controls during neuropathic and chronic inflammatory pain. Electrophysiologic recordings of DRG neurons from Slick KO mice revealed that Slick channels contribute to outward current, propensity to fire action potentials (APs), and to AP properties. Our data suggest that Slick channels restrain the excitability of CGRP-containing neurons, diminishing pain behavior after inflammation and injury.
RESUMO
Various epidemiological and preclinical studies have already established the cancer chemopreventive potential of vanadium. In addition, recent studies have also indicated the abilities of vanadium-based compounds to induce cell death selectively towards malignant cells. Therefore, the objective of the present investigation is to improve the therapeutic efficacy and toxicity profile of an alkylating agent, cyclophosphamide, by the concurrent use of an organovanadium compound, oxovanadium(IV)-l-cysteine methyl ester complex (VC-IV). In this study, VC-IV (1mg/kg b.w., p.o.) was administered alone as well as in combination with cyclophosphamide (25mg/kg b.w., i.p.) in concomitant and pretreatment schedules. The results showed that VC-IV in combination with cyclophosphamide resulted in an improved therapeutic efficacy as evidenced by reduction of tumor growth and prolongation of life span. The observed potentiation was mediated through generation of ROS in tumor cells, which ultimately led to significant DNA damage, and apoptosis in tumor cells. Further studies revealed that VC-IV sensitized tumor cells to cyclophosphamide therapy by down-regulating the anti-apoptotic protein Bcl-2 and by up-regulating molecules like p53, Bax, cytochrome c, caspases, which led to PARP cleavage and apoptosis. Significant inhibition of angiogenesis along with reduction in the levels of VEGF-A and MMP-9 in the tumor bed by VC-IV further contributed to the sensitization accomplished by VC-IV. Moreover, VC-IV ameliorated cyclophosphamide-induced hematopoietic, hepatic and genetic damages by modulating the antioxidant status in normal organs. Thus, the present study clearly demonstrated the sensitizing and protective efficacy of VC-IV and indicates it may serve as a promising adjuvant in cancer chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Ciclofosfamida/farmacologia , Neoplasias/tratamento farmacológico , Compostos de Vanádio/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Quimioterapia Adjuvante/métodos , Cisteína/análogos & derivados , Cisteína/farmacologia , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Camundongos , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Abstract: The sodium-activated potassium channel Slack (Kcnt1, Slo2.2) is highly expressed in dorsal root ganglion neurons where it regulates neuronal firing. Several studies have implicated the Slack channel in pain processing, but the precise mechanism or the levels within the sensory pathway where channels are involved remain unclear. Here, we furthered the behavioral characterization of Slack channel knockout mice and for the first time examined the role of Slack channels in the superficial, pain-processing lamina of the dorsal horn. We performed whole-cell recordings from spinal cord slices to examine the intrinsic and synaptic properties of putative inhibitory and excitatory lamina II interneurons. Slack channel deletion altered intrinsic properties and synaptic drive to favor an overall enhanced excitatory tone. We measured the amplitudes and paired pulse ratio of paired excitatory post-synaptic currents at primary afferent synapses evoked by electrical stimulation of the dorsal root entry zone. We found a substantial decrease in the paired pulse ratio at synapses in Slack deleted neurons compared to wildtype, indicating increased presynaptic release from primary afferents. Corroborating these data, plantar test showed Slack knockout mice have an enhanced nociceptive responsiveness to localized thermal stimuli compared to wildtype mice. Our findings suggest that Slack channels regulate synaptic transmission within the spinal cord dorsal horn and by doing so establishes the threshold for thermal nociception.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Vias Aferentes/metabolismo , Animais , Estimulação Elétrica/métodos , Gânglios Espinais/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Nociceptores/fisiologia , Dor/metabolismo , Células do Corno Posterior/metabolismo , Canais de Potássio/genética , Canais de Potássio Ativados por SódioRESUMO
Various epidemiological and preclinical studies have already established the cancer chemopreventive potential of vanadium-based compounds. In addition to its preventive efficacy, studies have also indicated the abilities of vanadium-based compounds to induce cell death selectively toward malignant cells. Therefore, the objective of the present investigation is to improve the therapeutic efficacy and toxicity profile of an alkylating agent, cyclophosphamide, by the concurrent use of an organovanadium complex, vanadium(III)-l-cysteine. In this study, vanadium(III)-l-cysteine (1 mg/kg body weight, per os) was administered alone as well as in combination with cyclophosphamide (25 mg/kg body weight, intraperitoneal) in concomitant and pretreatment schedule in mice bearing breast adenocarcinoma cells. The results showed that the combination treatment