Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m806891200
ISSN1083-351X
AutoresLudovic Gillet, Sébastien Roger, Pierre Besson, Fabien Lecaille, J. GORÉ, Philippe Bougnoux, Gilles Lalmanach, Jean‐Yves Le Guennec,
Tópico(s)Trace Elements in Health
ResumoVoltage-gated sodium channels (NaV) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of NaV1.5 and associated auxiliary β-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel®-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role (∼65%) in cancer invasiveness. Matrigel® invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on NaV function. Inhibiting NaV has no consequence for cathepsins at the transcription, translation, and secretion levels. However, NaV activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Nav enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases. Voltage-gated sodium channels (NaV) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of NaV1.5 and associated auxiliary β-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel®-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role (∼65%) in cancer invasiveness. Matrigel® invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on NaV function. Inhibiting NaV has no consequence for cathepsins at the transcription, translation, and secretion levels. However, NaV activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Nav enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases. Breast cancer is the most common female cancer and the primary cause of death in women by cancer worldwide (1Parkin D.M. Bray F. Ferlay J. Pisani P. CA-Cancer J. Clin. 2005; 55: 74-108Crossref PubMed Scopus (17337) Google Scholar). Deaths occur primarily after the development of metastases. The invasive potential of malignant cells is mainly linked to their capacity to degrade basement membranes and extracellular matrices by various proteases. Studies have mostly focused on metalloproteases, including matrix metalloproteinases and the closely related ADAMs (a disintegrin and metalloproteinase) and ADAMTs (a disintegrin and metalloproteinase with thrombospondin motifs) (2Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (973) Google Scholar), that are key factors in growth, invasion, and angiogenesis, and to a lesser extent on aspartyl and serine proteases. Pharmaceutical inhibitors of matrix metalloproteinases have been developed, but the results from clinical trials with these drugs have so far been disappointing (3Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5134) Google Scholar, 4Turk B. Nat. Rev. Drug Discov. 2006; 5: 785-799Crossref PubMed Scopus (1046) Google Scholar). On the other hand, cysteine cathepsins (Cat) 4The abbreviations used are: Cat, cathepsin(s); INa, sodium current; Nav, voltage-gated sodium channel(s); TTX, tetrodotoxin; AMC, 7-amino-4-methyl coumarin; CA-074, l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-l-isoleucyl-l-proline; E-64, l-3-carboxy-trans-2,3-epoxy-propionyl-leucylamide-(4-guanido)-butane; Z, benzyloxycarbonyl; DHPE, N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; BCECF, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; DMEM, Dulbecco's modified Eagle's medium; PSS, physiological saline solution; siRNA, small interfering RNA; siCTL, control siRNA. related to papain (family C1, clan CA), which have been confined for decades to housekeeping tasks including intracellular degradation of endocytosed proteins (5Turk V. Turk B. Turk D. EMBO J. 2001; 20: 4629-4633Crossref PubMed Scopus (637) Google Scholar), are now known to fulfill more specific functions in numerous biological processes (such as antigen presentation, enzyme or hormone maturation, bone resorption, etc.) and pathologies (6Chapman H.A. Riese R.J. Shi G.P. Annu. Rev. Physiol. 