RESUMO
Glioblastoma (GBM) is a highly malignant brain tumour characterised by limited treatment options and poor prognosis. The tumour microenvironment, particularly the central hypoxic region of the tumour, is known to play a pivotal role in GBM progression. Cells within this region adapt to hypoxia by stabilising transcription factor HIF1-α, which promotes cell proliferation, dedifferentiation and chemoresistance. In this study we sought to examine the effects of NNC-55-0396, a tetralol compound which overactivates the unfolded protein response inducing apoptosis, using the organ-on-chip technology. We identified an increased sensitivity of the hypoxic core of the chip to NNC, which correlates with decreasing levels of HIF1-α in vitro. Moreover, NNC blocks the macroautophagic process that is unleashed by hypoxia as revealed by increased levels of autophagosomal constituent LC3-II and autophagy chaperone p62/SQSTM1. The specific effects of NNC in the hypoxic microenvironment unveil additional anti-cancer abilities of this compound and further support investigations on its use in combined therapies against GBM.
Assuntos
Benzimidazóis , Neoplasias Encefálicas , Ciclopropanos , Glioblastoma , Naftalenos , Tetralonas , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Hipóxia/metabolismo , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Microambiente TumoralRESUMO
Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.
Assuntos
Glioblastoma , Animais , Proliferação de Células , Regulação para Baixo , GTP Fosfo-Hidrolases , Glioblastoma/genética , Humanos , CamundongosRESUMO
In the strongly polarized membranes of excitable cells, activation of T-type Ca2+ channels (TTCCs) by weak depolarizing stimuli allows the influx of Ca2+ which further amplifies membrane depolarization, thus "recruiting" higher threshold voltage-gated channels to promote action potential firing. Nonetheless, TTCCs perform other functions in the plasma membrane of both excitable and non-excitable cells, in which they regulate a number of biochemical pathways relevant for cell cycle and cell fate. Furthermore, data obtained in the last 20 years have shown the involvement of TTCCs in tumor biology, designating them as promising chemotherapeutic targets. However, their activity in the steadily-depolarized membranes of cancer cells, in which most voltage-gated channels are in the inactivated (nonconducting) state, is counter-intuitive. Here we discuss that in cancer cells weak hyperpolarizing stimuli increase the fraction of open TTCCs which, in association with Ca2+-dependent K+ channels, may critically boost membrane hyperpolarization and driving force for Ca2+ entry through different voltage-independent Ca2+ channels. Available evidence also shows that TTCCs participate in positive feedback circuits with signaling effectors, which may warrant a switch-like activation of pro-proliferative and pro-survival pathways in spite of their low availability. Unravelling TTCC modus operandi in the context of non-excitable membranes may facilitate the development of novel anticancer approaches.
Assuntos
Cálcio , Neoplasias , Potenciais de Ação/fisiologia , Cálcio/metabolismoRESUMO
Mibefradil and NNC-55-0396, tetralol derivatives with a proven -ability to block T-type calcium channels in excitable cells, reduce cancer cell viability in vitro, causing cell death. Furthermore, they reduce tumor growth in preclinical models of Glioblastoma multiforme (GBM), a brain tumor of poor prognosis. Here we found that GBM cells treated with cytotoxic concentrations of NNC-55-0396 paradoxically increased cytosolic calcium levels through the activation of inositol triphosphate receptors (IP3R) and ER stress. We used pharmacological inhibitors and gene silencing to dissect the cell death pathway stimulated by NNC-55-0396 in GBM cell lines and biopsy-derived cultures. Calcium chelation or IP3R inhibition prevented NNC-55-0396-mediated cytotoxicity, indicating that ER calcium efflux is the cause of cell death. Upstream of calcium mobilization, NNC-55-0396 activated the IRE1α arm of the Unfolded Protein Response (UPR) resulting in the nuclear translocation of pro-apoptotic CHOP. Consistent with these findings, silencing IRE1α or JNK1 rescued the cell death elicited by NNC-55-0396. Therefore, we demonstrate that activation of IRE1α and calcium signaling accounts for the cytotoxicity of NNC-55-0396 in GBM cells. The delineation of the signaling pathway that mediates the abrupt cell death triggered by this compound can help the development of new therapies for GBM.
