RESUMO
BACKGROUND: Several studies had suggested the potential role of calcium signaling in prostate cancer (PCa) prognosis and agressiveness. We aimed to investigate selected proteins contributing to calcium (Ca2+ ) signaling, (Orai, stromal interaction molecule (STIM), and transient receptor potential (TRP) channels) and involved in cancer hallmarks, as independent predictors of systemic recurrence after radical prostatectomy (RP). METHODS: A case-control study including 112 patients with clinically localized PCa treated by RP between 2002 and 2009 and with at least 6-years' follow-up. Patients were divided into two groups according to the absence or presence of systemic recurrence. Expression levels of 10 proteins involved in Ca2+ signaling (TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, STIM1, STIM2, Orai1, Orai2, and Orai3), were assessed by immunohistochemistry using tissue microarrays (TMAs) constructed from paraffin-embedded PCa specimens. The level of expression of the various transcripts in PCa was assessed using quantitative polymerase chain reaction (qPCR) analysis. RNA samples for qPCR were obtained from fresh frozen tissue samples of PCa after laser capture microdissection on RP specimens. Relative gene expression was analyzed using the 2-âµâµCt method. RESULTS: Multivariate analysis showed that increased expression of TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, and Orai2 was significantly associated with a lower risk of systemic recurrence after RP, independently of the prostate-specific antigen (PSA) level, percentage of positive biopsies, and surgical margin (SM) status (P = .007, P = .01, P < .001, P = .0065, P = .007, and P = .01, respectively). For TRPC4, TRPV5, and TRPV6, this association was also independent of Gleason score and pT stage. Moreover, overexpression of TRPV6 and Orai2 was significantly associated with longer time to recurrence after RP (P = .048 and .023, respectively). Overexpression of TRPC4, TRPV5, TRPV6, and Orai2 transcripts was observed in group R- (3.71-, 5.7-, 1.14-, and 2.65-fold increase, respectively). CONCLUSIONS: This is the first study to suggest the independent prognostic value of certain proteins involved in Ca2+ influx in systemic recurrence after RP: overexpression of TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, and Orai2 is associated with a lower risk of systemic recurrence. TRPC4, TRPV5, and TRPV6 appear to be particularly interesting, as they are independent of the five commonly used predictive factors, that is, PSA, percentage of positive biopsies, SM status, Gleason score, and pT stage.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/biossíntese , Sinalização do Cálcio , Recidiva Local de Neoplasia/metabolismo , Neoplasias da Próstata/metabolismo , Canais de Potencial de Receptor Transitório/biossíntese , Idoso , Biomarcadores Tumorais/biossíntese , Estudos de Casos e Controles , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/patologia , Valor Preditivo dos Testes , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , RiscoRESUMO
The remodeling of calcium homeostasis contributes to the cancer hallmarks and the molecular mechanisms involved in calcium channel regulation in tumors remain to be characterized. Here, we report that SigmaR1, a stress-activated chaperone, is required to increase calcium influx by triggering the coupling between SK3, a Ca2+-activated K+ channel (KCNN3) and the voltage-independent calcium channel Orai1. We show that SigmaR1 physically binds SK3 in BC cells. Inhibition of SigmaR1 activity, either by molecular silencing or by the use of sigma ligand (igmesine), decreased SK3 current and Ca2+ entry in breast cancer (BC) and colorectal cancer (CRC) cells. Interestingly, SigmaR1 inhibition diminished SK3 and/or Orai1 levels in lipid nanodomains isolated from BC cells. Analyses of tissue microarray from CRC patients showed higher SigmaR1 expression levels in cancer samples and a correlation with tumor grade. Moreover, the exploration of a cohort of 4937 BC patients indicated that high expression of SigmaR1 and Orai1 channels was significantly correlated to a lower overall survival. As the SK3/Orai1 tandem drives invasive process in CRC and bone metastasis progression in BC, our results may inaugurate innovative therapeutic approaches targeting SigmaR1 to control the remodeling of Ca2+ homeostasis in epithelial cancers.
