저자는 Thomas E. & Pamela M. Mischell Family Foundation에서 S.S.에 대한 지원과 Harold C. Schott Foundation이 UC 의과 대학의 Harold C. Schott 기부 의장에게 자금을 지원했음을 인정합니다.

1. 배양된 세포의 면역표지 이온 채널
<ol><li>세포 준비 및 실험 설정<ol><li>세포를 75cm2 배양 플라스크에서 활발하게 성장하는 배양물로 유지한다. 사용 중인 세포주의 배가 시간에 따라 50%-90% 합류할 때까지 세포를 한 번 계대합니다. 참고: 본 연구를 위해, 그룹 3 수모세포종 세포주인 D283 세포를 사용하였다.</li>
		<li>배양 플라스크에서 세포를 원심분리기 튜브(15mL 또는 50mL)에 모으고 0.2...

고형 종양을 치료하기 위한 이온 채널의 약리학적 표적화

<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. 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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. 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이온 채널 기능의 변화는 세포 내 신호 전달 캐스케이드를 변경하여 세포의 전반적인 기능에 영향을 줄 수 있습니다. 지난 10년 동안 이온 채널이 암세포 성장과 전이에 중요하다는 것이 점점 더 분명해졌습니다. 중요하게도, 많은 이온 채널이 광범위한 장애를 표적으로 하는 승인된 치료제의 주요 표적이다24. 연구자들은 이온 채널이 항암 표적이 될 수 있는지 여부를 조사했으?...

막 수송 단백질은 세포 간의 통신과 세포 항상성 유지에 중요합니다. 막 수송 단백질 중에서 이온 채널은 세포의 성장과 발달을 촉진하고 도전적이고 변화하는 환경에서 세포의 상태를 유지하는 역할을 합니다. 이온 채널은 또한 전신 및 중추신경계(CNS)에서 고형 종양의 발달을 유도하고 지원하는 것으로 보고되었습니다1,2. 예를 들어, KCa3.1 채널은 세포 주기 조절에 중요한 막 전위를 조절하고 세포 부피를 조절하는 역할을 합니다. 결함이 있는 KCa3.1 채널은 종양 세포의 비정상적인 증식에 기여하는 것으로 보고되었다3. 또한, 이온 채널은 암의 전이성 전파에 기여할 수 있습니다. 일시적인 수용체 전위 (TRP) 채널은 예를 들어 Ca2+ 및Mg2+ 유입에 관여하고; 이 유입은 종양을 둘러싼 세포외 기질을 조절하는 기능을 하는 여러 키나아제와 열 충격 단백질을 활성화시키며, 이는 차례로 암 전이를 시작하는 데 중요합니다4.
이온 채널은 암 발병에 기여할 수 있기 때문에 약물 관련 암 치료의 표적이 될 수도 있습니다. 예를 들어, 화학 요법 및 새로운 면역 요법을 포함한 치료 양식에 대한 내성은 이온 채널 기능 조절 장애와 관련이 있습니다 5,6,7. 또한, 이온 채널은 암의 성장과 발달을 방해하는 중요한 약물 표적으로 부상하고 있으며, 용도 변경 소분자(FDA 승인) 약물과 단클론 항체를 포함한 생체 고분자가 연구되고 있습니다 1,2,8,9. 이 분야에서 많은 진전이 있었지만 이온 채널 암 약물 발견은 아직 개발되지 않았습니다. 이것은 부분적으로 암세포에서 이온 채널을 연구하는 독특한 문제 때문입니다. 예를 들어, 느리게 작용하는 화합물에 대한 전기생리학 분석을 설정하는 데는 기술적 한계가 있고 채널 활성화 및 약물 작용의 시간적 차이가 있습니다. 또한, 화합물의 용해도는 또한 진행을 방해할 수 있는데, 이는 오늘날 일반적으로 사용되는 대부분의 자동화된 전기생리학 시스템이 소수성 기질을 사용하며, 이는 화합물 흡착의 결과로 인공물에 기여할 수 있기 때문입니다. 또한, 천연물, 펩타이드 및 단클론 항체와 같은 대형 생체 유기 분자 치료제는 기존의 전기생리학 분석법을 사용하여 스크리닝하기가 기술적으로 어렵다10. 마지막으로, 암세포의 생체 전기적 특성은 잘 알려져 있지 않다11.
한편, 이온 채널의 면역형광 염색은 종종 까다롭습니다. 이는 부분적으로 구조의 복잡성과 멤브레인의 맥락 때문이며, 이는 현미경 연구를 위해 항체를 생성하고 사용하는 능력에 영향을 미칩니다. 이온 채널을 염색하는 데 사용되는 항체의 특이성, 친화도 및 재현성을 검증하는 것이 특히 중요합니다. 이온 채널에 대한 상용 항체는 검증 전략 및 출판 기록에 따라 고려해야 합니다. 실험에는 표적 단백질의 녹다운 또는 녹아웃에 의한 비특이적 결합의 부족을 입증하기 위한 음성 대조군이 포함되어야 합니다. 대안적으로, mRNA 또는 단백질 결정에 기초하여 표적 단백질이 부재하거나 낮은 풍부도에 있는 세포주는 음성 대조군으로서 기능할 수 있다. 예를 들어, 이 연구는 수모세포종 세포주(D283)에서 (GABA) 수용체 서브유닛 Gabra5의 국소화를 보여줍니다. siRNA 녹다운을 갖는 D283 세포 및 또 다른 소뇌 수모세포종 세포주인 Daoy 세포를 Gabra5에 대해 염색하였고, 눈에 띄는 염색을 나타내지 않았다 (데이터는 나타내지 않음).
여기에서는 이온 채널 기능뿐만 아니라 이온 채널 조절제가 암세포에 미치는 영향을 분석하고 분석하는 방법을 제시합니다. 프로토콜은 (1) 이온 채널에 대한 세포 염색, (2) 미토콘드리아의 편광 상태 테스트, (3) 전기생리학을 사용한 이온 채널 기능 확립, (4) 시험관 내 약물 검증을 위해 제공됩니다. 이러한 프로토콜은 A형 감마-아미노부티르산(GABAA) 수용체 2,12,13,14,15,16, 염화물 음이온 채널 및 주요 억제성 신경전달물질 수용체에 대한 연구를 강조합니다. 그러나 여기에 제시된 방법은 다른 많은 암세포 및 이온 채널을 연구하는 데 적용됩니다.

