Type of Document Dissertation Author Suh, Yewseok Author's Email Address email@example.com URN etd-07072006-115648 Title Molecular Characterization of Tea Catechin Treated Human Prostate Cancer Cell Lines Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Dr. Hong Li Committee Member Dr. Joseph B. Schlenoff Committee Member Dr. Qing-Xiang Sang Committee Member Dr. Thomas C.S. Keller III Committee Member Keywords
Date of Defense 2006-05-12 Availability unrestricted AbstractProstate cancer is the most prevalent cancer diagnosed among men in the United States. The major green tea polyphenol epigallocatechin-3 gallate (EGCG) has been shown to exert remarkable preventive effects against various types of cancer including prostate cancer. Recent human clinical study proved that EGCG can prevent progression of high grade prostatic intraepithelial neoplasia (PIN) to prostate cancer. Cellular studies show that EGCG exhibits antiproliferative and apoptotic effects in androgen-responsive LNCaP and androgen-unresponsive DU145, PC3 prostate cancer cell lines. Previously, we have established a new type of prostate cancer line, androgen repressed carcinoma of prostate (ARCaP). ARCaP cells are highly invasive and metastatic and this cell line showed unique response to androgen since the hormone repressed the proliferation. In this study, we show that androgen-repressed ARCaP prostate cancer cell line, which represents more advanced and aggressive type of prostate cancer, is resistant to EGCG treatment.
In Western blot analyses, EGCG treated ARCaP cell line showed increase in phosphorylation of NF-ƒÛB and decrease in activation of p38 MAPK and Bax/Bcl-2 ratio. The levels of p21/CIP1/WAF1, cyclin-dependent kinases (CDKs) 2, 4, 6, activated forms of Akt and c-Jun NH2-terminal protein kinase (JNK) remain unchanged in EGCG treated ARCaP cells whereas decrease in active Akt, active JNK, and CDKs 2, 4, 6, and increased level of p21/CIP1/WAF1 were observed in LNCaP cells upon EGCG treatment. Moreover, EGCG treatment confers stronger adherence to types I, II, IV collagen extracellular matrix proteins on ARCaP cells. On the contrary, LNCaP cells lost the adhesion significantly to all extracellular matrix proteins tested, including collagens, fibronectin, laminin, vitronectin, and tenascin. Most importantly, ARCaP cells formed more colonies on soft agar in our anchorage-independent assay when treated with EGCG whereas the colony forming ability of LNCaP cells was totally abolished under the same condition. This study suggests that the use of tea catechin EGCG as anticancer agent may not be effective for treating patients with androgen repressed subtype of prostate cancer. This is the first study of apoptosis in ARCaP cell line.
The GeneChip microarray analysis revealed several genes that were differentially expressed when treated with EGCG. Among those, matrix metalloproteinases (MMPs) 1 and 3 were significantly up regulated in LNCaP cells upon EGCG treatment. Both RNA transcription and protein secretion/activation of these MMPs were observed by GeneChip assay, reverse transcription-polymerase chain reaction (RT-PCR) and by enzyme linked immunosorbent assay (ELISA) which can detect proMMP1 and total MMP3 in cell culture media. This feature is very unique in that (1) the MMPs are generally known to be involved in tumor invasion and metastasis not the cell death, and (2) the other EGCG sensitive prostate cancer cell lines, DU145 and PC3, did not display such characteristics. EGCG did not affect the expression of these MMPs in ARCaP cells also.
Using GeneChip analysis, we found several genes whose expressions were oppositely regulated in LNCaP and ARCaP cells upon EGCG treatment. These include early growth response -1 (EGR1), growth arrest and DNA damage inducible gene 45 (GADD45). The expression level of EGR1 and GADD45 were decreased in ARCaP cells but the level was increased in LNCaP cells after EGCG treatment. These results suggest that the proapoptotic EGR1 and GADD45 may play a role in EGCG induced apoptosis in LNCaP cells and thus may explain, at least in part, the resistance of ARCaP cells against such apoptotic stimuli. The role of these proteins in EGCG induced apoptosis is not known. The decreased level of topoisomerase II in EGCG treated LNCaP cells is also exciting. Topoisomerases are necessary in DNA replication and thus for survival of the organism. Since only LNCaP cells, but not ARCaP cells, displayed reduced expression of topoisomerase II during EGCG induced apoptosis and since ARCaP cells underwent apoptosis when treated with topoisomerase inhibitor etoposide, the function of this enzyme might be involved in life or death decision of ARCaP and LNCaP cells.
Elucidating the molecular effects of these proteins and the mechanisms of how these proteins function in ARCaP and LNCaP cell lines would help understanding the prostate cancer and may help with future design of cancer chemopreventive and chemotherapeutic agents.
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