1. Introduction 

Lung cancer is the most frequently diagnosed malignant cancer and the leading cause of cancer mortality worldwide.1,2 The two types of lung cancer are non-small cell lung cancer (NSCLC), which comprises approximately 85% of lung cancer cases, and small cell lung cancer (SCLC).3 Although clinical advances in prevention and therapeutics against lung cancer have progressed in recent years, the survival rate is still less than 20%.4 The poor survival of lung cancer patients is due to chemoresistance and the distant metastasis at the time of diagnosis.5 Therefore, novel approaches to chemotherapy, targeted therapy, and prevention of metastasis are urgently needed. Signaling pathways that regulate cell growth, cell survival, genomic instability, and angiogenesis are correlated with lung cancer progression and metastatic potential.6 V-Akt murine thymoma viral oncogene homolog (AKT) is a serine/threonine kinase that regulates cell survival, proliferation and many other biological responses through directly inducing the phosphorylation of downstream substrates.7 AKT isoforms (AKT1, AKT2 and AKT3) have a high sequence homology in the catalytic domains, but diverge in the hydrophobic motif (HM) domain and the pleckstrin homology (PH) domain.8

 It was recently discovered that the AKT signaling pathway induces epithelialemesenchymal transition (EMT) and migration through regulating E-cadherin expression.9 Dual-treatment with EGFR and AKT inhibitors was found to synergistically inhibit tumor growth and promote apoptosis in EGFRresistant NSCLC models.10 * Corresponding author. China-US (Henan) Hormel Cancer Institute, No. 127 Dongming Road, Zhengzhou, Henan, 450008, China. Fax: þ86 371 6558 7227. E-mail address: djkim@hci-cn.org (D.J. Kim). Peer review under responsibility of Japanese Pharmacological Society. Contents lists available at ScienceDirect Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs https://doi.org/10.1016/j.jphs.2021.01.003 1347-8613/© 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Journal of Pharmacological Sciences 145 (2021) 279e288 The p38 mitogen-activated protein kinase (MAPK) family consists of 4 isoforms (a, -b, -g and d) encoded by distinct genes with differences in tissue distribution and substrate specificity.11 Previously, it was observed that phosphorylated p38 MAPK protein levels are strongly increased in lung cancer tissues compared with normal lung tissues.12 Additionally, treatment of lung cancer patients with p38 MAPK inhibitors has been shown to suppress lung tumor growth.13 Therefore, AKT and p38 MAPK pathways might be potential therapeutic targets in lung cancer.

 Fermented wheat germ extract has been reported to show preventive and therapeutic activities in various cancer cells.14,15 2,6-Dimethoxy-1,4-benzoquinone (2,6-DMBQ), a major bioactive compound of fermented wheat germ extract, is a natural phytochemical present in various plants and can modulate several biological processes including oxidative phosphorylation, electron transport activity, and the inhibition of adipogenesis.16e18 2,6- DMBQ has also been reported to exert anti-inflammatory, antibacterial and anti-tumor activities.19,20 Recently, 2,6-DMBQ was shown to inhibit 3T3-L1 adipocyte differentiation via regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1).19 Our previous study also suggested that 2,6-DMBQ is an mTOR inhibitor that reduces gastric cancer growth.21 However, the efficacy of 2,6-DMBQ and its potential underlying mechanisms have not been investigated in NSCLC cells. Here, we report that 2,6-DMBQ strongly suppresses NSCLC cell proliferation and migration through inhibiting AKT and p38 MAPK signaling pathways. 2. Materials and methods 2.1.

 Cell lines H1299, H1650 and H358 human NSCLC cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cytogenetically tested and authenticated before generating cell stocks. Cells were cultured in RPMI 1640 medium (Biological Industries, Cromwell, CT, USA) supplemented with 10% FBS (Biological Industries) and 1% antibiotic-antimycotic solution (Solarbio, Beijing, China). All cells were maintained at 37 C in a 5% CO2 humidified incubator and cultured for a maximum of 8 weeks. 2.2. Reagents and antibodies 2,6-DMBQ was purchased from Shanghai Chemic Industry (Shanghai, China). AKT-I, SB203580 (p38-I) and GSK3b-I were purchased from MedChemExpress (Shanghai, China). Antibodies to detect phosphorylated AKT (S473) (Cat# 4060, 1:1000), p38 MAPK (T180/Y182) (Cat# 9211, 1:1000), MKK3/6 (S189/S207) (Cat# 12280, 1:1000), mTOR (S2481) (Cat# 2974, 1:1000), EGFR (Y1068) (Cat# 2220, 1:1000), GSK3b (S9) (Cat# 5558, 1:1000), -CDC2 (Cat# 9111, 1:1000), total AKT (Cat# 4691, 1:1000), GSK3b (Cat# 12456, 1:1000), -MKK3 (Cat# 8535, 1:1000), -mTOR (Cat# 2972, 1:1000), -ERK1/2 (Cat# 4695, 1:1000), -JAK1 (Cat# 3344, 1:1000), -EGFR (Cat# 2232, 1:1000), -CDC2 (Cat# 77055, 1:1000) and Cyclin B1 (Cat# 12231, 1:1000) were purchased from Cell Signaling Technology (Beverly, MA, USA). The b-actin (Cat# sc-47778, 1:3000) and E-cadherin (Cat# sc-7870, 1:1000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphorylated JAK1 (Y1022) (Cat# SAB4504446, 1:3000) was purchased from SigmaeAldrich (St Louis, MO, USA). COX2 (Cat# ab15191, 1:1000) 

