Bangladesh Journal of Pharmacology Research Article Kaempferol modulates the metasta-Kaempferol modulates the metasta-Kaempferol modulates the metasta- sis of human nonsis of human nonsis of human non---small cell lung small cell lung small cell lung cancer cells by inhibiting epithelialcancer cells by inhibiting epithelialcancer cells by inhibiting epithelial--- mesenchymal transitionmesenchymal transitionmesenchymal transition BJP Introduction Lung cancer is the most commonly diagnosed cancer type worldwide and a leading cause of cancer-related deaths. It can be categorized into two sub-types: non- small cell lung cancer (Koh et al., 2012) and small cell lung cancer. Despite many efforts to improve lung cancer outcome, long-term survival has not improved significantly over the last 20 years, with a 5-year cumulative survival rate that remains very dismal at only 15% (Ghosal et al., 2009). Current standard thera- pies limited to chemotherapy and radiotherapy or both rarely cure this disease, thus accentuating the need for more effective and alternate therapeutic strategies. Metastasis is a complex, multistep process and involves the invasion of cells from primary tumors into the circu- lation, migration to distant organs and finally infiltra- tion into tissues referred to as secondary metastatic sites (Gupta and Massagué, 2006). Epithelial–mesenchymal transition (EMT), a developmental program plays an important role in this process and involves down- regulation of epithelial markers like E-cadherin and up- regulation of mesenchymal markers like vimentin and fibronectin (Borthwick et al., 2009; Kalluri and Neilson, 2003). As a result, the epithelial cells acquire fibroblast- like properties, thus losing their defined cell-cell and cell-extracellular matrix contacts (Thiery et al., 2010). Thus, the EMT becomes a target to prevent tumor progression. Flavonoids are polyphenolic natural compounds pre- sent in a wide variety of fruits and vegetables (Bosetti et al., 2007) and in recent years their anti-tumor activities have been widely studied and recognized (Gonzalez and Riboli, 2006). Kaempferol (3,4’,5,7-tetrahydroxyfla- vone), is a natural polyphenol of the flavonoid family and exhibits various biological properties including anti -tumor activities. It induces apoptosis and cell cycle arrest in various cancer cell lines, including lung cancer cells (Nguyen et al., 2003), breast cancer cells (Kang et al., 2010), colon cancer cells (Li et al., 2009), besides inhibiting the migration and invasiveness of glioma cells (Shen et al., 2006). The effect of kaempferol on the growth and invasiveness of lung cancer is not yet deter- mined and the mechanism involved needs to be defi- ned. The present study examined the effects of kaemp- ferol on the metastasis and invasion of A549 non-small cell lung cancer cells. It was found that kaempferol markedly inhibited cell proliferation besides overcom- ing EMT and cell migration. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 267-70 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract The present study was done to determine whether kaempferol, a natural polyphenol of the flavonoid family, affects Epithelial-Mesenchymal Transition (EMT) in non-small cell lung cancer cells. Kaempferol not only inhibited cancer cell proliferation and migration in a dose-dependent manner but also modulated the expression of EMT-related proteins E-cadherin and vimentin which are indispensable to cellular motility, invasiveness and metastasis. These results indicate that kaempferol suppresses non-small cell lung cancer migration by modulating the expression of EMT proteins. Therefore, kaemp- ferol may be useful as a potential anticancer agent for non-small cell lung cancer. Article Info Received: 14 December 2014 Accepted: 22 January 2014 Available Online: 2 April 2015 DOI: 10.3329/bjp.v10i2.21739 Cite this article: Hang M, Zhao F, Chen SB, Sun Q, Zhang CX. Kaempferol modulates the metastasis of human non-small cell lung cancer cells by inhibiting epitheli- al-mesenchymal transition. Bangla- desh J Pharmacol. 