1997; 59: 63-88Crossref PubMed Scopus (690) Google Scholar, 7Lecaille F. Kaleta J. Bromme D. Chem. Rev. 2002; 102: 4459-4488Crossref PubMed Scopus (448) Google Scholar). Recently, they have emerged as important protagonists in cancer and have been reported to be involved in apoptosis, angiogenesis, proliferation, migration, and invasion (8Joyce J.A. Hanahan D. Cell Cycle. 2004; 3: 1516-1619Crossref PubMed Scopus (137) Google Scholar, 9Mohamed M.M. Sloane B.F. Nat. Rev. Cancer. 2006; 6: 764-775Crossref PubMed Scopus (1012) Google Scholar, 10Gocheva V. Joyce J.A. Cell Cycle. 2007; 6: 60-64Crossref PubMed Scopus (344) Google Scholar). Although Cat are predominantly expressed in acidic endosomal/lysosomal compartments, they are also found to be extracellularly active at physiological pH, as membrane-bound and soluble forms (11Punturieri A. Filippov S. Allen E. Caras I. Murray R. Reddy V. Weiss S.J. J. Exp. Med. 2000; 192: 789-799Crossref PubMed Scopus (190) Google Scholar, 12Roshy S. Sloane B.F. Moin K. Cancer Metastasis Rev. 2003; 22: 271-286Crossref PubMed Scopus (204) Google Scholar, 13Jane D.T. Morvay L. Dasilva L. Cavallo-Medved D. Sloane B.F. Dufresne M.J. Biol. Chem. 2006; 387: 223-234Crossref PubMed Scopus (34) Google Scholar, 14Brix K. Dunkhorst A. Mayer K. Jordans S. Biochimie (Paris). 2008; 90: 194-207Crossref PubMed Scopus (305) Google Scholar). Nevertheless knowledge still remains limited concerning transport pathways and regulation of extracellular Cat in cancer cells. The involvement of ion channels in carcinogenesis and tumor progression begins to be unraveled even though their roles are not totally understood. Presently, there is growing evidence emphasizing the abnormal expression and function of voltage-gated sodium channels (NaV) in cancer cells and their involvement in invasiveness (15Roger S. Potier M. Vandier C. Besson P. Le Guennec J.Y. Curr. Pharm. Des. 2006; 12: 3681-3695Crossref PubMed Scopus (87) Google Scholar). Indeed, NaV, which are up-regulated in human breast cancer tissues and associated with breast cancer progression, are functionally expressed in the highly metastatic MDA-MB-231 breast cancer cells in which they potentiate invasion (16Fraser S.P. Diss J.K. Chioni A.M. Mycielska M.E. Pan H. Yamaci R.F. Pani F. Siwy Z. Krasowska M. Grzywna Z. Brackenbury W.J. Theodorou D. Koyuturk M. Kaya H. Battaloglu E. De Bella M.T. Slade M.J. Tolhurst R. Palmieri C. Jiang J. Latchman D.S. Coombes R.C. Djamgoz M.B. Clin. Cancer Res. 2005; 11: 5381-5389Crossref PubMed Scopus (358) Google Scholar, 17Roger S. Besson P. Le Guennec J.Y. Biochim. Biophys. Acta. 2003; 1616: 107-111Crossref PubMed Scopus (128) Google Scholar). This makes MDA-MB-231 cells a good model for studying the specific molecular mechanisms linking the activity of NaV to invasive properties of cancer cells. In this context, the aim of the present study was to identify, among proteolytic enzymes involved in cancer cell invasion, those that are regulated by NaV activity. We demonstrated that Cat are the predominant proteases involved in MDA-MB-231 invasiveness and that the activity of NaV favors the pericellular activity of CatB and CatS, through the acidification of the perimembrane pH. We also performed experiments in non-small cell lung cancer cells (H460 cell line), which also express functional NaV involved in cellular invasiveness (18Roger S. Rollin J. Barascu A. Besson P. Raynal P.I. Iochmann S. Lei M. Bougnoux P. Gruel Y. Le Guennec J.Y. Int. J. Biochem. Cell Biol. 2007; 39: 774-786Crossref PubMed Scopus (137) Google Scholar). Similar results were obtained, highlighting the fact that Cat are partly regulated by NaV activity. This work presents a new general mechanism for highly metastatic cancer cell invasiveness. Inhibitors, Substrates, and Chemicals-Tetrodotoxin (TTX) was purchased from Latoxan (France) and was prepared in pH-buffered physiological saline solution at pH 7.4. Fluorescent dyes for measuring internal and perimembrane pH (BCECF-AM and DHPE, respectively) were purchased from Invitrogen (France). Protease inhibitors and substrates were purchased from Calbiochem (VWR International). The