Fig. 3. Camptothecin-mediated activation of pro-proliferative responses.
a HCT-116 and A549 cells were treated with CPT (250 nM) for 24, 36, and 48 h along with their respective vehicle controls; cells were analyzed for their invasion capability through Boyden chamber assay system. Images were taken under an inverted microscope (Nikon Eclipse 200) at ×10 magnification. Bar graphs represent the average number of migrated cells per field (n = 3, error bars ± s.d.); ***p < 0.001, **p < 0.0011, p = 0.9125 and p = 0.2657. b Spheroids were prepared with HCT-116 cells embedded in ECM matrix by hanging drop method and treated with indicated concentration of CPT for given time points. Migration of cells from the core of the spheroids was observed (upper panel) under inverted microscope at ×20 magnification. The corresponding threshold images (lower panel) were prepared by using Image j software. c HCT-116 cells treated for indicated time pulse with 250 nM of CPT; whole cell lysates were analyzed for the expression of MMP-2 and MMP-9 protein through immunoblot analysis. d Conditional media was collected from the above experiment and subjected to gelatin zymography to examine the gelatinase activity of MMP-2 and MMP-9; bovine serum albumin (BSA) was taken as a loading control. e HCT-116 cells were treated with indicated concentration of CPT for 0, 12, 24, 36, and 48 h; the whole cell lysates were subjected to western blotting for the analysis of NFκB, p-AKT, AKT, Survivin, and c-FLIPS/L proteins. f CPT (0.4, 0.8, and 1.2 mg/kg) treatment was orally given to induced AhCre-ErT Apcfl/fl mice for 24 and 48 h and the harvested intestinal tissues were subjected to immunohistochemistry analysis of NFκB protein. g HCT-116 cells were treated with vehicle, CPT (250 nM) and Paclitaxel (25 nM). Paclitaxel treatment was administrated 12 h before the treatment of CPT. Cycle analysis was performed through flow cytometry and the bar graph represents percentage of cells in different phases of cell cycle, (n = 3, error bars ± s.d.). h Boyden chamber assay was performed to check the invasion ability of HCT-116 cells treated with above set of treatment conditions