Manoalide Shows Mutual Interaction between Cellular and Mitochondrial Reactive Species with Apoptosis in Oral Cancer Cells

Figure 4

Effects of inhibitors for cellular reactive species, mitochondrial reactive species, and apoptosis (NAC, MT, and ZVAD) on flow cytometry of DCFH-DA (NO₂˙, CO₃˙¯, and ˙OH) in manoalide-treated oral cancer cells. Cells were pretreated with control (0.1% DMSO only), NAC (8 mM), MT (20 μM), and ZVAD (100 μM) for 2 h and posttreated with control and manoalide (10 μM) for 0 and 6 h. All experiments had the same concentration of DMSO. (a) Flow cytometry patterns for manoalide-treated oral cancer cells (Ca9-22 and CAL 27). (b) Statistics of suppression (fold). The suppression fold is defined in detail at Section 2.4. No suppression is defined at 1 (untreated control; 0.1% DMSO only). If the reactive species intensity determined by flow cytometry is decreased after inhibitor treatment, the suppression fold of inhibitors (NAC, MT, and ZVAD) is larger than 1. Results from different treatments are considered significantly different for multiple comparisons (indicated via different letters without overlapping) (). In the example of Ca9-22 cells, the suppression fold for control, NAC, MT, and ZVAD show letters at top for “D,” “A,” “C,” and “B” for Ca9-22 cells. Since they were marked with different letters without overlapping, all the treatments between each other (control, NAC, MT, or ZVAD) differ significantly. Data, ( independent experiments, each experiment collected with 10000 gated cell counts).

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