Fig. 1. Role of MAPK pathway elements in mediating effects of EGCG on p57 expression. (A) p57 is induced by EGCG in NHEK, but not OSC2 cells. Both OSC2 and NHEK cells were treated with 50 μM EGCG for the indicated time period. EGCG was added to replicate flasks at different times and then the cells were harvested at the same time (i.e., at the same stage of growth) to give the indicated exposure times. Only NHEK responded to EGCG by producing large amount of p57 protein at 24 h. (B) p38 inhibitor specifically blocked the EGCG-induced p57 expression. NHEK cells were treated with 50 μM EGCG for 24 h with or without specific inhibitors of p38 (SB203580), MEK (PD98059) or JNK (SP600125). Induction of p57 by EGCG was blocked specifically by the p38 inhibitor, but not by the JNK or MEK inhibitors. (Concentration of inhibitors: ⁺10 μM, ⁺⁺30 μM, added to the cell culture medium 30 min prior to EGCG exposure.)

Fig. 2. Role of MAPK elements in mediating effects of EGCG on growth and apoptosis in OSC2 cells. (A) EGCG-induced growth arrest and caspase 3 activity were blocked by JNK inhibitor. Inhibitors of MAPK elements MEK (PD98059), JNK (SP600125) and p38 (SB203580), were added to the cell culture medium of OSC2 cell cultures for 30 min, followed by exposure to 100 μM EGCG for 24 h prior to the assay analyses (concentrations of inhibitors: 10 μM SB203580, 100 μM PD98059, 25 μM SP600125, dissolved in DMSO as stocks). Only the JNK inhibitor blocked the effects of EGCG by restoring the cell growth and decreasing caspase 3 activity. (B) 100 μM EGCG induced JNK phosphorylation of both JNK1 and JNK2 proteins in OSC2 cells. JNK1/2 were hyper-phosphorylated at 60 min and phosphorylation remained elevated through 120 min.

Fig. 3. Comparison of p57 stably transfected (S2) and control vector-transfected (G6) OSC2 cells. (A) Protein levels of p57 and caspase 14 in OSC2, G6 and S subclones determined by Western blot. Basal levels of p57 and caspase 14 were detected in both OSC2 and G6 cells, while all the S subclones express high levels of p57 protein. Only the S2 subclone produces large amount of caspase 14 protein. (B) Western blot results of the mitochondrial and cytosolic fractions of G6 and S2 cells exposed to 100 μM EGCG. p57-expressing S2 cells did not exhibit translocation of BAX from the cytosol to the mitochondria, while BAX in control vector-transfected G6 cells translocated to the mitochondria (upper). S2 cells also failed to show cytochrome c release, while G6 cells showed extensive cytochrome c release into the cytosol (lower). (C) Western blot results of hyper-phosphorylated JNK in G6 and S2 cells after EGCG exposure. G6 cells showed JNK hyper-phosphorylation throughout the 120 min-period, while JNK phosphorylation was not sustained in S2 cells. Cells were incubated with 100 μM EGCG for time periods as indicated prior to Western analysis for phosphorylated JNK1 and JNK2.

Fig. 4. Xenograft of transfected OSC2 cells in nude mice and tumor development. (A) Result of xenograft experiments using OSC2 derived cells either expressing exogenous p57 (S2 and S5) or GFP (G6), plus the parental cells. Tumors developed in all but one parental OSC2-xenografted animals and all GFP-expressing G6-xenografted animals. In contrast, of the seven S2 xenografted animals, six were tumor-free, and only three out of seven S5-xenografted animals developed tumors. Each animal received 10 million cells subcutaneously and was monitored over a 3-week period. Among OSC2 and G6 cell-induced tumors, some of them invaded into the bone, verified by pathological analyses (data not shown). S2 and S5 cells did not form metastases (data not shown). (B) Frequencies of tumor-free animals xenografted with the cell lines. The tumor incidence was lower in the p57 transfectant subclones compared to OSC2 or G6 (χ² analysis, p < 0.025). (C) The mean peak volumes and the total peak tumor volumes from each group of animals (tumor-free animals received a 0 as tumor value) resulting from different cell lines.