Figures
Abstract
Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytotchrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7α, GSK3β and Thr58-Myc, which are involved in c-Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7α and GSK3β expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7α suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7α reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.
Citation: Chen X-m, Bai Y, Zhong Y-j, Xie X-l, Long H-w, Yang Y-y, et al. (2013) Wogonin Has Multiple Anti-Cancer Effects by Regulating c-Myc/SKP2/Fbw7α and HDAC1/HDAC2 Pathways and Inducing Apoptosis in Human Lung Adenocarcinoma Cell Line A549. PLoS ONE 8(11): e79201. https://doi.org/10.1371/journal.pone.0079201
Editor: Jian-Xin Gao, Shanghai Jiao Tong University School of Medicine, China
Received: January 23, 2013; Accepted: September 20, 2013; Published: November 12, 2013
Copyright: © 2013 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by grants from the Ministry of Science and Technology of Guizhou Province, China ([2012] 7006 and NY [2011]3072); Guangzhou Medical College (2012C16); the Foundation for Young Scientists of Guangzhou Educational Committee (2012C118). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Despite of the large number of clinical trials aimed at improving patient survival, lung cancer remains a leading cause of cancer-related mortality worldwide in both men and women. Approximately 85% of all lung cancer cases are categorized as non-small cell lung cancer (NSCLC), which is typically diagnosed at advanced stages [1]. Lung adenocarcinoma, the predominant histological subtype of NSCLC, accounts for 20 to 30% of primary lung cancer cases among subjects under 45 years of age, regardless of smoking history [2]. Most cases of NSCLC are unsuitable for surgery and chemotherapy remains the cornerstone of treatment for advanced disease.
Histone deacetylases (HDACs) are enzymes that remove histone acetylation products. This process compacts the structure of chromatin and represses transcription [3]. HDACs act on various nonhistone protein substrates which play a role in the regulation of gene expression, cell proliferation, cell migration, cell death and angiogenesis [4]. Data from preclinical studies have demonstrated that naturally occurring and synthetic histone deacetylase inhibitors have potent anticancer activity.
HDAC1 and HDAC2 belong to the Class I histone deacetylase (HDAC) family. In vivo, these enzymes form complexes with Sin3, NuRD and Co-REST [5]. HDAC1 and HDAC2 also bind directly to DNA binding proteins such as YY1, Rb binding protein-1 and Sp1 [5].
c-Myc is a transcription factor that is responsible for regulating an array of genes involved in cellular proliferation, growth, apoptosis and differentiation [6]. Deregulated c-Myc expression is observed in roughly 70% of all human tumors [10]. c-Myc expression is regulated by gene transcription, and is dependent on mRNA stability and posttranslational control of protein stability [7]–[9].
Posttranslational regulation of c-Myc can be mediated by Skp2 (S-phase kinase-associated protein 2) and Fbw7(F-box and WD repeat domain-containing 7) [11]. Skp2 and Fbw7 are two different recognition subunits of the SCF-type E3 ligase (SCF, Skp1/Cullin/F-box protein complexes) that recognize specific substrates for proteasomal degradation. Regulation of c-Myc involves phosphorylation of c-Myc at Thr58, resulting in Fbw7-mediated proteasomal degradation. Glycogen synthase kinase 3β(GSK3β) is the only kinase known to phosphorylate c-Myc at Thr58 [24].
Skp2 is a promising target for restricting cancer stem cell and cancer progression [12], it’s overexpression is frequently observed in human cancer and it may, therefore, act as an oncogene. In support of this hypothesis, Skp2 has been shown to recognize Cyclin-dependent kinase (Cdk) inhibitors and tumor suppressive proteins such as p27 Kip1 (Cyclin-dependent kinase inhibitor 1B), p57 Kip2 (Cyclin-dependent kinase inhibitor 1C), p130 (130 kDa retinoblastoma-associated protein) and Tob1 (transducer of ERBB-2 1), but not c-Myc [13]–[16]. Skp2-mediated formation of ubiquitin from c-Myc occurs independently of phosphorylation [25].
