Allyl ether of mansonone G as a potential anticancer agent for colorectal cancer

Abstract

Mansonone G (MG), a 1,2-naphthoquinone isolated from the heartwood of Mansonia gagei Drumm, exhibited several pharmacological activities such as anti-bacterial, anti-estrogenic and anti-adipogenic effect. This study evaluated the cytotoxicity of MG and its derivatives as well as determined the mechanism(s) underlying the cytotoxic activity of the most potent MG derivative on two CRC cell lines, HCT-116 cells carrying p53 wild-type and HT-29 cells carrying p53 mutant. We found that MG and its derivatives could inhibit viability of HCT-116 and HT-29 cells in a concentration-dependent manner. Of all semi-synthetic derivatives of MG, allyl ether mansonone G (MG7) was the most potent cytotoxic agent toward cancer cells and less toxic to normal cells. MG7 could induce ROS generation which was associated with cytotoxicity and apoptosis in both HCT-116 and HT-29 cells. Western blot analysis revealed that MG7 downregulated the expression of Bcl-2 and Bcl-xL proteins in both CRC cell lines and upregulated the expression of BAK protein in HT-29 cells. Moreover, MG7 inhibited AKT signaling pathway in both CRC cell lines and modulated ERK1/2 signaling pathway by inhibiting ERK1/2 phosphorylation in HCT-116 cells and activating ERK1/2 phosphorylation in HT-29 cells. Molecular docking revealed that MG7 could bind to the ATP-binding pocket of AKT and ERK1 via hydrophobic interactions.

Figure 1

Chemical structures of MG and its semisynthetic derivatives.

Figure 2

Effects of MG and its derivatives on viability of (A) HCT-116 and (B) HT-29 cells. The cells were treated with MG, semi-synthesized derivative of MGs or oxaliplatin at 0.1, 1, 10, and 100 µM for 48 h. Cell viability was evaluated using MTT assay. Data are presented as mean ± S.E.M of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to vehicle control (0.2% DMSO).

Figure 3

Effects of semi-synthetic derivatives of MGs on viability of (A) PCS201-010 and (B) CRL-1790 cells. The cells were treated with MG3, MG4, MG7 or MG10 at indicated concentrations for 48 h. Cell viability was evaluated using MTT assay. Data are presented as mean ± S.E.M of three independent experiments. **P < 0.01 and ***P < 0.001 compared to vehicle control (0.2% DMSO).

Figure 4

Effects of MG7 on apoptosis induction in CRC cells. (A) The percentages of apoptotic HCT-116 cells after being treated with MG7 at 2.5, 5 and 10 µM for 24 h were evaluated using annexin-V/PI staining followed by flow cytometry. (B) The expression ratio of cleaved PARP to total PARP proteins in HCT-116 cells was determined using western blot. (C) The percentage of apoptotic HT-29 cells and (D) the expression ratio of cleaved PARP to total proteins in HT-29 cells after being treated with MG7 at 5, 10 and 20 µM for 24 h. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to vehicle control (0.2% DMSO).

Figure 5

Effects of MG7 on the protein expression of Bcl-2 family members in HCT-116 cells. The cells were treated with MG7 at concentrations of 2.5, 5, and 10 µM for 24 h. The expression levels of (A) BAK, (B) BAX, (C) Bcl-2, (D) Bcl-xL and (E) cleaved caspase-3 proteins were determined using western blot. Data are expressed as mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 compared to vehicle control (0.2% DMSO).

Figure 6

Effects of MG7 on the protein expression of Bcl-2 family members in HT29 cells. The cells were treated with MG7 at concentrations of 5, 10 and 20 µM for 24 h. The expression levels of (A) BAK, (B) BAX, (C) Bcl-2, (D) Bcl-xL and (E) cleaved caspase-3 proteins were determined using western blot. Data are expressed as mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 compared to vehicle control (0.2% DMSO).

Figure 7

Roles of ROS on cytotoxic and apoptosis-inducing effects of MG7. The ROS levels in (A) HCT-116 and (B) HT-29 cells after being treated with MG7 at indicated concentrations or H₂O₂ at 200 µM were evaluated using DCFH₂-DA assay. The viability of (C) HCT-116 and (D) HT-29 cells and the numbers of annexin V-positive apoptotic (E) HCT-116 and (F) HT-29 cells after being treated with MG with or without NAC. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to vehicle control (0.2% DMSO).

Figure 8

Comprehensive transcriptome dynamics by the MG7 treatment in HCT-116 and HT-29 cells. Principal-component analysis (PCA) of directional RNA-seq data (left panel), heat map and hierarchical clustering of transcriptional pattern analysis (middle panel), and volcano plot indicating differentially expressed transcripts (right panel) after MG7 treatment by HCT-116 cells (A) and HT-29 cells (B). Heatmap describing fold changes for top 20 up-regulated and down-regulated transcripts indicating statistically significant differences between the MG7-treated group and the control group in HCT-116 (C) and HT-29 cells (D).

Figure 9

Effects of MG7 on ERK/MAPK and PI3K/AKT signaling pathways in HCT-116 and HT29 cells. The expression ratio of phosphorylated ERK to total ERK and phosphorylated AKT to total AKT in (A,B) HCT-116 and (C,D) HT-29 cells after being treated with MG7 at indicated concentrations for 24 h were determined using western blot. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to vehicle control (0.2% DMSO).

Figure 10

Predicted binding mode of MG7 against (A) AKT and (B) ERK1 signaling proteins and (C) ATPase domain of bacterial DNA gyrase obtained from molecular docking.