Human H69AR Lung Tumor Cell Growth Inhibition Assay - 86K Screen
Compounds that inhibit cell growth may have cytotoxic or cytostatic effects and vary widely in structure and mechanism of action. Most cancer chemotherapeutic drugs inhibit tumor cell growth by disrupting cell division, which often results in apoptotic cell death. Intrinsic or acquired drug resistance is a significant clinical obstacle, which limits the efficacy of many cancer chemotherapeutic more ..
BioActive Compounds: 5141
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
(Proposal number 1X01-MH077620-01) Submitted by Gary A. Piazza of Southern Research Institute
Compounds that inhibit cell growth may have cytotoxic or cytostatic effects and vary widely in structure and mechanism of action. Most cancer chemotherapeutic drugs inhibit tumor cell growth by disrupting cell division, which often results in apoptotic cell death. Intrinsic or acquired drug resistance is a significant clinical obstacle, which limits the efficacy of many cancer chemotherapeutic drugs. A major mechanism responsible for drug resistance involves the increased efflux of anticancer drugs outside of the cell by specific membrane transport proteins, thereby reducing the intracellular concentration and potency.
Here we describe a high throughput screening campaign to identify compounds that are capable of inhibiting the growth of the human H69AR small cell lung tumor cell line, which over-expresses the MRP-1 transport protein and displays resistance to a number of cancer chemotherapeutic drugs. Numerous classes of compounds may cause cytotoxicity by perturbing a variety of intracellular signaling pathways, interfering with mitosis, or disrupting various aspects of cell function, which will result in the reduction of viable cell number that can be measured by a variety of endpoints such as metabolic activity and ATP levels. Compounds that inhibit the growth of the H69AR line have the potential to evade efflux by the MRP-1 transporter and provide a preliminary indication of anticancer activity for treating patients with small cell lung carcinoma who may have failed cancer chemotherapy.
Cultured human H69AR lung tumor cells were treated with test compounds from a 10,000 compound subset of the NIH Small Molecule Repository for 72 hours at a final concentration of 10 micromolar. Following treatment, ATP was measured using a commercially available assay (Cell Titer Glo, Promega). The assay involves a recombinant luciferase enzyme that converts luciferin to oxyluciferin in the presence of Mg ions, oxygen, and ATP, and is accompanied by the emission of light, or chemiluminesence. Each plate contained 32 replicates of untreated cells which served as a negative control. Percent cell viability values were calculated based on two separate experiments.
Cell Culture: H69AR cells (ATCC) were subcultured every 3-4 days and grown in RPMI-1640 medium supplemented with 4.5 g/L glucose, 10 mM HEPES pH 7.4, 10% FBS, and 2 mM glutamine (growth medium). Cells were grown at 37C in 5% CO2, and maintained at a subconfluent density.
Plating: Twenty five microliters of cells were dispensed (1250 cells per 25 microliters) in complete growth medium containing 100 Units/ml penicillin and 100 micrograms/ml streptomcyin. Cells were maintained on ice during plating with stirring. Plates were incubated at room temperature for 1 hr prior to placing in incubator at 37C, 5% CO2, and high humidity overnight.
Dosing: Dilution media, carrier and positive control drug were dispensed to dilution plate. Compounds were diluted in complete growth medium to 7X final concentration (70 micromolar). Five microliters of dilution was transferred to the cell plate followed by 5 microliters complete growth medium. Plates were incubated for 72 hours at 37C, 5% CO2, and high humidity.
At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature. As per the manufacturer's protocol, an equal volume of Cell Titer Glo reagent was added and plates were incubated for an additional 10 minutes. At the end of the incubation, assay plates were analyzed using a Perkin Elmer Envision microplate reader with a read time of 0.1 seconds in luminescence mode.
Assay data were analyzed using ActivityBase from IDBS. Experimental data were reported as percent cell viability and calculated by dividing luminescence values from treated wells by the average luminescence values from untreated control wells. Any compound that exhibited lower % viability than the cutoff parameter was declared active.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Compounds that reduced cell viability to <=40% were assigned an outcome of Active. Compounds exhibiting cell viability values >40% were scored as inactive.
Because of the inherent error in all high throughput screens, especially involving single replicate and concentration testing, the compounds that were active were assigned a ranking of 100, while inactive compounds were assigned a rank of 0.
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Data Table (Concise)