Tremendous effort has been made to improve the anticancer value of tumor necrosis factor (TNF). In this study, we show that wogonin, a flavonoid isolated from Huang-Qin (Scutellaria baicalensis), synergistically sensitizes cancer cells derived from the cervix, ovary and lung to TNF-induced apoptosis, which was associated with inhibition of catalase activity and an increase of cellular hydrogen peroxide (H2O2). Wogonin-induced reactive oxygen species block TNF-induced NF-κB activation through inhibiting phosphorylation on the NF-κB p65 subunit and consequently the DNA binding of NF-κB. In addition, wogonin suppressed the expression of the antiapoptotic factor c-FLIP, which is accompanied with potentiation of TNF-induced caspase 8 activation that initiates apoptosis. Importantly, wogonin did not sensitize normal bronchial epithelial cells to TNF-induced cell death, which was associated with the defect in induction of H2O2. Thus, wogonin specifically sensitizes cancer cells to TNF-induced cytotoxicity through H2O2-mediated NF-κB suppression and apoptosis activation. Our data provide important insights into the molecular mechanism underlying wogonin’s anticancer activity, and suggest this common flavonoid could be used as a TNF adjuvant for cancer therapy. (Cancer Sci 2011; 102: 870–876)
Tumor necrosis factor (TNF, also referred to as TNFα) is a potential anti-cancer agent due to its cytotoxicity to cancer cells. However, most cancer cells are refractory to TNF-induced cell death, which is partly due to activation of nuclear factor-kappa B (NF-κB). Thus, developing NF-κB-blocking means is one of the main approaches for sensitizing cancer cells to TNF-induced cytotoxicity.(1–3)
NF-κB, typically a heterodimer consisting of the p65 (RelA) and p50 subunits is kept inactive in the cytoplasm by inhibitor of κB (IκB). TNF activates IκB kinase (IKK), which in turn triggers phosphorylation and degradation of IκB. The freed NF-κB migrates to the nucleus and activates its target genes. NF-κB’s transcriptional activity is further modulated by phosphorylation and acetylation of NF-κB subunits.(4–6) A number of NF-κB-stimulated genes, including c-FLIP, A20, cIAP-1, cIAP-2, Bcl-xL, XIAP and MnSOD could negatively regulate the TNF-induced apoptosis pathways.(7,8) Reactive oxygen species (ROS), a group of reactive oxygen-containing species including superoxide, hydrogen peroxide (H2O2) and hydroxyl radical are also important modulators of cellular signaling pathways. Although it is reported that intracellular ROS could play a positive role in TNF-induced NF-κB activation,(9) accumulating studies have shown that ROS can inhibit NF-kB activation to prevent transcription of survival genes, thus leading to cell death triggered by TNF.(10–14) Therefore, the redox status of cells may also determine, to a great extent, the biological response that TNF will induce in cells.
Wogonin, 5,7-dihydroxy-8-methoxyflavone, is a flavonoid isolated from Huang-Qin (Scutellaria baicalensis). Wogonin has been shown to exert antioxidant, antiviral, antithrombotic and anti-inflammatory activities in vitro as well as in vivo.(15–18) It was also reported that wogonin inhibited cell growth and induced apoptosis in various tumors in vitro as well as in vivo.(19–21) Importantly, wogonin shows no significant toxicity to normal cells, which makes it a good anti-cancer agent candidate.(19,20) Several recent studies have found that wogonin kills cancer cells through regulation of redox status in malignant cells.(22,23) However, the molecular mechanism by which wogonin exerts it anticancer effect is still elusive.
In this study, we found that wogonin synergistically sensitizes human cancer cells derived from the cervix, ovary and lung to TNF-induced apoptosis through H2O2-mediated suppression of NF-κB and activation of apoptosis. Our data provide important insights into the molecular mechanism underlying wogonin’s anticancer activity, and suggest this common flavonoid could be used as an adjuvant for improving TNF’s value in cancer therapy.
Materials and Methods
Reagents. Human TNF was from PeproTech Inc. (Rocky Hill, NJ, USA). Wogonin was from National Institute of the Control Pharmaceutical and Biological Products (Beijing, China). Butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) were from Sigma (St Louis, MO, USA). zVAD-fmk was from Calbiochem (La Jolla, CA, USA). Antibodies against active caspase-3, caspase-8 and poly (ADP-ribose) polymerase (PARP) were from BD Bioscience (San Diego, CA, USA). Anti-phospho-IκBα and -phospho-RelA/P65 were from Cell Signaling (Beverly, MA, USA). Anti-IκBα, anti-RelA/P65, anti-catalase and anti-c-FLIP were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-MnSOD and -β-actin were from Stressgene (Victoria, BC, Canada) and Protein Tech (Chicago, IL, USA), respectively. CM-H2DCFDA and dihydroethidium (DHE) were purchased from Molecular Probes (Eugene, OR, USA).