cell-impermeant Cat inhibitors are: E-64 (100 μm), a broad spectrum inhibitor (19Barrett A.J. Kembhavi A.A. Brown M.A. Kirschke H. Knight C.G. Tamai M. Hanada K. Biochem. J. 1982; 201: 189-198Crossref PubMed Scopus (920) Google Scholar), CA-074 (l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-l-isoleucyl-l-proline, 25 nm), a CatB inhibitor (20Towatari T. Nikawa T. Murata M. Yokoo C. Tamai M. Hanada K. Katunuma N. FEBS Lett. 1991; 280: 311-315Crossref PubMed Scopus (193) Google Scholar), N-(4-biphenylacetyl)-S-methylcysteine-(D)-Arg-Phe-β-phenethylamide, 200 nm), a CatL inhibitor (21Chowdhury S.F. Sivaraman J. Wang J. Devanathan G. Lachance P. Qi H. Menard R. Lefebvre J. Konishi Y. Cygler M. Sulea T. Purisima E.O. J. Med. Chem. 2002; 45: 5321-5329Crossref PubMed Scopus (73) Google Scholar), Z-l-NHNHCONHNH-LF-Boc (1-(N-benzyloxycarbonyl-leucyl)-5-(N-Boc-phenylalanyl-leucyl)carbohydrazide, 100 nm), a CatK inhibitor (22Wang D. Pechar M. Li W. Kopeckova P. Bromme D. Kopecek J. Biochemistry. 2002; 41: 8849-8859Crossref PubMed Scopus (43) Google Scholar), and Z-Phe-Leu-COCHO (2 nm), a slow, tight binding inhibitor of CatS (23Walker B. Lynas J.F. Meighan M.A. Bromme D. Biochem. Biophys. Res. Commun. 2000; 275: 401-405Crossref PubMed Scopus (35) Google Scholar). Fluorogenic substrates were Z-Phe-Arg-AMC (Z-FR-AMC), Z-Arg-Arg-AMC (Z-RR-AMC), Z-Leu-Arg-AMC (Z-LR-AMC), and Z-Gly-Pro-Arg-AMC (Z-GPR-AMC). Other drugs and chemicals were purchased from Sigma-Aldrich. Cancer Cell Culture and Colony Growth-The cancerous human cell lines, MDA-MB-231 (breast) and H460 (lung) were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM; Cambrex) supplemented with 5% fetal calf serum. When indicated, MDA-MB-231 cells were seeded on a planar film or in a three-dimensional matrix of Matrigel®. Experiments assessing the involvement of Na+ in the invasion process were performed in "Normo Na" and in "Low Na" culture media. These culture solutions were prepared from a DMEM solution (Cambrex) deprived of NaCl, KCl, and CaCl2. The Normo Na solution was prepared by adding 5.4 mm KCl, 2 mm CaCl2, and 110 mm NaCl. The Low Na solution was prepared by adding the same salts as above except that NaCl was replaced by 110 mm choline chloride. Under these conditions, the total concentrations of Na+ are, respectively, 155 and 45 mm (45 mm of Na+ being supplied in both conditions by 44 mm NaHCO3 and 1 mm NaH2PO4). These solutions were then supplemented with 5% fetal calf serum. Cell size, number, and size of colonies, and the number of cells spreading into the three-dimensional matrix were analyzed with the ImageJ software 1.38I (National Institutes of Health). The assessment of sizes for cells and cell colonies was done by evaluating cell surface from pictures coming from separate experiments. The numbers of colonies and cells were counted from pictures. Electrophysiology-Patch pipettes were pulled from borosilicate glass to a resistance of 4-6 MΩ. Currents were recorded, in whole cell configuration, under voltage clamp mode at room temperature using an Axopatch 200 B patch clamp amplifier (Axon Instruments). Analogue signals were filtered at 5 kHz and sampled at 10 kHz using a 1322A Digidata converter. Cell capacitance and series resistance were electronically compensated by about 60%. The P/2 subpulse correction of cell leakage and capacitance was used to study Na+ current (INa). Sodium currents were recorded by depolarizing the cells from a holding potential of -100 mV to a maximal test pulse of -5 mV for 30 ms every 500 ms. The protocol used to build sodium current-voltage (INa-V) relationships was as follows: from a holding potential of -100 mV, the membrane was stepped to potentials from -90 to +60 mV, with 5-mV increments, for 50 ms at a frequency of 2 Hz. Availability-voltage relationships were obtained by applying 50-ms prepulses using the INa-V curve procedure followed by a depolarizing pulse to -5 mV for 50 ms. In this