2015; 10: 267-70. Kaempferol modulates the metastasis of human non-small cell lung cancer cells by inhibiting epithelial-mesenchymal transition Meng Hang, Fan Zhao, Shu-Bo Chen, Qing Sun and Chun-Xiao Zhang Department of Oncology, Jiangsu Province Wuxi No. 2 People’s Hospital, Wuxi 214002, China. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Materials and Methods Cell culture and kaempferol treatment: A549 cells were maintained in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 100U/mL penicillin and 100 mg/mL streptomycin. The cells were grown under standard culture conditions at 37°C under 5% CO2 in a humidified incubator. The medium was changed every two days or until the cells became con- fluent and then used for further experimentation. Stock solution of 100 mM kaempferol (Sigma-Aldrich) was prepared in DMEM and diluted to different concentra- tions. Cell proliferation assay: The effect of kaempferol on A549 cells was evaluated using MTT cell proliferation assay. The cell suspension containing about 10,000 cells was seeded into each well of a 96 well plate. The cells were allowed to grow for 24 hours after which different concentrations of kaempferol were added and incubi- ted for 24 hours. MTT (Sigma-Aldrich) solution was added to the cells at a concentration of 0.1 mg/mL (dissolved in PBS) followed by incubation for 4 hours at 37°C in dark. The supernatant was removed and 100 µL of DMSO was added to dissolve the formazan crystals. The optical density was measured at 490 nm using a plate reader (Bio-Rad, Hercules, CA). The results were presented as the percentages relative to the controls. The percentage proliferation inhibition rate was calcula- ted as = (1 – OD sample/OD control) × 100%. Western blotting: Cells were lysed in ice cold RIPA buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5%Na-deoxycholate) supplemen- ted with protease inhibitors. The protein concentration was determined by BCA method (Thermo Scientific Pierce). Twenty five micrograms of each protein sample were separated on a 10% polyacrylamide gel and electro-transferred to PVDF membrane. The membrane was then blocked with 5% non-fat dried milk in PBS at room temperature for 2 hours, followed by incubation with primary antibodies against E-cadherin (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology) and β-actin (Sigma) at 4°C overnight. After washing, the blots were hybridized with secondary goat anti- rabbit antibodies (Sigma) for 1 hour at room tempera- ture and developed using an enhanced chemilumi- nescence detection system (Amersham). Wound healing assay: A549 cells were plated in 30 cm cell culture dishes and grown up to 80% confluence. The media was then removed and the cells scratched with 100 µL pipette tip. Cells were then washed with PBS to remove detached cells, photographed (t = 0 hour) and treated with kaempferol for 24 hours. Then the wounds were observed and photographed (t = 48 hours). Results The antiproliferative activity of kaempferol was tested by MTT assay against human non-small lung cancer cell line, A549. A549 cells showed a significant growth inhi- bition at all the tested concentrations of kaempferol. The effect of different concentrations on the prolifera- tion of A549 cells was seen to be dose-dependent. The results demonstrated that percentage of growth inhibi- tion increased with increasing concentration of kaemp- ferol from 10 to 140 μM with an IC50 value of 72 µM after 24 hours of incubation (Figure 1). Kaempferol exhibited maximum anti-proliferative potential at a concentration of 140 μM and lowest at 10 μM. Only such concentrations (10, 20, 40 and 60 µM) which exhibited lesser influence on the viability of A549 cells were used in all subsequent experiments In hi bi to ry e ffe ct Figure 1: Inhibitory effects of kaempferol on the proliferation of non-small cell lung cancer cells. A549 cells were treated with in- creasing concentrations of kaempferol for 24 hours Kaempferol concentration (µM) 268 Bangladesh J Pharmacol 2015; 10: 267-270 100 80 60 40 20 0 0 20 40 60 80 100 120 140 EMT imparts migratory abilities which in turn enhance the invasive capabilities of cancer cells. Once switched on, this process leads to unfavorable prognosis in many different types of cancers. We examined the effect of kaempferol on the expression of EMT-related proteins E-cadherin and vimentin