Introduction

Non-small-cell lung cancer (NSCLC) is one of the most common malignant diseases in humans worldwide [1]. T he three major types of NSCLC are adenocarcinoma (ADC), Squamous Cell Carcinoma (SCC) and Large Cell Carcinoma (LCC) [2,3]. However, current treatments have a drawback to cause a variety of side effects. For instance, cisplatin is known to induce adverse reactions such as anemia, joint pain, face swelling, convulsions and trouble in walking [4]. Recently molecular target therapy is attractive for effective cancer therapy via regulation of specific molecules.

It is well documented that the Myc family gene consisting of c-Myc, n-Myc and L-Myc work as oncogenes in several cancers, while p53 acts as one of tumor suppressors [5]. Furthermore, the cullin 4 subfamily of genes including CUL4A and CUL4B ubiquitin ligases or Cullin RING ligase (CRL) complexes are often overexpressed in human malignancies [6,7]. Also, exportin1 (XPO1) or Chromosome region maintenance 1 (CRM1), is often overexpressed or mutated in several cancer by regulation of nuclear export of proteins and RNAs, and ribosome biogenesis for cancer growth and survival [8,9]. Furthermore, in terms of lung cancer chemoprevention, some natural compounds are of interests to selectively induce apoptosis and growth arrest in lung cancer cells without severe cytotoxicity in normal cells [10]. For instance, sulforaphane [11], resveratrol [12], tanshinone IIA [13], shikonin [14] and quercetin [15].

In the same line, Fisetin, one of flavonol [16], was known to have anti-inflammatory [17], anti-aging [18], anti-angiogenic [19], anti-proliferative [20] and anti-tumor [21, 22] effects. In particular, Fisetin induced apoptosis in prostate [23], pancreatic [24], glioblastoma [25], gastric [26], breast [27], colon [28], ovarian [29] cancer cells. It also exerted antitumor effect in lung cancer cells via modulation of ERK1/2[30, 31] and AKT/PI3K/ mTOR [32] signaling pathway. Nevertheless, the underlying antitumor mechanism of Fisetin is not fully understood in nonsmall cell lung cancer cells so far. Thus, in the present study, the apoptotic mechanism of Fisetin was explored in association with c-Myc/p53 signaling mediated by XPO1 ribosomal protein and ubiquitin ligase CUL4A in non-small cell lung cancer cells.

Materials and Methods

Cell culture and reagents

H460 and A549 cells (human epithelial non-small cell lung cancers; NSCLCs) were purchased from the American Type Culture Collection (ATCC, USA). The cells were cultured in RPMI containing 10% FBS (Fetal bovine serum) and 1% antibiotic (Welgene, South Korea) and incubated at 37 ℃ with 5% CO₂. Fisetin ((3, 7, 3′, 4′-tetrahydroxyflavone) was purchased from Sigma Chemical Co., (St. Louis, MO, USA) and dissolved in DMSO (dimethyl sulfoxide) as a 100 mM stock.

Cell viability assay

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, St. Louis, MO, USA) according to the manufacturer’s instruction. After H460 and A549 cells were seeded onto 96-well microplate, the cells were treated by various concentrations (0, 10, 20, 40, 60, 80 μM) of Fisetin for 24 h. For time or concentration dependent study, the cells were exposed to Fisetin at 40 and 80 μM for 24 h, 48 h, and 72 h. MTT solution (1 mg/ml) was added and formazan was dissolved with DMSO (dimethyl sulfoxide). To measure the optical density, microplate reader (TECAN, Austria) was used at 570 nm. Cell viability was determined as a percentage of viable cells in Fisetin treated group versus untreated control.

Colony formation assay

H460 and A549 cells (1,000 cells) were seeded in 6-well plates RPMI 1640 medium containing 10% serum. The cells were treated with Fisetin (40, 80 μM) for 24 h and replaced by new media. The cells were incubated at 37 ℃ with 5% CO₂ for 10 days, the colonies were fixed and stained with Diff-Quick solution (Sysmex, Japan). Colonies containing ≥ 50 cells were counted using ImageJ software.

Annexin-V-FITC and propidium iodide staining for apoptotic portion analysis

H460 and A549 cells were treated with Fisetin (40, 80 μM) for 24 h. Then apoptosis was measured by Annexin V stainingbased FACS analysis by using FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Korea).

