Fig 1.
Chemical structure of flavokawain A, gymogrammene, flavokawain B, flavokawain C.
Fig 2.
Inhibition of cell proliferation and viability by FKC in human cancer cell lines and a normal cell line.
(A) Each cell lines were seeded at 4.5×103 cells per well in 96-well plates. After 24 hours, the cells were treated with various concentration of FKC (5–333μM) for 72 hours, and the results were expressed as inhibition of proliferation which was determined by SRB assay as described in Materials and Methods. (B) HCT 116 cells (4.5×103 cells/well) was seeded in a 96-well for 24 hours and then treated with FKC (20, 40 and 60μM) at increasing time points (6, 12, 24, 48 and 72 hours). All data shown are the mean±standard deviation (SD) of triplicates obtained from three independent experiments. Cell viability in FKC-treated cells was expressed as a percentage of viable cells compared to control cells.
Table 1.
Cytotoxic activities of FKC and GMM on various cancer cell lines and human normal cell (CCD-18Co) for 72 hours treatment in comparison to Cisplatin.
Fig 3.
FKC induces apoptosis in HCT 116 cells.
(A) HCT 116 cells were treated with FKC at the indicated concentrations for 48 hours, and subsequently observed under an inverted phase contrast microscope at magnification of 40× (Scale bar = 1μm). Control cells showing normal morphology (well spread and normal nuclei structure) while treated cells showing the typical morphological features of apoptosis include cell shrinkage, condensed or fragmented nuclei, membrane blebbing, vacuole formation and detachment of apoptotic bodies. (B) Representative fluorescence microscopy images of HCT 116 cells stained with Hoechst 34222 and PI after treated without or with the indicated concentrations of FKC for 48 hours and visualized using fluorescence microscope at magnification of 40× (Scale bar = 50μm). Arrows labelled with the following number and letter indicates: (1) viable cells with normal nuclei; (2) early apoptotic cells with highly condensed chromatin or fragmented chromatin (b); (3) late apoptotic cells with highly condensed chromatin or fragmented chromatin; (4) dead cells/necrosis. Untreated cells in 0.5% DMSO served as the control in (A) and (B).
Fig 4.
FKC induces phosphotidylserine (PS) externalization and DNA fragmentation in HCT 116 cells.
(A) HCT 116 cells were treated with increasing concentrations of FKC for 48 hours and stained with AnnexinV-FITC and PI, followed by flow cytometric analysis. Untreated cells in 0.5% DMSO served as the control. Each dot plot is the representative result of three independent experiments. Cell populations are distinguished based on the four quadrants from a dot plot: viable (bottom left), early apoptotic (bottom right), late apoptotic (top right), and dead cells/debris (top left) cells. (B) Quantification of number of early and late apoptotic cells (from total 10,000 cells) of HCT 116 cells measured by flow cytometry for 24 and 48 hours are presented as percentages in bar charts. (C) Cells were treated with absence or presence of FKC at the indicated concentrations for 48 hours, and assessed using TUNEL assay kit. The percentages of cells with fragmented DNA were analyzed using flow cytometry as indicated in the upper quadrant from a dot plot. Each dot plot is the representative result of three independent experiments. (D) Percentages of HCT 116 cells which showed positive DNA fragmentation measured by flow cytometry are presented in bar diagram. Values given are expressed as mean±SD of triplicates obtained from three independent experiments. The asterisk (*) shows statistically significant differences in comparison to the control, p<0.05. Untreated cells in 0.5% DMSO served as the control.
Fig 5.
FKC induces activation of caspases and cleavage of PARP in HCT 116 cells.
(A) and (B) Cells were treated with FKC (60μM) at the indicated time points. The levels of pro-caspase-3, -8 and cleaved PARP were analyzed by western blot and β-actin served as loading control. The results from representative experiments were expressed relative to the protein level at 0 hr after normalization to β-actin signals. (C) Activated caspase-3, -8 and -9 in HCT 116 cells were determined using flow cytometry. Cells were treated in the absence or presence of FKC at the indicated concentrations for 48 hours, and assessed using caspILLUME green Active caspase-3, -8 and -9 staining kit. The percentage of cells that showed positive for active caspase-3, -8 and -9 were quantified by flow cytometry and are presented in the bar chart. Values given are expressed as mean ± SD of triplicates obtained from three independent experiments. The asterisk (*) indicated p<0.05 when compared to the control. Untreated cells in 0.5% DMSO served as the control.
