Involvement of Exportin 1 Mediated c-Myc and Cullin 4A Signaling in Apoptotic Effect of Fisetin in Non-small Cell Lung Cancer Cells

of Exportin 1 Mediated c-Myc


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 [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.

Cell viability assay
Cell viability was assessed by using MTT (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).

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

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).

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).

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). 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).

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.

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).

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. It is well documented that the ubiquitin-dependent proteolysis system (UPS) for protein degradation is involved in the regulation of cell proliferation, differentiation and apoptosis via the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin-protein ligase (E3) [43,44]. S-phase kinase-associated protein 2 (Skp2) for a Skp2-SCF E3 ligase complex induces proteasome mediated proteolysis through conjugating K63-linked polyubiquitin chains to targeting Akt, LKB1 and Aurora B [45] CUL4A ubiquitin ligase that forms a complex with DNA damage binding protein 1 (DDB1) and ring of cullin 1(ROC1) is known as a promising target for cancer treatment [46]. Here Fisetin attenuated the expression of cMyc, SKP2, CUL4A in A549 and H460 cells. Of note, XPO1 depletion enhanced p53 activation, while CUL4A depletion enhanced PARP cleavage in Fisetin treated H460 cells, indicating the pivotal roles of XPO1 and CUL4A. However, considering that interaction scores were 0.998 between XPO1 and p53, 0.595 between CUL4A and p53 by String database, it can be assumed that XPO1 binds to p53, while CUL4A as E3 ubiquitine ligase enhances ubiquitination in H460 cells.

Conclusions
Fisetin increased cytotoxic and apoptotic effect by increasing the number of Annexin V/PI positive apoptotic portion and sub G1 accumulation, and the cleavages of PARP and caspase 3, activated p53/p21 and attenuating cyclin D1, cyclin E, CDK2, XPO1, cMyc, SKP2 and Cullin 4A in A549 and H460 cells. Conversely, depletion of XPO1 enhanced p53 and PARP cleavage, while depletion of UL4A promoted PARP cleavage without p53 upregulation in H460 cells. Taken together, our work strongly demonstrates the pivotal role of XPO1 mediated c-Myc and CUL4A signaling in Fisetin induced apoptotic effect in NSCLCs as a potent antitumor candidate ( Figure 6). Overall, our findings suggest that Fisetin remarkably supressed c-Myc and increased p53 in a concentration-dependent manner, along with decreased IPO7 and XPO1, suggesting the involvement of IPO7 and XPO1 linked c-Myc and p53 signaling in Fisetin-induced antiproliferative effect in NSCLCs.