筛选癌细胞中的离子通道
Summary
离子通道的药理学靶向是治疗实体瘤的一种有前途的方法。提供了详细的方案，用于表征癌细胞中的离子通道功能并分析离子通道调节剂对癌症活力的影响。
Abstract
离子通道对于细胞发育和维持细胞稳态至关重要。离子通道功能的扰动有助于各种疾病或离子通道病的发展。癌细胞利用离子通道来驱动自身的发育，以及作为肿瘤的改善，并在包括各种非癌细胞的微环境中同化。此外，肿瘤微环境中生长因子和激素水平的增加可导致离子通道表达增强，从而有助于癌细胞增殖和存活。因此，离子通道的药理学靶向是治疗实体恶性肿瘤（包括原发性和转移性脑癌）的潜在有前途的方法。本文描述了表征癌细胞中离子通道功能的协议以及分析离子通道调节剂以确定其对癌症活力的影响的方法。这些包括对细胞进行离子通道染色，测试线粒体的极化状态，使用电生理学建立离子通道功能，以及进行活力测定以评估药物效力。
Introduction
膜转运蛋白对于细胞之间的通讯以及维持细胞稳态至关重要。在膜转运蛋白中，离子通道用于驱动细胞的生长和发育，并在具有挑战性和不断变化的环境中维持细胞的状态。据报道，离子通道还可以驱动和支持实体瘤的发展，包括全身和中枢神经系统（CNS）1,2。例如，KCa3.1通道负责调节膜电位和控制细胞体积，这在细胞周期调节中很重要。据报道，有缺陷的KCa3.1通道有助于肿瘤细胞的异常增殖3。此外，离子通道可能有助于癌症的转移性播散。例如，瞬时受体电位 （TRP） 通道参与 Ca 2+ 和 Mg2+ 内流;这种涌入激活了几种激酶和热休克蛋白，它们的作用是调节肿瘤周围的细胞外基质，这反过来又对启动癌症转移很重要4。
由于离子通道有助于癌症的发展，它们也可能是药物相关癌症治疗的靶标。例如，对治疗方式（包括化疗和新型免疫疗法）的耐药性与离子通道功能失调有关5,6,7。此外，离子通道正在成为阻碍癌症生长和发展的重要药物靶点，正在研究重新利用的小分子（FDA批准）药物以及生物聚合物，包括单克隆抗体1,2,8,9。虽然在这方面取得了很大进展，但离子通道癌症药物的发现仍然不发达。这部分是由于研究癌细胞中离子通道的独特挑战。例如，在为慢效化合物设置电生理学测定方面存在技术限制，并且在通道活化和药物作用方面存在时间差异。此外，化合物的溶解度也会阻碍进展，因为目前常用的大多数自动化电生理系统都使用疏水底物，这可能会导致化合物吸附而导致伪影。此外，使用常规电生理学测定进行筛选大型生物有机分子疗法（如天然产物、肽和单克隆抗体）在技术上具有挑战性10。最后，癌细胞的生物电特性仍然知之甚少11。
同时，离子通道的免疫荧光染色通常具有挑战性。这部分是由于它们的结构和它们在膜中的环境的复杂性，这会影响产生和使用抗体进行显微镜研究的能力。尤其重要的是，对用于染色离子通道的抗体的特异性、亲和力和重现性进行验证。应根据离子通道的商业抗体的验证策略和发表记录来考虑。实验应包括阴性对照，以证明通过敲低或敲除目标蛋白缺乏非特异性结合。或者，基于mRNA或蛋白质测定的靶蛋白不存在或丰度低的细胞系可以作为阴性对照。例如，这项研究显示了（GABA）受体亚基Gabra5在髓母细胞瘤细胞系（D283）中的定位。对具有siRNA敲低的D283细胞和另一种小脑髓母细胞瘤细胞系Daoy细胞进行Gabra5染色，并且没有明显的染色（数据未显示）。
本文介绍了分析和测定离子通道功能以及离子通道调节剂对癌细胞的影响的方法。为（1）离子通道染色细胞，（2）测试线粒体的极化状态，（3）使用电生理学建立离子通道功能以及（4）体外药物验证提供了方案。这些协议强调A型γ-氨基丁酸（GABAA）受体2,12,13,14,15,16，氯化阴离子通道和主要抑制性神经递质受体的研究。然而，这里介绍的方法适用于研究许多其他癌细胞和离子通道。
Protocol
1. 培养细胞中的免疫标记离子通道 准备细胞和实验装置将细胞维持在75cm2 培养瓶中作为活跃生长的培养物。传代一次细胞，直到它们变得50%-90%汇合，具体取决于所用细胞系的倍增时间。注意：对于本研究，使用了D283细胞，即第3组髓母细胞瘤细胞系。 将培养瓶中的细胞收集到离心管（15 mL或50 mL）中，并加入2 mL的0.25%胰蛋白酶-EDTA以分离贴壁细胞。在室温（RT）下?…
Representative Results
以上是可用于表征癌细胞中离子通道的选择程序。第一个方案突出显示离子通道的染色。如前所述，在染色离子通道或就此而言，染色细胞外膜中存在的任何蛋白质时存在许多挑战。图1所示是五聚体GABAA受体亚基的染色。第二个协议强调了测试癌细胞中线粒体极化状态的结果。线粒体对细胞活力和增殖以及细胞死亡起着至关重要的作用。在哺乳动物细胞中，线粒体?…
Discussion
离子通道功能的变化会改变细胞内信号级联，从而影响细胞的整体功能。在过去的十年中，越来越清楚的是离子通道对癌细胞的生长和转移很重要。重要的是，许多离子通道是针对各种疾病的已获批疗法的主要靶标24。研究人员已经探索了离子通道是否可能是抗癌靶点，初步结果是有希望的2,16,25。该领域刚刚…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢Thomas E. & Pamela M. Mischell Family Foundation对S.S.的支持，以及Harold C. Schott基金会对加州大学医学院Harold C. Schott捐赠主席的资助。
Materials