Cell cycle analysis for sub G1 population

H460 and A549 cells were treated with Fisetin (40 or 80 μM) for 24 h. The cells were washed using PBS and fixed with ethanol (75%), incubated with 1mg/ml RNase A in PBS at 37 ^°C for 30 min, and stained in PBS containing 50 µg/mL PI (propidium iodide). The DNA contents of the stained cells were analyzed by using Cell Quest Software (BD Biosciences, San Jose, CA, USA) with the FACS Calibur flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Western blotting

H460 and A549 cells were exposed to various concentrations of Fisetin for 24h. The cells were lysed in RIPA buffer (50 mM Tris-

HCl, 150 mM NaCl, 2 mM EDTA, and 1% TritonX-100) containing protease inhibitors (Roche, Germany), and phosphatase inhibitors (Sigma, USA). Total cell lysates were separated in 8 to 15% SDS-PAGE (SDS-polyacrylamide gels), and electrophoretically transferred to nitrocellulose membranes. The antibodies against p53, cyclin D1, CDK4, CDK2, Bcl-2, XPO1 were obtained from Santa Cruz Biotechnology. Cyclin E, Poly (ADP-ribose) polymerase (PARP), and cleaved-PARP were purchased from Cell signaling (Beverly, MA, USA). β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA), and c-Myc was bought from Abcam (Cambridge, England). The antibodies were diluted in 3% BSA in PBS-Tween20 (1:500-1:2000), and the membranes were incubated overnight at 4 ^°C. Then the membranes were washed three times with PBS-Tween20, and incubated with HRPconjugated secondary antibodies. The expression was visualized by using ECL Western blotting detection reagent (GE Healthcare, UK).

Small interfering RNA (siRNA) transfection assay

H460 or A549 cells were transfected with scrambled siRNA, XPO1 siRNA or CUL4A siRNA (Bioneer, Korea) with Interferin^TM transfection reagent (Polyplus-transfection Inc., New York, NY, USA). The mixtures of XPO1 siRNA with Interferin^TM transfection reagent were incubated for 10 min at room temperature and the cells were incubated at 37 ^°C for 48 h before exposure to Fiseitn 80 µM for 24 h.

Statistical analysis

Data were presented as the means ± standard deviation. Statistical significance of the differences was determined by Student’s t-test using Sigma Plot Software (Systat Software Inc., USA).

Results

Fisetin exerted cytotoxic and antiproliferative effect in H460 and A549 non-small cell lung cancer cells

To evaluate the cytotoxic and antiproliferative effect of Fisetin (Figure 1A), MTT assay and colony formation assay were performed in H460 and A549 cells. Here Fisetin significantly reduced the viability of H460 and A549 cells in a concentration and time dependent fashion (Figure 1B and 1C). Also, colony formation assay showed that the sizes and number of colonies were reduced in Fisetin treated group compared to untreated control (Figure 1D).

Figure 1: Effect of Fisetin on cytotoxicity and colony formation in H460 and A549 cells. (A) Chemical structure of Fisetin (B). Concentration dependent cytotoxicity of Fisetin in H460 and A549 cells. The cell viability by Fisetin was assessed by MTT assay after exposure to Fisetin for 24 h. *, p<0.05, **, p<0.01 vs untreated control. (C) Time dependent cytotoxicity of Fisetin in H460 and A549 cells by MTT assay after exposure to Fisetin for 24 h. (D) Effect of Fisetin on colony formation in H460 and A549 cells. Ten days after exposure to Fisetin (40, and 80 μM) for 24 h, the colonies were stained with Diff Quick Solution to visualize, and then the colonies were counted. ***, p<0.001 vs untreated control.

Fisetin induced apoptosis and increased the accumulation of sub-G1 population in H460 and A549 cells

To examine the apoptotic effect of Fisetin, cell cycle analysis and Western blotting were carried out in Fisetin treated H460 and A549 cells. Apoptotic assay by using Annexin V/propidium iodide staining reveals that Fisetin increased apoptotic portion to 14.82% and 14.02% at 80 μM compared to untreated control in H460 and A549 cells, respectively (Figure 2A). Consistently, Fisetin increased sub G1 population to 22.5 and 12.87% cells compared to untreated control in H460 and A549 cells, respectively (Figure 2B). Also, to confirm above apoptotic effect of Fisetin, Western blotting was conducted. As expected, Fisetin effectively cleaved PARP and caspase 3 in H460 and A549 cells (Figure 2C, 2D).

Figure 2: Effect of Fisetin on Annexin V/PI stained apoptotic portion and sub G1 population in H460 and A549 cells.(A) Apoptotic effect of Fisetin on Sub G1 population in H460 and A549 cells. Sub G1 population was evaluated in Fisetin treated H460 and A549 cells by FACS analysis. The cells were pretreated with 40 and 80 μM of Fisetin for 24 h and stained with PI. Then the data were analyzed by using Cell Quest Software. (B) Apoptotic effect of Fisetin in H460 and A549 cells by flow cytometric analysis using Annexin V/PI. After H460 and A549 cells were treated by Fisetin at 40 and 80 μM for 24 h, the apoptotic portion was evaluated in H460 and A549 by FACS analysis. (C) Effect of Fisetin on apoptosis related proteins in H460 cells. The cells were treated with Fisetin for 24 h and were subjected to Western blotting. ***, p<0.001 vs untreated control. (D) Effect of Fisetin on apoptosis related proteins in A549 cells. The cells were treated with Fisetin for 24 h and were subjected to Western blotting. ***, p<0.001 vs untreated control.

Fisetin down-regulated cell cycle related proteins in H460 and A549 cells.