Fbw7 deficiency is thought to be involved in drug resistance in human cancers [22], [23]. It has been shown to be inactivated by mutation, deletion, or promoter hypermethylation in breast cancer [17], [18], colon cancer [19], [20], and leukemia [21]. Fbw7 is expressed as three different isoforms (designated α, β, and γ) respectively located in the α-nucleus, β-cytoplasm, and γ-nucleolus [26], [27]. As there are no antibodies for these three isoforms we used the best described and longest of the three isoforms Fbw7α, in our experiments with wogonin.
Wogonin (5, 7-dihydroxy-8-methoxyflavanon) is a naturally monoflavonoid extracted from Scutellaria baicalensis radix [28] that has been recognized as an anticancer drug candidate with potentially low toxicity [29]. The anticancer activity of wogonin has been reported various human cell lines including myeloma cell RPMI 8226 [30], hepatocellular carcinoma SK-HEP-1 [31] and SMMC-7721 [32], glioma cancer cells [33], nasopharyngeal carcinoma cells [34], human breast cancer cells [35], [36] and human cervical carcinoma HeLa cells [37]. Its activity is mediated by the induction of apoptosis and cell differentiation, and is regulated by various genes and proteins [38]–[41].
In this study, we evaluated the effects of wogonin on cell viability and apoptosis in the human lung adenocarcinoma epithelial cell line A549. We also assessed the regulation and function of c-Myc/Skp2/Fbw7α and HDAC1/HDAC2 pathways involved in its apoptotic effect.
Materials and Methods
Reagents and Antibodies
Wogonin was purchased from Guangzhou IDC (China) and MG132 (carbobenzoxy-Leu-Leu-leucinal) was obtained from (Beyotime, China). The following antibodies were used: Fbw7(Cdc4, H-300) and p-c-Myc (Thr 58) (Santa Cruz, USA); c-Myc, GSK3β, AIF (apoptosis inducing factor, Proteintech Group, Inc., USA); HDAC1, HDAC2, Skp2, Survivin, Bcl-2 (B-cell lymphoma 2), β-Actin (Boster, China); Mcl-1 (myeloid cell leukemia sequence 1), XIAP(X-linked inhibitor of apoptosis protein, Bioss, China); Cytochome c (KeyGEN, China); PARP (poly ADP-ribose polymerase, Sino Biological Inc., China).
Cell Culture
The human pulmonary adenocarcinoma cell line A549 was obtained from the Cell Bank of the Animal Experiment Center, North School Region, Sun Yat-Sen University. The cells used in the experiments were maintained in our laboratory in RPMI 1640 medium with 10% fetal bovine serum (Sijiqing, China) at 37°C and 5% CO2.
Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) Cell Vaibility Assay
Cells harvested with trypsin were seeded into 96-well plates at a density of 1×104 per well. After overnight incubation, the culture medium was removed and the cells were incubated with different concentrations of wogonin. After exposure to wogonin for 24, 48 or 72 h the cells were incubated with MTT at 37°C for an additional 4 h. This allowed mitochondrial dehydrogenase to convert MTT into insoluble formazan crystals. The culture medium was then discarded, and 100 µL of dimethylsulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The absorption of solubilized formazan was measured at 490 nm using a EL340 microplate reader (Bio-Tek. Instruments, Winooske, VT).
Nuclear Staining
A549 cells were stained with a DAPI (4′, 6-diamidino-2-phenylindole) staining kit (KeyGEN, China). After exposure to graded concentrations of wogonin for 48 h, the cells were washed and incubated with a DAPI working solution (1–2 µg/mL) for 15 min at 37°C. The cells were then rinsed with methanol and Buffer A (60% glycerol in 10 mM phosphate-buffered saline (PBS, pH7.6)) was added to the suspension. A549 cells were viewed using an Eclipse Ti Nikon microscope (Nikon, Japan).
Mitochondrial Membrane Potential
The mitochondrial membrane potential (Δψm) of A549 cells was measured usimg a fluorescent, lipophilic, cationic probe, JC-1 (Beyotime, China), according to the manufacturer’s directions. Briefly, cells exposed to wogonin were incubated with 1X JC-1 staining solution for 20 min at 37°C. They were then rinsed twice with JC-1 staining buffer and images were taken with an Eclipse Ti Nikon microscope (Nikon, Japan).