<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

이온 채널은 세포 발달과 세포 항상성 유지에 중요합니다. 이온 채널 기능의 섭동은 광범위한 장애 또는 채널병증의 발병에 기여합니다. 암세포는 이온 채널을 활용하여 자체 발달을 유도할 뿐만 아니라 종양으로 개선되고 다양한 비암성 세포를 포함하는 미세 환경에서 동화됩니다. 또한, 종양 미세 환경 내에서 성장 인자 및 호르몬 수준의 증가는 이온 채널 발현을 향상시켜 암세포 증식 및 생존에 기여할 수 있습니다. 따라서 이온 채널의 약리학적 표적화는 원발성 및 전이성 뇌암을 포함한 고형 악성 종양을 치료하는 유망한 접근 방식일 수 있습니다. 본원에서는 암 세포에서 이온 채널의 기능을 특성화하기 위한 프로토콜과 암 생존력에 미치는 영향을 결정하기 위해 이온 채널의 조절자를 분석하는 접근법을 설명합니다. 여기에는 이온 채널에 대한 세포 염색, 미토콘드리아의 편광 상태 테스트, 전기 생리학을 사용하여 이온 채널 기능 확립, 약물 효능을 평가하기 위한 생존력 분석 수행이 포함됩니다.

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.

저자 스포트라이트: 암에서 이온 채널의 역할 탐색: 특성화 및 잠재적 치료 접근법 

암세포의 이온 채널 스크리닝

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.

위는 암세포에서 이온 채널을 특성화하는 데 사용할 수 있는 선별된 절차입니다. 첫 번째 프로토콜은 이온 채널의 염색을 강조합니다. 자세히 설명했듯이 이온 채널 또는 세포외막에 존재하는 단백질을 염색할 때 많은 어려움이 있습니다. 도 1에 나타낸 것은 펜타머 GABAA 수용체의 서브유닛에 대한 염색이다. 두 번째 프로토콜은 암세포에서 미토콘드리아의 편광 상?...

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

이온 채널의 약리학적 표적화는 고형 종양을 치료하는 유망한 접근 방식입니다. 암세포에서 이온 채널 기능을 특성화하고 암 생존력에 대한 이온 채널 조절제의 효과를 분석하기 위한 자세한 프로토콜이 제공됩니다.

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

이온 채널의 기능은 세포 생물학에서 매우 중요하며 암 성장에 상당한 영향을 미칩니다. 우리 팀은 암 치료를 돕기 위해 이온 채널을 표적으로 삼는 데 중점을 두고 있습니다. 이 분야는 최근 종양 미세 환경과 암세포가 건강한 조직에서 어떻게 유지되는지에 대한 더 큰 이해를 얻었으며 암에서 이온 채널의 중요성을 강조했습니다.현재 실험에서 가장 큰 과제 중 하나는 치료 치료를 효과적으로 테스트할 수 있는 면역 능력이 있는 동물 모델의 가용성입니다. 우리는 GABA A 수용체가 이질적인 암에 대한 치료 적 취약성임을 입증하고 암세포를 효과적으로 손상시키는 새로운 종류의 GABA A 수용체 활성제를 확인했습니다. 이온 채널은 암세포 행동을 조절하는 데 중요한 역할을 하며 암 치료의 표적으로서 큰 잠재력을 가지고 있습니다.우리의 연구는 암을 효과적으로 치료하기 위해 확립 된 종류의 약물을 어떻게 사용할 수 있는지 강조했습니다.

암세포의 이온 채널 스크리닝

Abstract