RESULTS LY30 restores HeLa cells sensitivity to TRAIL-induced cell death Dose response curves for TRAIL and LY30 were established in HeLa cells by evaluating the effect of increasing doses of TRAIL (0 to 250 ng/mL) and LY30 (0 to 25 µM), alone or in combination, on cell viability after 16h by using the crystal violet assay (Figure 13A) HeLa cells did not exhibit a decrease in cell viability upon TRAIL treatment even at the highest dose used (250 ng/mL) However, preincubation with different doses of LY30 for 1h significantly reduced cell viability (50% viability with 25 !M LY30 and 20 ng/mL TRAIL in combination) as compared to both treatment alone (85% viability for 25 µM LY30 and 100% viability for 20 ng/mL), hence corroborating our earlier finding that LY30 is able to sensitize HeLa cells to TRAIL-induced cell death [156] Interestingly, the observed sensitization was enhanced in a dose dependent manner as a function of TRAIL dose but not of LY30 dose Further cell viability assays for TRAIL treatment alone were carried out over a longer time frame (up to 72h) (Figure 13B) HeLa cells viability was not decreased upon treatment with TRAIL for a longer period Only HeLa cells treated with 250 ng/mL of TRAIL for 72h showed a modest effect of TRAIL on cell viability For subsequent experiments, the chosen doses of LY30 and TRAIL were 25 !M and 20 ng/mL respectively At those doses, HeLa cells viability was decreased in a time dependent manner (Figure 13C) 84 LY30 and TRAIL combined treatment decreases HeLa cells ability to form colonies Given the ability of LY30 and TRAIL combination to induce cell death upon short-term treatment, we were interested in understanding the effect of LY30+TRAIL on the long-term survival/proliferative capacity of tumor cells This was done by evaluating the effect of the combined drug treatment on HeLa cells colony forming ability HeLa cells were exposed to LY30 for 1hr before incubation with TRAIL for 6h An equal number of cells were then seeded onto 100mm Petri dishes and allowed to form colonies over a period of 10 to 14 days After staining the cells with crystal violet, we observed a marked reduction in the number of colonies formed for cells treated with both compounds (Figure 14A) A significant decrease in clonogenic ability (35% of untreated cells) was observed for cells under combinatorial treatment as compared to cells treated singly with either TRAIL or LY30 (Figure 14B) Interestingly, TRAIL alone, and to a lesser extent LY30 alone, induced a significant decrease of the size of the colonies (40% when compared to control) (Figure 14C) The combined treatment further reduced the size of the colonies This observation explains the discrepancy between the actual number of colonies counted after TRAIL treatment and the apparent low number of colonies in the picture itself 85 Figure 13: Effect of LY30 treatment on TRAIL-mediated apoptosis Hela cells exposed to TRAIL (0-250 ng/mL) for 16h with or without 1h preincubation with various doses of LY30 (0-25 !M) (A), treated with TRAIL alone (0250 ng/mL) for 24h, 48h or 72h (B) or treated with 20ng/mL of TRAIL for different times (4h-24h) with or without 1h pre-incubation with 25 !M of LY30 (C) The percentage of cell survival was determined by crystal violet assay Data shown are the mean±S.D of three independent experiments NT: Non-treated; LY: treatment with LY30; T: treatment with TRAIL; LYT: treatment with LY30 and TRAIL 86 Figure 14: Effect of LY30 and TRAIL treatment on colony forming ability of HeLa cells HeLa cells were exposed to 20 ng/ml TRAIL for 6h with or without pre-incubation with 25 !M LY30 The cells were then re-seeded onto 100 mm Petri dishes and allowed to form colonies over 10 to 14 days, followed by staining with crystal violet (A) for colony counts (B) and colony size (C) determination Data shown are the mean±S.D of three independent experiments *p