significantly decreased the tumor burden and enhanced survivability of tumor-bearing mice through generation of reactive oxygen species in tumor cells. These ultimately led to DNA damage, depolarization of mitochondrial membrane potential, and apoptosis in tumor cells. Further insight into the molecular pathway disclosed that the combination treatment caused upregulation of p53 and Bax and suppression of Bcl-2 followed by the activation of caspase cascade and poly (ADP-ribose) polymerase cleavage. Administration of vanadium(III)-l-cysteine also resulted in significant attenuation of peritoneal vasculature and sprouting of the blood vessels by decreasing the levels of vascular endothelial growth factor A and matrix metalloproteinase 9 in the ascites fluid of tumor-bearing mice. Furthermore, vanadium(III)-l-cysteine significantly attenuated cyclophosphamide-induced hematopoietic, hepatic, and genetic damages and provided additional survival advantages. Hence, this study suggested that vanadium(III)-l-cysteine may offer potential therapeutic benefit in combination with cyclophosphamide by augmenting anticancer efficacy and diminishing toxicity to the host.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Complexos de Coordenação/administração & dosagem , Cisteína/análogos & derivados , Neoplasias Mamárias Animais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclofosfamida/administração & dosagem , Cisteína/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Neoplasias Mamárias Animais/patologia , Camundongos , Neovascularização Patológica/patologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Sodium-activated potassium (KNa) channels contribute to firing frequency adaptation and slow after hyperpolarization. The KCNT1 gene (also known as SLACK) encodes a KNa subunit that is expressed throughout the central and peripheral nervous systems. Missense mutations of the SLACK C-terminus have been reported in several patients with rare forms of early onset epilepsy and in some cases severely delayed myelination. To date, such mutations identified in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), epilepsy of infancy with migrating focal seizures (EIMFS) and Ohtahara syndrome (OS) have been reported to be gain-of-function mutations (Villa and Combi, 2016). An exome sequencing study identified a p.Phe932Ile KCNT1 mutation as the disease-causing change in a child with severe early infantile epileptic encephalopathy and abnormal myelination (Vanderver et al., 2014). We characterized an analogous mutation in the rat Slack channel and unexpectedly found this mutation to produce a loss-of-function phenotype. In an effort to restore current, we tested the known Slack channel opener loxapine. Loxapine exhibited no effect, indicating that this mutation either caused the channel to be insensitive to this established opener or proper translation and trafficking to the membrane was disrupted. Protein analysis confirmed that while total mutant protein did not differ from wild type, membrane expression of the mutant channel was substantially reduced. Although gain-of-function mutations to the Slack channel are linked to epileptic phenotypes, this is the first reported loss-of-function mutation linked to severe epilepsy and delayed myelination.
Assuntos
Epilepsia do Lobo Frontal/genética , Leucoencefalopatias/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Animais , Células CHO/metabolismo , Cricetulus , Modelos Animais de Doenças , Epilepsia do Lobo Frontal/metabolismo , Leucoencefalopatias/genética , Proteínas do Tecido Nervoso/genética , Fenótipo , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , RatosRESUMO
Cyclophosphamide (CP) is one of the widely used anticancer agents; however, it has serious deleterious effects on normal host cells due to its nonspecific action. The essential trace element Selenium (Se) is suggested to have chemopreventive and chemotherapeutic efficacy and currently used in pharmaceutical formulations. Previous report had shown Nano-Se could protect CP-induced hepatotoxicity and genotoxicity in normal Swiss albino mice; however, its role in cancer management is still not clear. The aim of present study is to investigate the chemoprotective efficacy of Nano-Se against CP-induced toxicity as well as its chemoenhancing capability when used along with CP in Swiss albino mice against Ehrlich's ascites carcinoma (EAC) cells. CP was administered (25 mg/kg b.w., i.p.) and Nano-Se was given (2 mg Se/kg b.w., p.o.) in concomitant and pretreatment schedule. Increase levels of serum hepatic marker, hepatic lipid peroxidation, DNA damage, and chromosomal aberration in CP-treated mice were significantly (P < 0.05) reversed by Nano-Se. The lowered status of various antioxidant enzymes in tumor-bearing mice after CP treatment was also effectively increased by Nano-Se. Administration of Nano-Se along with CP caused a significant reduction in tumor volume, packed cell volume, viable tumor cell count, and increased the survivability of the tumor-bearing hosts. The results suggest that Nano-Se exhibits significant antitumor and antioxidant effects in EAC-bearing mice. The potential for Nano-Se to ameliorate the CP-evoked toxicity as well as to improve the chemotherapeutic effect could have beneficial implications for patients undergoing chemotherapy with CP.
