Induction of Apoptosis and Cell Cycle Arrest by Flavokawain C on HT-29 Human Colon Adenocarcinoma via Enhancement of Reactive Oxygen Species Generation, Upregulation of p21, p27, and GADD153, and Inactivation of Inhibitor of Apoptosis Proteins

Abstract

Chalcones have been shown to exhibit anti-cancer properties by targeting multiple molecular pathways. It was, therefore, of interest to investigate flavokawain C (FKC), a naturally occurring chalcone, which can be isolated from Kava (Piper methysticum Forst) root extract. The aim of this study was to investigate the inhibitory effect of FKC on the growth of HT-29 cells and its underlying mechanism of action. Cell viability of HT-29 cells was assessed by Sulforhodamine B assay after FKC treatment. Induction of apoptosis was examined by established morphological and biochemical assays. ROS generation was determined by dichlorofluorescein fluorescence staining, and superoxide dismutase activity was measured using the spectrophotometric method. Western blotting was used to examine the changes in the protein levels. FKC markedly decreased the cell viability of HT-29 cells and the cells showed dramatic changes in cellular and nuclear morphologies with typical apoptotic features. The induction of apoptosis correlated well with the externalization of phosphatidylserine, DNA fragmentation, decreased mitochondrial membrane potential, activation of caspases, and PARP cleavage. This was associated with an increase in reactive oxygen species and a decrease in SOD activity. The protein levels of XIAP, c-IAP1, and c-IAP2 were downregulated, whereas the GADD153 was upregulated after FKC treatment. FKC induced cell cycle arrest at the G₁ and G₂/M phases via upregulation of p21 and p27 in a p53-independent manner. Our results provide evidence that FKC has the potential to be developed into chemotherapeutic drug for the treatment of colon adenocarcinoma.

Summary: Flavokawain C inhibited the growth of HT-29 human colon adenocarcinoma cellsFlavokawain C induced apoptosis in HT-29 cells, associated with an increase in reactive oxygen species and a decrease in SOD activityFlavokawain C induced cell cycle arrest at the G₁ and G₂/M phases via upregulation of p21 and p27 in HT-29 cellsHT-29 cells treated with flavokawain C caused downregulation of XIAP, c-IAP1, and c-IAP2, and upregulation of GADD153. Abbreviations used: FKC: Flavokawain C; SRB: Sulforhodamine B; ROS: Reactive oxygen species; SOD: Superoxide dismutase; PARP: Poly(ADP-ribose) polymerase; ER: Endoplasmic reticulum; IAPs: Inhibitor of apoptosis proteins; TUNEL: Transferase dUTP nick end labeling; Annexin V-FITC: Annexin V conjugated with fluorescein isothicyanate.

Keywords: Apoptosis; HT-29; ROS; cell cycle; colon adenocarcinoma; flavokawain C.

Figure 1

Structure of flavokawain C.

Figure 2

(A) The inhibitory effect of FKC on cell viability of HT-29 cells. In comparison to the control, the inhibition of cell proliferation was significantly increased in dose- and time-dependent manner. (B) The effect of FKC on the cell cycle in HT-29 cells. Data given are expressed as mean ± standard deviation of triplicates obtained from three independent experiments. *P<0.05 compared to the control.

Figure 3

(A) Changes in morphology of HT-29 after FKC treatment for 48 h were detected by inverted phase contrast microscopy (400×). Cells became irregular, shrunken, and began to detach from the culture plate in a dose-dependent manner. (B) Changes in nuclear morphology of HT-29 cells were detected by the fluorescence microscope (400×). The arrows indicate the formation of chromatin fragmentation and apoptotic body.

Figure 4

(A) The number of early and late apoptotic cells (from total 10,000 cells) of HT-29 cells was increased following FKC treatment for 24 and 48 h. *P<0.05 compared to the control. (B) The effect of FKC on the mitochondrial membrane potential (Δ Ψm) of HT-29 cells. Δ Ψm of HT-29 cells was significantly reduced by FKC in a dose- and time-dependent manner. *P<0.05 compared to the control. (C) FKC induces DNA fragmentation in HT-29 cells. Percentages of HT-29 cells that showed positive DNA fragmentation are represented in the upper quadrant from a dot plot.

Figure 5

(A) FKC treatment increased the activity of caspase-3, -8, and -9 in HT-29 cells. Values given are expressed as mean ± standard deviation of triplicates obtained from three independent experiments. * P<0.05 compared to the control. (B) FKC caused the cleavage of PARP-1 in HT-29 cells.

Figure 6

Concentration-dependent effect of FKC on ROS generation (A) and SOD activity (B) in HCT 116 and HT-29 cells. The data are presented as the mean ± standard deviation for three independent experiments. * P<0.05 compared to control.

Figure 7

(A) Effect of FKC on the protein levels of p53, p21, and p27 in HT-29 and HCT 116 cells. (B) The changes in the protein levels of GADD153, c-IAP1, c-IAP2, and XIAP in HT-29 cells treated with FKC were analyzed by western blot.