Os autores agradecem o apoio da Thomas E. & Pamela M. Mischell Family Foundation à S.S. e do financiamento da Harold C. Schott Foundation da Harold C. Schott Endowed Chair, UC College of Medicine, à S.S.

Direcionamento farmacológico de canais iônicos para tratar tumores sólidos

<ol>
	<li>Prevarskaya, N., Skryma, R., Shuba, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+in+cancer:+Are+cancer+hallmarks+oncochannelopathies.">Ion channels in cancer: Are cancer hallmarks oncochannelopathies.</a> Physiological Reviews. 98 (2), 559-621 (2018).</li><li>Rao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-gated+neurotransmitter+receptors+as+targets+for+treatment+and+management+of+cancers.">Ligand-gated neurotransmitter receptors as targets for treatment and management of cancers.</a> Frontiers in Physiology. 13, 839437 (2022).</li><li>Mohr, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+intermediate+conductance+Ca2+-activated+K++channel+KCa3.1.">Cancer-associated intermediate conductance Ca2+-activated K+ channel KCa3.1.</a> Cancers. 11 (1), 109 (2019).</li><li>Fels, B., Bulk, E., Petho, Z., Schwab, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TRP+channels+in+the+metastatic+cascade.">The role of TRP channels in the metastatic cascade.</a> Pharmaceuticals. 11 (2), 48 (2018).</li><li>Eil, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+immune+suppression+within+the+tumour+microenvironment+limits+T+cell+effector+function.">Ionic immune suppression within the tumour microenvironment limits T cell effector function.</a> Nature. 537 (7621), 539-543 (2016).</li><li>Haustrate, A., Hantute-Ghesquier, A., Prevarskaya, N., Lehen'kyi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+targeting+ion+channels+and+their+therapeutic+potential.">Monoclonal antibodies targeting ion channels and their therapeutic potential.</a> Frontiers in Pharmacology. 10, 606 (2019).</li><li>Kischel, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels:+New+actors+playing+in+chemotherapeutic+resistance.">Ion channels: New actors playing in chemotherapeutic resistance.</a> Cancers. 11 (3), 376 (2019).</li><li>Tuszynski, J., Tilli, T. M., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+and+neurotransmitter+modulators+as+electroceutical+approaches+to+the+control+of+cancer.">Ion channel and neurotransmitter modulators as electroceutical approaches to the control of cancer.</a> Current Pharmaceutical Design. 23 (32), 4827-4841 (2017).</li><li>Kale, V. P., Amin, S. G., Pandey, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+ion+channels+for+cancer+therapy+by+repurposing+the+approved+drugs.">Targeting ion channels for cancer therapy by repurposing the approved drugs.</a> Biochimica Biophysica Acta. 1848 (10), 2747-2755 (2015).</li><li>Wickenden, A., Priest, B., Erdemli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+drug+discovery:+Challenges+and+future+directions.">Ion channel drug discovery: Challenges and future directions.</a> Future Medicinal Chemistry. 4 (5), 661-679 (2012).</li><li>Rocha, P. R. F., Elghajiji, A., Tosh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+system+for+electrophysiology+of+cancer+cell+populations:+A+review.">Ultrasensitive system for electrophysiology of cancer cell populations: A review.</a> Bioelectricity. 1 (3), 131-138 (2019).</li><li>Sengupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;5-GABAA+receptors+negatively+regulate+MYC-amplified+medulloblastoma+growth.">&#945;5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth.</a> Acta Neuropathologica. 127 (4), 593-603 (2014).</li><li>Jonas, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+in+vivo+testing+of+compounds+targeting+Group+3+medulloblastomas+using+an+implantable+microdevice+as+a+new+paradigm+for+drug+development.">First in vivo testing of compounds targeting Group 3 medulloblastomas using an implantable microdevice as a new paradigm for drug development.</a> Journal of Biomedical Nanotechnology. 12 (6), 1297-1302 (2016).</li><li>Kallay, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+native+GABAA+receptors+in+medulloblastoma+with+positive+allosteric+benzodiazepine-derivatives+induces+cell+death.">Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.</a> Journal of Neurooncology. 142 (3), 411-422 (2019).</li><li>Pomeranz Krummel, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+cell+intrinsic+GABAA+receptor+enhancement+potentiates+radiation+and+immune+checkpoint+response+by+promoting+direct+and+T+cell-mediated+anti-tumor+activity.">Melanoma cell intrinsic GABAA receptor enhancement potentiates radiation and immune checkpoint response by promoting direct and T cell-mediated anti-tumor activity.</a> International Journal of Radiation Oncology, Biology, Physics. 109 (4), P1040-P1053 (2021).</li><li>Bhattacharya, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutically+leveraging+GABAA+receptors+in+cancer.">Therapeutically leveraging GABAA receptors in cancer.</a> Experimental Biology and Medicine. 246 (19), 2128-2135 (2021).