To more clearly understand the molecular mechanisms involved in synergistic enhancement of cancer preventive activity with the green tea polyphenol (2)-epigallocatechin gallate (EGCG), we examined the effects of cotreatment with EGCG plus celecoxib, a cyclooxygenase-2 selective inhibitor. We specifically looked for induction of apoptosis and expression of apoptosis related genes, with emphasis on growth arrest and DNA damage-inducible 153 (GADD153) gene, in human lung cancer cell line PC-9: Cotreatment with EGCG plus celecoxib strongly induced the expression of both GADD153 mRNA level and protein in PC-9 cells, while neither EGCG nor celecoxib alone did. However, cotreatment did not induce expression of other apoptosis related genes, p21 WAF1 and GADD45. Judging by upregulation of GADD153, only cotreatment with EGCG plus celecoxib synergistically induced apoptosis of PC-9 cells. Synergistic effects with the combination were also observed in 2 other lung cancer cell lines, A549 and ChaGo K-1. Furthermore, EGCG did not enhance GADD153 gene expression or apoptosis induction in PC-9 cells in combination with N-(4-hydroxyphenyl)retinamide or with aspirin. Thus, upregulation of GADD153 is closely correlated with synergistic enhancement of apoptosis with EGCG. Cotreatment also activated the mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38 MAPK: Preteatment with PD98059 (ERK1/2 inhibitor) and UO126 (selective MEK inhibitor) abrogated both upregulation of GADD153 and synergistic induction of apoptosis of PC-9 cells, while SB203580 (p38 MAPK inhibitor) did not do so, indicating that GADD153 expression was mediated through the ERK signaling pathway. These findings indicate that high upregulation of GADD153 is a key requirement for cancer prevention in combination with EGCG. ' 2006 Wiley-Liss, Inc.Key words: EGCG; apoptosis; ERK1/2; prevention (2)-Epigallocatechin gallate (EGCG), a main constituent of green tea polyphenol, can act synergistically with cancer preventive agents (such as sulindac and tamoxifen) in inducing apoptosis of lung, colon and breast cancer cells. 1-3 Since EGCG and green tea have a wide range of target organs and are nontoxic for animals and humans, there is growing interest in increasing the antitumor activity of cancer preventive agents in combination with EGCG or green tea. [4][5][6] However, the molecular mechanisms responsible for the synergistic effects of cotreatment with EGCG plus cancer preventive agents are not known. Using human cDNA cancer expression array, we have found upregulated expressions of 2 genes, growth arrest and DNA damage-inducible gene 153 (GADD153) and p21 WAF1 , and downregulated expressions of 4 genes, T-plasminogen activator, TIMP3, IL-1b and integrin b4: All of these genes are associated with synergistic induction of apoptosis of human non-small-cell lung carcinoma (NSCLC) cell line PC-9 by cotreatment with EGCG plus sulindac. 7 Treatment with either EGCG or sulindac alone did not affect these gene expressions. Since upregulation of GADD153 gene expressi...

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL‐60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL‐60 cells with Zn for 6 h in the presence of Py (1 µm) exhibited cytotoxicity in a Zn dose‐dependent manner (25–200 µm). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose‐dependent fashion (50–100 µm), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase‐mediated dUTP‐biotin nick end‐labeling method, peaked at 25 µm, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N′,N′‐tetrakis (2‐pyridylmethyl)ethylenediamine, a cell‐permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 µm Py and 25 µm Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP‐ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen‐activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z‐Asp‐CH2‐DCB, a broad‐spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK‐specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z‐Asp‐CH2‐DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.

The transition metal zinc (Zn) is an endogenous regulator of apoptosis. The ability of Zn to modulate apoptosis is believed to be mediated by the regulation of caspase activity. Previously, we reported that an acute influx of labile Zn induced apoptosis via activation of caspase in human leukemia HL-60 cells treated with a Zn ionophore (Py, pyrithione) and Zn at 1 and 25 m mM, respectively. In the present study, we investigated the involvement of caspase-3 in Py (1 m mM)/Zn (25 m mM)-induced apoptosis in HL-60 cells. Pro-caspase-3 is an inactive form of caspase-3. The processing of pro-caspase-3, a sign of caspase-3 activation, occurred 6 h after treatment with Py/Zn. Proteolysis of poly (ADP-ribose) polymerase (PARP), a substrate of caspase-3, was also observed 6 h after treatment with Py/Zn. We also confirmed the elevation of caspase-3 activity as an index of the cleavage of amino acid sequences recognized by activated caspase-3. An inhibitor of caspase-3 attenuated the appearance of the DNA ladder. Taken together, these results indicate that the activation of caspase-3 is partly responsible for the induction of apoptosis in Py/Zn-treated HL-60 cells.

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