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The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.
Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.
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. Defect....
1. Immunolabeling ion channels in cultured cells
Above are select procedures that can be employed to characterize ion channels in cancerous cells. The first protocol highlights the staining of an ion channel. As detailed, there are many challenges when staining an ion channel or, for that matter, any protein that is present in the extracellular membrane. Shown in Figure 1 is the staining for a subunit of the pentameric GABAA receptor. The second protocol highlights the results of testing the polarized state of mitochondria in ca.......
Changes in ion channel function alter intracellular signaling cascades, which can impact the overall functioning of a cell. Over the past decade, it has become increasingly clear that ion channels are important to cancer cell growth and metastasis. Importantly, many ion channels are primary targets for approved therapeutics targeting a broad range of disorders24. Investigators have probed whether ion channels could be anti-cancer targets, and the initial results are promising2
The authors acknowledge support from the Thomas E. & Pamela M. Mischell Family Foundation to S.S. and the Harold C. Schott Foundation funding of the Harold C. Schott Endowed Chair, UC College of Medicine, to S.S.
....Name | Company | Catalog Number | Comments |
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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