Cell culture and cell death assay. HeLa (a cervical cancer cell line), SKOV3 (an ovarian cancer cell line), A549 (a non-small-cell lung cancer cell line) were from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI 1640 or DMEM supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). A549 cells stably transfected with a NF-κB–responsive luciferase reporter were cultured in RPMI 1640 supplemented with 100 μg/mL hygromycin. Beas-2B, an immortalized human bronchial epithelial cell line, was cultured in keratinocyte serum-free medium. Cell death was assessed based on the release of lactate dehydrogenase (LDH) using a cytotoxicity detection kit (Promega, Madison, WI, USA) as previously described.(24) All experiments were repeated three to five times and the average is shown in each figure. For the morphological study of cell death, cells were stained with 50 μg/mL of acridine orange and 50 μg/mL of ethidium bromide and photographed under a fluorescence microscope.
Immunoblot analysis. Whole cell lysate were collected by lysing cells in M2 buffer as previously described.(24) Nuclear protein extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instruction. The proteins were seen by enhanced chemiluminescence (Millipore, Billerica, MA, USA) using a BIO-RAD Image station. Each experiment was repeated at least three times and representative results are shown in each figure.
Luciferase reporter assay. Cells cultured in a 24-well plate were transfected with p5xκB-Luc and pRSV-LacZ by using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, cells were treated as indicated in each figure legend. Luciferase activity was measured using a luciferase assay kit (Promega Corporation) and normalized to the β-galactosidase activity of each sample as previously described.(2)
Electrophoretic mobility shift assay (EMSA). NF-κB-DNA interactions were detected using the non-radioactive Light Shift Chemiluminescent EMSA kit (Thermo Scientific) following the manufacturer’s instructions. Streptavidin–horseradish peroxidase conjugate and the LightShift Chemiluminescent substrate were used to detect the biotin end-labeled DNA.
Detection of ROS. Cells cultured in 12-well plates were treated as indicated in each figure legend. Thirty minutes before collecting cells, H2O2-sensitive fluorescent dye CM-H2DCFDA (5 μM) or O2−-sensitive dye DHE (5 μM) was added. ROS were detected by flow cytometry with a Beckman coulter cell as previously reported.(25)
Detection of catalase activity. Catalase activity was detected using the catalase activity detection kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instructions. Catalase activity was calculated by normalizing the absorbance reading at 520 nm to the total protein amount of each sample, with the untreated sample taken as 100%. The experiment was repeated three times.
Statistical analysis. Data are expressed as mean ± SD. Statistical significance was examined by Student’s paired-sample t test using SPSS statistics software package (IBM SPSS, Chicago, IL, USA) and P < 0.05 was used for significance. The “(−)” in the figures means the cells were exposed to the solvent DMSO.
Wogonin sensitizes cancer cells to TNF-induced cytotoxicity. It has been reported that wogonin treatment significantly sensitizes TNF-resistant leukemia cells to TNF-induced apoptosis.(22) However, whether wogonin impacts the TNF-induced cellular effect in solid cancers has never been addressed. First, HeLa cells were pre-treated with increasing concentration of wogonin for 1 h followed by human TNF treatment (20 ng/mL) for an additional 48 h and cell death was detected by a LDH release assay. Wogonin or TNF alone caused moderate cell death. However, co-treatment with TNF and wogonin resulted in a synergistic increase of cytotoxicity in a dose-dependent manner (Fig. 1a). A similar dose-dependent synergistic effect was also observed with a fixed wogonin dose (20 μM) and increasing concentrations of TNF (Fig. 1b). SKOV3 and A549 cells showed similar synergistic cytotoxicity after wogonin and TNF co-treatment (Fig. 1c). It is noteworthy that Beas-2B, an immortalized but non-transformed human bronchial epithelial cell line, is insensitive to wogonin and TNF cotreatment-induced cytotoxicity (Fig. 1c). Human bronchial epithelial cells (HBEC-2),(24) which were immortalized by insertion of cyclin-dependent kinase 4 and human telomerase reverse transcriptase, were also resistant to wogonin and TNF co-treatment-induced cell death (Fig. S1a). As a whole, these results suggest that wogonin can sensitize cancer cells derived from the cervix, ovary and lung to TNF-induced cell death.