Assuntos
Carcinoma de Ehrlich/tratamento farmacológico , Ciclofosfamida/farmacologia , Nanopartículas Metálicas/química , Selênio/farmacologia , Animais , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patologia , Masculino , Camundongos , Selênio/químicaRESUMO
Cisplatin (CDDP) is one of the first-line anticancer drugs that has gained widespread use against various forms of human malignancies. But, the therapeutic outcome of CDDP therapy is limited due to its adverse effects including myelotoxicity and DNA damage which may lead to the subsequent risk of developing secondary cancer. Hence, in search of a suitable cytoprotectant, this study investigated the probable protective efficacy of an oxovanadium(IV) complex, namely oxovanadium(IV)-L-cysteine methyl ester complex (VC-IV) against CDDP-induced myelosuppression and genotoxic damage in the bone marrow cells of Swiss albino mice. CDDP was administered intraperitoneally (5 mg/kg b.w.) and VC-IV was administered orally (1 mg/kg b.w.) in concomitant and 7 d pretreatment schedule. Treatment with VC-IV in CDDP-treated mice significantly (p < 0.01) enhanced bone marrow cell proliferation and inhibited cell death in the bone marrow niche indicating improvement of CDDP-induced myelotoxicity. The organovanadium compound also significantly (p < 0.01) reduced the percentage of chromosomal aberrations, the frequency of micronuclei formation, and the extent of DNA damage. The observed chemoprotective effect of VC-IV was attributed to its anti-oxidant efficacy which significantly (p < 0.01) attenuated CDDP-induced generation of free radicals, and restored (p < 0.01) the levels of oxidized and reduced glutathione. Hence, VC-IV may serve as a promising candidate for future development to decrease the deleterious effects of CDDP in the bone marrow cells of cancer patients and associated secondary complications.
Assuntos
Antineoplásicos/toxicidade , Células da Medula Óssea/efeitos dos fármacos , Cisplatino/toxicidade , Dano ao DNA/efeitos dos fármacos , Compostos Organometálicos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Vanadatos/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Cisteína/análogos & derivados , Cisteína/química , Relação Dose-Resposta a Droga , Feminino , Camundongos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Testes para Micronúcleos , Compostos Organometálicos/química , Substâncias Protetoras/química , Vanadatos/químicaRESUMO
OBJECTIVE: The present study was designed to investigate the chemoprotective efficacy of an L-cysteine-based oxovanadium (IV) complex, namely, oxovanadium (IV)-L-cysteine methyl ester complex (VC-IV) against cisplatin (CDDP)-induced renal injury in Swiss albino mice. METHODS: CDDP was administered intraperitoneally (5 mg/kg body weight) and VC-IV was administered orally (1 mg/kg body weight) in concomitant and 7 days pre-treatment schedule. RESULTS: CDDP-treated mice showed marked kidney damage and renal failure. Administration of VC-IV caused significant attenuation of renal oxidative stress and elevation of antioxidant status. VC-IV also significantly decreased serum levels of creatinine and blood urea nitrogen, and improved histopathological lesions. Western blot analysis of the kidneys showed that VC-IV treatment resulted in nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) through modulation of cytosolic Kelch-like ECH-associated protein 1. Thus, VC-IV stimulated Nrf2-mediated activation of antioxidant response element (ARE) pathway and promoted expression of ARE-driven cytoprotective proteins, heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1, and enhanced activity of antioxidant enzymes. Interestingly, VC-IV did not alter the bioavailability and renal accumulation of CDDP in mice. DISCUSSION: In this study, VC-IV exhibited strong nephroprotective efficacy by restoring antioxidant defense mechanisms and hence may serve as a promising chemoprotectant in cancer chemotherapy.
Assuntos
Cisplatino/toxicidade , Nefropatias/tratamento farmacológico , Rim/efeitos dos fármacos , Rim/metabolismo , Vanadatos/uso terapêutico , Compostos de Vanádio/uso terapêutico , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Feminino , Nefropatias/induzido quimicamente , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria Atômica , Vanadatos/administração & dosagem , Compostos de Vanádio/administração & dosagemRESUMO
p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Potássio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Gânglios Espinais/enzimologia , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Neurônios/enzimologia , Fosforilação , Canais de Potássio Ativados por Sódio , Ratos Sprague-DawleyRESUMO
Cisplatin (CDDP) is one of the first-line anticancer drugs; however, the major limitation of CDDP therapy is development of nephrotoxicity (25-35% cases), whose precise mechanism mainly involves oxidative stress, inflammation and cell death. Therefore, in search of a potential chemoprotectant, an organovanadium complex, viz., vanadium(III)-L-cysteine (VC-III) was evaluated against CDDP-induced nephropathy in mice. CDDP was administered intraperitoneally (5 mg/kg b.w.) and VC-III was given by oral gavage (1 mg/kg b.w.) in concomitant and pre-treatment schedule. The results showed that VC-III administration reduced (p < 0.001) serum creatinine and blood urea nitrogen levels, suggesting amelioration of renal dysfunction. VC-III treatment also significantly (p < 0.001) prevented CDDP-induced generation of reactive oxygen