Herbal Formulation C168 Attenuates Proliferation and Induces Apoptosis in HCT 116 Human Colorectal Carcinoma Cells: Role of Oxidative Stress and DNA Damage

Abstract

The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME) on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action.

Figure 1

CME exerted selective cytotoxic effect on colorectal carcinoma cells. Percentage viability of (a) HCT 116 colorectal carcinoma cells, (b) HepG2 hepatocellular carcinoma cells, (c) Jurkat E6.1 lymphoblastic leukemic cells, (d) V79-4 Chinese hamster lung fibroblasts, and (e) CCD-841-CoN normal colon epithelial cells following 24-hour treatment with CME. Results are expressed as the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups by using one-way ANOVA.

Figure 2

CME inhibited proliferation of HCT 116 cells. Cell count of HCT 116 cells exposed to different concentrations of CME for 24 hours. The results represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups by one-way ANOVA.

Figure 3

CME induced accumulation of cell population in G₂/M phase. Cell-cycle analysis of HCT 116 cells treated with 500 µg/mL of CME. The results represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups by one-way ANOVA.

Figure 4

CME induced apoptosis in HCT 116 cells. (a) Mode of cell death investigation on HCT 116 cells treated with different concentrations of CME for 72 hours. (b) Representative dot plots for the (i) control group and the treatment groups treated with (ii) 125, (iii) 250, (iv) 500, and (v) 1000 µg/mL of CME. Data represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups.

Figure 5

CME induced superoxide anion increment in HCT 116 cells during early time point. Percentages of ethidium positive cells indicate the population with superoxide anion accumulation. The results represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups.

Figure 6

CME induced decrease of intracellular glutathione in HCT 116 cells. HCT 116 cells were treated with 500 µg/mL CME for various time points. The results represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups.

Figure 7

CME induced early DNA damage in HCT 116 cells. HCT 116 cells treated with 500 µg/mL CME at various time points and their tail moments. The results represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups.

Figure 8

CME increased phosphorylated H2AX level in HCT 116 cells. Level of phosphorylated H2AX in HCT 116 cells treated with 500 µg/mL of CME for various time points, assessed by immunoblot analysis.

Figure 9

CME induced MMP loss in HCT 116 cells. HCT 116 cells treated with 500 µg/mL CME at various time points and subjected to TMRE staining. The results represent the means ± SEM of three independent experiments. ^∗ p < 0.05. Data were compared between the untreated negative control and the treatment groups.