Theaflavin-3, 3'-digallate induces apoptosis and G2 cell cycle arrest through the Akt/MDM2/p53 pathway in cisplatin-resistant ovarian cancer A2780/CP70 cells - PubMed (original) (raw)

Theaflavin-3, 3'-digallate induces apoptosis and G2 cell cycle arrest through the Akt/MDM2/p53 pathway in cisplatin-resistant ovarian cancer A2780/CP70 cells

Youying Tu et al. Int J Oncol. 2016 Jun.

Abstract

Ovarian cancer is the most lethal gynecological cancer among women worldwide. Adverse side effects and acquired resistance to conventional platinum based chemotherapy are major impediments in ovarian cancer treatment, and drive the development of more selective anticancer drugs that target cancer-specific defects. In this study, theaflavin-3, 3'-digallate (TF3), the major theaflavin monomer in black tea, exhibited a potent growth inhibitory effect on the cisplatin-resistant ovarian cancer A2780/CP70 cells (IC50, 23.81 µM), and was less cytotoxic to a normal ovarian IOSE‑364 cells (IC50, 59.58 µM) than to the cancer cells. Flow cytometry analysis indicated that TF3 induced preferential apoptosis and G2 cell cycle arrest in A2780/CP70 cells with respect to IOSE‑364 cells. TF3 induced apoptosis through both the intrinsic and extrinsic apoptotic pathways, and caused G2 cell cycle arrest via cyclin B1 in A2780/CP70 cells. The p53 protein played an important role in TF3-induced apoptosis and G2 cell cycle arrest. TF3 might upregulate the p53 expression via the Akt/MDM2 pathway. Our findings help elucidate the mechanisms by which TF3 may contribute to the prevention and treatment of platinum-resistant ovarian cancer.

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Figures

Figure 1

Figure 1

Chemical structure of TF3 used in this study.

Figure 2

Figure 2

Effect of TF3 on cell growth in human ovarian cancer cells A2780/ CP70 and normal ovarian cells IOSE-364. Cells (1×104/well) were seeded in 96-well plates, incubated overnight, and then treated with TF3 for 24 h. Cell viability was determined by MTT assay. Data represent means ± SD from three independent experiments. Significant differences among the treatments are indicated by different letters (p<0.05).

Figure 3

Figure 3

TF3 induces apoptosis in A2780/CP70 and IOSE-364 cells. (A) Flow cytometry of TF3 treated A2780/CP70 and IOSE-364 cells using a double-staining method with FITC-conjugated Annexin V and PI. The upper left and low left quadrant indicate the percentage of dead and live cells, respectively, and the upper right and low right quadrants indicate the percentage of late and early apoptotic cells, respectively. (B) Caspase-3/7 activity levels with TF3-treatment for 24 h. The caspase-3/7 activity of the control cells after treatment was arbitrarily expressed as 100%. Data represent means ± SD of three independent experiments. Significant differences among the treatments are indicated by different letters (p<0.05).

Figure 4

Figure 4

TF3 induces cell cycle arrest in A2780/CP70 and IOSE-364 cells. Cells were treated with various concentrations (0, 5, 10 and 20 μM) of TF3 for 24 h, fixed in 70% ethanol, and stained with propidium iodide. DNA contents were determined by flow cytometry.

Figure 5

Figure 5

Effect of TF3 on the intrinsic apoptotic pathway in A2780/CP70 cells. Protein lysates were prepared from A2780/CP70 cells after treatment with various concentrations (0, 5, 10 and 20 μM) of TF3 for 24 h. Puma, Bax, Bad, Bcl-xL and caspase-9 protein levels were analyzed by western blotting. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among the treatments are indicated by different letters (p<0.05).

Figure 6

Figure 6

Effect of TF3 on the extrinsic apoptotic pathway in A2780/CP70 cells. Protein lysates were prepared from A2780/CP70 cells after treatment with various concentrations (0, 5, 10 and 20 μM) of TF3 for 24 h. DR5, FADD, procaspase-8 and cleaved caspase-8 protein levels were analyzed by western blotting. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among different treatments are indicated by different letters (p<0.05).

Figure 7

Figure 7

Effect of TF3 on cell cycle G2-related proteins in A2780/CP70 cells. Protein lysates were prepared from A2780/CP70 cells after treatment with various concentrations (0, 5, 10 and 20 μM) of TF3 for 24 h. p21, cyclin B1, phospho-cdc2 and total-cdc2 protein expression levels were analyzed by western blotting. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among the treatments are indicated by different letters (p<0.05).

Figure 8

Figure 8

Role of p53 in TF3-induced apoptosis and G2 cell cycle arrest of A2780/CP70 cells. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. (A) The effects of TF3 on the protein expression of p53 determined by western blotting. Significant differences among the treatments are indicated by different letters (p<0.05). (B) The effects of p53 siRNA (50 nM) on the protein expression of procaspase-8, procaspase-9 and cyclin B1 determined by western blotting. *p<0.05, **p<0.01, compared with respective controls.

Figure 9

Figure 9

Effect of TF3 on Akt/MDM2 pathway in A2780/CP70 cells. Protein lysates were prepared from A2780/CP70 cells after treatment with various concentrations (0, 5, 10 and 20 μM) of TF3 for 24 h. phospho-Akt, total-Akt and MDM2 protein expression levels were analyzed by western blotting. The quantification histograms are shown with error bars. Data represent means ± SD from three independent experiments. Significant differences among the treatments are indicated by different letters (p<0.05).

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