FIG. 3.
Resveratrol and equol induce apoptosis. (A) Cells infected with Ad5 (22Rv1 and DU145, 100 ppc; PC-3, 300 ppc) (open diamond dashed line) and equol at 100 μM (open square dashed line) or resveratrol at 10 μM (open circle dashed line) were added alone or in combination with Ad5 (equol; black square solid line or resveratrol black circle solid line). Cells were stained with tetramethylrhodamine ethyl ester perchlorate (TMRE) and 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by flow cytometry after 24–96 hr. Cells that retained TMRE staining (intact, active mitochondria) and remained negative for DAPI (intact cellular membrane) were considered live, means of duplicates±SD, n=3, expressed as percent of live cells (Δψm). Two-way ANOVA with Bonferroni post-tests, significant differences indicated at 96 hr: 22Rv1; °°combination-treated or ***single agent treated compared to untreated cells, DU145; °°°combination-treated or ***single-agent treated compared to untreated or virus infected cells, PC-3: °°°combination-treated compared to each single agent treatment, and ***single agent treatments compared to untreated cells. B) Cells infected with Ad5 and treated with phytochemicals as described in A, analyzed by flow cytometry for active caspase 3, averages±SD of one experiment representative of three independent studies. Significant results at 96 hr, two-way ANOVA with Bonferroni post-tests: 22Rv1; *single agents compared to untreated cells, PC-3 and DU145; °°°combination-treated compared to each single agent treated; and ***single agents compared to untreated cells. (C) Cells were infected with Ad5 as described in A and treated with equol at 50 or 100 μM or resveratrol at 5 or 10 μM. After 24–96 hr, cells were stained with Alexa fluor 488–conjugated annexin V and propidium iodide (PI). Cells that were negative for annexin V and PI cells were plotted as live cells under each condition. Results are means of duplicates from two to four experiments±SD. Significant results at 96–120 hr, two-way ANOVA with Bonferroni post-tests: DU145; *treatments compared to single agent or to °untreated, PC-3; Eq combinations compared to single agent treated, Res combinations compared to virus-infected cells. (D) Cells infected with Ad5 (0.256–10,000 ppc), with or without the pan-caspase inhibitor zVAD-fmk at 25 μM, and with or without equol 100 μM or resveratrol 10 μM. Cell viability was assessed 6 days post-infection, one representative study out of two to three. Two-way ANOVA with Bonferroni post-tests on the corresponding average EC50 values; ***Ad5 EC50 versus both phytochemicals in all three cell lines, sensitization was significantly reduced with zVADfmk in DU145 (***) and PC-3 cells (**).