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

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.


Introduction
Despite of the large number of clinical trials aimed at improving patient survival, lung cancer remains a leading cause of cancerrelated 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][8][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 Fbw7mediated proteasomal degradation. Glycogen synthase kinase 3b(GSK3b) is the only kinase known to phosphorylate c-Myc at Thr58 [24].
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 a, b, and c) respectively located in the a-nucleus, b-cytoplasm, and c-nucleolus [26,27]. As there are no antibodies for these three isoforms we used the best described and longest of the three isoforms Fbw7a, in our experiments with wogonin.
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/Fbw7a and HDAC1/HDAC2 pathways involved in its apoptotic effect.

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 37uC and 5% CO 2 .

Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) Cell Vaibility Assay
Cells harvested with trypsin were seeded into 96-well plates at a density of 1610 4 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 37uC for an additional 4 h. This allowed mitochondrial dehydrogenase to convert MTT into insoluble formazan crystals. The culture medium was then discarded, and 100 mL 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 (49, 6-diamidino-2phenylindole) 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 mg/mL) for 15 min at 37uC. 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 (Dym) 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 37uC. 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 10 5 cells were mixed with 5 mL of Annexin V-FITC and 5 mL 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 PrimeScriptH RT Master Mix kit (TAKARA, Japan). Gene-specific primers were combined with SYBRH Premix Ex Taq TM (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 2DDCt method. The primer sequences used are listed in Table 1.
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 HRPconjugated 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 6 SEM) from at least three independent experiments. Oneway analysis of variance (ANOVA) was used to determine statistical significance between groups. Values of P,0.05 were considered statistically significant.

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 timedependent manner (Fig. 1B). This was confirmed by results of flow cytometric analysis using AnnexinV-PI (Fig. 1C).
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 mg/ mL group. DAPI staining (Fig. 1D) identified condensed and cleaved nuclei in cells exposed to 35 mg/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 dosedependent decrease in mitochondria potential (Dym) 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).

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.

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 mg/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, Fbw7a, which targets c-Myc.

Wogonin Decreased Fbw7a and GSK3b, and Increased Thr58-Myc at the Protein Level
Protein levels of Fbw7a 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 GSK3b. However, GSK3b expression decreased at both the mRNA (0.78-fold at 35 mg/mL) (Fig. 5A) and protein level (Fig. 5B). These findings suggest that phophorylation of c-Myc at Thr58 may occur independently of GSK3b. However, this requires further study.
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 mg/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][31][32][33][34][35][36][37], and to be regulated by various genes and proteins [38][39][40][41]. It is known that the c-Myc/Skp2/Fbw7a 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 (IC 50 ,35 mg/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 IC 50 (35 mg/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 (Dym). 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 Fbw7a, another proteasome recognition subunit that targets c-Myc.
Thr58 phophorylation of c-Myc is required for Fbw7a mediated c-Myc degradation [24]. As Thr58 is phophorylated by GSK3b, we evaluated expression levels of Fbw7a, Thr58 c-Myc and GSK3b. In these experiments Fbw7a expression decreased at the protein level but not at the mRNA level. Thr58 phophorylation of c-Myc increased to some degree and GSK3b expression at both the mRNA and protein level decreased. These results highlight lack of conformity between Fbw7a mRNA and protein levels, the decreased expression of GSK3b, and the increased phophorylation of c-Myc at Thr58.
A previous study demonstrated that phosphorylation of Thr58 in A549 cells occurred independently of GSK3b [42]. However, GSK3b 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 Fbw7a 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 Fbw7a 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.