| ABS SpectraMax Plate Reader | Molecular Devices | ABS | |
| Accutase | Invitrogen | 00-4555-56 | |
| Alexa Flor 488 | Invitrogen | A32723 | Goat Anti-Rabbit |
| Antibiotic-Antimycotic | Gibco | 15240-062 | 100x |
| B27 Supplement | Gibco | 12587-010 | Lacks vitamin A |
| Biosafety Cabinet | LABCONCO | 302381101 | Class II, Type A2 |
| Bovine Serum Albumin | Fisher Scientific | BP1606-100 | |
| CO2 Incubator | Fisher Scientific | 13-998-211 | Heracell VIOS 160i |
| Calcium Chloride | Fisher Scientific | C7902 | Dihydrate |
| Cell Culture Dishes, 150 mm | Fisher Scientific | 12-600-004 | Cell culture treated |
| Cell Culture Flasks, 75 cm2 | Fisher Scientific | 430641U | Cell culture treated |
| Cell Culture Plates, 6 well | Fisher Scientific | 353046 | Cell culture treated |
| Cell Culture Plates, 96 well | Fisher Scientific | 353072 | Cell culture treated |
| Centrifuge | Eppendorf | EP-5804R | Refrigerated |
| Corning CoolCell | Fisher Scientific | 07-210-0006 | |
| Coverslips, 22 x 22 mm | Fisher Scientific | 12-553-450 | Corning brand |
| D283 Med | ATCC | HTB-185 | |
| DABCO Mounting Media | EMS | 17989-97 | |
| D-Glucose | Sigma Life Sciences | D9434 | |
| Dimethyl Sulfoxide | Sigma Aldrich | D2650 | Cell culture grade |
| DMEM/F12, base media | Fisher Scientific | 11330-032 | With phenol red |
| DMEM/F12, phenol red free | Fisher Scientific | 21041-025 | |
| EGTA | Sigma Aldrich | E4378 | |
| Epidermal Growth Factor | STEMCELL | 78006.1 | |
| FCCP | Abcam | AB120081 | |
| Fetal Bovine Serum, Qualified | Gibco | 10437-028 | |
| Fibroblast Growth Factor, Basic | Millipore | GF003 | |
| GARBA5 Antibody | Aviva | ARP30687_P050 | Rabbit Polyclonal |
| Glutamax | Gibco | 35050-061 | |
| Glycerol Mounting Medium | EMS | 17989-60 | With DAPI+DABCO |
| Hemocytometer | Millipore Sigma | ||
| Heparin | STEMCELL | 7980 | |
| HEPES | HyClone | SH3023701 | Solution |
| HEPES | Fisher Scientific | BP310-500 | Solid |
| ImageJ | Open platform | With Fiji plugins | |
| Immuno Mount DAPI | EMS | 17989-97 | |
| KRM-II-08 | experimental compounds not available from a commercial source | ||
| Leica Application Suite X | Leica Microsystems | ||
| Leukemia Inhibitory Factor | Novus | N276314100U | |
| L-Glutamine | Gibco | 25030-081 | |
| Magnesium Chloride | Sigma Aldrich | M9272 | Hexahydrate |
