A Critical Role of Luteolin-Induced Reactive Oxygen Species in Blockage of Tumor Necrosis Factor-Activated Nuclear Factor- B Pathway and Sensitization of Apoptosis in Lung Cancer Cells

Nuclear factor B (NFB) activated by tumor necrosis factor (TNF) attenuates the TNF-induced apoptosis pathway. Therefore, blockage of NFB should improve the anticancer activity of TNF. Luteolin, a naturally occurring polyphenol flavonoid, has been reported to sensitize colorectal cancer cells to TNF-induced apoptosis through suppression of NFB; however, the mechanisms of this effect have not been well elucidated. In this article, we provide evidence showing a critical role of reactive oxygen species (ROS) accumulation induced by luteolin in modulating TNF-activated pathways in lung cancer cells. Luteolin effectively suppressed NFB, whereas it potentiated the c-Jun N-terminal kinase (JNK) to increase apoptosis induced by TNF in lung cancer cells. Our results further demonstrate that luteolin induced an early phase ROS accumulation via suppression of the cellular superoxide dismutase activity. It is noteworthy that suppression of ROS accumulation by ROS scavengers butylated hydroxyanisole, and N-acetyl-L-cysteine prevented the luteolin-induced suppression of NFB and potentiation of JNK and significantly suppressed the synergistic cytotoxicity seen with cotreatment of luteolin and TNF. Taken together, these results suggest that the accumulation of ROS induced by luteolin plays a pivotal role in suppression of NFB and potentiation of JNK to sensitize lung cancer cells to undergo TNF-induced apoptosis. Tumor necrosis factor (TNF) can induce both survival and death signals, depending on cell context and environment (Aggarwal, 2003; Wajant et al., 2003). Most cancer cells are resistant to TNF-induced death, which is believed to involve survival signals such as nuclear factor B (NFB) that blunt the apoptotic pathway (Wajant et al., 2003). Therefore, interventions that inhibit TNF-induced survival signals may sensitize cancer cells to TNF-induced apoptosis. The binding of TNF to TNF receptor 1 (TNFR1) initiates a sequential recruitment of adaptor proteins to form a dynamic complex that leads to activation of diverse signaling pathways (Wajant et al., 2003). The activation of the transcription factor NFB is critical for cell survival and proliferation (Wajant et al., 2003). The role of c-Jun N-terminal kinase (JNK) activation in cell death regulation is controversial, but recent studies suggested that sustained JNK activation is proapoptotic (Lin and Dibling, 2002; Ventura et al., 2004). There is cross-talk between the NFB and JNK activation pathways that controls the outcome of the cells in response to TNF (Lin and Dibling, 2002; Kamata et al., 2005). The caspase cascade can be activated, resulting in apoptotic cell death (Wajant et al., 2003). Therefore, the balance of TNFinduced survivaland death-signaling is pivotal in determining the fate of TNF-exposed cells. During TNFR1 signaling, the I B kinase (IKK) is recruited to the TNFR1 signaling complex through TNF receptor-assoThis work was partly supported by National Institutes of Health grants CA095568 and P30-ES012072. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.032185. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: TNF, tumor necrosis factor; NFB, nuclear factor B; ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; NAC, N-acetyl-L-cysteine; BHA, butylated hydroxyanisole; TNFR1, tumor necrosis factor receptor 1; IKK, I B kinase; MnSOD, manganese superoxide dismutase; SOD, superoxide dismutase; CCS, copper chaperon for superoxide dismutase-1; LDH, lactase dehydrogenase; O2, OH, singlet oxygen; LOO , peroxyl radical; Cu-ZnSOD, copper-zinc superoxide dismutase; PARP, poly(ADP-ribose) polymerase; CM-H2DCFDA, 5-(and -6)-chloromethyl-2 , 7 -dichlorodihydro-fluorescein diacetate acetyl ester; DHE, dihydroethidium; PAGE, polyacrylamide gel electrophoresis; MKP, mitogen-activated protein kinase phosphatase; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoro-methylketone. 0026-895X/07/7105-1381–1388$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 5 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 32185/3200604 Mol Pharmacol 71:1381–1388, 2007 Printed in U.S.A. 1381 http://molpharm.aspetjournals.org/content/suppl/2007/02/13/mol.106.032185.