Assuntos
Glioblastoma , Apoptose , Benzimidazóis , Cálcio/metabolismo , Sinalização do Cálcio , Morte Celular , Ciclopropanos , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Naftalenos , Proteínas Serina-Treonina Quinases/genética , Tetralonas , Resposta a Proteínas não DobradasRESUMO
Over the past 20 years, various studies have demonstrated a pivotal role of T-type calcium channels (TTCCs) in tumor progression. Cytotoxic effects of TTCC pharmacological blockers have been reported in vitro and in preclinical models. However, their roles in cancer physiology are only beginning to be understood. In this review, we discuss evidence for the signaling pathways and cellular processes stemming from TTCC activity, mainly inferred by inverse reasoning from pharmacological blocks and, only in a few studies, by gene silencing or channel activation. A thorough analysis indicates that drug-induced cytotoxicity is partially an off-target effect. Dissection of on/off-target activity is paramount to elucidate the physiological roles of TTCCs, and to deliver efficacious therapies suited to different cancer types and stages.
Assuntos
Antineoplásicos , Canais de Cálcio Tipo T , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de SinaisRESUMO
Focal adhesion kinase (FAK) is a central component of focal adhesions that regulate cancer cell proliferation and migration. Here, we studied the effects of FAK inhibition in glioblastoma (GBM), a fast growing brain tumor that has a poor prognosis. Treating GBM cells with the FAK inhibitor PF-573228 induced a proliferative arrest and increased cell size. PF-573228 also reduced the growth of GBM neurospheres. These effects were associated with increased p27/CDKN1B levels and ß-galactosidase activity, compatible with acquisition of senescence. Interestingly, FAK inhibition repressed the expression of the autophagy cargo receptor p62/SQSTM-1. Moreover, depleting p62 in GBM cells also induced a senescent-like phenotype through transcriptional upregulation of p27. Our results indicate that FAK inhibition arrests GBM cell proliferation, resulting in cell senescence, and pinpoint p62 as being key to this process. These findings highlight the possible therapeutic value of targeting FAK in GBM.
RESUMO
Hyperactivation of the Mitogen Activated Protein Kinase (MAPK) pathway is prevalent in melanoma, principally due to mutations in the BRAF and NRAS genes. MAPK inhibitors are effective only short-term, and recurrence occurs due to functional redundancies or intertwined pathways. The remodeling of Ca2+ signaling is also common in melanoma cells, partly through the increased expression of T-type channels (TTCCs). Here we summarize current knowledge about the prognostic value and molecular targeting of TTCCs. Furthermore, we discuss recent evidence pointing to TTCCs as molecular switches for melanoma chemoresistance, which set the grounds for novel combined therapies against the advanced disease.
Assuntos
Antineoplásicos/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Melanoma/mortalidade , Melanoma/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Mutação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
T-Type calcium channels (TTCCs) are key regulators of membrane excitability, which is the reason why TTCC pharmacology is subject to intensive research in the neurological and cardiovascular fields. TTCCs also play a role in cancer physiology, and pharmacological blockers such as tetralols and dihydroquinazolines (DHQs) reduce the viability of cancer cells in vitro and slow tumor growth in murine xenografts. However, the available compounds are better suited to blocking TTCCs in excitable membranes rather than TTCCs contributing window currents at steady potentials. Consistently, tetralols and dihydroquinazolines exhibit cytostatic/cytotoxic activities at higher concentrations than those required for TTCC blockade, which may involve off-target effects. Gene silencing experiments highlight the targetability of TTCCs, but further pharmacological research is required for TTCC blockade to become a chemotherapeutic option.
