Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines

Figure 4

Induction of apoptosis in HeLa and Caco-2 cells by cyclodipeptides from Pseudomonas aeruginosa. HeLa and Caco-2 cells were incubated in CM medium after treatment with the CDP mix for 24 h. HeLa cells were stained with annexin V and propidium iodide and analyzed by flow cytometry. ((a)–(h)) Schematic diagrams of dot plots, showing the quadrant divisions for the determination of apoptosis using the annexin V and PI probes. The populations of cells for each of the treatments were gated in the forward scatter and side scatter analyses, in order to eliminate dead cells and cell debris. Populations corresponding to autofluorescence or basal fluorescence are located in the lower-left quadrants. Cells with increased fluorescence emission of at least one log unit are located in the lower-right quadrants. The percentage of fluorescent cells is indicated in the dot plots. HeLa cell treatment: (a) DMSO (0.05%; negative control); (b) actinomycin D (50 mg/mL; positive control); (c) 0.01 mg/mL CDP mix; (d) 1.0 mg/mL CDP mix. Caco-2 cell treatment: (e) DMSO (0.05%; negative control); (f) actinomycin D (50 mg/mL; positive control); (g) 0.01 mg/mL CDP mix; (h) 0.1 mg/mL CDP mix. (i) Dose-response plot of apoptotic cell induction by CDP treatment. Percentages of fluorescent cells, determined from dot plots, were used in this analysis. Bars represent the mean value ± the standard error (SE) of three independent experiments. One-way analysis of variance was carried out, with Tukey’s post hoc test; . Values for SE () are shown in lowercase letters. (j) Nonlinear regression analysis of dose-response for the induction of apoptosis by the CDP mix; 95% confidence interval, . HeLa: 50% inhibitory concentration (IC₅₀) = 6.5 × 10⁻⁵mg/mL; = 0.92. Caco-2: IC₅₀ = 1.8 × 10⁻⁴mg/mL; = 0.99.

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