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 3, 2017 m oharm .aspeurnals.org D ow nladed from ciated factor 2 and activated through a receptor-interaction protein-mediated mechanism that involves mitogen-activated protein kinase kinase kinase 3 (Devin et al., 2000; Yang et al., 2001). The activated IKK in turn phosphorylates the I Bs, which retain NFB in the cytoplasm, to trigger their rapid polyubiquitination followed by degradation in the 26S proteasome. This process allows NFB’s nuclear translocation and binding to the promoters of its target genes. Several of NFB’s target genes are found to have antiapoptotic properties (Karin et al., 2004). Induction of the antioxidant manganese superoxide dismutase (MnSOD) by NFB is also implicated to be antiapoptotic (Kamata et al., 2005). The transcriptional activity of NFB is further regulated by phosphorylation and acetylation (Wajant et al., 2003). Therefore, multiple steps affecting post-translational modification of NFB could be targets for regulating its activity. Reactive oxygen species (ROS) are a group of reactive, short-lived, oxygen-containing species such as superoxide (O2 .), hydrogen peroxide (H2O2), hydroxyl radical ( OH), singlet oxygen (O2,), and peroxyl radical (LOO ). Cells have developed effective mechanisms to reduce cellular ROS levels. The superoxide dismutase (SOD) converts O2 . to oxygen (O2) and H2O2. Catalase reduces H2O2 to H2O and O2. There are two types of SOD in the cell. MnSOD/ SOD-2 functions in the mitochondria, whereas copper-zinc SOD (Cu-ZnSOD)/SOD1 is present mainly in the cytosol (Curtin et al., 2002; Nimnual et al., 2003). The copper chaperon for SOD-1 (CCS) is important to maintain the activity of Cu-ZnSOD (Culotta et al., 1997). In addition, glutathione and glutathione peroxidases provide another mechanism in scavenging H2O2 (Curtin et al., 2002). In addition to its direct effect of damaging cellular components, ROS can mediate signal transduction (Rhee, 2006; Shen and Pervaiz, 2006). For example, ROS were found to play a pivotal role in activation of the JNK pathway and nonapoptotic cell death induced by TNF (Lin et al., 2004; Kamata et al., 2005). The role of ROS in TNF induction of the NFB pathway is somewhat controversial, because they were reported to activate, inhibit, or have no effect on this pathway. This diversity in effect may be influenced by cell type and the nature of ROS-inducing agents that were studied (Panopoulos et al., 2005; Shen and Pervaiz, 2006). Luteolin, 3 ,4 ,5,7-tetrahydroxyflavone, a common flavonoid that exists in many types of fruits, vegetables, and medicinal plants (Ross and Kasum, 2002), has been used as an anti-inflammatory agent (Ueda et al., 2002; Bagli et al., 2004; Kanadaswami et al., 2005). Although it was generally believed to be an antioxidant, luteolin was found to induce ROS accumulation (Matsuo et al., 2005). However, the mechanism by which luteolin induces ROS has not been addressed. Luteolin has been shown to sensitize colorectal cancer cell lines to TNF-induced apoptosis via inhibition of NFB and augmentation of JNK (Shi et al., 2004). However, the mechanism of luteolin-induced NFB suppression and JNK potentiation has not been elucidated. In this study, we demonstrate that luteolin induces ROS through suppression of SOD activity, and ROS are crucial in inhibiting the NFB and potentiating the JNK pathways and subsequently sensitizing lung cancer cells to TNF-induced apoptosis. Materials and Methods Plasmids and Reagents. Reporter plasmids 2x B-Luc and pRSV-LacZ have been described previously (Lin et al., 1999). Luteolin was purchased from Sigma (St. Louis, MO). Human TNF was from R&D Systems (Minneapolis, MN). Butylated hydroxyanisol (BHA) and N-acetyl-L-cysteine (NAC) were purchased from Sigma. The JNK inhibitor SP600125 and pan-caspase inhibitor zVAD-fmk were purchased from Calbiochem (La Jolla, CA). Antibodies against I B , JNK1, Cu-ZnSOD, CCS, and caspase-3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-A20 and anti-MnSOD were from BD Biosciences (San Diego, CA). Anti-phospho-JNK, -actin, and -poly(ADP-ribose) polymerase (PARP) antibodies were purchased from BioSource (Camarillo, CA), Sigma, and Biomol (Plymouth Meeting, PA), respectively. Antibodies for BcL-xL, XIAP, and phospho-I B were from Cell Signaling Technology (Danvers, MA). 5-(and -6)-chloromethyl-2 , 7 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) and dihydroethidium (DHE) were purchased from Invitrogen (Carlsbad, CA). SOD activity detection kit was purchased from Cayman Chemical Company (Ann Arbor, MI). Cell Culture. Lung cancer cell lines H23, H2009, H460, and A549 were obtained from American Type Culture Collection (Manassas, VA). All cancer cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM glutamate, 100 U/ml penicillin, and 100 g/ml streptomycin. Cell Death Assays. Cytotoxicity was assessed with a lactase dehydrogenase (LDH) release detection kit (Roche, Penzberg, Germany) (Wang et al., 2006). Cells were seeded in 24-well plates at 70 to 80% confluence and cultured overnight. Then cells were treated as indicated in each figure legend. Culture medium from each well was collected and transferred to 96-well flat-bottomed plates. LDH activity was determined by adding equal volumes of reaction mixture to each well. The absorbance of the samples was measured at 490 nm using a plate reader. All of the experiments were repeated three to five times, and the average is shown in each figure. Cell death was calculated using the following formula: