Binding of (-)-epigallocatechin-3-gallate to the Hsp70 ATPase domain may promote apoptosis in colorectal cancer
University of Chester
23 Sep 2007
17 Dec 2007
1 Mar 2008
colorectal cancer, (-)-epigallocatechin-3-gallate, Hsp70, apoptosis
Colorectal cancer (CRC) patients frequently have a poor prognosis because of metastases and drug resistance. Heat-shock protein 70 (Hsp70) over-expression in cancer may exacerbate these factors by inhibiting apoptosis, thus providing a potential therapeutic target. The green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG) induces apoptosis in numerous cancer cell lines by unclear mechanisms. This study proposed and investigated a novel mechanism by which EGCG might inhibit the anti-apoptotic activity of Hsp70, namely, by competing with adenosine triphosphate (ATP) for binding to the Hsp70 ATPase domain. It also examined the impact of EGCG on the CRC cell line HT-29, which is known to over-express Hsp70.
HT-29 cells were treated with 10–300 µM EGCG for 8, 24 and/or 48 h, then analysed using an MTS proliferation-assay or flow cytometry with Annexin V-FITC and propidium iodide. The competitive binding of EGCG to the Hsp70 ATPase domain was assessed using ATP-agarose, a dot blot and chemiluminescence techniques.
EGCG significantly (P < 0.001) and dose-dependently inhibited HT-29 cell viability. Viability was inhibited by 50% (IC50) at 89 µM and 74 µM after 24- and 48-h treatments, respectively, also suggesting a time-dependent effect. Apoptosis was induced both dose- and time-dependently, commencing at 50 µM after 8- and 24-h treatments. Further, apoptosis correlated significantly (P < 0.01) with reduced viability as measured by MTS at 24 h, indicating 98% causality. The dot blot suggested 200 µM EGCG competed with ATP for binding to Hsp70, presumably by binding its ATPase domain, theoretically implying this flavonoid could inhibit the anti-apoptotic effect of Hsp70.