Assuntos
Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Ativação do Canal Iônico , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/química , Sobrevivência Celular/efeitos dos fármacos , Suscetibilidade a Doenças , Técnicas de Silenciamento de Genes , Humanos , Transdução de SinaisRESUMO
Glioblastoma (GBM) is a highly invasive brain neoplasia with an elevated recurrence rate after surgical resection. The cyclin D1 (Ccnd1)/Cdk4-retinoblastoma 1 (RB1) axis is frequently altered in GBM, leading to overproliferation by RB1 deletion or by Ccnd1-Cdk4 overactivation. High levels of Ccnd1-Cdk4 also promote GBM cell invasion by mechanisms that are not so well understood. The purpose of this work is to elucidate the in vivo role of cytoplasmic Ccnd1-Cdk4 activity in the dissemination of GBM. We show that Ccnd1 activates the invasion of primary human GBM cells through cytoplasmic RB1-independent mechanisms. By using GBM mouse models, we observed that evaded GBM cells showed cytoplasmic Ccnd1 colocalizing with regulators of cell invasion such as RalA and paxillin. Our genetic data strongly suggest that, in GBM cells, the Ccnd1-Cdk4 complex is acting upstream of those regulators. Accordingly, expression of Ccnd1 induces focal adhesion kinase, RalA and Rac1 activities. Finally, in vivo experiments demonstrated increased GBM dissemination after expression of membrane-targeted Ccnd1. We conclude that Ccnd1-Cdk4 activity promotes GBM dissemination through cytoplasmic and RB1-independent mechanisms. Therefore, inhibition of Ccnd1-Cdk4 activity may be useful to hinder the dissemination of recurrent GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Ciclina D1/metabolismo , Citoplasma/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos SCID , Invasividade NeoplásicaRESUMO
T-type Ca2+ channels (TTCC) have been identified as key regulators of cancer cell cycle and survival. In vivo studies in glioblastoma (GBM) murine xenografts have shown that drugs able to block TTCC in vitro (such as tetralol derivatives mibefradil/NNC-55-096, or different 3,4-dihydroquinazolines) slow tumor progression. However, currently available TTCC pharmacologic blockers have limited selectivity for TTCC and are unable to distinguish between TTCC isoforms. Here we analyzed the expression of TTCC transcripts in human GBM cells and show a prevalence of Cacna1g/Cav3.1 mRNAs. Infection of GBM cells with lentiviral particles carrying short hairpin RNA against Cav3.1 resulted in GBM cell death by apoptosis. We generated a murine GBM xenograft via subcutaneous injection of U87-MG GBM cells and found that tumor size was reduced when Cav3.1 expression was silenced. Furthermore, we developed an in vitro model of temozolomide-resistant GBM that showed increased expression of Cav3.1 accompanied by the activation of macroautophagy. We confirmed a positive correlation between Cav3.1 and autophagic markers in both GBM cultures and biopsies. Of note, Cav3.1 knockdown resulted in transcriptional downregulation of p62/SQSTM1 and deficient autophagy. Together, these data identify Cav3.1 channels as potential targets for slowing GBM progression and recurrence based on their role in regulating autophagy. SIGNIFICANCE: These findings identify Cav3.1 calcium channels as a molecular target to regulate autophagy and prevent progression and chemotherapeutic resistance in glioblastoma.
Assuntos
Neoplasias Encefálicas/patologia , Canais de Cálcio Tipo T/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Temozolomida/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Canais de Cálcio Tipo T/genética , Proliferação de Células , Progressão da Doença , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).
Assuntos
Canais de Cálcio Tipo T/metabolismo , Movimento Celular , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Substituição de Aminoácidos , Canais de Cálcio Tipo T/genética , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas Associadas aos Microtúbulos/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Transcrição da Família Snail/genéticaRESUMO
In the past decade, T-type Ca2+ channels (TTCC) have been unveiled as key regulators of cancer cell biology and thus have been proposed as chemotherapeutic targets. Indeed, in vitro and in vivo studies indicate that TTCC pharmacologic blockers have a negative impact on the viability of cancer cells and reduce tumor size, respectively. Consequently mibefradil, a TTCC blocker approved in 1997 as an antihypertensive agent but withdrawn in 1998 because of drug-drug interactions, was granted 10 years later the orphan drug status by the FDA to investigate its efficacy against brain, ovary, and pancreatic cancer. However, the existence of different channel isoforms with distinct physiologic roles, together with the lack of selective pharmacologic agents, has hindered a conclusive chemotherapeutic evaluation. Here, we review the available evidence on TTCC expression, value as prognostic markers, and effectiveness of their pharmacologic blockade on cancer cells in vitro and in preclinical models. We additionally summarize the status of clinical trials using mibefradil against glioblastoma multiforme. Finally, we discuss the future perspectives and the importance of further development of multidisciplinary research efforts on the consideration of TTCCs as biomarkers or targetable molecules in cancer. Cancer Res; 78(3); 603-9. ©2018 AACR.
Assuntos
Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/química , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Humanos , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidence indicates that macroautophagy/autophagy regulates WNT signaling. Here we investigated the impact of inhibiting WNT signaling on autophagy in glioblastoma (GBM), a devastating brain tumor. Inhibiting TCF, or silencing TCF4 or CTNNB1/ß-catenin upregulated SQSTM1/p62 in GBM at transcriptional and protein levels and, in turn, autophagy. DKK1/Dickkopf1, a canonical WNT receptor antagonist, also induced autophagic flux. Importantly, TCF inhibition regulated autophagy through MTOR inhibition and dephosphorylation, and nuclear translocation of TFEB, a master regulator of lysosomal biogenesis and autophagy. TCF inhibition or silencing additionally affected GBM cell proliferation and migration. Autophagy induction followed by its blockade can promote cancer cell death. In agreement with this notion, halting both TCF-CTNNB1 and autophagy pathways decreased cell viability and induced apoptosis of GBM cells through a SQSTM1-dependent mechanism involving CASP8 (caspase 8). In vivo experiments further underline the therapeutic potential of such dual targeting in GBM.