| Microscope, Confocal | Leica | SP8 | |
| Microscope, Light | VWR | 76382-982 | DMiL Inverted |
| MTS – Promega One Step | Promega | G3581 | |
| Multi-channel pipette, 0.5-10 µL | Eppendorf | Z683914 | |
| Multi-channel pipette, 10-100 µL | Eppendorf | Z683930 | |
| Multi-channel pipette, 30-300 µL | Eppendorf | Z683957 | |
| Nest-O-Patch | Heka | ||
| Neurobasal-A Medium | Gibco | 10888022 | Without vitamin A |
| Neurobasal-A Medium | Gibco | 12348-017 | Phenol red free |
| Non-Essential Amino Acids | Gibco | 11140-050 | |
| NOR-QH-II-66 | experimental compounds not available from a commercial source | ||
| Parafilm | Fisher Scientific | 50-998-944 | 4 inch width |
| Paraformaldehyde | EMS | RT-15710 | |
| PATHCHMASTER | Heka | ||
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| Perfusion System | Nanion | 4000120 | |
| PFA | EMS | RT-15710 | |
| Phosphate Bufered Saline | Fisher Scientific | AAJ75889K2 | Reagent grade |
| Poly-D-Lysine | Fisher Scientific | A3890401 | |
| Poly-L-Lysine | Sigma Life Sciences | P4707 | |
| Port-a-Patch | Nanion | 21000072 | |
| Potassium Chloride | Sigma Life Sciences | P5405 | |
| Primary Antibody | Invitrogen | MA5-34653 | Rabbit Monoclonal |
| Prism | GraphPad | ||
| Propofol | Fisher Scientific | NC0758676 | 1 mL ampule |
| QH-II-66 | experimental compounds not available from a commercial source | ||
| Reagent Reservoirs | VWR | 89094-664 | Sterile |
| Slides, 75 x 25 mm | Fisher Scientific | 12-544-7 | Frosted one side |
| Sodium Bicarbonate | Corning | 25-035-Cl | |
| Sodium Chloride | Fisher Scientific | S271-3 | |
| Sodium Pyruvate | Gibco | 11360-070 | |
| Synth-a-Freeze Medium | Gibco | R00550 | Cryopreservation |
| TMRE | Fisher Scientific | 50-196-4741 | Reagent |
| TMRE Kit | Abcam | AB113852 | Kit |
| Triton X-100 | Sigma Aldrich | NC0704309 | |
| Trypan Blue | Gibco | 15-250-061 | Solution, 0.4% |
| Trypsin/EDTA | Gibco | 25200-072 | Solution, 0.25% |
| Vortex Mixer | VWR | 97043-562 | |
| Whatman Filter Paper | Fisher Scientific | 09-927-841 |
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Citazione di questo articolo
Kallay, L., Gawali, V. S., Toukam, D. K., Bhattacharya, D., Jenkins, A., Sengupta, S., Pomeranz Krummel, D. A. Screening Ion Channels in Cancer Cells. J. Vis. Exp. (196), e65427, doi:10.3791/65427 (2023).