Apoptosis Assay
Cells were labeled with FITC-labeled Annexin V and propidium iodide (PI) using an Annexin V-FITC apoptosis detection kit (KeyGEN BioTECH, China), according to the manufacturer’s instructions. Briefly, after 48 h exposure to different concentrations of wogonin, the cells were washed with cold PBS and resuspended in 1X binding buffer. Aliquots of 105 cells were mixed with 5 µL of Annexin V-FITC and 5 µL of PI for 15 min at room temperature in the dark. Fluorescence (530 nm) was detected by flow cytometry (FACS Aria, BD Biosciences, USA) within 1 h.
Real-time PCR Analysis
Quantitative RT-PCR was undertaken using a SYBR Green reporter. A549 cells exposed to wogonin were washed with PBS and total RNA was purified by using RNAiso Plus (TAKARA, Japan). The resultant RNA was first reverse transcribed into cDNA using a PrimeScript® RT Master Mix kit (TAKARA, Japan). Gene-specific primers were combined with SYBR® Premix Ex Taq™ (TAKARA, Japan) and amplified using an ABI 7500 real-time PCR machine (Applied Biosystems, USA). All qPCR reactions were performed independently on five samples. The relative mRNA expression was calculated using the 2−ΔΔCt method. The primer sequences used are listed in Table 1.
Western Blot Analysis
Cells washed with PBS were lysed with RIPA (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA and leupeptin, (Beyotime, China) supplemented with protease inhibitor PMSF (Beyotime, China). Cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN BioTECH, China).
The soluble protein concentration was determined with a BCA protein assay kit (Beyotime, China). The cell lysates were boiled for 5 min in loading buffer and separated on an SDS-PAGE gel. After electrophoresis, the proteins were transferred to a PVDF membrane (Millipore, USA). The membranes were blocked with nonfat milk, probed with various primary antibodies and HRP-conjugated secondary antibodies, and visualized with enhanced chemiluminescence (ECL) detection reagents (Beyotime, China).
Statistical Analysis
Statistical analyses was undertaken using SPSS version 17.0 software. Results are expressed as means and standard errors (Mean ± SEM) from at least three independent experiments. One-way analysis of variance (ANOVA) was used to determine statistical significance between groups. Values of P<0.05 were considered statistically significant.
Results
Wogonin Inhibits Cell Viability and Induces Cell Apoptosis
Cell viability was assessed using an MTT assay in association with DAPI staining and flow cytometric analysis after exposure to different concentrations of wogonin. MTT analysis indicated that wogonin inhibited cell viability in a dose-dependent and time-dependent manner (Fig. 1B). This was confirmed by results of flow cytometric analysis using AnnexinV-PI (Fig. 1C).
(A) Chemical structure of wogonin (C16H12O5, MW: 284.27). (B) Cell viability was analyzed using the MTT assay. Cells were incubated with wogonin for 24 h, 48 h or 72 h. The bars represent the mean values ± SEM (n = 3). (C) Apoptosis assessed by Annexin V/PI staining in A549 cells. Cells were incubated with wogonin at concentrations of 0, 15, 25, 35 µg/mL, for 24 h, 48 h and 72 h. (D) Nuclei stained by DAPI and observed by fluorescence microscope. Apoptotic cells are indicated by arrows. *P<0.05 vs control group, **P<0.01 vs control group.
Compared with the control group, the rate of both early and late stage apoptosis increased after exposure to all concentrations of wogonin. The total apoptosis rate exceeded 50% in the 35 µg/mL group.
DAPI staining (Fig. 1D) identified condensed and cleaved nuclei in cells exposed to 35 µg/mL wogonin, while only clear nuclei with pale blue staining were observed in the control group.
As shown in Fig. 2A, Wogonin was associated with a dose-dependent decrease in mitochondria potential (Δψm) which resulted in decreased red fluorescence (JC-1 polymer) and increased of green fluorescence (JC-1 monomer). This may have been related to down-regulation of Mcl-1 as there was no obvious decrease in Bcl-2 levels. Wogonin also promoted the release of AIF and cytochrome C into the cytoplasm providing further evidence of mitochondrial damage (Fig. 2C). Down-regulation of XIAP, survivin, and of cleaved fragments from PARP indicated that the process of apoptosis continued after mitochondria damage (Fig. 2B).