</li><li>Mazia, D., Schatten, G., Sale, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+cells+to+surfaces+coated+with+polylysine.+Applications+to+electron+microscopy.">Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.</a> Journal of Cell Biology. 66 (1), 198-200 (1975).</li><li>Wiatrak, B., Kubis-Kubiak, A., Piwowar, A., Barg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PC12+cell+line:+Cell+types,+coating+of+culture+vessels,+differentiation+and+other+culture+conditions.">PC12 cell line: Cell types, coating of culture vessels, differentiation and other culture conditions.</a> Cells. 9 (4), 958 (2020).</li><li>Baker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+in+cytochemistry+and+electron-microscopy.">Fixation in cytochemistry and electron-microscopy.</a> Journal of Histochemistry and Cytochemistry. 6 (5), 303-308 (1958).</li><li>Chung, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histomorphological+and+molecular+assessments+of+the+fixation+times+comparing+formalin+and+ethanol-based+fixatives.">Histomorphological and molecular assessments of the fixation times comparing formalin and ethanol-based fixatives.</a> Journal of Histochemistry and Cytochemistry. 66 (2), 121-135 (2018).</li><li>Crowley, L. C., Christensen, M. E., Waterhouse, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+transmembrane+potential+by+TMRE+staining.">Measuring mitochondrial transmembrane potential by TMRE staining.</a> Cold Spring Harbor Protocols. 2016 (12), (2016).</li><li>Cory, A. H., Owen, T. C., Barltrop, J. A., Cory, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+an+aqueous+soluble+tetrazolium/formazan+assay+for+cell+growth+assays+in+culture.">Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.</a> Cancer Communications. 3 (7), 207-212 (1991).</li><li>Maro, B., Marty, M. C., Bornens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+effects+of+the+mitochondrial+uncoupler+FCCP+on+microtubules.">In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.</a> EMBO Journal. 1 (11), 1347-1352 (1982).</li><li>Zheng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+regulation+of+melanoma+cell+death+via+activation+of+thermo-TRPV4+and+TRPV.">Mechanism for regulation of melanoma cell death via activation of thermo-TRPV4 and TRPV.</a> Journal of Oncology. 2019, 7362875 (2019).</li><li>Konno, K., Watanabe, M., Luj&#225;n, R., Ciruela, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+for+ion+channels+and+their+interacting+molecules:+Tips+for+improving+antibody+accessibility.">Immunohistochemistry for ion channels and their interacting molecules: Tips for improving antibody accessibility.</a> Receptor and Ion Channel Detection in the Brain. , (2016).</li><li>Mortensen, M., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-channel+recording+of+ligand-gated+ion+channels.">Single-channel recording of ligand-gated ion channels.</a> Nature Protocols. 2 (11), 2826-2841 (2007).</li><li>Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+of+cells+in+vitro.">Clonogenic assay of cells in vitro.</a> Nature Protocols. 1 (5), 2315-2319 (2006).</li><li>Rafehi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay:+Adherent+cells.">Clonogenic assay: Adherent cells.</a> Journal of Visualized Experiments. (49), 2573 (2011).</li><li>Scudiero, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+soluble+tetrazolium/formazan+assay+for+cell+growth+and+drug+sensitivity+in+culture+using+human+and+other+tumor+cell+lines.">Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.</a> Cancer Research. 48 (17), 4827-4833 (1988).</li><li>Wang, P., Henning, S. M., Heber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+MTT+and+MTS-based+assays+for+measurement+of+antiproliferative+activity+of+green+tea+polyphenols.">Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.</a> PLoS One. 5, e10202 (2010).</li><li>Berridge, M. V., Tan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+cellular+reduction+of+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium+bromide+(MTT):+subcellular+localization,+substrate+dependence,+and+involvement+of+mitochondrial+electron+transport+in+MTT+reduction.">Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.</a> Archives of Biochemistry and Biophysics. 303 (2), 474-482 (1993).</li><li>Plumb, J. A., Milroy, R., Kaye, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+pH+dependence+of+3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium+bromide-formazan+absorption+on+chemosensitivity+determined+by+a+novel+tetrazolium-based+assay.">Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay.</a> Cancer Research. 49 (16), 4435-4440 (1989).</li><li>Chakrabarti, R., Kundu, S., Kumar, S., Chakrabarti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+as+an+enzyme+that+catalyzes+the+reduction+of+MTT+to+formazan+by+vitamin+C.">Vitamin A as an enzyme that catalyzes the reduction of MTT to formazan by vitamin C.</a> Journal of Cellular Biochemistry. 80 (1), 133-138 (2000).</li><li>Dong, G. 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D.A.P.K. é cofundador, presidente e CEO da Amlal Pharmaceuticals Inc.