case, the currents were normalized to the amplitude of the test current without a prepulse. Conductance through Na+ channels (gNa) was calculated as already described (17Roger S. Besson P. Le Guennec J.Y. Biochim. Biophys. Acta. 2003; 1616: 107-111Crossref PubMed Scopus (128) Google Scholar). The current amplitudes were normalized to cell capacitance and expressed as current density (pA/pF). The physiological saline solution (PSS) had the following composition: 140 mm NaCl, 4 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 11.1 mm d-glucose, and 10 mm HEPES, adjusted to pH 7.4 with NaOH (1 m). The intrapipette solution had the following composition: 125 potassium glutamate, 20 mm KCl, 0.37 mm CaCl2, 1 mm MgCl2, 1 mm Mg-ATP, 1 mm EGTA, 10 mm HEPES, adjusted to pH 7.2. Assessment of Gelatinolytic Activity by Confocal Microscopy-MDA-MB-231 cells were cultured in a three-dimensional Matrigel® matrix containing 25 μg/ml of DQ-Gelatin® (Invitrogen) on glass coverslips for 24 h and were then fixed for 10 min in 4% paraformaldehyde in phosphate-buffered saline at room temperature. Confocal microscopy was performed with an Olympus Fluoview 500 instrument. The samples were excited at 495 nm, and emission light was recorded at 515 nm. Fluorescence density was quantified by image analysis thanks to the ImageJ software 1.38I. Cell Survival and Proliferation-The cells were seeded at 4 × 104 cells/well in a 24-well plate coated or not with Matrigel® and were grown for a total of 5 or 6 days. The culture medium and TTX and/or Cat inhibitors were changed every other day. Growth and viability of cells were measured as a whole by the tetrazolium salt assay (24Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46516) Google Scholar) as previously described (17Roger S. Besson P. Le Guennec J.Y. Biochim. Biophys. Acta. 2003; 1616: 107-111Crossref PubMed Scopus (128) Google Scholar). Viable cell number was assessed at 570 nm (formazan absorbance) and normalized to the control condition, without TTX (on the same day of the experiment). Migration and in Vitro Invasion Assay-Migration was analyzed in 24-well plates receiving 8-μm pore size polyethylene terephtalate membrane cell culture inserts (BD Biosciences). The upper compartment was seeded with 4 × 104 cells in DMEM supplemented with 5% fetal bovine serum. The lower compartment was filled with DMEM supplemented with 10% fetal bovine serum, as a chemoattractant. After 24 h at 37 °C, the remaining cells were removed from the upper side of the membrane. The cells that had migrated and were attached to the lower side were stained with hematoxylin and counted in the whole insert, using a light microscope (200× magnification). In vitro invasion was assessed using the same inserts and the same protocol as above but with the membrane covered with a film of Matrigel® (extracellular-mimicking matrix; BD Biosciences). Migration and invasion assays were performed in triplicate in eight separate experiments. For easier comparison between cells lines, the results obtained for migration and invasion were normalized to the control condition. In addition, invasion experiments were repeated in Normo Na+ (155 mm) or in Low Na+ solutions (45 mm). Cell Line RNA Extraction, Reverse Transcription, Quantitative, and Conventional PCR-Total RNA extraction from MDA-MB-231 cells was performed by using RNAgents® total RNA isolation system (Promega). RNA yield and purity were determined by spectrophotometry, and only samples with an A260/A280 ratio above 1.6 were kept for further experiments. Total RNA were reverse-transcribed with the RT kits Ready-to-go® You-prime First-Strand Beads (Amersham Biosciences). Random hexamers pd(N)6 5′-Phosphate (0.2 μg; Amersham Biosciences) were added, and the reaction mixture was incubated at 37 °C for 60 min. Quantitative (real time) PCR experiments were performed with an iCycler™ system (Bio-Rad). The PCR protocol consisted of a denaturation step at 95 °C for 2 