Assuntos
Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Movimento Celular , Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores sigma/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Cálcio/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Receptores sigma/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Receptor Sigma-1RESUMO
Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca(2+)-activated K(+) channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Glioblastoma/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Sesterterpenos/farmacologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Técnicas de Patch-Clamp , Potássio/metabolismoRESUMO
Many studies have reported changes in potassium channel expression in many cancers and the involvement of these channels in various stages of cancer progression. By contrast, data concerning SKCa channels (small conductance calcium-activated potassium channels) have only recently become available. This review aims i) to present the structure and physiology of SKCa channels, ii) to provide an overview of published data concerning the SKCa proteins produced in tumor cells, and, whenever possible, the biological function assigned to them and iii) to review previous and novel modulators of SKCa channels. SKCa channels are activated by low concentrations of intracellular calcium and consist of homo- or heteromeric assemblies of α-subunits named SK1, SK2 and SK3. SK2-3 channels are expressed in tumors and have been assigned a biological function in cancer cells: the enhancement of cell proliferation and cell migration by hijacking the functions of SK2 and SK3 channels, respectively. Two major classes of SKCa modulators have been described: toxins (apamin) and small synthetic molecules. Most SKCa blockers are pore blockers, but some modify the calcium sensitivity of SKCa channels without interacting with the apamin binding site. In this review, we present edelfosine and ohmline as atypical anticancer agents and novel SK3 inhibitors. Edelfosine and ohmline are synthetic alkyl-lipids with structures different from all previously described SKCa modulators. They should pave the way for the development of a new class of migration-targeted anticancer agents. We believe that such blockers have potential for use in the prevention or treatment of metastasis.
Assuntos
Neoplasias/tratamento farmacológico , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Antineoplásicos , Apamina , Humanos , Terapia de Alvo Molecular , Inibidores de Fosfodiesterase , Éteres Fosfolipídicos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/química , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologiaRESUMO
Edelfosine is an inhibitor of SK3 channel mediated cell migration. However, this compound bears adverse in vivo side effects. Using cell SK3 dependent cell-migration assay, patch-clamp, (125)I-apamin binding, and in vivo experiments we tested the ability of 15 lipid derivatives with chemical structures inspired from edelfosine to inhibit SK3 channels. Using a structure-activity relationship approach we identified an edelfosine analog named Ohmline (1-O-hexadecyl- 2-O-methyl-sn-glycero-3-lactose) with potent inhibitory effects on the SK3 channel. Its potency was greater for SK3 channels than for SK1 channels; it did not affect IKCa channels and only slightly but not significantly affected SK2 channels. This is the first SKCa channel blocker that can be used to discriminate between SK2 and SK1/SK3 channels and represents a useful tool to investigate the functional role of SK3 channels in peripheral tissues (that do not express SK1 channels). This compound, which acts with an IC(50) of 300 nM, did not displace apamin from SKCa channels and had no effect on non-specific edelfosine targets such as protein kinase C (PKC), receptors for platelet activating factor (PAF) and lysophosphatidic acid (LPA), as well as non-cancerous cells. This is promising because the pitfalls associated with the use of edelfosine-like compounds have been that their effective and high concentrations are often cytotoxic due to their detergent-like character causing normal cell lysis. Finally, Ohmline reduced metastasis development in a mice model of tumor indicating that this compound could become a lead compound for the first class of lipid-antimetastatic agent.
Assuntos
Movimento Celular/efeitos dos fármacos , Glicolipídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaios de Migração Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Glicolipídeos/química , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Éteres Fosfolipídicos/química , Éteres Fosfolipídicos/farmacologia , Glicoproteínas da Membrana de Plaquetas/agonistas , Proteína Quinase C/antagonistas & inibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores de Ácidos Lisofosfatídicos/agonistas , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND PURPOSE: The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function. EXPERIMENTAL APPROACH: Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and ¹²5I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca²(+) -activated K(+) channel, SK3/K(Ca)2.3. KEY RESULTS: Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/K(Ca)2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit ¹²5I-Apamin binding to this SK(Ca) channel; rather, it reduced the calcium sensitivity of SK3/K(Ca)2.3 channel and dramatically decreased intracellular Ca²(+) concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SK(Ca) channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/K(Ca)2.3 protein in a SK3/K(Ca)2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine. CONCLUSIONS AND IMPLICATIONS: Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/K(Ca)2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Éteres Fosfolipídicos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Antineoplásicos/metabolismo , Apamina/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Ensaios de Migração Celular , Endopeptidase K/metabolismo , Células Epiteliais , Feminino , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Terapia de Alvo Molecular , Éteres Fosfolipídicos/metabolismoAssuntos
