All four compounds at 10 M inhibited the proliferation of A2780-SP cells by more than 3-fold (Figure 2D)
January 21, 2022
All four compounds at 10 M inhibited the proliferation of A2780-SP cells by more than 3-fold (Figure 2D). paclitaxel showed enhanced effect in ovarian CSCs xenograft mouse models. Our results suggested that four CCBs may be potential therapeutic drugs for preventing ovarian cancer recurrence. and than those in A2780 cells (Figure S1B). Next, using A2780-SP cells, we screened the FDA-approved compound library to identify drug candidates that inhibit proliferation of ovarian CSCs. The library was first A 740003 screened for compounds selective for CSCs through sphere viability and sphere formation assay using Rabbit Polyclonal to CKS2 a high-throughput screening system, followed by cytotoxicity testing A 740003 (Figure 1A). Open in a separate window Figure 1 Screening of four CCBs and their effects on CSC sphere formation. (A) Schematic of the screening stage. (B) A2780 and A2780-SP cells were seeded in 96-well plates. After 24 h, 10 M compound was added to the cells. After 3 days (A2780) or 8 days (A2780-SP), ATP-based cell viability was detected by luminescence assay. (C) A2780 and A2780-SP cells were seeded at 10,000 cells per A 740003 well in 6-well plates. After 3 days, 10 M compound was added to the cells in each well. At 7 days after compound treatment, they were stained with 5% crystal violet (left panel). Dye was extracted using 0.1% SDS and then quantified using a spectrophotometer at 590nm (right panel). (D) A2780-SP cells were seeded in ultra-low attachment round bottom 96-well plates. After 24 h, apigenin and four CCBs were added to the cells at each concentration. After 8 days, sphere cells were imaged under a microscope (left panel) and the sphere size was quantified (right panel). (E) SKOV3-SP cells were seeded in ultra-low attachment round bottom 96-well plates. After 24h, apigenin and four CCBs were added to the cells at 10 M. After 8 days, ATP-based cell viability was detected by luminescence assay. Data are expressed as mean SD of three independent experiments; * 0.05, ** 0.01, *** 0.001; nsnot significant compared with DMSO. For sphere viability and sphere formation assay, A2780-SP cells were seeded to form a sphere and then treated with 1018 FDA-approved compounds at a concentration of 10 M. Next, the sphere size and viability were measured after 8 days of incubation (Figure S2A,B). Apigenin, a natural flavone known to reverse drug resistance in CSCs and inhibit the growth of SKOV3-derived sphere cells, was used as a reference compound . We identified 104 compounds that reduced sphere size to more than 90% compared with DMSO control (Figure S2B, left). The result of the ATP-based cell viability test also showed that 127 compounds reduced sphere viability to more than 90% compared with DMSO control (Figure S2B, right). Collectively, we selected 97 compounds that reduced both sphere size and viability to more than 90% compared with DMSO control. Next, we performed cytotoxicity tests to exclude relatively cytotoxic substances from the 97 selected compounds (Figure S2C). Cytotoxicity tests were performed by treating two normal fibroblast cells, NIH-3T3 and BJ6, with the selected compounds and reference compounds, including 5FU and doxorubicin. From the result, we selected 21 compounds that resulted in more than 80% viability in both BJ6 and NIH-3T3 cells (Figure S2C). Among the 21 compounds selected for the subsequent experiments, 15 compounds were orally available drugs and 4 were calcium channel blockers (Figure 1A). 2.2. Calcium Channel Blockers (CCBs) Inhibit Sphere Formation and Viability Four out of the 15 selected compounds target calcium channels, and the remaining 11 compounds each have different targets. This suggests that calcium channels are important for maintaining ovarian CSCs and the effect of these compounds on ovarian CSCs can be originated by targeting calcium channels directly. Therefore, we selected the four CCBs to investigate their role and mode of action in CSCs. We first explored the effects of these four CCBs on.