Alterações na função do canal iônico alteram as cascatas de sinalização intracelular, o que pode afetar o funcionamento geral de uma célula. Na última década, tornou-se cada vez mais claro que os canais iônicos são importantes para o crescimento de células cancerígenas e metástases. É importante ressaltar que muitos canais iônicos são alvos primários de terapêuticas aprovadas visando uma ampla gama de distúrbios24. Pesquisadores investigaram se os canais iônicos poderiam ser ...

As proteínas transportadoras de membrana são fundamentais para a comunicação entre as células, bem como para a manutenção da homeostase celular. Entre as proteínas de transporte de membrana, os canais iônicos servem para impulsionar o crescimento e desenvolvimento das células e para manter o estado das células em ambientes desafiadores e em mudança. Também tem sido relatado que canais iônicos conduzem e apoiam o desenvolvimento de tumores sólidos, tanto sistemicamente quanto no sistema nervoso central (SNC)1,2. Por exemplo, os canais de KCa3.1 são responsáveis por regular o potencial de membrana e controlar o volume celular, o que é importante na regulação do ciclo celular. Canais defeituosos de KCa3.1 têm sido relatados como contribuintes para a proliferação anormal de célulastumorais3. Além disso, os canais iônicos podem contribuir para a disseminação metastática de cânceres. Canais de potencial receptor transitório (TRP), por exemplo, estão envolvidos no influxo de Ca 2+ e Mg2+; Esse influxo ativa várias quinases e proteínas de choque térmico que têm a função de regular a matriz extracelular ao redor de um tumor, o que é, por sua vez, importante para iniciar a metástase docâncer4.
Uma vez que os canais iónicos podem contribuir para o desenvolvimento de cancros, podem também ser alvos para o tratamento do cancro relacionado com medicamentos. Por exemplo, a resistência às modalidades de tratamento, incluindo quimioterapia e nova imunoterapia, está relacionada à desregulação da função dos canais iônicos 5,6,7. Além disso, canais iônicos estão emergindo como importantes alvos de fármacos para impedir o crescimento e desenvolvimento de cânceres, sendo examinados fármacos reaproveitados de pequenas moléculas (aprovados pela FDA), bem como biopolímeros, incluindo anticorposmonoclonais 1,2,8,9. Embora tenha havido muito progresso nessa frente, a descoberta de medicamentos para o câncer de canal iônico permanece subdesenvolvida. Isto é em parte devido aos desafios únicos de estudar os canais iônicos em células cancerosas. Por exemplo, existem limitações técnicas na montagem de ensaios eletrofisiológicos para compostos de ação lenta e diferenças temporais na ativação de canais e ação de drogas. Além disso, a solubilidade dos compostos também pode impedir o progresso, já que a maioria dos sistemas de eletrofisiologia automatizados comumente usados atualmente utilizam substratos hidrofóbicos, que podem contribuir para artefatos como resultado da adsorção de compostos. Além disso, grandes terapêuticas moleculares bioorgânicas, como produtos naturais, peptídeos e anticorpos monoclonais, são tecnicamente desafiadoras de rastrear usando ensaios eletrofisiológicos convencionais10. Finalmente, as propriedades bioelétricas das células cancerosas permanecem pouco compreendidas11.
Enquanto isso, a coloração por imunofluorescência dos canais iônicos é muitas vezes desafiadora. Isso se deve, em parte, à complexidade de suas estruturas e seu contexto na membrana, que impactam a capacidade de gerar e empregar anticorpos para estudos de microscopia. É especialmente importante que os anticorpos usados para corar os canais iônicos sejam validados quanto à especificidade, afinidade e reprodutibilidade. Anticorpos comerciais para canais iônicos devem ser considerados com base em sua estratégia de validação e registro de publicação. Os experimentos devem incluir controles negativos para demonstrar a falta de ligação inespecífica por knockdown ou knockout da proteína-alvo. Alternativamente, linhagens celulares nas quais a proteína-alvo está ausente ou em baixa abundância com base em determinações de RNAm ou proteínas podem servir como controles negativos. Por exemplo, este estudo mostra a localização da subunidade do receptor (GABA) Gabra5 em uma linhagem celular de meduloblastoma (D283). Células D283 com knockdown de siRNA e células Daoy, outra linhagem celular de meduloblastoma cerebelar, foram coradas para Gabra5 e não apresentaram coloração apreciável (dados não mostrados).