species, reactive nitrogen species, and onset of lipid peroxidation in kidney tissues of the experimental mice. In addition, VC-III also substantially (p < 0.001) restored CDDP-induced depleted activities of the renal antioxidant enzymes such as, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, and glutathione (reduced) level. Furthermore, histopathological study also confirmed the renoprotective efficacy of VC-III. Western blotting analysis appended by immunohistochemical data showed that VC-III treatment quite effectively reduced the expression of proinflammatory mediators such as, NFκß, COX-2 and IL-6. VC-III administration also stimulated Nrf2-mediated antioxidant defense system by promotion of downstream antioxidant enzymes, such as HO-1. Moreover, treatment with VC-III significantly (p < 0.001) enhanced CDDP-mediated cytotoxicity in MCF-7 and NCI-H520 human cancer cell lines. Thus, VC-III can serve as a suitable chemoprotectant and increase the therapeutic window of CDDP in cancer patients.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Complexos de Coordenação/uso terapêutico , Cisteína/análogos & derivados , Heme Oxigenase-1/metabolismo , Nefropatias/induzido quimicamente , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Antioxidantes/metabolismo , Nitrogênio da Ureia Sanguínea , Linhagem Celular Tumoral , Complexos de Coordenação/química , Creatinina/sangue , Cisteína/química , Cisteína/uso terapêutico , Feminino , Humanos , Nefropatias/sangue , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Transdução de SinaisRESUMO
Vanadium has been emerged as a promising agent owing to its ability to prevent several types of cancer. This study was aimed to investigate the protective role of an organovanadium complex, viz., oxovanadium(IV)-L-cysteine methyl ester (VC-IV) against cyclophosphamide (CP)-induced hepatotoxicity and genotoxicity in mice. Oral administration of VC-IV quite effectively ameliorated CP-induced histopathological lesions and reduced levels of alanine transaminase, aspartate transaminase and alkaline phosphatase. In addition, VC-IV significantly attenuated CP-induced oxidative stress in the liver as evident from levels of reactive oxygen species, nitric oxide and lipid peroxidation. Restoration of glutathione level and activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase) were also observed upon VC-IV administration. Moreover, VC-IV significantly mitigated CP-induced chromosomal aberrations, micronuclei formation, DNA fragmentation and apoptosis in bone marrow cells and DNA damage in lymphocytes. The present study showed that VC-IV could provide adequate protection against CP-induced hepatotoxicity and genotoxicity in vivo.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Aberrações Cromossômicas/efeitos dos fármacos , Ciclofosfamida/administração & dosagem , Cisteína/química , Compostos Organometálicos/administração & dosagem , Vanadatos/química , Alanina Transaminase/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Aberrações Cromossômicas/induzido quimicamente , Ciclofosfamida/toxicidade , Dano ao DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Compostos Organometálicos/química , Estresse Oxidativo/efeitos dos fármacosRESUMO
Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.
Assuntos
NF-kappa B/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Transcrição Gênica , Animais , Hipóxia Celular/genética , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Humanos , NF-kappa B/genética , Células PC12 , Canais de Potássio/biossíntese , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Sódio/metabolismoRESUMO
Chemotherapy is an integral part of modern day treatment regimen but anticancer drugs fail to demarcate between cancerous and normal cells thereby causing severe form of systemic toxicity. Among which pulmonary toxicity is a dreadful complication developed in cancer patients upon cyclophosphamide (CP) therapy. Oxidative stress, fibrosis, and apoptosis are the major patho-mechanisms involved in CP-induced pulmonary toxicity. In the present study, we have synthesized Nano-Se, nanotechnology-based new form of elemental selenium which has significantly lower toxicity and acceptable bioavailability. In order to meet the need of effective drugs against CP-induced adverse effects, nano selenium (Nano-Se) was tested for its possible protective efficacy on CP-induced pulmonary toxicity and bone marrow toxicity. CP intoxication resulted in structural and functional lung impairment which was revealed by massive histopathological changes. Lung injury was associated with oxidative stress/lipid peroxidation as evident by increased in reactive oxygen species, nitric oxide level, and malondialdehyde (MDA) formation with decreased in level of antioxidants such as reduced glutathione, glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, and catalase. Furthermore, CP at a dose of 25 mg/kg b.w. increased pulmonary DNA damage ('comet tail') and triggered DNA fragmentation and apoptosis in mouse bone marrow cells. On the other hand, Nano-Se at a dose of 2 mg Se/kg b.w., significantly inhibited CP-induced DNA damage in bronchoalveolar lavage cells, and decreased the apoptosis and percentage of DNA fragmentation in bone marrow cells and also antagonized the reduction of the activities of antioxidant enzymes and the increase level of MDA. Thus, our results suggest that Nano-Se in pre- and co-administration may serve as a promising preventive strategy against CP-induced pulmonary toxicity.