(A) Analysis of the mitochondrial membrane potential (ΔΨm) using JC-1 staining after exposure to wogonin for 48 h. A fluorescence microscope was used to visualize the results. Mitochondrial depolarization was indicated by an increase in green fluorescence and a decrease in red fluorescence intensity. (B) Protein levels of Bcl-2, Mcl-1, XIAP, survivin and PARP assayed by western blot. (C) Cytoplasmic proteins were extracted for western blot analysis of released Cytochrome c and AIF. In these experiments, cells were exposed to wogonin 0, 15, 25, 35 µg/mL for 48 h. *P<0.05 vs control group, **P<0.01 vs control group.
Wogonin Down-regulates HDAC1 and HDAC2 at Both mRNA and Protein Levels
Protein levels of HDAC1 and HDAC2 were down-regulated in a dose-dependent manner after exposure to different concentrations of wogonin for 48 h (Fig. 3B). This result is consistent with the down-regulation of mRNA detected by qPCR showing that HDAC1 was decreased by 0.69-fold and HDAC2 by 0.73-fold, (uncertainties related to fold-changes were <2) (Fig. 3A). These changes may result in a proportional increase in histone acetylation and promote the expression of tumor suppressive proteins.
(A) Relative mRNA levels of HDAC1 and HDAC2 were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean ± SEM of five independent experiments. #P<0.01 vs control group. (B) Protein levels of HDAC1 and HDAC2 assayed by western blot. In these experiments, cells were exposed to wogonin 0, 15, 25 and 35 µg/mL for 48 h. *P<0.05 vs control group, **P<0.01 vs control group.
Wogonin Down-regulates c-Myc and Skp2 at the Protein Level, and Increases the mRNA Level of c-Myc
Both c-Myc and Skp2 were down-regulated at the protein level following exposure to wogonin (0, 15, 25, 35 µg/mL) for 48 h, (Fig. 4B). As shown in Fig. 4A, the mRNA level of Skp2 decreased 0.81-fold, whereas the mRNA level of c-Myc increased approximately 1.6-fold (uncertainties related to fold-changes both <2). These findings indicate that a proteasomal degradation pathway may be involved in the regulation of c-Myc and Skp2. However, as wogonin resulted in decreased protein expression in Skp2, further experiments focused on the proteasome recognition subunit, Fbw7α, which targets c-Myc.
(A) Relative mRNA levels of c-Myc and Skp2 were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean ± SEM of five independent experiments. #P<0.01 vs control group. (B) Protein levels of c-Myc and Skp2 assayed by western blot. In these experiments, cells were exposed to wogonin 0, 15, 25 and 35 µg/mL for 48 h. *P<0.05 vs control group, **P<0.01 vs control group.
Wogonin Decreased Fbw7α and GSK3β, and Increased Thr58-Myc at the Protein Level
Protein levels of Fbw7α decreased following exposure to wogonin, (Fig. 5B), but there was no corresponding decrease in mRNA expression (Fig. 5A). Thr58 phophorylation of c-Myc increased (Fig. 5B). Thr58 phophorylation of c-Myc is a required component of its degradation and is mediated by GSK3β. However, GSK3β expression decreased at both the mRNA (0.78-fold at 35 µg/mL) (Fig. 5A) and protein level (Fig. 5B). These findings suggest that phophorylation of c-Myc at Thr58 may occur independently of GSK3β. However, this requires further study.
(A) Relative mRNA levels of Fbw7α and GSK3β were detected using real-time PCR with GAPDH as an internal control. Results are expressed as the mean ± SEM of five independent experiments. #P<0.01 vs control group. (B) Protein levels of Fbw7α, Thr58-Myc and GSK3β assayed by western blot. (C) Protein levels of c-Myc assayed by western blot. In these experiments, 1 µM MG132 was added incubated with or without 25 µg/mL wogonin for 48 h. *P<0.05 vs control group, **P<0.01 vs control group.
Experiments were conducted with the proteasome inhibitor MG132 to further understand the proteasomal degradation pathway involved in the regulation of c-Myc. The results in Fig. 5C show that MG132 was unable to reverse c-Myc degradation induced by 25 µg/mL wogonin. Further research is therefore needed to define the exact pathway involved in c-Myc degradation.
