Name: In Vitro Drug Validation to Assay Ion Channel Function in Cancer Cells
Uploaded: 2023-06-16T12:00:00
Description: Watch this Scientific Journal Video about In Vitro Drug Validation to Assay Ion Channel Function in Cancer Cells at JoVE.com

in vitro drug validation to assay ion channel function in cancer cells

Pharmacological Targeting of Ion Channels to Treat Solid Tumors

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.

Author Spotlight: Exploring the Role of Ion Channels in Cancer: Characterization and Potential Treatment Approaches 

Screening Ion Channels in Cancer Cells

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.

Watch this Scientific Journal Video about In Vitro Drug Validation to Assay Ion Channel Function in Cancer Cells at JoVE.com

In Vitro Drug Validation to Assay Ion Channel Function in Cancer Cells  

To begin, plate the cells in 75 microliters per well of antibiotic-free medium without HEPES buffer to determine the IC50. On the second day, prepare the control solution and the test solutions by performing serial dilutions of high-concentration stock solutions. After adding control and test solutions to the cells, incubate the cells at 37 degrees Celsius and 5%carbon dioxide in a humidified environment for 48 hours.Begin the cell proliferation assay by first vortexing the MTS reagent to fully dissolve any precipitated reagent before usage on day three of the experiment transfer the thawed MTS reagents into a sterile, 25-milliliter reagent reservoir. Pipette 20 microliters of MTS reagent into each well of the 96-well plate containing samples and 100 microliters of culture medium. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide in a humidified environment for one to four hours.Then, centrifuge the plate at room temperature to remove any bubbles that may interfere with the absorbance reading. Measure the absorbance at 490 nanometers with a microplate reader or spectrophotometer. A 96-well plate showing the calorimetric results of an MTS assay with increasing drug concentrations is presented.The dose response curve showed that the test agent impairs the viability of the cancer cells in a dose-dependent manner.

Research

JoVE Journal

Cancer Research

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