 was purchased from Abcam (Chembridge Science Park, Chembridge, UK). Phosphorylated ERK1/2 (T202/Y204) (Cat#700012, 1:1000) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Goat anti-rabbit IgG (H þ L) (Cat# ZB2301, 1:10000) and goat anti-mouse IgG (H þ L) (Cat# ZB2305, 1:10000) were purchased from Beijing Zhongshan Jinqiao Biotechnology Co. LTD (Beijing, China). 2.3. Western blotting Proteins were measured by BCA kit (Solarbio) following the manufacturer's suggested protocol. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). Membranes were then blocked with 5% nonfat dry milk (Solarbio) in TBST (TBS with 1% Tween 20) at room temperature for 1 h. After blocking, the membranes were washed three times with TBST and incubated overnight with appropriate primary antibodies at 4 C. The next day, the membranes were washed with TBST three times and then incubated with an appropriate horseradish peroxidaseelinked secondary antibody for 1 h. The membranes were washed three times with TBST and the immuno-reactive proteins were detected by Thermo Scientific SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using the ImageQuant LA S4000 system (GE Healthcare, Piscataway, NJ, USA). 2.4. Cell proliferation assay Cells were seeded (2 103 cells per well) in 96-well plates with 100 ml complete growth medium and incubated for 24 h at 37 C. Cells were treated with various concentrations of 2,6- DMBQ diluted in 100 ml of complete growth medium for 48 h. After the incubation period, 20 ml of MTT solution (Solarbio) were added to each well and the cells were incubated for an additional 2 h at 37 C. 

The cell culture medium was then discarded and replaced with 150 ml of DMSO (Kermel, Tianjin, China). Formazan crystals were dissolved by gentle agitation. Cell proliferation was measured at 570 nm wavelength using a Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA, USA). To determine the IC50value of all compounds used within this study, we chose the 72 h MTT result to calculate the half maximal inhibitory concentration using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). We used the calculated IC50 value of AKT-I or SB203580 or GSK3b-I for cotreatment with 2,6-DMBQ. 2.5. Anchorage-independent cell growth Cells (8 103 per well) suspended in complete growth medium -RPMI 1640 supplemented with 10% FBS and 10 mg/ml gentamycin (Solarbio) were added to 0.3% agar (Becton, Dickinson and Company, NJ, USA) with or without various concentrations of 2,6-DMBQ in a top layer over a base layer of 0.6% agar with or without various concentrations of 2,6-DMBQ. The culture dishes were maintained at 37 C in a 5% CO2 incubator for 2 weeks. Colonies were photographed using a wide-field microscope and processed for analysis with the Image-Pro Plus software (v.6) program (Media Cybernetics, Rockville, MD, USA). 2.6. Colony formation assay Cells were seeded (500 cells per well) in 6-well plates with 2 ml complete growth medium (RPMI 1640 supplemented with 10% FBS and 1% antibiotic-antimycotic solution) and incubated for 24 h. Cells were treated with different concentrations of 2,6-DMBQ in 2 ml of complete growth medium and incubated for 1 week. Cells were stained with 0.5% Coomassie brilliant blue (Solarbio) for 20 min and then imaged