Discussion
Wogonin is a naturally occurring monoflavonoid extracted from Scutellaria baicalensis radix [28]. It has been reported to have antineoplastic activity in various types of cancer by the induction of apoptosis and cell differentiation [30]–[37], and to be regulated by various genes and proteins [38]–[41]. It is known that the c-Myc/Skp2/Fbw7α and HDAC1/HDAC2 pathways, are associated with tumor progression. Here we investigate their role in the anticancer effects of wogonin in NSCLC A549 cells.
We first evaluated the anti-viability and apoptotic effects of wogonin using MTT and Annexin V-PI double staining assays. Our results indicated that wogonin caused dose-dependent and time-dependent inhibition of cell viability (IC50<35 µg/mL at 48 h). The early increase in apoptosis rate in response to wogonin occurred in parallel, with Annexin V-positive cells gradually becoming Annexin-V negative.
At the IC50 (35 µg/mL) cells showed evidence of marked apoptosis, consistent with the nuclear morphology changes seen with DAPI staining.
The expression of apoptosis related proteins, such as Bcl-2, Mcl-1, PARP, XIAP, Survivin, cytochrome c and AIF was evaluated to further identify the apoptotic effects of wogonin at the protein level. The results indicate that wogonin is able to influence mitochondrial membrane stability and decrease mitochondria membrane potential (Δψm). This was evidenced by JC-1 staining, decreased mcl-1 expression and the release of cytochrome C and AIF into the cytoplasm. Cleaved PARP and decreased XIAP and survivin expression may also have contributed to the progression of apoptosis.
HDAC1 and HDAC2 are Class I HDACs that deacetylate histone and non-histone proteins [5]. They suppress gene expression, and modify tumor specific proteins involved in progression [4]. Our results show that the mRNA and protein levels of HDAC1 and HDAC2 were both decreased in the presence of wogonin, indicating that acetylated histone protein may promote expression of tumor suppressive proteins and thereby inhibit tumor progression.
An inter-relationship exists between c-Myc and Skp2 such that c-Myc promotes Skp2 expression, and Skp2 targets c-Myc for ubiquitin-dependent degradation [11], [12], [24], [25]. In our experiments, wogonin down-regulated c-Myc and Skp2 at the protein level, but the mRNA expression of c-Myc increased 1.6-fold. These findings suggest that the proteasomal degradation pathway may be related to the reversal of c-Myc. However, since Skp2 expression decreased, we focused our further experiments on Fbw7α, another proteasome recognition subunit that targets c-Myc.
Thr58 phophorylation of c-Myc is required for Fbw7α mediated c-Myc degradation [24]. As Thr58 is phophorylated by GSK3β, we evaluated expression levels of Fbw7α, Thr58 c-Myc and GSK3β. In these experiments Fbw7α expression decreased at the protein level but not at the mRNA level. Thr58 phophorylation of c-Myc increased to some degree and GSK3β expression at both the mRNA and protein level decreased. These results highlight lack of conformity between Fbw7α mRNA and protein levels, the decreased expression of GSK3β, and the increased phophorylation of c-Myc at Thr58.
A previous study demonstrated that phosphorylation of Thr58 in A549 cells occurred independently of GSK3β [42]. However, GSK3β is the only known kinase that phosphorylates c-Myc at Thr58. In our studies the proteasome inhibitor MG132 was unable to prevent the degradation of c-Myc suggesting that decreased Fbw7α and Skp2 might be involved in this process. However, the exact mechanism involved requires further research.
Taken together our findings suggest that the ability of wogonin to influence different biochemical pathways may explain its activity against a variety of different cancers. Our data may also in part explain why some gene deficient cells (e.g. those with Fbw7α deficiency) are resistant to wogonin.
Author Contributions
Conceived and designed the experiments: QJ XHW. Performed the experiments: YJZ YB XMC XLX HWL. Contributed reagents/materials/analysis tools: XMC XLX HWL. Wrote the paper: XMC YB YJZ. Analyzed the data and interpreted the results: YYY SGW. Read and approved the final manuscript: XMC YB YJZ XLX HWL YYY SGW GJ XHW.
