Serum starvation enhances EGCG-induced cell death independent of caspase.

(A) Serum deprivation promotes EGCG-induced cell death in a concentration-dependent and time-course manner. HepG2 cells were treated with different doses of EGCG in full or serum-free medium for 12 h (left panel) or with 60 µM EGCG for different time as indicated (right panel). The cell viability was determined by Hoechst-PI double staining (n = 3, mean ± SD). (B) Representative pictures of Hoechst-PI double staining. HepG2 cells were cultured in full medium (as a control); treated with 60 µM EGCG for 12 h in serum-free medium; or incubated with 20 ng/ml TNFα and 10 µg/ml CHX for 12 h in full medium (as a positive control for apoptosis). (C) EGCG induces caspase-independent cell death. HepG2 cells were treated with EGCG (60 µM×24 h) or in the absence or presence of 40 µM z-VAD-fmk. The co-treatment with TNFα (20 ng/ml) and CHX (10 µg/ml) for 12 h was used as a positive control. Cell viability was determined as described in Panel A. **p<0.005 comparing to the group without z-VAD (Student's t-test, n = 3). (D) No caspase-3 activation and PARP cleavage cause by EGCG-induced cell death. Cells were treated with EGCG or TNF/CHX as described in panel C, and cell lysates were collected and subject to western blot.