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Cancer Research

Screening Ion Channels in Cancer Cells

Published: June 16th, 2023

DOI:

10.3791/65427

1Department of Neurology and Rehabilitation Medicine, Division of Neuro-Oncology, University of Cincinnati College of Medicine, 2Department of Pharmaceutical Sciences, School of Pharmacy, University of Saint Joseph, 3The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine

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....

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1. Immunolabeling ion channels in cultured cells

  1. Preparing the cells and experimental set-up
    1. Maintain the cells as an actively growing culture in 75 cm2 culture flasks. Passage the cells once until they become 50%-90% confluent, depending on the doubling time of the cell line being used.
      NOTE: For the present study, D283 cells, a Group 3 medulloblastoma cell line, were used.
    2. Collect the cells from the culture flask into a centrifuge tube (15 mL or 50 mL), and add 2 mL.......

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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.......

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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

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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.

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NameCompanyCatalog NumberComments
ABS SpectraMax Plate ReaderMolecular DevicesABS
AccutaseInvitrogen00-4555-56
Alexa Flor 488InvitrogenA32723Goat Anti-Rabbit
Antibiotic-AntimycoticGibco15240-062100x
B27 SupplementGibco12587-010Lacks vitamin A
Biosafety CabinetLABCONCO302381101Class II, Type A2
Bovine Serum AlbuminFisher ScientificBP1606-100
CO2 IncubatorFisher Scientific13-998-211Heracell VIOS 160i
Calcium ChlorideFisher ScientificC7902Dihydrate
Cell Culture Dishes, 150 mmFisher Scientific12-600-004Cell culture treated
Cell Culture Flasks, 75 cm2Fisher Scientific430641UCell culture treated
Cell Culture Plates, 6 wellFisher Scientific353046Cell culture treated
Cell Culture Plates, 96 wellFisher Scientific353072Cell culture treated
CentrifugeEppendorfEP-5804RRefrigerated
Corning CoolCellFisher Scientific07-210-0006
Coverslips, 22 x 22 mmFisher Scientific12-553-450Corning brand
D283 MedATCCHTB-185
DABCO Mounting MediaEMS17989-97
D-GlucoseSigma Life SciencesD9434
Dimethyl SulfoxideSigma AldrichD2650Cell culture grade
DMEM/F12, base mediaFisher Scientific11330-032With phenol red
DMEM/F12, phenol red freeFisher Scientific21041-025
EGTASigma AldrichE4378
Epidermal Growth FactorSTEMCELL78006.1
FCCPAbcamAB120081
Fetal Bovine Serum, QualifiedGibco10437-028
Fibroblast Growth Factor, BasicMilliporeGF003
GARBA5 AntibodyAvivaARP30687_P050Rabbit Polyclonal
GlutamaxGibco35050-061
Glycerol Mounting MediumEMS17989-60With DAPI+DABCO
HemocytometerMillipore Sigma
HeparinSTEMCELL7980
HEPESHyCloneSH3023701Solution
HEPESFisher ScientificBP310-500Solid
ImageJOpen platformWith Fiji plugins
Immuno Mount DAPIEMS17989-97
KRM-II-08experimental compounds not available from a commercial source
Leica Application Suite XLeica Microsystems
Leukemia Inhibitory FactorNovusN276314100U
L-GlutamineGibco25030-081
Magnesium ChlorideSigma AldrichM9272Hexahydrate
Microscope, ConfocalLeicaSP8
Microscope, LightVWR76382-982DMiL Inverted
MTS - Promega One StepPromegaG3581
Multi-channel pipette, 0.5-10 µLEppendorfZ683914
Multi-channel pipette, 10-100 µLEppendorfZ683930
Multi-channel pipette, 30-300 µLEppendorfZ683957
Nest-O-PatchHeka
Neurobasal-A MediumGibco10888022Without vitamin A
Neurobasal-A MediumGibco12348-017Phenol red free
Non-Essential Amino AcidsGibco11140-050
NOR-QH-II-66experimental compounds not available from a commercial source
ParafilmFisher Scientific50-998-9444 inch width
ParaformaldehydeEMSRT-15710
PATHCHMASTERHeka
Penicillin-StreptomycinGibco15140-122
Perfusion SystemNanion4000120
PFAEMSRT-15710
Phosphate Bufered SalineFisher ScientificAAJ75889K2Reagent grade
Poly-D-LysineFisher ScientificA3890401
Poly-L-LysineSigma Life SciencesP4707
Port-a-PatchNanion21000072
Potassium ChlorideSigma Life SciencesP5405
Primary AntibodyInvitrogenMA5-34653Rabbit Monoclonal
PrismGraphPad
PropofolFisher ScientificNC07586761 mL ampule
QH-II-66experimental compounds not available from a commercial source
Reagent ReservoirsVWR89094-664Sterile
Slides, 75 x 25 mmFisher Scientific12-544-7Frosted one side
Sodium BicarbonateCorning25-035-Cl
Sodium ChlorideFisher ScientificS271-3
Sodium PyruvateGibco11360-070
Synth-a-Freeze MediumGibcoR00550Cryopreservation
TMREFisher Scientific50-196-4741Reagent
TMRE KitAbcamAB113852Kit
Triton X-100Sigma AldrichNC0704309
Trypan BlueGibco15-250-061Solution, 0.4%
Trypsin/EDTAGibco25200-072Solution, 0.25%
Vortex MixerVWR97043-562
Whatman Filter PaperFisher Scientific09-927-841

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