References
- 1. Juergens R, Brahmer J (2007) Targeting the epidermal growth factor receptor in non-small-cell lung cancer: who, which, when, and how? Curr Oncol Rep 4: 255–264.
- 2. Crocetti E, Paci E (2002) Trends in lung adenocarcinoma incidence and survival, Lung Cancer. 2: 215–216.
- 3. Ruthenburg AJ, Li H, Patel DJ, Allis CD (2007) Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev Mol Cell Biol 8: 983–994.
- 4. Marks PA, Xu WS (2009) Histone deacetylase inhibitors: potential in cancer therapy, J Cell Biochem. 107: 600–608.
- 5. de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. BioChem J 370: 739–749.
- 6. Dang CV, Resar LM, Emison E, Kim S, Li Q, et al. (1999) Function of the c-Myc oncogenic transcription factor. Exp Cell Res 253: 63–77.
- 7. Kelly K, Cochran H, Stiles CD, Leder P (1983) Cell-specific regulation of the c-Myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35: 603–610.
- 8. Flinn EM, Busch CM, Wright AP (1998) Myc boxes, which are conserved in Myc family proteins, are signals for protein degradation via the proteasome. Mol Cell Biol 18: 5961–5969.
- 9. Jones TR, Cole MD (1987) Rapid cytoplasmic turnover of c-Myc mRNA: requirement of the 3′ untranslated sequences. Mol Cell Biol 7: 4513–4521.
- 10. Nesbit CE, Tersak JM, Prochownik EV (1999) MYC oncogenes and human neoplastic disease. Oncogene 18: 3004–3016.
- 11. Amati B (2004) Myc degradation: dancing with ubiquitin ligases. Proc Natl Acad Sci 101: 8843–8844.
- 12. Chan CH, Morrow JK, Li CF, Gao Y, Jin G, et al. (2013) Pharmacological Inactivation of Skp2 SCF Ubiquitin Ligase Restricts Cancer Stem Cell Traits and Cancer Progression. Cell 3: 556–68.
- 13. Carrano AC, Eytan E, Hershko A, Pagano M (1999) Skp2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1: 193–199.
- 14. Kamura T, Hara T, Kotoshiba S, Yada M, Ishida N, et al. (2003) Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation. Proc Natl Acad Sci 100: 10231–10236.
- 15. Tedesco D, Lukas J, Reed SI (2002) The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2). Genes Dev 16: 2946–2957.
- 16. Hiramatsu Y, Kitagawa K, Suzuki T, Uchida C, Hattori T, et al. (2006) Degradation of Tob1 mediated by SCFSkp2-dependent ubiquitination. Cancer Res 66: 8477–8483.
- 17. Zhao D, Zheng HQ, Zhou Z, Chen C (2010) The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation. Cancer Res 70: 4728–4738.
- 18. Akhoondi S, Lindstrom L, Widschwendter M, Corcoran M, Bergh J, et al. (2010) Inactivation of FBXW7/hCDC4-beta expression by promoter hypermethylation is associated with favorable prognosis in primary breast cancer. Breast Cancer Res 12: R105.
- 19. Inuzuka H, Shaik S, Onoyama I, Gao D, Tseng A, et al. (2011) SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature 471: 104–109.
- 20. Sancho R, Jandke A, Davis H, Diefenbacher ME, Tomlinson I, et al. (2010) F-box and WD repeat domain-containing 7 regulates intestinal cell lineage commitment and is a haploinsufficient tumor suppressor. Gastroenterology 139: 929–941.
- 21. O’Neil J, Look AT (2007) Mechanisms of transcription factor deregulation in lymphoid cell transformation. Oncogene 26: 6838–6849.
- 22. Wang Z, Fukushima H, Gao D, Inuzuka H, Wan L, et al. (2011) The two faces of FBW7 in cancer drug resistance. Bioessays 11: 851–859.
- 23. Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, et al. (2011) Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature 471: 110–114.
- 24. Pulverer BJ, Fisher C, Vousden K, Littlewood T, Evan G, et al. (1994) Site-specific modulation of c-Myc cotransformation by residues phosphorylated in vivo. Oncogene 9: 59–70.
