Polyphenols Extracted from Chinese Hickory (Carya cathayensis) Promote Apoptosis and Inhibit Proliferation through the p53-Dependent Intrinsic and HIF-1α-VEGF Pathways in Ovarian Cancer Cells

Abstract

Ovarian cancer is the second most common gynecologic cancer with an estimated 13,940 mortalities across the United States in 2020. Natural polyphenols have been shown to double the survival time of some cancer patients due to their anticancer properties. Therefore, the effect of polyphenols extracted from Chinese hickory seed skin Carya cathayensis (CHSP) on ovarian cancer was investigated in the present study. Cell viability results showed that CHSP is more effective in inhibiting ovarian cancer cells than normal ovarian cells, with the IC50 value for inhibition of cell proliferation of Ovarian cancer cells (OVCAR-3) being 10.33 ± 0.166 μg/mL for a 24 h treatment. Flow cytometry results showed that the apoptosis rate was significantly increased to 44.21% after 24 h treatment with 20 μg/mL of CHSP. Western blot analysis showed that CHSP induced apoptosis of ovarian cancer cells through a p53-dependent intrinsic pathway. Compared with control values, levels of VEGF excreted by OVCAR-3 cancer cells were reduced to 7.87% with a 40 μg/mL CHSP treatment. Consistent with our previous reports, CHSP inhibits vascular endothelial growth factor (VEGF) secretion by regulating the HIF-1α-VEGF pathway. In addition, we also found that the inhibitory effect of CHSP on ovarian cancer is related to the up-regulation of Phosphatase and tension homolog (PTEN) and down-regulation of nuclear factor kappa-B (NF-kappa B). These findings provide some evidence of the anti-ovarian cancer properties of CHSP and support the polyphenols as potential candidates for ovarian cancer adjuvant therapy.

Keywords: Chinese hickory; apoptosis; ovarian cancer; p53; polyphenol.

Figure 1.

Effect of Chinese hickory seed skin Carya cathayensis (CHSP) on cell viability of ovarian cancer cells (OVCAR-3, A2780/CP70) and normal ovarian cells (IOSE 364). * p < 0.05 as compared to control. ** p < 0.01 as compared to control.

Figure 2.

CHSP induced apoptosis in ovarian cancer cells (OVCAR-3) and normal ovarian cells (IOSE 364). (a) CHSP induced apoptosis was analyzed by flow cytometry. Cells treated with CHSP for 24 h were collected, stained with PI and Annexin V, and analyzed by flow cytometry. (b) Calculation of the apoptotic rate of the data in A. (c) Induction of caspase by CHSP. OVCAR-3 cells were treated with CHSP for 4 h, and caspase-3/7 enzymatic activity was determined using a Caspase-Glo 3/7 assay kit. * p < 0.05 as compared to control. ** p < 0.01 as compared to control.

Figure 3.

CHSP activated the p53-dependent intrinsic pathway. (a) CHSP increased the levels of Puma, Bax, Bad, cleaved PARP, and p53 in OVCAR-3 cells. (b) p53. (c) Puma. (d) Bax. (e) Bad. (f) cleaved PARP. * p < 0.05 as compared to control. ** p < 0.01 as compared to control.

Figure 4.

CHSP inhibited cell proliferation via the HIF-1α/VEGF pathway in OVACAR-3 cells. (a) Cells were treated with CHSP for 24 h. Culture supernatants were collected for the VEGF assay by ELISA with a Quantikine Human VEGF Immunoassay kit. (b) CHSP decreased the levels of HIF-1α and HIF-1α bands were quantitated with NIH Image J software for analysis. (c) Cells were transfected with VEGF luciferase reporter and HIF-1α plasmids. After transfection, all the cells were treated with or without CHSP (10 μg/mL) for 16 h and the luciferase activity was determined. * p < 0.05 as compared to control. ** p < 0.01 as compared to control.

Figure 5.

CHSP up-regulated the expression of PTEN and down-regulated NF-kappa B protein in OVACAR-3 cells. (a) CHSP increased the levels of PTEN and decreased the levels of NF-kappa B in OVCAR-3 cells. (b) PTEN. (c) NF-kappa B. * p < 0.05 as compared to control. ** p < 0.01 as compared to control.