min, followed by 35 cycles of amplification at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 10 s. The experiments were performed in triplicate, and negative controls containing water instead of first strand cDNA were done. The results were calculated with the ΔCt method, where the parameter Ct (threshold cycle) is defined as the fractional cycle number at which the PCR reporter signal passes a fixed threshold. For each sample ΔCt values were determined by subtracting the average Ct value of the investigated transcript under control conditions (cells grown in normal culture medium) from the average Ct value of the same transcript when the cells were grown for 24 h in presence of 30 μm TTX. For conventional PCR, the temperature profile was 2 min at 94 °C, followed by 35 cycles of amplification which consisted of 30 s of DNA denaturation at 94 °C, 60 s of primers hybridization at 60 °C, and 20 s of polymerization at 72 °C and a final extension for 2 min at 72 °C. PCR products were then analyzed by electrophoresis in a 2% agarose gel containing ethidium bromide and visualized by UV trans-illumination (Gel Doc 2000 system; Bio-Rad). Primers used for PCR experiments had the following sequences (expected sizes): NaV1.5, forward 5′-CACGCGTTCACTTTCCTTC-3′ and reverse 5′-CATCAGCCAGCTTCTTCACA-3′ (208 bp); β1, forward 5′-GAAAACTACGAGCACAACACCA-3′ and reverse 5′-GGCAGTATTGCTTTACCCATCA-3′ (510 bp); β2, forward 5′-TGACCCACTCTCTTCCATCC-3′ and reverse 5′-GGTCCTCTCTGAAGCCACTG-3′ (216 bp); β3, forward 5′-TCAACGTCACTCTGAACGACTC-3′ and reverse 5′-CATGTCACACTGCTCCTGTTCT-3′ (346 bp); β4, forward 5′-ACAGCAGTGACGCATTCAAG-3′ and reverse 5′-CACATGGCAGGTGTATTTGC-3′ (188 bp); CatB, forward 5′-ACAGCCCGACCTACAAACAG-3′ and reverse 5′-CCAGTAGGGTGTGCCATTCT-3′ (239 bp); CatS, forward 5′-TCTCTCAGTGCCCAGAACCT-3′ and reverse 5′-GCCACAGCTTCTTTCAGGAC-3′ (248 bp); CatK, forward 5′-CCTTGAGGCTTCTCTTGGTG-3′ and reverse 5′-GGGCTCTACCTTCCCATTCT-3′ (134 bp); CatL, forward 5′-AGGAGAGCAGTGTGGGAGAA-3′ and reverse 5′-TGGGCTTACGGTTTTGAAAG-3′ (160 bp); Cystatin C, forward 5′-GATCGTAGCTGGGGTGAACT-3′ and reverse 5′-CCTTTTCAGATGTGGCTGGT-3′ (115 bp); Cystatin M, forward 5′-CTTCCTGACGATGGAGATGG-3′ and reverse 5′-GGAACCACAAGGACCTCAAA-3′ (141 bp); and Stefin B, forward 5′-TAGGAGAGCGTGGCTGTTTT-3′ and reverse 5′-TGATGCTCCCTCTTCTGTCC-3′ (128 bp). The efficacy of the primers for NaV1.5 and β-subunits was checked in human tissues where the various isoforms are known to be expressed, i.e. in cardiac muscle for NaV1.5 and in the central nervous system for β1 to β4. siRNA Transfection Protocol-MDA-MB-231 breast cancer cells were transfected with siRNA directed against SCN5A mRNA (siNaV1.5) or scramble siRNA-A as a control (siCTL), which were purchased from Santa Cruz, Tebu-Bio (France). Briefly, cells in suspension were transfected with 20 nm siRNA by using Lipofectamine RNAi max (Invitrogen). The experiments (invasion assays and patch clamp) were performed 24 h after transfection. Fluorescence Measurement of Intracellular pH and Perimembrane pH-Intracellular and perimembrane pH were measured using ratiometric methods (Cairn Optoscan) with BCECF (excitation, 400/490 nm; emission, 535 nm) and fluorescein DHPE (excitation, 440/485 nm; emission, 535 nm) respectively. The cells were incubated for 45 min at room temperature in PSS containing 5 μm BCECF-AM. Perimembrane pH was monitored by incubating the cells with 1 μg/ml of fluorescein DHPE in serum-free DMEM for 1 h at 37 °C. In both cases, excess dye was removed by rinsing the cells twice with PSS. Calibration for each dye was performed by perifusing each studied cell with varying pH (pH 6.9-7.6). For determining internal pH, 0.01 mm nigericin was added to the solution. All of the data were corrected for background fluorescence. Western Blots-The specific primary antibodies were rabbit anti-human Cat B (Fitzgerald) and cathepsin S, K, and L (Calbiochem). MDA-MB-231 cells in their subconfluent state cultivated on