In vitro and in vivo effects of water extract of white cocoa tea (Camellia ptilophylla) against human prostate cancer

Abstract

Purpose: We evaluated the chemotherapeutic effect of water extract of white cocoa tea (WCTE) against human prostate cancer (PCa) in vitro and in vivo.

Methods: Cell viability and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Western blotting was performed to determine changes in levels of various proteins. Effect of WCTE was determined in athymic nude mice implanted with PC-3 cells.

Results: Treatment with WCTE (100-150 microg/ml) inhibited cell proliferation, which correlated with G2/M phase arrest in PC-3 cells. WCTE treatment to PC-3 cells resulted in (1) induction of WAF1/p21 and KIP1/p27, (2) decrease in cyclins D1, D2 and E, (3) decrease in cyclin-dependent kinase (cdk) 2, 4 and 6, (4) induction of Bax and down-regulation of Bcl-2, (5) decrease in procaspase-3, -8, (6) inhibition of nuclear translocation and phosphorylation of NF-kappaB and activation of IKKalpha, and (7) inhibition of phosphorylation and degradation of IkappaBalpha. Oral administration of WCTE (0.1 and 0.2%, wt/vol) to athymic nude mice resulted in greater than 50% inhibition of tumor growth. There was a decrease in expressions of cyclin D1, Bcl-2 and p-NF-kappaB and an increase in WAF1/p21 and Bax in tumor tissues of mice.

Conclusion: WCTE can be a useful chemotherapeutic agent against human PCa.

Fig. 1

(A) Effect of WCTE on cell growth. As detailed in Materials and Methods, PC-3 cells were treated with WCTE and the viablility of cells was determined by the MTT assay. The data are expressed as the percentage of cell viability and represent the means ± SE of three experiment in which each treatment was performed in multiple wells. (B) Effect of WCTE on apoptosis in PC-3 cells. (C) Effect of WCTE on cell cycle distribution in PC-3 cells. The cells treated with WCTE were collected and stained with PI by using an apoptosis APO-DIRECT kit obtained from Phoenix Flow systems as per vendor’s protocol followed by flow cytometry. Following FACS analysis, cellular DNA histograms were further analyzed by ModfitLT V3.0. The data are representative example for duplicate tests. The details are described in Materials and Methods.

Fig. 2

(A) Effect of WCTE on protein expression of WAF1/p21 and KIP1/p27 in PC-3 cells. (B) Effect of WCTE on protein expression of cyclin D1, cyclin D2 and E in PC-3 cells. (C) Effect of WCTE on protein expression of cdk2, cdk4 and cdk6 in PC-3 cells. As detailed in Materials and Methods, the cells were treated with WCTE (100–150 μg/ml) and then harvested. Total cell lysates were prepared and 50 μg protein was subjected to SDS-PAGE followed by western blotting analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the western blotting and reprobing it for β-actin. The western blotting shown here are representative of three independent experiments with similar results. The values above the figures represent relative density of the bands normalized to β-actin.

Fig. 3

(A) Effect of WCTE on protein expression of Bax, Bcl-2 in PC-3 cells. (B) Bax to Bcl-2 ratio. (C) Effect of WCTE on protein expression of caspase-3 and -8 in PC-3 cells. (D) Effect of WCTE on IKKα and phosphorylation and degradation of IκBα in PCa PC-3 cells. (E) Effect of WCTE on activation of NF-κB PCa PC-3 cells by western blotting analysis. As detailed in Materials and Methods, the cells were treated with WCTE (100–150 μg/ml) and then harvested. Total cell lysates were prepared and 50 μg protein was subjected to SDS-PAGE followed by western blotting analysis and chemiluminescence detection. Equal loading of protein was confirmed by stripping the western blot and reprobing it for β-actin. The western blots shown here are representative of three independent experiments with similar results. The values above the figures represent relative density of the bands normalized to β-actin.

Fig. 4

Effect of oral administration of WCTE on PC-3 tumor growth in athymic nude mice. Approximately 1 million PC-3 cells were s.c. injected in each flank of the mouse to initiate tumor growth. 24 h after cell implantation, the control group of animals continued to receive drinking water whereas animals of group 2 and 3 received 0.1 and 0.2% WCTE, respectively, in the same drinking water ad libitum. Water bottles were changed every other day. Once tumors stated to grow, their sizes were measured twice weekly and the tumor volume was calculated. (A) Average tumor volume of water-fed, 0.1 and 0.2% WCTE-fed mice plotted over days after tumor cell inoculation. (B) Number of mice remaining with tumor volumes ≤1,200 mm³ after they consumed 0.1 and 0.2% WCTE for the indicated days. Values represent mean ± SE of six animals. *p<0.01, **p<0.001 versus the water-fed group of mice; ***p<0.01 versus the 0.1% WCTE-fed group of mice. Details are described in Materials and Methods. (C) photographs of excised tumors form each group. (D) Protein levels of Bax, Bcl-2, cyclin D1, p21 and phospho-NF-κB/p65 as determined by western blotting analysis in pooled tumors excised from mice treated with WCTE. Equal loading of protein was confirmed by stripping and reprobing the blots with β-actin antibody. Western blotting analysis was conducted in all animals of each group, and only representative blots of two animals from each group are shown. (E) Bax to Bcl-2 ratio.