WYC-209 inhibits proliferation of malignant murine melanoma tumor-repopulating cells (TRCs, IC50: 0.19 uM). It targets the retinoic acid receptor (RAR).

TRC:0.19 μM

WYC-209 inhibits proliferation of malignant murine melanoma tumor-repopulating cells (TRCs), with an IC50 of 0.19?μM in a dose-dependent manner. WYC-209 also inhibits proliferation of TRCs of human melanoma, lung cancer, ovarian cancer, and breast cancer in culture.

WYC-209 abrogates 87.5% of lung metastases of melanoma TRCs in immune-competent wild-type C57BL/6 mice at 0.22?mg?kg-1 without showing apparent toxicity.

Cell proliferation rate was measured by using MTT colorimetric assay. Cells were seeded in 3D soft fibrin gel in 96-well microplates at a density to maintain control (untreated) cells in an exponential phase of growth during the entire experiment. Cells were incubated with various concentrations of compounds for each time point followed by incubation with 50?μL MTT reagent for 3?h at 37?°C. After incubation, 150?μL MTT solvent was added and absorbance was measured at 570?nm. All experiments were repeated at least three times. The percentage of viable cells was calculated and averaged for each well: percent growth?=?(OD-treated cells/OD control cells)?×?100, cell proliferation at each time point is normalized to base line cell survival at the time of initial compound treatment (day 0).

Four- to six-week-old female and male C57BL/6 mice were used in mice experiment. Mice were randomized into different groups. In metastasis experiment, B16-F1 cell spheroids were selected from 3D 90-Pa fibrin gels and pipetted to single cells. These TRCs were harvested and the cell number was counted under microscopy. The cells were then suspended in PBS with appropriate cell density. Thirty thousand TRCs were intravenously injected into the tail vein of each wild-type C57BL/6 mouse. Five days later, inoculated mice were intravenous implanted with 0.022?mg/kg WYC-209, 0.22?mg/kg WYC-209, or 0.1% DMSO every two days. The mice were euthanized and examined for lung tumor formation at day 30. In treated TRCs injected in vivo experiments, B16-F1 cells were seeded into 3D 90-Pa fibrin gels and treated with 10?μM WYC-209 or 0.1% DMSO on day 3, then isolated treated TRCs colonies on day 5 and pipetted into single cell. 30,000 of TRCs were intravenously injected into the tail vein of each wild-type C57BL/6 mouse. No blinding was performed in the mice experiments.