RESUMO
The P2X7 receptor is an exceptional member of the P2X purinergic receptor family, with its activation requiring high concentrations of extracellular adenosine 5'-triphosphate (ATP) that are often associated with tissue damage and inflammation. In the central nervous system (CNS), it is highly expressed in glial cells, particularly in microglia. In this review, we discuss the role and mechanisms of the P2X7 receptor in mediating neuroinflammation and other pathogenic events in a variety of traumatic CNS damage conditions, which lead to loss of neurological and cognitive functions. We raise the perspective on the steady progress in developing CNS-penetrant P2X7 receptor-specific antagonists that leverage the ATP-P2X7 receptor signaling axis as a potential therapeutic strategy to alleviate traumatic CNS damage and related complications.
Assuntos
Sistema Nervoso Central , Receptores Purinérgicos P2X7 , Humanos , Microglia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Trifosfato de AdenosinaRESUMO
Voltage-gated sodium channels (VGSC) are a well-established drug target for anti-epileptic, anti-arrhythmic and pain medications due to their presence and the important roles that they play in excitable cells. Recently, their presence has been recognized in non-excitable cells such as cancer cells and their overexpression has been shown to be associated with metastatic behavior in a variety of human cancers. The neonatal isoform of the VGSC subtype, Nav1.5 (nNav1.5) is overexpressed in the highly aggressive human breast cancer cell line, MDA-MB-231. The activity of nNav1.5 is known to promote the breast cancer cell invasion in vitro and metastasis in vivo, and its expression in primary mammary tumors has been associated with metastasis and patient death. Metastasis development is responsible for the high mortality of breast cancer and currently there is no treatment available to specifically prevent or inhibit breast cancer metastasis. In the present study, a 3D-QSAR model is used to assist the development of low micromolar small molecule VGSC blockers. Using this model, we have designed, synthesized and evaluated five small molecule compounds as blockers of nNav1.5-dependent inward currents in whole-cell patch-clamp experiments in MDA-MB-231 cells. The most active compound identified from these studies blocked sodium currents by 34.9⯱â¯6.6% at 1⯵M. This compound also inhibited the invasion of MDA-MB-231 cells by 30.3⯱â¯4.5% at 1⯵M concentration without affecting the cell viability. The potent small molecule compounds presented here have the potential to be developed as drugs for breast cancer metastasis treatment.
Assuntos
Antineoplásicos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Invasividade Neoplásica/prevenção & controle , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Metástase Neoplásica/prevenção & controle , Relação Quantitativa Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/químicaRESUMO
BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.
RESUMO
Previous studies in native T-type currents have suggested the existence of distinct isoforms with dissimilar pharmacology. Amiloride was the first organic blocker to selectively block the native T-type calcium channel, but the potency and mechanism of block of this drug on the three recombinant T-type calcium channels (Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of Ca(V)3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in Ca(V)3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human Ca(V)3 channels, we found that amiloride blocked the human Ca(V)3 channels in a concentration-response manner; the IC50 for Ca(V)3.2 channels (62 µM) was 13-fold lower than that for Ca(V)3.1 and Ca(V)3.3. Block is voltage-independent (except for Ca(V)3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in Ca(V)3.1 channels. In addition, amiloride block of Ca(V)3.2 channels is mainly due to an extracellular effect, whereas in Ca(V)3.1 and Ca(V)3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human Ca(V)3 channels differentially through a mechanism involving mainly the closed state of the channel and suggest a negative allosteric interaction with at least two putative binding sites with different affinities. The preferential block of Ca(V)3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.
Assuntos
Amilorida/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/fisiologia , Diurético Poupador de Potássio/farmacologia , Células HEK293 , Humanos , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
Colorectal cancer (CRC) is the second leading cause of death worldwide, with 0.9 million deaths per year. The metastatic stage of the disease is identified in about 20% of cases at the first diagnosis and is associated with low patient-survival rates. Voltage-gated sodium channels (NaV) are abnormally overexpressed in several carcinomas including CRC and are strongly associated with the metastatic behavior of cancer cells. Acidification of the extracellular space by Na+/H+ exchangers (NHE) contributes to extracellular matrix degradation and cell invasiveness. In this study, we assessed the expression levels of pore-forming α-subunits of NaV channels and NHE exchangers in tumor and adjacent non-malignant tissues from colorectal cancer patients, CRC cell lines and primary tumor cells. In all cases, SCN5A (gene encoding for NaV1.5) was overexpressed and positively correlated with cancer stage and poor survival prognosis for patients. In addition, we identified an anatomical differential expression of SCN5A and SLC9A1 (gene encoding for NHE-1) being particularly relevant for tumors that originated on the sigmoid colon epithelium. The functional activity of NaV1.5 channels was characterized in CRC cell lines and the primary cells of colon tumors obtained using tumor explant methodologies. Furthermore, we assessed the performance of two new small-molecule NaV1.5 inhibitors on the reduction of sodium currents, as well as showed that silencing SCN5A and SLC9A1 substantially reduced the 2D invasive capabilities of cancer cells. Thus, our findings show that both NaV1.5 and NHE-1 represent two promising targetable membrane proteins against the metastatic progression of CRC.
RESUMO
Metastatic progression is a major burden for breast cancer patients and is associated with the ability of cancer cells to overcome stressful conditions, such as nutrients deprivation and hypoxia, and to gain invasive properties. Autophagy and epithelial-to-mesenchymal transition are critical contributors to these processes. Here, we show that the P2X4 purinergic receptor is upregulated in breast cancer biopsies from patients and it is primarily localised in endolysosomes. We demonstrate that P2X4 enhanced invasion in vitro, as well as mammary tumour growth and metastasis in vivo. The pro-malignant role of P2X4 was mediated by the regulation of lysosome acidity, the promotion of autophagy and cell survival. Furthermore, the autophagic activity was associated with epithelial-to-mesenchymal transition (EMT), and this role of P2X4 was even more pronounced under metabolic challenges. Pharmacological and gene silencing of P2X4 inhibited both autophagy and EMT, whereas its rescue in knocked-down cells led to the restoration of the aggressive phenotype. Together, our results demonstrate a previously unappreciated role for P2X4 in regulating lysosomal functions and fate, promoting breast cancer progression and aggressiveness.