Aqui, são apresentados métodos para analisar e testar a função dos canais iônicos, bem como o efeito dos moduladores dos canais iônicos sobre as células cancerosas. São fornecidos protocolos para (1) coloração de células para um canal iônico, (2) teste do estado polarizado das mitocôndrias, (3) estabelecimento da função do canal iônico usando eletrofisiologia e (4) validação in vitro de fármacos. Esses protocolos enfatizam estudos do receptor tipo A do ácido gama-aminobutírico (GABAA) 2,12,13,14,15,16, um canal de ânion cloreto e do principal receptor inibitório de neurotransmissores. No entanto, os métodos apresentados aqui se aplicam ao estudo de muitas outras células cancerosas e canais iônicos.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABS SpectraMax Plate Reader</td>
			<td>Molecular Devices</td>
			<td>ABS</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Invitrogen</td>
			<td>00-4555-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Flor 488</td>
			<td>Invitrogen</td>
			<td>A32723</td>
			<td>Goat Anti-Rabbit</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td>Lacks vitamin A</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>LABCONCO</td>
			<td>302381101</td>
			<td>Class II, Type A2</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1606-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13-998-211</td>
			<td>Heracell VIOS 160i</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Fisher Scientific</td>
			<td>C7902</td>
			<td>Dihydrate</td>
		</tr>
		<tr>
			<td>Cell Culture Dishes, 150 mm</td>
			<td>Fisher Scientific</td>
			<td>12-600-004</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Flasks, 75 cm2</td>
			<td>Fisher Scientific</td>
			<td>430641U</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Plates, 6 well</td>
			<td>Fisher Scientific</td>
			<td>353046</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Cell Culture Plates, 96 well</td>
			<td>Fisher Scientific</td>
			<td>353072</td>
			<td>Cell culture treated</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>EP-5804R</td>
			<td>Refrigerated</td>
		</tr>
		<tr>
			<td>Corning CoolCell</td>
			<td>Fisher Scientific</td>
			<td>07-210-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 22 mm</td>
			<td>Fisher Scientific</td>
			<td>12-553-450</td>
			<td>Corning brand</td>
		</tr>
		<tr>
			<td>D283 Med</td>
			<td>ATCC</td>
			<td>HTB-185</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO Mounting Media</td>
			<td>EMS</td>
			<td>17989-97</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Life Sciences</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>DMEM/F12, base media</td>
			<td>Fisher Scientific</td>
			<td>11330-032</td>
			<td>With phenol red</td>
		</tr>
		<tr>
			<td>DMEM/F12, phenol red free</td>
			<td>Fisher Scientific</td>
			<td>21041-025</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>STEMCELL</td>
			<td>78006.1</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Abcam</td>
			<td>AB120081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor, Basic</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>GARBA5 Antibody</td>
			<td>Aviva</td>
			<td>ARP30687_P050</td>
			<td>Rabbit Polyclonal</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Mounting Medium</td>
			<td>EMS</td>
			<td>17989-60</td>
