9. Induction of Apoptosis in Cancer Cells by GCP

Apoptosis, defined as a programmed cell death, plays a very important role in many physiological and pathological conditions such as embryo and organ development, immune responses, tumor development and growth. Apoptosis is characterized by many biological and morphological changes; such as, change of mitochondrial membrane potential, activation of caspases, DNA fragmentation, membrane blebbing, and formation of apoptotic bodies. Based on these changes, various assays are designed to detect or quantify apoptotic cells. Typical assays including Annexin-V binding, caspase enzyme activity, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling) and DNA gel electrophoresis.

A) GCP induced apoptosis of cancer cells as detected by Annexin V Staining

To reveal the effect of GCP on induction of apoptosis, we first detected GCP by Annexin –V binding assay. The method is based on the relocation of PhosphatidylSerine (PS) to the outer cell membrane when the cells were on the early stage of apoptosis. Annexin –V conjugates bind to PS on the cell surface and identify apoptotic cells. The B-16 mouse melanoma cells (1x10⁶/ml) were treated with GCP (100 g/ml, contains 6 g/ml of genistein) for 48 hours, the purified genistein (6g/ml) was taken as the positive control. The cells were then stained with Annexin –V and propidium Iodide (PI). The early apoptotic cells were Annexin –V positive and PI negative; the late apoptotic cells were Annexin –V and PI double positive.

Result: The apoptotic cell at early stage (Annexin –V positive and PI negative cells) increased from 0.01% to 12.34 % after GCP treatment. Moreover, GCP induced 33.43 % apoptotic cells (including early and late stages) whereas purified genistein caused only 15.18 % apoptotic cells even though both of them were under equal concentration of genistein. Results suggest that GCP was more effective than purified genistein in induction of apoptosis.

B) GCP Induces Cancer Cells To Apoptosis Through p53 – p21 Pathway.

p53 is a transcription factor that participates in cell cycle checkpoint processes and apoptosis. Among the possible molecular mechanisms of apoptosis, the p53 gene encodes a transcription factor that contributes to several different cellular activities, including apoptosis. The wild-type p53-activated fragment 1 (WAF1 also known as p21) is an important downstream effector in the p53-specific growth arrest pathway. p21 is a cell cycle protein that regulates and can arrest the cell cycle in G1 or S phase (either dependent or independent of p53). Its role may be pivotal in many cell processes including differentiation and apoptosis. The p53 and p21 genes are associated with G1 arrest during the cell cycle and with apoptosis, both of which have a close relationship with the effect of chemotherapy.

To reveal the mechanism by which GCP induces apoptosis, we treated human breast cancer MDA-MB-231 cells with various concentration of GCP (from 100 to 400 g/ml) for 48 hours and detected their p53 and p21 protein expressions in the cell lysates by Western-blot analysis.

Result: GCP dose-dependently increased the expression of p53 and p21 proteins. This suggests GCP induces cancer cells to apoptosis through p53- p21 pathway.

C) GCP Induce Cancer Cells to Apoptosis –DNA Fragmentation Assay

Cell death by apoptosis is characterized by DNA fragmentation. The fragmentation occurs when nuclear enzymes become active and destroy the normal chromatin structure of DNA. Further internucleosomal DNA fragmentation in 180 – 200 base pair may also occur. Such characteristics have been used to distinguish apoptotic cells from normal or necrotic cells. We analyzed the DNA from the tumors treated with GCP and compared them with non-treated tumors.

Result. GCP treatment caused DNA fragments in the tumor treated with GCP (1.0 g/kg, one week, p.o.). And, there were no DNA fragments found in the non-treated tumors.

D) GCP Induces Cancer Cells to Apoptosis: TUNEL Assay

In the late phase of apoptisis, exogenous TdT incorporates dUTP to the ends of DNA fragments which can be detected with the TUNEL assay. We treated the cancer cells with GCP (100 g/ml) for 48 hours and stained the cells with FITC-conjugated Br-dUTP and checked apoptotic cells by fluorescence microscope.

Result. There were many fluorescent apoptotic cells found in GCP treated slides while only a few apoptotic cells found in non-treated control cells.

Summary: The evidence above strongly indicates that GCP can induce early to late stage cancer cells to apoptosis. GCP was more powerful than the purified genistein even though they were in equal concentration. GCP’s mechanisms for induction of apoptosis might involve the p53-p21 pathway.