Synthetic Homoisoflavane Derivatives of Cremastranone Suppress Growth of Colorectal Cancer Cells through Cell Cycle Arrest and Induction of Apoptosis

Abstract

Colorectal cancer is diagnosed as the third most prevalent cancer; thus, effective therapeutic agents are urgently required. In this study, we synthesized six homoisoflavane derivatives of cremastranone and investigated their cytotoxic effects on the human colorectal cancer cell lines HCT116 and LoVo. We further examined the related mechanisms of action using two of the potent compounds, SH-19027 and SHA-035. They substantially reduced the cell viability and proliferation in a dose-dependent manner. Treatment with SH-19027 and SHA-035 induced cell cycle arrest at the G2/M phase and increased expression of p21 both of which are implicated in cell cycle control. In addition, the apoptotic cell population and apoptosis-associated marker expression were accordingly increased. These results suggest that the synthesized cremastranone derivatives have anticancer effects through the suppression of cell proliferation and induction of apoptosis. Therefore, the synthesized cremastranone derivatives could be applied as novel therapeutic agents against colorectal cancer.

Keywords: Anticancer effect; Apoptosis; Cell cycle; Colorectal cancer; Homoisoflavane derivatives of cremastranone.

Fig. 1

Structure of cremastranone and the homoisoflavane derivatives. (A) Natural product cremastranone and synthetic homoisoflavane SH-17059, (B) A-ring modification of SH-17059, and (C) B-ring modification of SH-17059.

Fig. 2

Cell viability of colorectal cancer (CRC) cells is reduced by treatment with the homoisoflavane derivatives in a time- and dose-dependent manner. HCT116 (A) and LoVo (B) cells were treated with the indicated doses of SH-17059, SH-19021, SH-19027, and SHA-035 for the indicated times. A WST-based cell viability assay was performed. Values are the mean ± SD. *p<0.05, **p<0.01, ***p<0.005.

Fig. 3

Effects of SH-19027 and SHA-035 on proliferation and cell cycle in CRC cells. (A) BrdU colorimetric incorporation assay was performed in HCT116 and LoVo cells after treatment with the indicated doses of SH-19027 and SHA-035 for the indicated times. (B) HCT116 cells were treated with the indicated doses of SH-19027 or SHA-035 for 48 h, and the cell lysates were subjected to western blot analysis. β-actin was used as a loading control. (C) Cell cycle of HCT116 and LoVo cells were examined by flow cytometry after treatment with the indicated doses of SH-19027 or SHA-035 for 24 h (HCT116) or 48 h (LoVo). Percentages of cells in the G0/G1, S, and G2/M phase of the cell cycle are shown as a graph. Values are the mean ± SD. *p<0.05, **p<0.01, ***p<0.005.

Fig. 4

SH-19027 and SHA-035 induce apoptosis in the CRC cells. (A) After treatment with SH-19027 or SHA-035 for 24 h in HCT116 and 48 h in LoVo cells, the apoptosis assay was performed by Annexin V-FITC/PI staining. Percentage of apoptotic cells is the sum of annexin V positive cells. (B) HCT116 cells were treated with SH-19027 or SHA-035 for 48 h, followed by western blot analysis performed for detecting apoptosis marker expression. β-actin was used as a loading control. (C) Caspase activity in SH-19027- and SHA-035-treated HCT116 cells was measured using caspase substrates. Values are the mean ± SD. *p<0.05, **p<0.01, ***p<0.005.