Phytochemical Analysis and Anticancer Properties of Drimia maritima Bulb Extracts on Colorectal Cancer Cells

Abstract

Cancer is a worldwide health problem and is the second leading cause of death after heart disease. Due to the high cost and severe side effects associated with chemotherapy treatments, natural products with anticancer therapeutic potential may play a promising role in anticancer therapy. The purpose of this study was to investigate the cytotoxic and apoptotic characteristics of the aqueous Drimia maritima bulb extract on Caco-2 and COLO-205 colorectal cancer cells. In order to reach such a purpose, the chemical composition was examined using the GC-MS method, and the selective antiproliferative effect was determined in colon cancer cell lines in normal gingival fibroblasts. The intracellular ROS, mitochondrial membrane potential, and gene expression changes in selected genes (CASP8, TNF-α, and IL-6 genes) were assessed to determine the molecular mechanism of the antitumor effect of the extract. GC-MS results revealed the presence of fifty-seven compounds, and Proscillaridin A was the predominant secondary metabolite in the extract. The IC₅₀ of D. maritima bulb extract on Caco-2, COLO-205, and the normal human gingival fibroblasts were obtained at 0.9 µg/mL, 2.3 µg/mL, and 13.1 µg/mL, respectively. The apoptotic effect assay indicated that the bulb extract induced apoptosis in both colon cancer cell lines. D. maritima bulb extract was only able to induce statistically significant ROS levels in COLO-205 cells in a dose-dependent manner. The mitochondrial membrane potential (MMP) revealed a significant decrease in the MMP of Caco-2 and COLO-205 to various concentrations of the bulb extract. At the molecular level, RT-qPCR was used to assess gene expression of CASP8, TNF-α, and IL-6 genes in Caco-2 and COLO-205 cancer cells. The results showed that the expression of pro-inflammatory genes TNF-α and IL-6 were upregulated. The apoptotic initiator gene CASP8 was also upregulated in the Caco-2 cell line and did not reach significance in COLO-205 cells. These results lead to the conclusion that D. maritima extract induced cell death in both cell lines and may have the potential to be used in CRC therapy in the future.

Keywords: Drimia maritima; anticancer; colorectal.

Figure 1

Gas chromatography and mass spectrometry chromatogram showing the major components of the aqueous bulb extract of Drimia maritima. * a.u.: arbitrary unit.

Figure 2

Cell viability MTT assay of Caco-2 and COLO-205 colon cancer cells and hGFs after treatment with the following: (A) increasing concentration of aqueous Drimia maritima bulb extract (0.122–31.25 µg/mL); (B) Proscillaridin (0.0024–1.32 μg/mL); (C) Doxorubicin (from 50 to 0.195 µg/mL). The IC₅₀ values were calculated using log-probit analysis. Each value is presented as the mean ± SD of an average of three independent experiments.

Figure 2

Cell viability MTT assay of Caco-2 and COLO-205 colon cancer cells and hGFs after treatment with the following: (A) increasing concentration of aqueous Drimia maritima bulb extract (0.122–31.25 µg/mL); (B) Proscillaridin (0.0024–1.32 μg/mL); (C) Doxorubicin (from 50 to 0.195 µg/mL). The IC₅₀ values were calculated using log-probit analysis. Each value is presented as the mean ± SD of an average of three independent experiments.

Figure 3

Flow cytometry analysis of D. maritima (DM) bulb extract effect on COLO-205 and Caco-2 cells. (A) Representative flow cytometer plots are presented for the untreated group (control) and DM-treated groups (4 μg/Ml, 3.5 μg/mL, 2.75 μg/mL, 2 μg/mL, 1 μg/mL and 0.5 μg/mL) in COLO205 cells. (B) The bar graph represents the percentage of early and late apoptotic cells detected by flow cytometer from three separate experiments (mean ± SD, n = 3). (C) Representative flow cytometery plots are presented for the control group (Untreated) and DM treated groups (2 μg/mL, 1 μg/mL, 0.75 μg/mL, and 0.5 μg/mL) in Caco-2 cells. (D) The bar graph represents the percentage of early and late apoptotic cells detected by flow cytometer from three different individual experiments (mean ± SD, n = 3). ** Significant differences were observed between the DM-treated (2 μg/mL and 1 μg/mL) and untreated control group (p-values *** < 0.001, ** < 0.01 and * < 0.05).

Figure 4

Production of reactive oxygen species (ROS) in COLO-205 and Caco-2 cells incubated for 48 h with D. maritima (DM) bulb extract: (A,B) Histograms and bar graphs of ROS production in COLO-205 cells were obtained by flow cytometer in the FITC channel in different groups. The bar graph shows a remarkable increase in the level of intracellular ROS in the treated group. (C,D) Representative histograms and bar graphs from Caco-2 cells treated with indicated concentrations of D. maritima extract on intracellular ROS levels production in comparison to control (untreated cells) detected by flow cytometer from three separate experiments (mean ± SD, n = 3). p-values *** < 0.001, ** < 0.01.

Figure 5

Assessment of the mitochondrial membrane potential (ΔΨm) in COLO-205 and Caco-2 cells after treatment with D. maritima (DM) bulb extract for 48 h: (A,B) Histograms and bar graphs show changes in COLO-205 cells ΔΨm response to various concentrations of DM extract (3.5 μg/mL, 2.75 μg/mL, 2 μg/mL, 1 μg/mL, and 0.5 μg/mL) in comparison to untreated cells. (C,D) Histogram and bar graphs show loss of ΔΨm following exposure to different concentrations of DM extract (2 μg/mL, 1.5 μg/mL, 1 μg/mL, 0.75 μg/mL, and 0.5 μg/mL) compared to control (untreated cells). All data are expressed as mean  ±  SD of three separate experiments. All data are expressed as mean ± SD of three separate experiments. (p-values *** < 0.001 ** < 0.01, and * < 0.05).

Figure 6

Gene expression analysis of apoptotic initiator gene CASP8 and two inflammatory cytokine genes TNF-α and IL-6 in COLO-205 and Caco-2 cells after treatment with various concentrations of D. maritima (DM) bulb extract for 48 h as examined by quantitative RT–PCR: (A,D) The graphs reveal fold changes in the expression of CASP8 in COLO-205 and Caco-2 cells, respectively. (B,E) TNF-α gene in COLO-205 and Caco-2 cells at various extract concentrations (2.0, 1.5, 1.0, 0.75, and 0.5 μg/mL). (C,F) IL-6 in COLO-205 and Caco-2 cells treated with different extract concentrations (1.5, 1.0, 0.75, and 0.5 μg/mL). Fold change was calculated using the ΔΔCt method. Untreated cells were used as the control; therefore, the fold change for untreated cells is 1 in all plots. GAPDH gene served as an internal reference gene. The error bars indicate the standard deviation from triplicate experiments (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).