Standardized Saponin Extract from Baiye No.1 Tea (Camellia sinensis) Flowers Induced S Phase Cell Cycle Arrest and Apoptosis via AKT-MDM2-p53 Signaling Pathway in Ovarian Cancer Cells

Abstract

Ovarian cancer is considered to be one of the most serious malignant tumors in women. Natural compounds have been considered as important sources in the search for new anti-cancer agents. Saponins are characteristic components of tea (Camellia sinensis) flower and have various biological activities, including anti-tumor effects. In this study, a high purity standardized saponin extract, namely Baiye No.1 tea flower saponin (BTFS), which contained Floratheasaponin A and Floratheasaponin D, were isolated from tea (Camellia sinensis cv. Baiye 1) flowers by macroporous resin and preparative liquid chromatography. Then, the component and purity were detected by UPLC-Q-TOF/MS/MS. This high purity BTFS inhibited the proliferation of A2780/CP70 cancer cells dose-dependently, which is evidenced by the inhibition of cell viability, reduction of colony formation ability, and suppression of PCNA protein expression. Further research found BTFS induced S phase cell cycle arrest by up-regulating p21 proteins expression and down-regulating Cyclin A2, CDK2, and Cdc25A protein expression. Furthermore, BTFS caused DNA damage and activated the ATM-Chk2 signaling pathway to block cell cycle progression. Moreover, BTFS trigged both extrinsic and intrinsic apoptosis-BTFS up-regulated the expression of death receptor pathway-related proteins DR5, Fas, and FADD and increased the ratio of pro-apoptotic/anti-apoptotic proteins of the Bcl-2 family. BTFS-induced apoptosis seems to be related to the AKT-MDM2-p53 signaling pathway. In summary, our results demonstrate that BTFS has the potential to be used as a nutraceutical for the prevention and treatment of ovarian cancer.

Keywords: A2780/CP70 ovarian cancer cells; BTFS; S phase cell cycle arrest; apoptosis; tea (Camellia sinensis) flowers.

Figure 1

Typical chromatograms of BTFS. (A) Ultraviolet chromatograms. (B) Total ion chromatograms.

Figure 2

BTFS inhibits proliferation of A2780/CP70 cells. (A) BTFS inhibited the cell viability of A2780/CP70 and showed moderate effect on IOSE-364 cells. (B) The effect of BTFS on colony formation of A2780/CP70 cells. (C) Statistical histogram. *p < 0.05; **p < 0.01 versus control. (D)Effects of BTFS on protein expression of PNCA in A2780/CP70 cells. (E) Statistical histogram of protein quantization. * p < 0.05; ** p < 0.01 versus control.

Figure 3

BTFS-induced cell cycle arrest at S phase in A2780/CP70 cells and regulated proteins expression related with S phase. (A,B) BTFS induced cell cycle arrest at S phase by flow cytometry. Statistical analysis bar chart, * p < 0.05 and ** p < 0.01 versus control. (C,D) Effects of BTFS on the expression of cell cycle-related proteins in A2780/CP70 cancer cells. Statistical histogram of protein quantization, * p < 0.05; ** p < 0.01 versus control.

Figure 4

BTFS-induced apoptosis in A2780/CP70 cells. (A) Hoechst 33342 staining confirmed the apoptotic effect induced by BTFS in A2780/CP70 cells. (B) The effect of BTFS on mitochondrial membrane potential in A2780/CP70 cells was determined by JC-1 staining. ** p < 0.01 versus control. (C,D) BTFS-induced apoptosis in A2780/CP70 cells evidenced by flow cytometry. Statistical analysis bar chart, ** p < 0.01 versus control.

Figure 5

BTFS trigged both extrinsic and intrinsic pathways. (A) Effects of BTFS on the activity of caspase-3/7, 8 and 9. * p < 0.05 and ** p < 0.01 versus control. (B,C) Effects of BTFS on extrinsic apoptosis-related protein expression in A2780/CP70 cells. (D,E) Effect of BTFS on intrinsic apoptosis-related protein expression in A2780/CP70 cells.

Figure 6

BTFS caused DNA damage in A2780/CP70 cells. (A) The effect of BTFS 3 on ROS production in A2780/CP70 cells. * p < 0.05; ** p < 0.01 versus control. (B,C) The effect of BTFS on the expression of DNA damage-related proteins in A2780/CP70 cells. * p < 0.05; ** p < 0.01 versus control.

Figure 7

Effect of BTFS on AKT/MDM/P53 pathway related proteins. (A) Western blot image (B) Statistical histogram of protein quantization. * p < 0.05; ** p < 0.01 versus control.