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

מיקוד פרמקולוגי של תעלות יונים לטיפול בגידולים מוצקים

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.

מחבר זרקור: חקר תפקידן של תעלות יונים בסרטן: אפיון וגישות טיפול פוטנציאליות 

בדיקת תעלות יונים בתאים סרטניים

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  

תיקוף תרופתי חוץ גופי לבדיקת תפקוד תעלת היונים בתאים סרטניים

כדי להתחיל, צלחת את התאים 75 מיקרוליטר לכל באר של תווך ללא אנטיביוטיקה ללא חיץ HEPES כדי לקבוע IC50. ביום השני, הכינו את פתרון הבקרה ואת פתרונות הבדיקה על ידי ביצוע דילולים סדרתיים של תמיסות מלאי בריכוז גבוה. לאחר הוספת תמיסות בקרה ובדיקה לתאים, יש לדגור על התאים בטמפרטורה של 37 מעלות צלזיוס ופחמן דו חמצני של 5% בסביבה לחה למשך 48 שעות.התחל את בדיקת התפשטות התאים על ידי מערבול תחילה של מגיב MTS כדי להמיס באופן מלא כל מגיב מואץ לפני השימוש ביום השלישי של הניסוי להעביר את ריאגנטי MTS המופשרים למאגר מגיבים סטרילי של 25 מיליליטר. פיפטה 20 מיקרוליטר של מגיב MTS לכל באר של צלחת 96 באר המכילה דגימות ו -100 מיקרוליטר של מדיום תרבית. דוגרים על הצלחת בטמפרטורה של 37 מעלות צלזיוס ו-5% פחמן דו-חמצני בסביבה לחה למשך שעה עד ארבע שעות.לאחר מכן, צנטריפוגו את הצלחת בטמפרטורת החדר כדי להסיר בועות שעלולות להפריע לקריאת הספיגה. מדוד את הספיגה ב 490 ננומטר עם קורא microplate או ספקטרופוטומטר. צלחת 96 בארות המציגה את התוצאות הקלורימטריות של בדיקת MTS עם ריכוזי תרופות הולכים וגדלים מוצגת.עקומת תגובת המינון הראתה כי סוכן הבדיקה פוגע ביכולת הקיום של התאים הסרטניים באופן תלוי מינון.

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Research

JoVE Journal

Cancer Research

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

450 Views.  University of Cincinnati College of Medicine. כדי להתחיל, צלחת את התאים 75 מיקרוליטר לכל באר של תווך ללא אנטיביוטיקה ללא חיץ HEPES כדי לקבוע IC50. ביום השני, הכינו את פתרון הבקרה ואת פתרונות הבדיקה על ידי ביצוע דילולים סדרתיים של תמיסות מלאי בריכוז גבוה. לאחר הוספת תמיסות בקרה ובדיקה לתאים, יש לדגור על התאים בטמפרטורה של 37 מעל...

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

Screening Ion Channels in Cancer Cells

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.

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 ...

Testing the Polarized State of Mitochondria to Analyze the Ion Channel Function in Cancer Cells 

To begin, maintain cells in a 75-square-centimeter culture flask. Aspirate the culture medium and rinse the cells with PBS without calcium and magnesium. Next, add two milliliters of 0.25%trypsin EDTA to detach adherence cells.Incubate for five minutes at room temperature and resuspend the cells in 10 milliliters of medium. Next, collect the cells from the flask into a 15-milliliter centrifuge tube. Centrifuge at 480 G for five minutes at room temperature and aspirate the medium.Resuspend the cells in 5 to 10 milliliters of medium based on the original confluency of the culture. Remove 100 microliters of cells and add to a 1.5-milliliter tube with 100 microliters of 0.4%trypan blue. Add the cells to the hemocytometer, and count them by observing under the microscope and using the manual tally counter.Dilute the cells to three times 10 to the fourth cells per milliliter in the culture medium without phenol red. Add 2.5 milliliters of the cell suspension to each well of a six-well plate with poly-D-lysine-coated cover slip per the manufacturer's directions. Grow the cells overnight at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator to enable cell attachment to the cover slip.The next day, examine cell attachment under an inverted microscope using a 20X objective. Prepare the treatment stock solutions at the desired concentrations and create a media-only control, a vehicle-only control at a concentration that matches the test compound, and a control with the ionophore FCCP and the test compounds. Working with one cover slip at a time, add 1.8 milliliters of medium for the condition under the study to the cells.Incubate the cells treated with either the control or test compound at 37 degrees Celsius and 5%carbon dioxide in a humidified environment for the desired time. Following the treatment period, add 200 microliters of 10X TMRE to the cells. Incubate for 15 to 30 minutes at 37 degrees Celsius in a 5%carbon dioxide humidified environment.Gently aspirate the medium and rinse the cells two times with PBS to minimize background interference. Then invert the cover slip onto a microscope slide labeled with the treatment conditions. Image the cells live at 549 nanometers excitation and 575 nanometers emission.Utilize a confocal microscope to capture several Z-stack fields with high-speed image capture before the cell integrity is lost. Save and store the image file for later analysis. The cells with active mitochondria retained TMRE staining and showed high fluorescence.Depolarized or inactive mitochondria have reduced membrane potential, fail to retain TMRE, and show a low fluorescence signal.

בדיקת המצב המקוטב של המיטוכונדריה לניתוח תפקוד תעלת היונים בתאים סרטניים 

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.