Assuntos
Autofagia/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteína Sequestossoma-1/metabolismo , beta Catenina/metabolismo , Autofagia/fisiologia , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição 4/genética , beta Catenina/efeitos dos fármacosRESUMO
ß-catenin is a central component of adherent junctions and a key effector of canonical Wnt signaling, in which dephosphorylated Ser/Thr ß-catenin regulates gene transcription. ß-catenin phosphorylation at Tyr142 (PTyr142 ß-catenin), which is induced by receptor and Src family Tyr kinases, represents a previously described ß-catenin switch from adhesive to migratory roles. In addition to classical ß-catenin roles, phosphorylated Ser/Thr ß-catenin and total ß-catenin were involved in centrosomal functions, including mitotic spindle formation and centrosome separation. Here we find that PTyr142 ß-catenin is present in centrosomes in non-transformed and glioblastoma cells and that, in contrast to the Ser/Thr phosphorylated ß-catenin, PTyr142 ß-catenin centrosomal levels drop in mitosis. Furthermore, we show that the inhibitor of Spleen Tyrosine Kinase (Syk) piceatannol decreases centrosomal PTyr142 ß-catenin levels, indicating that Syk regulates centrosome PTyr142 ß-catenin. Our findings suggest that PTyr142 ß-catenin and Syk may regulate centrosomal cohesion. This study highlights the contribution of different phosphorylated ß-catenin forms to the cell and centrosome cycles.
Assuntos
Centrossomo/metabolismo , Quinase Syk/metabolismo , beta Catenina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Imunoprecipitação , Camundongos , Fosforilação , Tubulina (Proteína)/metabolismoRESUMO
Glioblastoma multiforme (GBM) is a fast growing brain tumor characterized by extensive infiltration into the surrounding tissue and one of the most aggressive cancers. GBM is the most common glioma (originating from glial-derived cells) that either evolves from a low grade astrocytoma or appears de novo. Wnt/ß-catenin and Hepatocyte Growth Factor (HGF)/c-Met signaling are hyperactive in human gliomas, where they regulate cell proliferation, migration and stem cell behavior. We previously demonstrated that ß-catenin is phosphorylated at Y142 by recombinant c-Met kinase and downstream of HGF signaling in neurons. Here we studied phosphoY142 (PY142) ß-catenin and dephospho S/T ß-catenin (a classical Wnt transducer) in glioma biopsies, GBM cell lines and biopsy-derived glioma cell cultures. We found that PY142 ß-catenin mainly localizes in the nucleus and signals through transcriptional activation in GBM cells. Tissue microarray analysis confirmed strong nuclear PY142 ß-catenin immunostaining in astrocytoma and GBM biopsies. By contrast, active ß-catenin showed nuclear localization only in GBM samples. Western blot analysis of tumor biopsies further indicated that PY142 and active ß-catenin accumulate independently, correlating with the expression of Snail/Slug (an epithelial-mesenchymal transition marker) and Cyclin-D1 (a regulator of cell cycle progression), respectively, in high grade astrocytomas and GBMs. Moreover, GBM cells stimulated with HGF showed increasing levels of PY142 ß-catenin and Snail/Slug. Importantly, the expression of mutant Y142F ß-catenin decreased cell detachment and invasion induced by HGF in GBM cell lines and biopsy-derived cell cultures. Our results identify PY142 ß-catenin as a nuclear ß-catenin signaling form that downregulates adhesion and promotes GBM cell invasion.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , beta Catenina/genética , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/ultraestrutura , Proliferação de Células , Transição Epitelial-Mesenquimal , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Voltage-gated calcium channel blockers are widely used for the management of cardiovascular diseases, however little is known about their effects on cardiac cells in vitro. We challenged neonatal ventricular cardiomyocytes (CMs) with therapeutic L-type and T-type Ca(2+) channel blockers (nifedipine and mibefradil, respectively), and measured their effects on cell stress and survival, using fluorescent microscopy, Q-PCR and Western blot. Both nifedipine and mibefradil induced a low-level and partially transient up-regulation of three key mediators of the Unfolded Protein Response (UPR), indicative of endoplasmic (ER) reticulum stress. Furthermore, nifedipine triggered the activation of macroautophagy, as evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), decreased levels of polyubiquitin-binding protein p62/SQSTM1 and ubiquitinated protein aggregates, that was followed by cell death. In contrast, mibefradil inhibited CMs constitutive macroautophagy and did not promote cell death. The siRNA-mediated gene silencing approach confirmed the pharmacological findings for T-type channels. We conclude that L-type and T-type Ca(2+) channel blockers induce ER stress, which is divergently transduced into macroautophagy induction and inhibition, respectively, with relevance for cell viability. Our work identifies VGCCs as novel regulators of autophagy in the heart muscle and provides new insights into the effects of VGCC blockers on CMs homeostasis, that may underlie both noxious and cardioprotective effects.