PMC

Structures of cranberry flavonoids quercetin aglycone and PAC DP-9. (A) HPLC chromatograph of quercetin aglycone; (B) HPLC chromatograph of PAC DP-9; (C) MS spectrum of quercetin aglycone (m/z=301); (D) Structures of quercetin algycone and PAC DP-9.

Effect of quercetin aglycone and PAC DP-9 (12-h treatment) on cellular expression of MEK (A), phospho-ERK1/2 (B), phospho-histone H3 (C), cyclin D1 (D), DNA-PK (E) and p21 (F) in SKOV-3 and OVCAR-8 cells. Cells were treated with either DMSO vehicle, PAC DP-9 (80 μg/ml), or quercetin aglycone (80 μg/ml) for 12 h and stained for target proteins. MEK, cyclin D1 and p21 were probed by DyLight 488 secondary antibody (green); phospho-ERK1/2, phospho-histone H3 and DNA-PK were probed by DyLight 594 secondary antibody (red).

Cytotoxicity of individual cranberry flavonols and PACs in SKOV-3 and OVCAR-8 ovarian cancer cells. Cytotoxicity was determined by MTS cell viability assay; cells were treated with DMSO vehicle or different concentrations of cranberry flavonoids (25–200 μg/ml) for 72 h. Experiments were performed in triplicate; data are expressed as mean ± SD in percent of cell viability of untreated cells (100%). M-Gal, myricetin-3-galactoside; Q-Gal, quercetin-3-galactoside; Q-Glu, quercetin-3-glucoside; Q-A-P, quercetin-3-arabinopyranoside; Q-A-F, quercetin-3-arabinofuranoside; Q-Xylo, quercetin-3-xylopyranoside; Q-Rhamno, quercetin-3-rhamnopyranoside;Q-Aglycone, quercetin aglycone.

Effects of quercetin aglycone and PAC DP-9 on expression, activation and localization of EGFR in SKOV-3 and OVCAR-8 cells. (A) Expression of total and activated EGFR on cranberry flavonoid treated SKOV-3 and OVCAR-8 cells. Cells were serum-deprived for 4 h and then treated with 5, 20 or 40 μg/ml quercetin aglycone or PAC DP-9 in serum-free DMEM medium for 12 h. Actin was probed as internal loading control. (B) Effects of PAC DP-9 on EGFR expression and localization in SKOV-3 cells. Cells were treated with different concentrations (12.5–200 μg/ml) of PAC DP-9 for 3 h before fluorescent microscopic analysis. EGFR was visualized by Alexa Fluor 594 secondary antibody (red), and nuclei were stained with DAPI.

Effect of PAC DP-9 and quercetin aglycone on cell cycle regulation in SKOV-3 and OVCAR-8 cells. (A) Western blot analysis of ERK-MAPK signal components and cell cycle regulatory factors in SKOV-3 and OVCAR-8 cells after treatment with quercetin aglycone or PAC DP-9 (80 μg/ml) for 0–24 h. Tubulin was probed as an internal loading control; (B) EGFR co-immunoprecipitation on SKOV-3 and OVCAR-8 cell lysates. 1, SKOV-3 untreated input control; 2, OVCAR-8 untreated input control; 3, SKOV-3 EGFR-co-immunoprecipitation; 4, OVCAR-8 EGFR-co-immunoprecipitation; 5, SKOV-3 IgG-co-immunoprecipitation control; 6, OVCAR-8 IgG-co-immunoprecipitation control. (C and D) Determination of cell cycle progression by FACS analysis in SKOV-3 and OVCAR-8 cells treated with 50 or 100 μg/ml quercetin aglycone or PAC DP-9 for 24–48 h. Data are presented as relative fluorescence intensity of G0/G1, S and G2/M phase subpopulations in 2-D charts (C) or tables (D).

Induction of apoptosis (A and B) and sensitization to cisplatin (C) by quercetin aglycone and PAC DP-9 in SKOV-3 and OVCAR-8 cells. (A) Expression of cleaved caspase-3 and cleaved PARP after treatment with PAC DP-9 and quercetin aglycone. Ovarian cancer cells were treated with PAC DP-9 or quercetin aglycone (80 μg/ml) for 0–24 h. Actin or tubulin were probed as internal loading controls; (B) TUNEL assay in PAC DP-9 and quercetin aglycone (Q-A)-treated SKOV-3 and OVCAR-8 cells. Cells were treated with PAC DP-9 (50 μg/ml), quercetin aglycone (25 μg/ml) or DMSO vehicle for 12 h. DNA strand breaks due to apoptosis were detected and labelled by fluorescein-labelled nucleotides (green). (C) Cell viability of cranberry flavonoid and cisplatin-treated SKOV-3 and OVCAR-8 cells. Cells were pre-treated with either DMSO vehicle, PAC DP-9 (25 μg/ml), or quercetin aglycone (10 μg/ml) for 6 h in complete DMEM media, followed by cisplatin treatment alone or cisplatin + PAC DP-9/quercetin aglycone. Cells were incubated overnight for 12 h and analyzed via MTS for viability. Experiments were performed in triplicate; data are expressed as mean ± SD in percent of cell viability of untreated cells (100%).