a Matrigel® basement membrane were washed twice with phosphate-buffered saline. The cells were scraped and lysed in presence of radioimmunoprecipitation assay lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EDTA), containing 1% Triton X-100 and protease inhibitors (Complete; Roche Applied Science) for 1 h at 4 °C. Cell lysates were centrifuged at 16,000 × g for 10 min to remove insoluble debris. Total proteins concentrations were determined using the bicinchoninic acid method (Bio-Rad). Alternatively, culture media from cells cultivated on a Matrigel® basement membrane were centrifuged to remove floating cells and then concentrated by centrifugation with Amicon Ultra-4 columns (Millipore) at 4,000 × g for 30 min. Protein samples were diluted in the sample buffer under reducing conditions, boiled for 3 min, separated by SDS-PAGE on 12% gels (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar), and then transferred onto a polyvinylidene difluoride membrane (Millipore). After saturation for 2 h in 5% nonfat milk Tris-buffered saline solution containing 0.5% Tween 20 (TTBS), the membrane was incubated overnight at 4 °C with the primary antibody (1/1,000) in a 2% nonfat milk TTBS solution. The membrane was further incubated for 1 h at room temperature, with a goat anti-rabbit (1/6,000) horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). ECL (Amersham Biosciences) was used for immunodetection. Densitometric analyses were performed using QuantityOne software v4.6.3 (Bio-Rad). Cathepsin Activity Assays-After growing MDA-MB-231 cells on Matrigel®-coated flasks, total cell extracts were obtained by a series of seven freeze/thaw cycles in liquid nitrogen and a 30 °C water bath. Concentrated supernatants were prepared as described above. For the assessment of membrane-associated Cat activity, the cells were scraped in phosphate-buffered saline. A panel of AMC-derived fluorogenic substrates were used to measure proteolytic activities. The activation buffer was 0.1 m sodium acetate buffer, pH 5.5, containing 2 mm EDTA and 2 mm dithiothreitol. After activation for 5 min, kinetic measurements were continuously recorded with a microtiter plate reader at 30 °C with λex = 350 nm and λem = 460 nm (Gemini spectrofluorimeter; Molecular Devices). The activities of CatB/K/L/S were measured using Z-FR-AMC (10 μm), that of CatB using Z-RR-AMC (10 μm), those of CatK/S (K > S > L) were measured using Z-LR-AMC (10 μm), and those of CatK/B (K ≫ B) were measured using Z-GPR-AMC (10 μm). Experiments with Z-FR-AMC were also performed at pH 8.0 in a Tris-HCl buffer containing 2 mm EDTA and 2 mm dithiothreitol. Specificity of the Cat-dependent hydrolysis was checked by preincubation with E-64 (100 μm). Measurement of the overall Cat activity in concentrated supernatants was performed using Z-FR-AMC (25 μm) after 2 h of incubation at 30 °C at pH 5.5 in acetate buffer in presence of dextran sulfate (40 μg/ml). Total Cat were titrated by using increasing concentrations of E-64 (0-50 nm). The same experiment was repeated in the presence of CA-074 (0-25 nm) for titration of CatB. Statistical Analyses-The data are displayed as the means ± S.E. of the mean (n = number of cells/experiments). One-way analysis of variance on ranks followed by a Student-Newmann-Keul's test were used to compare cell proliferation, migration and invasion, secretion of Cat, and intracellular and perimembrane pH in control conditions or in the presence of the different drugs and inhibitors. t tests were used to compare cell numbers, colony numbers and sizes, and the number of cells escaping from colonies. Alternatively, a Mann-Whitney rank sum test was used when the variance homogeneity test failed. Statistical significance is indicated: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). NaV1.5 Expression in Cancer Cells and Role in Invasion in Vitro-MDA-MB-231 breast cancer cells express transcripts for the cardiac NaV1.5 isoform (Fig. 1A), which