Adenosina/farmacologia , Dióxido de Carbono/metabolismo , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/metabolismo , Animais , Animais Recém-Nascidos , Corpo Carotídeo/efeitos dos fármacos , Corpo Carotídeo/metabolismo , Hipercapnia/metabolismo , Hipóxia/metabolismo , Técnicas In Vitro , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Reflexo/efeitos dos fármacos , Respiração/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
1. Plasma adenosine concentration increases during hypoxia to a level that excites carotid body chemoreceptors by an undetermined mechanism. We have examined this further by determining the electrophysiological responses to exogenous adenosine of sinus nerve chemoafferents in vitro and of whole-cell currents in isolated type I cells. 2. Steady-state, single-fibre chemoafferent discharge was increased approximately 5-fold above basal levels by 100 microM adenosine. This adenosine-stimulated discharge was reversibly and increasingly reduced by methoxyverapamil (D600, 100 microM), by application of nickel chloride (Ni2+, 2 mM) and by removal of extracellular Ca2+. These effects strongly suggest a presynaptic, excitatory action of adenosine on type I cells of the carotid body. 3. Adenosine decreased whole-cell outward currents at membrane potentials above -40 mV in isolated type I cells recorded during superfusion with bicarbonate-buffered saline solution at 34-36 C. This effect was reversible and concentration dependent with a maximal effect at 10 microM. 4. The degree of current inhibition induced by 10 microM adenosine was voltage independent (45.39 +/- 2. 55 % (mean +/- s.e.m.) between -40 and +30 mV) and largely ( approximately 75 %), but not entirely, Ca2+ independent. 4-Aminopyridine (4-AP, 5 mM) decreased the amplitude of the control outward current by 80.60 +/- 3.67 % and abolished the effect of adenosine. 5. Adenosine was without effect upon currents near the resting membrane potential of approximately -55 mV and did not induce depolarization in current-clamp experiments. 6. We conclude that adenosine acts to inhibit a 4-AP-sensitive current in isolated type I cells of the rat carotid body and suggest that this mechanism contributes to the chemoexcitatory effect of adenosine in the whole carotid body.
Assuntos
4-Aminopiridina/farmacologia , Adenosina/farmacologia , Corpo Carotídeo/efeitos dos fármacos , Corpo Carotídeo/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Adenosina/antagonistas & inibidores , Animais , Cálcio/metabolismo , Corpo Carotídeo/citologia , Relação Dose-Resposta a Droga , Condutividade Elétrica , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos WistarRESUMO
Decreases in intracellular pH (pHi) potently dilate coronary resistance arteries but constrict small pulmonary arteries. To define the ionic mechanisms of these responses, this study investigated whether acute decreases in pHi differentially regulate K+ currents in single vascular smooth muscle (VSM) cells isolated from rat coronary and pulmonary resistance arteries. In patch-clamp studies, whole cell K+ currents were elicited by 10-mV depolarizing steps between -60 and 0 mV in VSM cells obtained from 50- to 150-micrometers-OD arterial branches, and pHi was lowered by altering the NH4Cl gradient across the cell membrane. Progressively lowering pHi from calculated values of 7.0 to 6.7 and 6.4 increased the peak amplitude of K+ current in coronary VSM cells by 15 +/- 5 and 23 +/- 3% but reduced K+ current in pulmonary VSM cells by 18 +/- 3 and 21 +/- 3%, respectively. These changes were reversed by returning cells to the control pHi of 7.0 and were eliminated by dialyzing cells with pipette solution containing 50 mmol/l HEPES to buffer NH4Cl-induced changes in pHi. Pharmacological block of ATP-sensitive K+ channels and Ca2+-activated K+ channels by 1 micromol/l glibenclamide and 100 nmol/l iberiotoxin, respectively, did not prevent changes in K+ current levels induced by acidotic pHi. However, block of voltage-gated K+ channels by 3 mmol/l 4-aminopyridine abolished acidosis-induced changes in K+ current amplitudes in both VSM cell types. Interestingly, alpha-dendrotoxin (100 nmol/l), which blocks only select subtypes of voltage-gated K+ channels, abolished the acidosis-induced decrease in K+ current in pulmonary VSM cells but did not affect the acidosis-induced increase in K+ current observed in coronary VSM cells. These findings suggest that opposing, tissue-specific effects of pHi on distinct subtypes of voltage-gated K+ channels in coronary and pulmonary VSM membranes may differentially regulate vascular reactivity in these two circulations under conditions of acidotic stress.