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Autofagia/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Macrolídeos/farmacologia , Mibefradil/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Nifedipino/farmacologia , Ratos , Proteína Sequestossoma-1 , Transdução de Sinais , Tapsigargina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
We have recently reported that human melanoma cells express a variety of voltage-gated calcium (Ca(2+) ) channel types, including low-voltage-activated T-type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T-type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G1 -S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in-depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca(2+) homeostasis, autophagy, and cell death were mimicked by T-type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/metabolismo , Melanoma/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/enzimologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Neoplasias Cutâneas , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Melanoma Maligno CutâneoRESUMO
Axon morphogenesis is a complex process regulated by a variety of secreted molecules, including morphogens and growth factors, resulting in the establishment of the neuronal circuitry. Our previous work demonstrated that growth factors [Neurotrophins (NT) and Hepatocyte Growth Factor (HGF)] signal through ß-catenin during axon morphogenesis. HGF signaling promotes axon outgrowth and branching by inducing ß-catenin phosphorylation at Y142 and transcriptional regulation of T-Cell Factor (TCF) target genes. Here, we asked which genes are regulated by HGF signaling during axon morphogenesis. An array screening indicated that HGF signaling elevates the expression of chemokines of the CC and CXC families. In line with this, CCL7, CCL20, and CXCL2 significantly increase axon outgrowth in hippocampal neurons. Experiments using blocking antibodies and chemokine receptor antagonists demonstrate that chemokines act downstream of HGF signaling during axon morphogenesis. In addition, qPCR data demonstrates that CXCL2 and CCL5 expression is stimulated by HGF through Met/b-catenin/TCF pathway. These results identify CC family members and CXCL2 chemokines as novel regulators of axon morphogenesis downstream of HGF signaling.
RESUMO
Glioblastoma multiforme (GBM) is a commonly occurring brain tumor with a poor prognosis. GBM can develop both "de novo" or evolve from a previous astrocytoma and is characterized by high proliferation and infiltration into the surrounding tissue. Following treatment (surgery, radiotherapy, and chemotherapy), tumors often reappear. Glioma-initiating cells (GICs) have been identified in GBM and are thought to be responsible for tumors initiation, their continued growth, and recurrence. ß-catenin, a component of the cell-cell adhesion complex and of the canonical Wnt pathway, regulates proliferation, adhesion, and migration in different cell types. ß-catenin and components of the Wnt canonical pathway are commonly overexpressed in GBM. Here, we review previous work on the role of Wnt/ß-catenin signalling in glioma initiation, proliferation, and invasion. Understanding the molecular mechanisms regulating GIC biology and glioma progression may help in identifying novel therapeutic targets for GBM treatment.
RESUMO
Wnt factors regulate neural stem cell development and neuronal connectivity. Here we investigated whether Wnt-3a and Wnt-3, expressed in the developing spinal cord, regulate proliferation and the neuronal differentiation of spinal cord neural precursors (SCNP). Wnt-3a promoted a sustained increase of SCNP proliferation and decreased the expression of cyclin-dependent kinase inhibitors. In contrast, Wnt-3 transiently enhanced SCNP proliferation and increased neurogenesis through ß-catenin signaling. Furthermore, both Wnt-3a and Wnt-3 stimulated neurite outgrowth in SCNP-derived neurons through ß-catenin- and TCF4-dependent transcription. Glycogen synthase kinase-3ß inhibitors mimicked Wnt signaling and promoted neurite outgrowth in established cultures. We conclude that Wnt-3a and Wnt-3 factors signal through the canonical Wnt/ß-catenin pathway to regulate different aspects of SCNP development. These findings may be of therapeutic interest for the treatment of neurodegenerative diseases and nerve injury.