Abstract
Epigallocatechin 3-gallate (EGCG), the major polyphenol found in green tea, exerts antiproliferative and proapoptotic effects in many cancer cells. However, we found that among many cancer cells human lung adenocarcinoma A549 cells are markedly resistant to apoptosis induction by EGCG (even at 100 microm for 72 h). Heme oxygenase-1 (HO-1) induced by stress stimuli represents a prime cellular defense mechanism, but it may be associated with enhanced cell proliferation and chemoresistance in some cancer cells. Because we found that A549 cells constitutively overexpress HO-1 and its associated transcription factor Nrf2, we tested an hypothesis that EGCG resistance in these cells may be linked with Nrf2-mediated HO-1 overexpression. HO-1 inhibition with tin-protoporphyrin IX and silencing with RNA interference rendered cells more sensitive to apoptosis induction by EGCG and classical prooxidants. Interestingly, EGCG at high concentration (>200 microm) induced apoptosis by suppressing expression of HO-1 protein and mRNA, and this effect correlated with a decrease in both Nrf2-ARE binding and HO-1-ARE-luciferase activity, suggesting Nrf2-driven transcriptional activation of ho-1. Because we observed notably high levels of phosphorylated protein kinase C alpha and its suppression by EGCG and deferoxamine (an iron chelator), a possible mechanism involving phosphorylated protein kinase C alpha and iron in Nrf2-HO-1 activation was further investigated. Collectively, our findings suggest that Nrf2-mediated HO-1 overexpression confers resistance to apoptosis induction by EGCG; therefore, its inactivation may be a target for overcoming the resistance to chemoprevention and chemotherapy.
Highlights
Heme oxygenase-1 (HO-1) is known to be highly induced by a variety of stress stimuli and many cancer chemopreventive agents, and it represents a prime cellular defense mechanism against oxidative stress via antioxidant function of its catalytic products like bilirubin and carbon monoxide (CO) with concomitant induction of iron sequestering ferritin [3, 54]
While comparing Epigallocatechin 3-gallate (EGCG)-mediated cytotoxicity in various cancer cells, we observed that human lung adenocarcinoma A549 cells, which belong to non-small cell lung cancer (NSCLC), were significantly resistant to the induction of apoptosis by EGCG
To determine whether HO-1 overexpression is a general property of lung cancer cells or unique to NSCLC represented by A549 cells, and if EGCG resistance is specific to lung-derived cancer cells, we explored this possibility with additional lung cancer cells (NSCLC H441 cells and SCLC H196 cells) and primary Normal human bronchial epithelial cells (NHBEs)
Summary
Chemicals and Reagents—A purified preparation of EGCG (Ͼ98% pure) was procured from Sigma, and aliquots of stock solution (20 mM) prepared in Me2SO were stored at Ϫ80 °C for further use. Cell Treatment and Viability—The A549, LNCaP, DU145, PC-3, AsPC-1, HCC1419, SKOV-3, and HT-29, H196, and H441 human cancer cells were obtained from American Type Culture Collection (Manassas, VA) and cultured according to the manufacturer’s protocol. Immunocytochemistry of Nrf2—A549 cells grown on culture slides and treated with EGCG as indicated were fixed in 100% methanol and incubated with monoclonal rabbit anti-Nrf antibody (2.5 g/ml) in phosphate-buffered saline containing 1.5% bovine serum albumin for 60 min at room temperature. At 40 M of treatment for 48 h, significant cytotoxicity (p Ͻ 0.01– 0.001) was noted for SKOV-3, HT-29, HCC1419, and PC-3 cells, whereas moderate toxicity at the same concentration of EGCG was observed for AsPC-1 and LNCaP cells. A549 cells were found to express (PerkinElmer Life Sciences) by the random hexamer priming high levels of GCL and thioredoxine (Trx) but not ferritin system using a RediprimeTM II kit (Amersham Biosciences). and glutathione peroxidase (Gpx) as compared with other
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