To confirm whether Fisetin affects cell cycle related proteins, Western blotting was performed. As shown in Figure 3A, Fisetin activated p53 and p21, and attenuated the expression of cyclin D1, cyclin E, CDK2 compared to untreated control in H460 and A549 cells (Figure 3B).

Figure 3: Effect of Fisetin on cell cycle related proteins in H460 and A549 cells.(A) Effect of Fisetin on cell cycle related proteins in H460 cells. After exposure to Fisetin at 40 and 80 μM for 24 h, cell cycle related proteins were evaluated in H460 cells by Western blotting. ***, p<0.001 vs untreated control. (B) Effect of Fisetin on cell cycle related proteins in A549 cells. The expression of cell cycle and ribosomal biogenesis related proteins was assessed in Fisetin treated A549 cells by Western blotting. ***, p<0.001 vs untreated control.

Fisetin attenuated the ubiquitination related proteins in H460 and A549 cells

Exportin-1 (XPO1) controls nuclear-cytoplasmic transport of protein, ribosomal RNA and certain mRNAs for ribosomal biogenesis, which is closely associated with ubiquitination [5,33]. The c-Myc is known an unstable protein susceptible to ubiquitination [34], while SKP2 (S-phase kinase-associated protein 2) acts as the F-box protein of the E3 ubiquitin ligase complex [35] and Cullin 4A (CUL4A), an E3 ubiquitin ligase, is overexpressed in several cancers [36]. Hence, Western blotting was performed to assess the effect of Fisetin on ubiquitination related proteins in A549 and H460 cells. Here Fisetin attenuated the expression of XPO1, c-Myc, SKP2 and CUL4A compared to untreated control in A549 and H460 cells (Figure 4A and 4B). However, H460 cells were more susceptible to Fisetin than A549 cells.

Figure 4: Effect of Fisetin on ubiquitination related proteins in H460 and A549 cells.(A) Effect of Fisetin on ubiquitination related proteins in H460 cells. After exposure to Fisetin at 40 and 80 μM for 24 h, ubiquitination related proteins were evaluated in H460 cells by Western blotting. **, p<0.01, ***, p<0.001 vs untreated control. (B) Effect of Fisetin on ubiquitination related proteins in A549 cells. *, p<0.05, ***, p<0.001 vs untreated control.

XPO1 or CUL4A depletion reduced the capacity of Fisetin to cleave PARP and induce p53 in H460 cells

To confirm the important role of XPO1 and IPO7 in Fisetin-induced apoptosis in H460 cells, Western blotting was performed in H460 cells transfected with siRNA vector, XPO1 siRNA plasmids. Herein XPO1 depletion enhanced p53 activation, while CUL4A depletion enhanced PARP cleavage in Fisetin treated H460 cells (Figure 5A and 5B).

Figure 5: The pivotal role of XPO1 or CUL4A in Fisetin induced apoptosis in H460 cells. (A) PPI binding score between XPO1 or CUL4A and p53 by String database (B) Effect of XPO1 depletion on apoptosis related proteins in Fisetin treated H460 cells.*, p<0.05, ***, p<0.001 vs untreated control. (C) Effect of CUL4A depletion on apoptosis related proteins in Fisetin treated H460 cells ***, p<0.001 vs untreated control.

Figure 6: Apoptotic mechanism of Fisetin via modulation of XPO1 and CUL4A in non-small lung cancer cells.

Discussion

In the current study, the underlying mechanisms of Fisetin were explored in H460 and A549 cells in association with ribosomal protein XPO1 and CUL4A signaling axisis. Herein we found that Fisetin significantly suppressed the cell proliferation A549 and H460 lung cancer cells by MTT assay and colony formation assay, implying antitumor potential of Fisetin in non-small cell lung cancer cells. Also, Fisetin significantly increased the Annexin V/PI positive apoptotic portion and sub G1 portion in H460 and A549 cells, indicating the cytotoxicity of Fisetin is mediated by apoptosis in H460 and A549 cells. Consistently, several papers reported the capacity of Fisetin to induce cell cycle arrest in several cancer cells [5,37]. Additionally, Fisetin attenuated the expression of G1 related proteins such as cyclin D1, cyclin E, CDK2 and activated p53 and p21, implying G1 arrest leading to cell death by Fisetin in H460 and A549 cells.

Ribosome biogenesis is known the course of producing a variety of ribosomes in the cytoplasm and nucleus to organize the production and processing of ribosomal RNAs (rRNAs) to sustain cell homeostasis and cell survival [38]. Also, the important role of ribosomal proteins was reported as the regulators of cell cycle and proliferation [38], though ribosomal proteins require the assistance of nuclear import factors during the active nuclear import process [9]. To date, exportin 1 (XPO1) is known to mediate the nuclear export of various proteins including p53 and RPL5, RPL4,RPL6 and RPL23A [39], since ribosomal proteins increase the cell proliferation of colon cancer cells through c-Myc [40,41] and p53 [39,42]. Our data demonstrate that Fisetin effectively suppressed the expression of XPO1 and c-Myc and activated the expression of p53 in A549 and H460 cells.