- 25. Kim SY, Herbst A, Tworkowski KA, Salghetti SE, Tansey WP (2003) Skp2 regulates Myc protein stability and activity. Mol Cell 11: 1177–1188.
- 26. Spruck CH, Strohmaier H, Sangfelt O, Müller HM, Hubalek M, et al. (2002) hCDC4 gene mutations in endometrial cancer. Cancer Res 62: 4535–4539.
- 27. Welcker M, Orian A, Grim JE, Eisenman RN, Clurman BE (2004) A nucleolar isoform of the Fbw7 ubiquitin ligase regulates c-Myc and cell size. Curr Biol 14: 1852–1857.
- 28. Chi YS, Cheon BS, Kim HP (2001) Effect of wogonin, a plant flavone from Scutellaria radix, on the suppression of cyclooxygenase-2 and the induction of inducible nitric oxide synthase in lipopolysaccharide-treated RAW 264.7 cells. Biochem Pharmacol 61: 1195–1203.
- 29. Li-Weber M (2009) New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents wogonin, baicalein and baicalin. Cancer Treat Rev 35: 57–68.
- 30. Zhang M, Liu LP, Chen Y, Tian XY, Qin J, et al. (2013) Wogonin induces apoptosis in RPMI 8226, a human myeloma cell line, by downregulating phospho-Akt and overexpressing Bax. Life Sci 1: 55–62.
- 31. Chen YC, Shen SC, Lee WR, Lin HY, Ko CH, et al. (2002) Wogonin and fisetin induction of apoptosis through activation of caspase 3 cascade and alternative expression of p21Cip1 protein in hepatocellular carcinoma cells SK-HEP-1. Arch Toxicol 76: 351–359.
- 32. Wang W, Guo Q, You Q, Zhang K, Yang Y, et al. (2006) Involvement of bax/bcl-2 in wogonin-induced apoptosis of human hepatoma cell line SMMC-7721. Anticancer Drugs 17: 797–805.
- 33. Tsai CF, Yeh WL, Huang SM, Tan TW, Lu DY (2012) Wogonin induces reactive oxygen species production and cell apoptosis in human glioma cancer cells. Int J Mol 8: 9877–92.
- 34. Chow SE, Chang YL, Chuang SF, Wang JS (2011) Wogonin induced apoptosis in human nasopharyngeal carcinoma cells by targeting GSK-3b and DNp63. Cancer Chemother Pharmacol 68: 835–845.
- 35. Yu JS, Kim AK (2011) Wogonin induces apoptosis by activation of ERK and p38 MAPKs signaling pathways and generation of reactive oxygen species in human breast cancer cells. Mol Cells 31: 327–335.
- 36. Chen P, Lu N, Ling Y, Chen Y, Hui H, et al. (2011) Inhibitory effects of wogonin on the invasion of human breast carcinoma cells by down regulating the expression and activity of matrix metalloproteinase-9. Toxicology 282: 122–128.
- 37. Yang L, Zhang HW, Hu R, Yang Y, Qi Q, et al. (2009) Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells. Biochem Cell Biol 87: 933–942.
- 38. Lee E, Enomoto R, Koshiba C, Hirano H (2009) Inhibition of P-glycoprotein by wogonin is involved with the potentiation of etoposide-induced apoptosis in cancer cells. Ann NY Acad Sci 1171: 132–136.
- 39. Ding J, Polier G, Köhler R, Giaisi M, Krammer PH, et al. (2012) Wogonin and related natural flavones overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein resistance of tumors by down-regulation of c-FLIP protein and up-regulation of TRAIL receptor 2 expression. J Biol Chem 1: 641–9.
- 40. Song X, Yao J, Wang F, Zhou M, Zhou Y, et al. (2013) Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein. Toxicol Appl Pharmacol 2: 144–55.
- 41. Yang L, Wang Q, Li D, Zhou Y, Zheng X, et al. (2013) Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins. Apoptosis 5: 618–26.
- 42. Li Q, Kluz T, Sun H, Costa M (2009) Mechanisms of c-Myc Degradation by Nickel Compounds and Hypoxia. PLoS ONE 4: e8531.