after incubation of cells with different concentrations (10, 20, 40 and 60 µM) for 24 hours. Western blotting showed that kaempferol treatment in a dose-dependent manner enhanced the expression of E-cadherin with concomi- tant decrease in expression of vimentin, thereby limi- ting EMT (Figure 2). Since the concentration of 40 µM was very effective in modulating the expression of E-cadherin and vimentin, we therefore tried to evaluate whether kaempferol at similar concentration could also affect the metastatic behaviour of A549 cells. A549 cells were grown to sub- confluency, wounded and then treated with 40 µM kaempferol for 24 hours. Kaempferol significantly over- came migratory abilities of A549 cells in comparison to control (untreated) as was reflected by attenuated wound healing (Figure 3). Discussion There is considerable epidemiological evidence which shows that cancer risk decreases with higher intake of vegetables, fruits, and grains (Witte et al., 1996). Flavonoids, commonly found in fruits and vegetables have been widely recognized for their remarkable anti- Figure 2: Effects of kaempferol on the expression of EMT markers in non-small cell lung cancer cells. A549 cells were treated with increasing concentrations of kaempferol for 24 hours and the protein expression levels of E-cadherin and vimentin were detected Figure 3: Effects of kaempferol on the migration of non-small cell lung cancer cells. A549 cells were either treated with 40 µM of kaempferol or left untreated (control) for 24 hours and the wound healing was observed after 48 hours Bangladesh J Pharmacol 2015; 10: 267-270 269 0 hour 48 hours Kaempferol (µM) 0 10 20 40 60 E-Cadherin GAPDH Kaempferol (µM) 0 10 20 40 60 Vimentin GAPDH Kaempferol (40 µM) Untreated carcinogenic properties (Hoensch et al., 2005). Kaemp- ferol, is a member of the flavonoid family and exhibits potential anti-tumor activities. Previous studies demons -trated that kaempferol inhibits growth and prolifera- tion of cancer cells by various mechanisms including apoptosis (Sharma et al., 2007) cell cycle arrest (Cho et al., 2013) and inhibition of tyrosine phosphorylation (Lee et al., 1998). However, studies focusing on the potential of kaempferol in overcoming invasiveness of cancer cells particularly lung cancer have rarely been done. Therefore, the present study was performed to evaluate the potential of kaempferol in overcoming the invasiveness of human non-small cell lung cancer cells. In this study, kaempferol was potent in inhibiting the proliferation of A549 cells. This effect was dose depen- dent with an IC50 value of 72 µM. To assess the efficacy of kaempferol in the lung cancer progression, A549 cells were treated with different concentrations of kaemp- ferol for 24 hours and the expression levels of EMT markers E-cadherin and vimentin monitored by western blotting. Kaemferol increased the expression of the epithelial marker, E-cadherin in A549 cells in a dose -dependent manner. In addition, there was down- regulation of mesenchymal protein marker, vimentin by kaempferol treatment. There was inhibition of A549 cell migration when treated with the most efficacious concentration of kaempferol, as evaluated by the wound-healing assay. The finding suggests the role of kaempferol in impeding the EMT in A549 cells and provides an evidence for the use of kaempferol against non-small cell lung cancer progression. References Borthwick LA, Parker SM, Brougham KA et al. Epithelial to Mesenchymal Transition (EMT) and airway remodelling after human lung transplantation. Thorax 2009; 64: 770–77. Bosetti C, Rossi M, McLaughlin JK, Negri E, Talamini R, Lagiou P, Montella M, Ramazzotti V, Franceschi S, LaVecchia C. Flavonoids and the risk of renal cell carcinoma. Cancer Epidemiol Biomarkers Prev. 2007; 16: 98- 101. Cho HJ, Han J, Park Y. 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Am J Epidemiol. 1996; 144: 1015-25. 270 Bangladesh J Pharmacol 2015; 10: 267-270 Author Info Chun-Xiao Zhang (Principal contact) e-mail: zhangchunxiao98@hotmail.com nonsmall cell lung cancer progression: Author Info ChunXiao Zhang Principal contact email zhangchunxiao98hotmailcom: DatePrinted: This article was downloaded by you on: Jul 23, 2017