Hydrogen peroxide accumulation contributes to the synergistic cytotoxicity induced by wogonin plus TNF. Wogonin has been reported to be either anti- or pro-oxidant in different cell types.(15,20) In this regard, it is important to determine which redox modulating action of wogonin is executed, and the role of which in cytotoxicity induced by wogonin plus TNF in cancer cells. Wogonin dose-dependently induced rapid H2O2 accumulation in HeLa cells while TNF only induced moderate and transient H2O2 accumulation at 10 min (Fig. 2a,b). Treatment with wogonin plus TNF retained similar strong H2O2 induction as the treatment with wogonin alone (Fig. 2b). In contrast, wogonin and TNF had a marginal effect on superoxide (Fig. 2c). Interestingly, wogonin induced marginal H2O2 accumulation in Beas-2B cells (Fig. 2d) as well as in HBEC-2 cells (Fig. S1b). ROS scavengers BHA and NAC were then used to remove H2O2. These scavengers effectively suppressed the synergistic cytotoxicity in both HeLa and A549 cells (Fig. 3a,b), which is associated with significant reduction of H2O2 levels in cells (Fig. 3c). Notably, when it was added into the culture medium, H2O2 could sensitize HeLa cells and Beas-2B cells to TNF-induced cytotoxicity (Fig. S2a,b). Although mechanisms other than H2O2 accumulation may also be involved, these observations strongly suggest that wogonin-induced H2O2 substantially contributes to the potentiated cytotoxicity caused by TNF and wogonin cotreatment.
Wogonin induces ROS through suppression of catalase activity. H2O2 is detoxified by catalase-mediated conversion to H2O and O2. We examined if wogonin inhibits catalase activity, thereby resulting in elevated H2O2 levels in cancer cells. As shown in Figure 4(a), wogonin alone or combined with TNF caused significant suppression of catalase activity in HeLa cells, starting as early as 5 min, which is well correlated with the induction of H2O2. There were no detectable changes in the expression level of catalase in wogonin-treated cells (Fig. 4b). A similar inhibitory effect on catalase activity by wogonin was also detected in SKOV3 cells and A549 cells (Fig. S3). Consistent with its marginal effect on H2O2 levels in normal bronchial epithelial cells, wogonin moderately affected catalase activity in Beas-2B cells (Fig. 4c).
Wogonin enhances the TNF-induced apoptosis pathway in cancer cells. Typical apoptotic changes such as cell shrinkage, cell membrane blebbing and nuclear condensation were detected microscopically in wogonin and TNF co-treated HeLa cells after staining with acridine orange and ethidium bromide (Fig. 5a). In the co-treated cells, activation of caspases-8 and -3, as well as cleavage of the caspase-3 substrate PARP, were enhanced (Fig. 5b). Furthermore, the pan-caspase inhibitor z-VAD-fmk effectively suppressed cytotoxicity induced by wogonin and TNF co-treatment (Fig. 5c). Collectively, all these results suggest that the TNF-induced extrinsic apoptosis pathway was enhanced by wogonin.
Wogonin inhibits TNF-induced NF-κB activation in cancer cells. Because TNF-induced NF-κB activation plays an important role in protecting cells against TNF-induced apoptosis, we examined whether wogonin inhibits TNF-induced NF-κB activation. TNF robustly stimulated the NF-κB-driven luciferase reporter activity in HeLa and A549 cells, which was effectively blocked by wogonin (Fig. 6a). The molecular target of wogonin in the TNF-induced NF-κB activation pathway is likely downstream of IκBα, because TNF-induced IκBα phosphorylation and degradation were not altered by wogonin treatment (Fig. 6b,c). However, the recovery of IκBα, which occurred following NF-κB activation, was completely blocked by wogonin (Fig. 6c). Consistently, the RelA/p65 nuclear translocation was not affected by wogonin (Fig. 6d). However, wogonin suppressed TNF-induced p65 phosphorylation at Ser 536 in both HeLa and A549 cells (Fig. 6e and data not shown) and also suppressed TNF-induced NF-κB DNA-binding activity (Fig. 6f, compare TNF alone versus TNF and wogonin co-treatment). These results suggest that wogonin blocks TNF-induced NF-κB activation through inhibiting its DNA binding presumably by suppression of p65 phosphorylation at Ser536. TNF-induced expression of the NF-κB targets cFLIP/L and MnSOD was blocked by wogonin (Fig. 6g). Because cFLIP/L and MnSOD are important antiapoptotic molecules, it is likely that their repression by wogonin helps to shift TNF-induced signaling from survival to death.