			<td>With DAPI+DABCO</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>STEMCELL</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>HyClone</td>
			<td>SH3023701</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Open platform</td>
			<td></td>
			<td>With Fiji plugins</td>
		</tr>
		<tr>
			<td>Immuno Mount DAPI</td>
			<td>EMS</td>
			<td>17989-97</td>
			<td></td>
		</tr>
		<tr>
			<td>KRM-II-08</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Leica Application Suite X</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Novus</td>
			<td>N276314100U</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td>Hexahydrate</td>
		</tr>
		<tr>
			<td>Microscope, Confocal</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, Light</td>
			<td>VWR</td>
			<td>76382-982</td>
			<td>DMiL Inverted</td>
		</tr>
		<tr>
			<td>MTS - Promega One Step</td>
			<td>Promega</td>
			<td>G3581</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 0.5-10 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683914</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 10-100 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683930</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipette, 30-300 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683957</td>
			<td></td>
		</tr>
		<tr>
			<td>Nest-O-Patch</td>
			<td>Heka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Gibco</td>
			<td>10888022</td>
			<td>Without vitamin A</td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Gibco</td>
			<td>12348-017</td>
			<td>Phenol red free</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>NOR-QH-II-66</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>50-998-944</td>
			<td>4 inch width</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>EMS</td>
			<td>RT-15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PATHCHMASTER</td>
			<td>Heka</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion System</td>
			<td>Nanion</td>
			<td>4000120</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>EMS</td>
			<td>RT-15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufered Saline</td>
			<td>Fisher Scientific</td>
			<td>AAJ75889K2</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Fisher Scientific</td>
			<td>A3890401</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma Life Sciences</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Port-a-Patch</td>
			<td>Nanion</td>
			<td>21000072</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Life Sciences</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibody</td>
			<td>Invitrogen</td>
			<td>MA5-34653</td>
			<td>Rabbit Monoclonal</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fisher Scientific</td>
			<td>NC0758676</td>
			<td>1 mL ampule</td>
		</tr>
		<tr>
			<td>QH-II-66</td>
			<td></td>
			<td></td>
			<td>experimental compounds not available from a commercial source</td>
		</tr>
		<tr>
			<td>Reagent Reservoirs</td>
			<td>VWR</td>
			<td>89094-664</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Slides, 75 x 25 mm</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td>Frosted one side</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Corning</td>
			<td>25-035-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Synth-a-Freeze Medium</td>
			<td>Gibco</td>
			<td>R00550</td>
			<td>Cryopreservation</td>
		</tr>
		<tr>
			<td>TMRE</td>
			<td>Fisher Scientific</td>
			<td>50-196-4741</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>TMRE Kit</td>
			<td>Abcam</td>
			<td>AB113852</td>
			<td>Kit</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>NC0704309</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td>Solution, 0.4%</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Solution, 0.25%</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper</td>
			<td>Fisher Scientific</td>
			<td>09-927-841</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening ion channels in cancer cells

Os canais iônicos são críticos para o desenvolvimento celular e manutenção da homeostase celular. A perturbação da função dos canais iônicos contribui para o desenvolvimento de uma ampla gama de distúrbios ou canalopatias. As células cancerosas utilizam canais iônicos para impulsionar seu próprio desenvolvimento, bem como para melhorar como um tumor e assimilar em um microambiente que inclui várias células não cancerosas. Além disso, aumentos nos níveis de fatores de crescimento e hormônios dentro do microambiente tumoral podem resultar em maior expressão de canais iônicos, o que contribui para a proliferação e sobrevivência das células cancerígenas. Assim, o direcionamento farmacológico de canais iônicos é potencialmente uma abordagem promissora para o tratamento de neoplasias sólidas, incluindo cânceres cerebrais primários e metastáticos. Neste trabalho, são descritos protocolos para caracterizar a função de canais iônicos em células cancerosas e abordagens para analisar moduladores de canais iônicos para determinar seu impacto na viabilidade do câncer. Estes incluem a coloração de uma célula (s) para um canal iônico (s), testar o estado polarizado da mitocôndria, estabelecer a função do canal iônico usando eletrofisiologia e realizar ensaios de viabilidade para avaliar a potência da droga.

Membrane transport proteins are critical for communication between cells, as well as for maintaining cellular homeostasis. Amongst the membrane transport proteins, ion channels serve to drive the growth and development of cells and to maintain the state of cells in challenging and changing environments. Ion channels have also been reported to drive and support the development of solid tumors, both systemically and in the central nervous system (CNS)1,2. For example, KCa3.1 channels are responsible for regulating membrane potential and controlling cell volume, which is important in cell-cycle regulation. Defective KCa3.1 channels have been reported to contribute to the abnormal proliferation of tumor cells3. Further, ion channels may contribute to the metastatic dissemination of cancers. Transient receptor potential (TRP) channels, for example, are involved in Ca2+ and Mg2+ influx; this influx activates several kinases and heat shock proteins that function to regulate the extracellular matrix surrounding a tumor, which is, in turn, important for initiating cancer metastasis4.
Since ion channels can contribute to the development of cancers, they may also be targets for drug-related cancer treatment. For example, resistance to treatment modalities, including chemotherapy and novel immunotherapy, is related to ion channel function dysregulation5,6,7. In addition, ion channels are emerging as important drug targets to impede the growth and development of cancers, with repurposed small molecule (FDA-approved) drugs being examined, as well as biopolymers, including monoclonal antibodies1,2,8,9. While there has been much progress on this front, ion channel cancer drug discovery remains underdeveloped. This is partly due to the unique challenges of studying ion channels in cancer cells. For example, there are technical limitations in setting up electrophysiology assays for slow-acting compounds and temporal differences in channel activation and drug action. Further, the solubility of compounds can also impede progress, as most of the automated electrophysiology systems commonly in use today utilize hydrophobic substrates, which may contribute to artifacts as a result of compound adsorption. In addition, large bioorganic molecular therapeutics such as natural products, peptides, and monoclonal antibodies are technically challenging to screen using conventional electrophysiology assays10. Finally, the bioelectrical properties of cancer cells remain poorly understood11.
Meanwhile, the immunofluorescence staining of ion channels is often challenging. This is due, in part, to the complexity of their structures and their context in the membrane, which impact the ability to both generate and employ antibodies for microscopy studies. It is especially important that the antibodies used to stain ion channels are validated for specificity, affinity, and reproducibility. Commercial antibodies for ion channels should be considered based on their validation strategy and publication record. Experiments should include negative controls to demonstrate the lack of nonspecific binding by either knockdown or knockout of the target protein. Alternatively, cell lines in which the target protein is absent or in low abundance based on mRNA or protein determinations may serve as negative controls. For example, this study shows the localization of the (GABA) receptor&#160;subunit Gabra5 in a medulloblastoma cell line (D283). D283 cells with an siRNA knockdown and Daoy&#160;cells, another cerebellar medulloblastoma cell line, were stained for Gabra5 and showed no appreciatable staining (data not shown).
Here, methods are presented to analyze and assay ion channel function, as well as the effect of ion channel modulators on cancer cells. Protocols are provided for (1) staining cells for an ion channel, (2) testing the polarized state of mitochondria, (3) establishing ion channel function using electrophysiology, and (4) in vitro drug validation. These protocols emphasize studies of the type A gamma-aminobutyric acid (GABAA) receptor2,12,13,14,15,16, a chloride anion channel and major inhibitory neurotransmitter receptor. However, the methods presented here apply to studying many other cancer cells and ion channels.