Assuntos
Neoplasias da Mama , Receptores Purinérgicos P2X4 , Autofagia/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismoRESUMO
Voltage-gated sodium (NaV) channels, initially characterized in excitable cells, have been shown to be aberrantly expressed in non-excitable cancer tissues and cells from epithelial origins such as in breast, lung, prostate, colon, and cervix, whereas they are not expressed in cognate non-cancer tissues. Their activity was demonstrated to promote aggressive and invasive potencies of cancer cells, both in vitro and in vivo, whereas their deregulated expression in cancer tissues has been associated with metastatic progression and cancer-related death. This review proposes NaV channels as pharmacological targets for anticancer treatments providing opportunities for repurposing existing NaV-inhibitors or developing new pharmacological and nutritional interventions.
RESUMO
The SCN4B gene, coding for the NaVß4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVß4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVß4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of ß-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navß4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.
Assuntos
Células Epiteliais/metabolismo , Glândulas Mamárias Animais/citologia , Subunidades Proteicas/metabolismo , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Regulação para Baixo , Células Epiteliais/citologia , Feminino , Humanos , Mesoderma/metabolismo , Fenótipo , Proteólise , beta Catenina/metabolismoRESUMO
The P2X7 receptor is an ATP-gated cation channel with a still ambiguous role in cancer progression, proposed to be either pro- or anti-cancerous, depending on the cancer or cell type in the tumour. Its role in mammary cancer progression is not yet defined. Here, we show that P2X7 receptor is functional in highly aggressive mammary cancer cells, and induces a change in cell morphology with fast F-actin reorganization and formation of filopodia, and promotes cancer cell invasiveness through both 2- and 3-dimensional extracellular matrices in vitro. Furthermore, P2X7 receptor sustains Cdc42 activity and the acquisition of a mesenchymal phenotype. In an immunocompetent mouse mammary cancer model, we reveal that the expression of P2X7 receptor in cancer cells, but not in the host mice, promotes tumour growth and metastasis development, which were reduced by treatment with specific P2X7 antagonists. Our results demonstrate that P2X7 receptor drives mammary tumour progression and represents a pertinent target for mammary cancer treatment.
RESUMO
The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3- and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of NaV1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive NaV1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.
Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Invasividade Neoplásica/patologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Loss of epithelial polarity and gain in invasiveness by carcinoma cells are critical events in the aggressive progression of cancers and depend on phenotypic transition programs such as the epithelial-to-mesenchymal transition (EMT). Many studies have reported the aberrant expression of voltage-gated sodium channels (NaV) in carcinomas and specifically the NaV1.5 isoform, encoded by the SCN5A gene, in breast cancer. NaV1.5 activity, through an entry of sodium ions, in breast cancer cells is associated with increased invasiveness, but its participation to the EMT has to be clarified. In this study, we show that reducing the expression of NaV1.5 in highly aggressive human MDA-MB-231 breast cancer cells reverted the mesenchymal phenotype, reduced cancer cell invasiveness and the expression of the EMT-promoting transcription factor SNAI1. The heterologous expression of NaV1.5 in weakly invasive MCF-7 breast cancer cells induced their expression of both SNAI1 and ZEB1 and increased their invasive capacities. In MCF-7 cells the stimulation with the EMT-activator signal TGF-ß1 increased the expression of SCN5A. Moreover, the reduction of the salt-inducible kinase 1 (SIK1) expression promoted NaV1.5-dependent invasiveness and expression of EMT-associated transcription factor SNAI1. Altogether, these results indicated a prominent role of SIK1 in regulating NaV1.5-dependent EMT and invasiveness.
Assuntos
Neoplasias da Mama/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Proteínas Serina-Treonina Quinases/genética , Fator de Crescimento Transformador beta1/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fatores de Transcrição da Família Snail/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Voltage-gated sodium (NaV) channels have been related with cell migration and invasiveness in human cancers. We previously reported the contribution of NaV1.6 channels activity with the invasion capacity of cervical cancer (CeCa) positive to Human Papilloma Virus type 16 (HPV16), which accounts for 50% of all CeCa cases. Here, we show that NaV1.6 gene (SCN8A) overexpression is a general characteristic of CeCa, regardless of the HPV type. In contrast, no differences were observed in NaV1.6 channel expression between samples of non-cancerous and cervical intraepithelial neoplasia. Additionally, we found that CeCa cell lines, C33A, SiHa, CaSki and HeLa, express mainly the splice variant of SCN8A that lacks exon 18, shown to encode for an intracellularly localized NaV1.6 channel, whereas the full-length adult form was present in CeCa biopsies. Correlatively, patch-clamp experiments showed no evidence of whole-cell sodium currents (INa) in CeCa cell lines. Heterologous expression of full-length NaV1.6 isoform in C33A cells produced INa, which were sufficient to significantly increase invasion capacity and matrix metalloproteinase type 2 (MMP-2) activity. These data suggest that upregulation of NaV1.6 channel expression occurs when cervical epithelium have been transformed into cancer cells, and that NaV1.6-mediated invasiveness of CeCa cells involves MMP-2 activity. Thus, our findings support the notion about using NaV channels as therapeutic targets against cancer metastasis.