8. The Anti-tumor Mechanisms of GCP

Effects of GCP on cell cycle

Cell cycle is a series of processes of cell growth, differentiation, division. Nuclear DNA encodes most of the information needed to produce cell constituents. During cell growth or S phase, when the DNA is replicated, the nuclear DNA increases from the G1 level (period preceding DNA synthesis) until it reaches twice the amount of DNA phase (gap between the end of synthesis and beginning of mitosis).

Genistein has been shown to inhibit cell proliferation of various cancer cell lines in vitro including both estrogen receptor-positive and estrogen receptor-negative breast carcinoma cell lines. It is generally accepted that genistein can arrest the cells at G2-M phase of the cell cycle, but a recent report has shown that genistein can also cause G0-G1 arrest in a mouse fibroblast cell line. Collectively, these reports suggest that cell cycle arrest caused by genistein may be attributable to both G0-G1 and G2-M arrest, depending on the cell lines and experimental conditions. To investigate the effect of GCP on cell cycle, we performed the experiment as follows:

A) Cell culture

Human breast cancer cell line MDA-MB-231 (1x10⁶/ml) x 10 ml were cultured in 10 cm culture dishes in a DMEM medium containing 10% FBS for 24 hours. After changing to a fresh medium, the samples were added as follows: Control- DMSO 10 l (the final concentration was less than 0.1 %). GCP treated- GCP 100mg/ml dissolved in DMSO, added 10 l in 10 ml cell culturing medium, the final concentration of GCP was 100 g/ml. The cells were incubated for an additional 48 hours and then the cell proliferation was photographed with Trypan blue staining to count the number of cells.

B) Flow Cytometry Analysis

The harvested cell suspensions were fixed by ice-cold 70% ethanol for 24 hours. The cells were then re-suspended with phosphate-buffer saline containing 0.1% glucose and RNase (100 U/ml) for 30 min at room temperature. The cells were stained with propidium iodide (PI, 50 g/ml) for 10 minutes just before the flow cytometry analysis. The cell cycles of GCP-treated MDMAB-231 cells were analyzed by using the flow cytometry (Becton Dickinson FACScan, CA, USA).

C) Western Blot Analysis for Cell Cycle Related Proteins

The samples were applied onto 30g protein and run to 12% SDS- PAGE. The proteins were transferred to PVDF membrane and blocked by 5% non-fat milk for 1 hour at room temperature (RT). The membranes were then incubated with 1000:1 diluted anti-human p53 (SantaCruz, sc-126); p21 (SantaCruz, sc-397) and CDK4 (SantaCruz, sc-749), respectively overnight at 4 ℃. After washing, the membranes were incubated with HRP-linked second antibodies for 1 hour at RT. The immunoblots were developed using ECL reagent (Amersham) and the chemiluminescence were visualized with X-film (Kodak).

A) GCP inhibit MDMAB-231Cell Proliferation: Morphological Analysis

The figure shows that GCP arrested cell proliferation, and enlarged and prolonged the cell soma. The number of cells per dish was much less than in the GCP treated group than in the control.

B) GCP inhibit MDMAB-231Cell Proliferation as well as Lead Cell Death

Result: GCP inhibited more than 50% of the MDMAB-231 cell proliferation as well as lead to 30 % cell death [Control: Total=6/128 x10⁶ (Dead=4.69%); GCP:Total=18/60 x10⁶ (Dead=30%)] . * Total Cell Number = x10⁶; Death Cell % = Dead cells / Total cells x100%.

This result suggests that GCP inhibited cell proliferation as well as induced cancer cells to apoptosis.

C) GCP Arrests G1S Phase and G2M Phase of Cancer Cells: Cell Cycle Analysis

Result: The figure below shows a cell cycle analysis of MDMAB-231 cells treated with GCP (100 g/ml, 48h), the vertical represents the cell number counted by flow cytometry, the horizontal represents the fluorescence density of PI-positive staining (600 nm). M1 contains apoptotic cells; M2 (G1 phase) contains the cells which are preparing DNA synthesis, M3 (S phase) contains the cells which are in the DNA synthesis phase; M4 (G2M phase) contains the cells which are preparing to go the mitosis process. The figure showed GCP treatment induced the apoptotic cells (M1) increased from 5.86% to 19.34%; GCP induced into G2M arrest in the cell cycle and could not go into mitosis. The DNA content in G1S phase (M2 + M3) decreased from 73.13 % to 14.91% and G2M phase (M4) increased from 20.21 % to 45.27 %. The results suggest that GCP inhibited DNA synthesis and induced apoptosis in MDMAB-231 cells through G1S phase and G2M phase arrest manner.

D) Induction of Cell Cycle Related Molecules by GCP Treatment

Result: The figure below shows that GCP dose-dependently increased expression of cdk4 protein in MDA-MB-231 cells from 100 to 400 g/ml after 48 hour treatment. The combined results of this data showing the apoptosis related evidence suggests that GCP activated p53 and p21^waf1 leads to the dephosphorylation of Cyclin/cdk4, the wild type activated p53 fragment 1, and the activated p21^waf1 (increased cdk protein subunit), and finally inhibits the cell cycle.

Summary:

The data above indicates that GCP inhibits cancer cell proliferation and cell cycle through G1S phase and G2M phase arrest manner. The cycle related protein p53 and p21^waf1 and cdk4 were involved in the GCP induced cell cycle arrest.