Autor em destaque: Explorando o papel dos canais iônicos no câncer: caracterização e possíveis abordagens de tratamento 

Rastreamento de Canais Iônicos em Células Cancerosas

The function of ion channels is crucial in cell biology and significantly impacts cancer growth. Our team is focused on targeting ion channels to aid cancer treatments. The field has recently gained a greater understanding of the tumor microenvironment and how cancer cells take hold in healthy tissue, highlighting the importance of ion channels in cancer.One of the biggest challenges in current experiment is the availability of immune-competent animal model that can effectively test therapeutic treatments. We have established that GABA A receptor is a therapeutic vulnerability to disparate cancers and identified a new class of GABA A receptor activators that effectively impair cancer cells. Ion channels play a crucial role in regulating cancer cell behavior, and they hold great potential as target for cancer treatment.Our research has highlighted how an established class of drug can be employed to treat cancer effectively.

Acima estão selecionados procedimentos que podem ser empregados para caracterizar canais iônicos em células cancerosas. O primeiro protocolo destaca a coloração de um canal iônico. Como detalhado, existem muitos desafios ao colorir um canal iônico ou, para esse assunto, qualquer proteína que esteja presente na membrana extracelular. A Figura 1 mostra a coloração para uma subunidade do receptor GABAA pentamérica. O segundo protocolo destaca os resultados dos testes do es...

Watch this Scientific Journal Video about Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches at JoVE.com

O direcionamento farmacológico dos canais iônicos é uma abordagem promissora para o tratamento de tumores sólidos. Protocolos detalhados são fornecidos para caracterizar a função de canais iônicos em células cancerosas e avaliar os efeitos de moduladores de canais iônicos na viabilidade do câncer.

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development ...

A função dos canais iônicos é crucial na biologia celular e impacta significativamente o crescimento do câncer. Nossa equipe está focada em direcionar canais iônicos para ajudar no tratamento do câncer. Recentemente, o campo ganhou uma maior compreensão do microambiente tumoral e de como as células cancerosas se instalam no tecido saudável, destacando a importância dos canais iônicos no câncer.Um dos maiores desafios no experimento atual é a disponibilidade de um modelo animal imunocompetente que possa efetivamente testar tratamentos terapêuticos. Nós estabelecemos que o receptor GABA A é uma vulnerabilidade terapêutica para cânceres díspares e identificamos uma nova classe de ativadores do receptor GABA A que efetivamente prejudicam as células cancerosas. Os canais iônicos desempenham um papel crucial na regulação do comportamento das células cancerígenas, e eles têm grande potencial como alvo para o tratamento do câncer.Nossa pesquisa destacou como uma classe estabelecida de drogas pode ser empregada para tratar o câncer de forma eficaz.

Research

JoVE Journal

Cancer Research

Screening Ion Channels in Cancer Cells

1.4K visualizações.  University of Cincinnati College of Medicine. A função dos canais iônicos é crucial na biologia celular e impacta significativamente o crescimento do câncer. Nossa equipe está focada em direcionar canais iônicos para ajudar no tratamento do câncer. Recentemente, o campo ganhou uma maior compreensão do microambiente tumoral e de como as células cancerosas se instalam no tecido saudável, destacando a importância dos canais iônicos no câncer.Um dos maiores desafios no experimento atual é a disponibilidade de um modelo ...