Constitutive overexpression of Nrf2-dependent HO-1 confers resistance to apoptosis by EGCG in human lung adenocarcinoma A549 cells

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Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea is a widely studied cancer chemopreventive and anticancer agent. The major mechanism of EGCG-mediated anticancer effects appears to be related to induce apoptosis of cancer cells. Studies have shown differential sensitivity to EGCG between tumor cells or tumor cells versus normal cells. EGCG has been shown to modulate multiple but different signal transduction pathways depending on the cell types. These may be due to the differential oxidative status imposed by EGCG or differential expression level of endogenous antioxidant defense enzymes in different cells. Heme oxygenase-1 (HO-1) is known to be induced by a variety of stress stimuli and represents a key enzyme for the protection of cells against oxidative tissue injury. However, in cancer cells HO-1 may contribute to growth advantage and cellular resistance against chemotherapy. We found that human lung adenocarcinoma A549 cells are markedly resistant to apoptosis induction by EGCG even at high concentrations (∼100 μM; 72 h). The lung cancer cells were found to express high constitutive HO-1 and Nrf2, as compared to other human epithelial cancer cells where EGCG leads to apoptosis. We investigated whether the constitutively elevated-HO-1 expression in A549 cells is associated with the observed exceptional resistance to apoptosis by EGCG. Employing two approaches: selective HO-1 inhibition with tin protoporphyrin (SnPPIX) and siRNA-mediated knockdown of HO-1 gene, we show that HO-1 blocking in A549 cells i) reduces cell proliferation; ii) upregulates p53, p21, and Bax; iii) sensitizes lung cancer cells to apoptosis induction by EGCG as well as by hydrogen peroxide and tert-butylhydroperoxide, suggesting a protective role of HO-1 against ROS-mediated apoptosis. In parallel with HO-1 upregulation, cellular Nrf2 was shown to be located in the nucleus and bound to antioxidant response element sequence, which suggests that the constitutive HO-1 expression is mediated by Nrf2 transcriptional activation. Additionally, we found that the transcriptional activation of Nrf2-targeted HO-1 mRNA and protein expression as well as cellular resistance to EGCG were decreased by inhibition of protein kinase C alpha (PKCα) and extracellular signal-regulated kinase (ERK). These effects were more profound by PKCα inhibitor, Ro317549, than by ERK inhibitor, PD98059, suggesting the involvement of an oxidative signaling pathway in the elavation of HO-1 expression. More importantly, we observed that EGCG at higher concentrations (>200 μM) could induce apoptosis and completely blocks HO-1 protein and mRNA by inhibition of PKCα phosphorylation as well as translocation and transactivation of Nrf2. Collectively, our results suggest a possibility that HO-1-, Nrf2- and PKCα-signaling pathways could be targets for lung cancer chemoprevention and chemotherapy.