. X. Xie, X. Zu, K. Laster et al. Journal of Pharmacological Sciences 145 (2021) 279e288 280 2.7. Cell cycle analysis Cells were seeded (8 104 cells per dish) into 60 mm culture dishes. After incubation for 24 h, cells were treated with various concentrations of 2,6-DMBQ for 24 h and harvested. Cells were then washed with cold phosphate buffered saline (PBS) and fixed in 1 ml 70% cold ethanol. After rehydration, cells were permeabilized with 0.6% Triton X-100 (Solarbio) and digested with 100 mg/ml RNase A (Solarbio) for 1 h. Cells were subsequently stained with 20 mg/ml propidium iodide (Clontech, Palo Alto, CA, USA) for 15 min before analysis by flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). 2.8. Wound healing assay Cells were seeded (1 105 cells per well) in 6-well plates with 100 ml complete growth medium. After incubation for 24 h, the cells were gently scratched using a plastic micro pipette tip and then washed three times with PBS. Cells were treated with various concentrations of 2,6-DMBQ for 24 h or 48 h. The 6-well plates containing the scratch-wounds were photographed in three different fields of each well. The average width of each scratchwounds was measured and calculated using ImageJ (National Institutes of Health, Bethesda, MD, USA). 2.9. Migration assay The lower compartment of transwell chambers (Corning, Bedford, MA, USA) were coated with Matrigel (Becton, Dickinson and Company, NJ, USA) and incubated for 30 min at room temperature under sterile conditions. The lower compartment of each chamber was then filled with 600 ml complete growth medium. Fifty thousand cells suspended in 200 ml complete growth medium with or without 2,6-DMBQ were added to the upper compartment. 

The chambers were incubated for 24 h at 37 C in a 5% CO2 atmosphere. Migrated cells were fixed with methanol (Tianjin Zhiyuan Chemical Reagent Co. LTD, Tianjin, China) and stained with hematoxylin (Baso Diagnostics INC, Zhuhai, China) and eosin (Baso Diagnostics INC) prior to imaging. Photos of the stained cells were analyzed using the Image-Pro Plus software (v.6) program (Media Cybernetics). 2.10. Statistical analysis All quantitative results are indicated as mean values ± S.D. Statistically significant differences were determined using the Student's t test or by one-way ANOVA (p < 0.05). Statistical significance was determined using the Statistical Package for Social Science (SPSS) 21.0 (Xishu software, Shanghai, China). 3. Results 3.1. 2,6-DMBQ suppresses anchorage-dependent and -independent growth of NSCLC cells 2,6-DMBQ is a benzoquinone compound (Fig. 1A). We first investigated whether 2,6-DMBQ could affect the growth of NSCLC cells. Cells were treated with 2,6-DMBQ at different doses and incubated for 48 h and before proliferation was analyzed with an MTT assay. Results indicated that anchorage-dependent growth of NSCLC cells was significantly suppressed by 2,6-DMBQ treatment in a dose-dependent manner (Fig. 1B).

 Specifically, we observed that 2,6-DMBQ strongly inhibited cell growth in H1299 and H1650 cells compared with H358 cells (Fig. 1B). Furthermore, we investigated whether 2,6-DMBQ could induce cell apoptosis in NSCLC cells. Cells were treated with 2,6-DMBQ for 48 h and then cell apoptosis was analyzed by flow cytometry. Results showed that late cell apoptosis by 2,6-DMBQ was strongly increased in H1299 and H1650 cells, but not H358 cells (Supplemental Fig. 1A and B). Therefore, we used H1299 and H1650 NSCLC cells for further study. We next investigated the effect of 2,6-DMBQ on anchorage-dependent growth (Fig. 1C) and anchorage-independent growth (Fig. 1D) in NSCLC cells using foci formation and soft agar assays, respectively. Results indicated that treatment with 2,6-DMBQ strongly inhibited foci number and anchorage-independent growth relative to untreated controls (Fig. 1C and D). 3.2. 2,6-DMBQ increases G2 phase cell cycle arrest in lung cancer cells We next performed flow cytometry analysis to determine the effect of 2,6-DMBQ on cell cycle progression after treatment with 2,6-DMBQ for 24 h. The results showed that 2,6-DMBQ significantly increased the fraction of cells in G2 phase (Fig. 2A and B).

 We also examined whether 2,6-DMBQ could affect the expression levels of G2 phase marker proteins. After treatment with 2,6-DMBQ for 24 h, the expression levels of the G2 phase marker proteins cyclin B1 and phosphorylated CDC2 were detected by Western blotting. Our findings indicated that 2,6-DMBQ treatment strongly inhibited the expression levels of the cyclin B1and phosphorylation of CDC2 in NSCLC cell lines (Fig. 2C). 3.3. 2,6-DMBQ inhibits the migration of NSCLC cells We next performed a wound healing assay to determine the effect of 2,6-DMBQ on lung cancer cell migration. Cells were scratched using a pipette tip and treated with 2,6-DMBQ for 24 or 48 h prior to visualization under microscope. The results indicated that 2,6-DMBQ significantly suppressed cell migration into the wound area compared with the untreated control cells (Fig. 3A). We next performed a series of transwell migration assays to verify the inhibition of cell migration observed upon treatment with 2,6- DMBQ. Cells were treated with 2,6-DMBQ for 24 or 48 h in the transwell chambers; migrated cells were fixed and counted after each time point. The results of the transwell migration assay experiments showed that 2,6-DMBQ strongly inhibited the number of migrated cells compared with untreated control cells (Fig. 3B).