Figure 2.
A. Compound‐induced formation of γ‐H2AX in Cal27 cells. Cells were treated with an IC50 or fivefold IC50 (from MTT assay) for 24 h. γ‐H2AX formation was analyzed by immunohistochemistry. 100 μM cisplatin served as positive control and was set as 100 %. “control” is vehicle control. Data are the mean ± SD, n ≥3. T‐test was used to analyse for significant differences between compounds and control or as indicated. ns (p>0.05); ** (p ≤0.01). B. Synthesized hit compound 3 n and its control compound without N‐lost‐functionality (3 o) and their cytotoxicity. C. Induction of apoptosis shown as subG1 nuclei induced by 3 n and the nitrogen mustard‐free compound 3 o in Cal27 cells. Cells were treated with indicated compounds and concentrations (MTT‐IC50) for 24 h, and sub‐G1 cell fractions were analysed by flow cytometry. 100 μM cisplatin served as positive control for apoptosis induction. “control” is vehicle control. Data are the mean ± SD, n=3. T‐test was used to analyze for significant differences between compounds and control or as indicated. ns (p>0.05); * (p ≤0.05). D. Compound‐induced caspase3/7 activation in Cal27 cells. Cells were treated with a single, double, or fivefold IC50 (from MTT assay) for 24 h. 100 μM Cisplatin was added as positive control. “control” is vehicle control. Data are the mean ± SD, n=3. t‐test was used to analyze for significant differences between compounds and control or as indicated. ns (p>0.05); *** (p ≤0.001). E. 3 n is significantly more effective than the sum of the effects of 3 o and CAB on caspase3/7 activation (left) and on formation of γH2AX (right). Compounds were used in concentrations reflecting the fivefold IC50 (MTT assay). Green and grey bars show sum of the effects of 3 o and CAB. Light blue bars show the effect of 3 n. Vehicle control was subtracted. Data are mean ± SD, n=3. T‐test was used to analyze for significant differences. ** (p ≤0.01); *** (p ≤0.001).The analysis was performed as previously described. [20]