Fig 6.
FKC causes dissipation of mitochondrial membrane potential in HCT 116 cells.
(A) Cells were treated with the indicated concentrations of FKC for 48 hours. Cells were then incubated with JC-1 probe, and then analyzed using flow cytometry. Untreated cells in 0.5% DMSO served as the control. Upper quadrant indicates percentage of cells with polarized mitochondrial membranes which emit red fluorescence whereas bottom quadrant indicates percentages of cells with depolarized mitochondrial membranes which emit green fluorescence. Each dot plot is the representative result of three independent experiments. (B) The bar charts showing the ratio of mean intensity of JC-1 red fluorescence to JC-1 green fluorescence in HCT 116 cells treated with various concentrations for 24 and 48 hours. Values given are expressed as mean±SD of triplicates obtained from three independent experiments. The asterisk (*) indicated p<0.05 when compared to the control. (C) After treatment with FKC, whole cell lysate was fractionated into cytosolic and mitochondrial portions. Western blotting was used to examine the levels of cytochrome c, smac/DIABLO and AIF in both fractions of HCT 116 cells at indicated time points after FKC treatment (60μM). β-actin and COX IV were served as loading control for cytoplasm and mitochondria, respectively.
Fig 7.
Effects of FKC on the levels of pro-apoptotic and anti-apopotic proteins in HCT 116 cells.
HCT 116 cells were treated with FKC (60μM) for indicated time points, and followed by protein extraction and western blot analysis. (A) Effect of FKC on the levels of Bcl-2 family proteins in HCT 116 cells. (B) FKC inhibits the levels of c-IAP-1, XIAP, c-FLIPL and survivin. The results from representative experiments were expressed relative to the protein level at 0 hr after normalization to β-actin signals.
Fig 8.
Effect of FKC on the cell cycle and the cell cycle regulatory proteins in HCT 116 cells.
(A) Cells were treated with the indicated concentrations of FKC for 24 and 48 hours, and stained with PI. DNA content of HCT 116 was analyzed using flow cytometry and quantification of cell cycle distribution(sub-G1, G0/G1, S and G2/M phases)was performed using ModFit software. Untreated cells in 0.5% DMSO served as the control. Each histogram is the representative cell cycle profile of three independent experiments. (B) The quantitative data of mean of percentages of cells in each phase for HCT 116 cells for 24 and 48 hours are presented in bar chart. Values given are expressed as mean ± SD of triplicates obtained from three independent experiments. The asterisk (*) indicated p<0.05 when compared to the control. (C) Cells were incubated in the absence or presence of FKC (60μM) for the indicated times, and followed by protein extraction. Changes in levels of the cyclins (cyclin D1 and E), cyclin dependent kinase (Cdk2 and 4), Cdk inhibitors (p21Cip1 and p27Kip1), and RB phosphorylation were analyzed by Western blot. Untreated cells in 0.5% DMSO served as the control. β-actin was used as the loading control. The results from representative experiments were expressed relative to the proteins level at 0 hr after normalization to β-actin signals.
Fig 9.
Effect of FKC on the protein levels involved in endoplasmic reticulum stress, MAPKs and Akt signaling pathways.
(A) Cells were incubated with FKC (60μM) for the indicated time points and followed by protein extraction. The change in level of GADD153/CHOP were evaluated by western blot. (B) and (C) Cells were incubated with FKC (60μM) for the indicated time points and followed by protein extraction. Cell lysates were subjected to western blot analysis using antibodies indicated to detect the levels of phosphorylated or total proteins of ERK and Akt. Untreated cells in 0.5% DMSO served as the control. β-actin in the western blot was used as the internal control. The results from representative experiments were expressed relative to the proteins level at 0 hr after normalization to β-actin signals.