Blocking TNF-induced NF-κB activation by wogonin depends on H2O2. We next examined whether H2O2 accumulation was involved in suppression of TNF-induced NF-κB by wogonin. Both BHA and NAC effectively attenuated the suppression effect of wogonin on TNF-induced NF-κB reporter activity (Fig. 7a). Consistently, preincubation with BHA or NAC restored IκBα recovery, p65 phosphorylation and NF-κB DNA binding in HeLa and A549 cells (Figs 6f,7b,c and data not shown). Together with the results showing that H2O2 inhibited TNF-induced IκBα recovery and phosphorylation of p65 at Ser536 in HeLa cells (Fig. S4a,b), these results indicate that wogonin-induced ROS accumulation is crucial for suppression of TNF-induced NF-κB activation and subsequent sensitization of TNF-induced cytotoxicity in cancer cells. The results are consistent with previous reports that hydrogen peroxide suppresses NF-κB under certain conditions.(25–27)
In the present study we demonstrated that wogonin synergistically sensitizes human cancer cells derived from the cervix, ovary and lung to TNF-induced apoptosis by inhibiting NF-κB activation via a ROS-dependent mechanism. First, wogonin dose-dependently induced a rapid H2O2 accumulation in cancer cells through suppression of catalase activity. Second, pre-treatment with wogonin inhibited TNF-induced NF-κB activation, which in turn led to reduced expression of anti-apoptotic NF-κB target genes and enhanced activation of the apoptotic pathway. Finally, ROS scavengers effectively attenuated the wogonin-induced inhibition of NF-κB and significantly inhibited the synergistic cytotoxicity in cells co-treated with wogonin and TNF.
Activation of caspase 3, Bax, p53 and p21, as well as suppression of Akt and Cyclin D1 has been proposed for wogonin’s anti-cancer effect in various tumors.(19,21,28) However, the role of redox regulation in wogonin’s anticancer activity, particularly in solid tumors, is not understood. In the present study, we clearly demonstrate that induction of H2O2 is critical for sensitizing TNF-induced cytotoxicity in a variety of human cancer cells derived from cervical, lung and ovarian tumors. Wogonin-induced H2O2 accumulation is likely through suppression of catalase activity, because the inhibition of this H2O2 detoxifying enzyme occurs at an early phase that is well correlated with H2O2 accumulation. Because wogonin did not obviously reduce the superoxide concentration, it is unlikely that wogonin mimics SOD to convert superoxide to H2O2 as proposed in leukemia cells.(22) The action of wogonin-induced H2O2 in sensitizing TNF-induced cytotoxicity is likely to be twofold: to block the survival signal NF-κB and to activate caspases, tipping the cell survival and death balance to the side of death.
It has been shown that wogonin inhibits diverse stimuli-induced NF-κB activation.(29–31) However, the molecular mechanism underlying the suppression effect of wogonin on NF-κB has not been elucidated. We clearly showed that the phosphorylation of p65 at Ser536 and DNA binding of NF-κB was inhibited by wogonin in a ROS-dependent manner. Because phosphorylation of p65 at Ser536 is important for NF-κB’s DNA binding, coactivator interaction and transcription activity,(4,32) determining if wogonin suppresses the p65 phosphorylation kinase or activates the p65 phosphatase will help to understand the defined mechanism for wogonin’s NF-κB suppression. It was reported that wogonin slightly inhibited TNF-induced NF-κB activation through moderately inhibiting phosphorylation and degradation of IκBα in leukemia cells.(22) This discrepancy is currently unknown, but may be due to different cell types and drug concentrations. Nevertheless, we showed for the first time that wogonin induces H2O2 to inhibit NF-κB activation by targeting p65.
It is noteworthy that wogonin has little toxic effect on non-transformed cells, which is consistent with others’ reports that wogonin could selectively kill malignant cells but not normal cells.(19,20) This property renders wogonin a good choice as an adjuvant for cancer therapy. In addition, recent studies have suggested TNF as an endogenous tumor promoter that facilitates inflammation-associated carcinogenesis through NF-κB-mediated cell survival and proliferation in different organs.(33–35) Thus, blocking NF-κB by wogonin would convert TNF from a tumor promoter to a tumor suppressor. In this regard, wogonin may have a potential for chemoprevention.
This study was supported in part by grants from the National Natural Science Foundation of China (30772539 and 30973403), a grant from the Young Scientist Fund of Science & Technology Department of Sichuan Province, China (2010JQ0012) and a grant from the Scientific Research Foundation for the Returned Overseas Chinese Scholar, State Education Ministry of China.