Fig. 1

Selective killing effects of a131 in cancer cells. a Structure of a131. b Normal and transformed BJ cells were treated with a131, paclitaxel or nocodazole at a range of different concentrations for 72 h in triplicate and cell viability was determined by MTT assay in comparison with indicated antiproliferative agents, hence a reduction in cell viability would imply cell cycle arrest and cell death. Mean values with ± standard deviation (S.D.) are shown (n = 3). c, d Normal and transformed BJ cells were treated with a131 at 2.5 μM for 48 h. c FACS analysis using BrdU and PI double staining of the indicated cells. Percentages of BrdU-positive (S) population indicating cell cycle arrest and subG1 (<2N) population indicating cell death are shown. Representative FACS analysis shown (n = 3). d Immunoblot analysis of cleaved PARP and caspase-3 (Cas-3) indicating a131-induced apoptosis in transformed BJ cells (lanes 5, 6), but not normal counterparts (lanes 2, 3). Representative immunoblots shown (n = 3). e MTT assay of human normal and cancer cell lines treated with a131 for 72 h (n = 3). Mean concentration values for a131 to achieve 50% growth inhibition (GI₅₀) in each cell line are plotted with ± S.D. f FACS analysis of a series of engineered BJ-derived fibroblasts treated with a131 at 5 µM for 48 h. Mean values of subG1 (<2N) population ± S.D. are shown (n > 3). g, h Normal and transformed BJ cells stably expressing GFP-histone H2B were treated with a131 at 2.5 μM for 32 h prior to fixation. g Immunofluorescence analysis with representative images (n = 3). h Quantification of cells (n > 100 per condition) with the indicated centrosome numbers. Mean values ± S.D. are shown (n = 3). i Mice bearing established tumor xenografts were treated orally (PO) or intraperitoneally (IP) twice daily with the indicated doses of a131 or its derivative b5. Tumor volumes were calculated periodically as indicated. Paclitaxel (PTX) was administered intravenously for twice every 4 days. Mean tumor volumes ± S.D. from six mice are shown. Two-way ANOVA was performed to determine statistical significance compared to vehicle control. j, k TUNEL staining (j) or immunohistochemical analysis (k) of HCT-15 tumor sections on Day 12 with representative images (top). j The percentage of TUNEL-positive cells were calculated from tumor sections. Note that detection of apoptosis by TUNEL and scanning of the entire sections of each slides, which contained both tumors and adjacent normal tissues, was performed in a completely unbiased manner. Moreover, for unbiased quantification, we used five images from each sections of >6 sections from each group (bottom). k Mean values of cell populations (n > 100 per condition) with multipolar mitotic-spindles ± S.D. are shown (bottom) with representative images (top). White bars, 5 μm. Where indicated, two-tailed unpaired t tests were performed to determine statistical significance