has been shown to be the only functional NaV α-subunit in these cells (16Fraser S.P. Diss J.K. Chioni A.M. Mycielska M.E. Pan H. Yamaci R.F. Pani F. Siwy Z. Krasowska M. Grzywna Z. Brackenbury W.J. Theodorou D. Koyuturk M. Kaya H. Battaloglu E. De Bella M.T. Slade M.J. Tolhurst R. Palmieri C. Jiang J. Latchman D.S. Coombes R.C. Djamgoz M.B. Clin. Cancer Res. 2005; 11: 5381-5389Crossref PubMed Scopus (358) Google Scholar, 17Roger S. Besson P. Le Guennec J.Y. Biochim. Biophys. Acta. 2003; 1616: 107-111Crossref PubMed Scopus (128) Google Scholar, 26Jude S. Roger S. Martel E. Besson P. Richard S. Bougnoux P. Champeroux P. Le Guennec J.Y. Prog. Biophys. Mol. Biol. 2006; 90: 299-325Crossref PubMed Scopus (101) Google Scholar). Although it has been proposed that α-subunits alone are sufficient to form functional channels (27Catterall W.A. Annu. Rev. Biochem. 1986; 55: 953-985Crossref PubMed Google Scholar), mRNA for the β1, β2, and β4 auxiliary subunits are also expressed (Fig. 1A), suggesting that a complete NaV complex could exist, made up of an α-subunit associated with multiple auxiliary β-subunits. In all studied cells, voltage-dependent sodium currents were recorded. The mean current-voltage (INa-V) relationship of the sodium current is shown on Fig. 1B (n = 80 cells). The activation threshold takes place around -60 mV, and the maximal current density, obtained from a depolarizing pulse from -100 to -10 mV, is seen at -11.29 ± 0.89 pA/pF. Conductance-voltage and availability-voltage relationships show that at voltages between -60 and -20 mV, i.e. in the range of the membrane potentials of these cells (Em = -36.8 ± 1.5 mV; n = 105 cells), NaV are partially activated and not fully inactivated (Fig. 1C). This leads to a sustained "window" inward current at -30 mV, which is inhibited by 30 μm TTX (Fig. 1D). This sustained inward current can be increased by using the sodium channel opener veratridine (Fig. 2A). Blocking the sodium current with TTX decreased the invasiveness by about 40% (Fig. 2B). On the other hand, veratridine dose-dependently enhanced the invasiveness of MDA-MB-231 cells. TTX, veratridine (10 and 50 μm), and a mixture of both had no effect on cancer cell migration (supplemental Fig. S1A). To validate the influence of Na+ entry on invasion through NaV, the assays were repeated with decreasing the extracellular Na+ concentration from 155 to 45 mm (Fig. 2C). This resulted in a 3-fold reduction of INa (Fig. 2C, panel a) and a slight membrane potential hyperpolarization (-4.00 ± 1.62 mV, n = 4). Reducing the Na+ concentration of the culture medium decreased invasion by ∼34%, whereas it had no effect on cell viability for 24 h and no effect on NaV1.5 expression (supplemental Fig. S2). Likewise, TTX was approximately three times less effective in reducing invasion in the Low Na+ culture medium (Fig. 2C, panel b). This highlights the crucial role of the inward sodium gradient in cell invasion. We then knocked down the expression of NaV1.5 by using a siRNA protocol (Fig. 3). The transfection of siRNA directed against NaV1.5 mRNA was assessed by quantitative reverse transcription-PCR and resulted in a 65% reduction of expression compared with the transfection of siCTL (Fig. 3A). This reduction in mRNA level was associated to a ∼70% reduction of the maximal current recorded when the cells were submitted to a depolarization from -100 to -5 mV (Fig. 3B). When NaV1.5 expression was knocked down, MDA-MB-231 cells invasiveness was reduced by about 35% and was no longer significantly reduced by TTX (Fig. 3C).FIGURE 2Involvement of the sodium influx through NaV in invasion. A, effect of veratridine (50 μm) on a sodium current elicited by a depolarization to -5 mV from a holding potential of -100 mV. The inset emphasizes the veratridine-induced increase of the sustained current. B, effect of 30 μm TTX, alone or in combination with veratridine (Ver, 10 or
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