Surfactin from Bacillus subtilis attenuates ambient air particulate matter- promoted human oral cancer cells metastatic potential Thi Thuy Tien Vo1,#, Chiang-Wen Lee2,3,4,5,#, Ching-Zong Wu1, Ju-Fang Liu6, Wei- Ning Lin7, Yuh-Lien Chen8, Lee-Fen Hsu9,10, Ming-Horng Tsai11,12, I-Ta Lee1,* 1School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 2Department of Orthopaedic Surgery, Chang Gung Memorial Hospital, Puzi City, Chiayi County 61363, Taiwan 3Department of Nursing, Division of Basic Medical Sciences, and Chronic Diseases and Health Promotion Research Center, and Research Center for Chinese Herbal Medicine, Chang Gung University of Science and Technology, Puzi City, Chiayi County 61363, Taiwan 4Department of Safety Health and Environmental Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan 5College of Medicine, Chang Gung University, Guishan Dist., Taoyuan City 33303, Taiwan 6School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 7Graduate Institute of Biomedical and Pharmaceutical Science, Fu Jen Catholic University, New Taipei City, Taiwan 8Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 9Department of Respiratory Care, Chang Gung University of Science and Technology, Puzi City, Chiayi County 613, Taiwan 10Division of Neurosurgery, Department of Surgery, Chang Gung Memorial Hospital, Puzi City, Chiayi County 613, Taiwan 11Department of Pediatrics, Division of Pediatric Hematology/Oncology and Neonatology, Yunlin Chang Gung Memorial Hospital, Yunlin, Taiwan 12College of Medicine, Chang Gung University, Taoyuan, Taiwan #These authors contributed equally to this work Running Title: Inhibition of oral cancer cell invasion by surfactin *Corresponding Author: I-Ta Lee School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan 250 Wuxing St. Taipei 11031, Taiwan Tel: +886-2-27361661 ext. 5162 1 Fax: +886-2-27362295 E-mail addresses: [email protected] 2 Abstract Recently, many studies have indicated that ambient air particulate matter (PM) can increase the risk of oral cancer. The most common malignant tumor in the oral cavity is oral squamous cell carcinoma (OSCC). Usually, cancer cell migration/invasion is the most important cause of cancer mortality. Matrix metalloproteinase-2 (MMP-2) and MMP-9 have been shown to play important roles in regulating metastasis and the tumor microenvironment. Here, we studied the anti-cancer effects of surfactin, a cyclic lipopeptide generated by Bacillus subtilis, on cancer cell migration and invasion. Surfactin suppressed PM-promoted cell migration and invasion and colony formation of SCC4 and SCC25 human oral squamous cell carcinoma cell lines. We observed that PM induced MMP-2 and MMP-9 expression, which was inhibited by surfactin. Transfection with p65, p50, c-Jun, c-Fos, p85, p110, Akt, mammalian target of rapamycin (mTOR), or interleukin-6 (IL-6) siRNA markedly inhibited PM-induced MMP-2 and MMP-9 expression. Moreover, surfactin could reduce Akt, mTOR, p65, and c-Jun activation and IL-6 secretion induced by PM. Finally, we proved that transfection with Akt, p65, or c-Jun siRNA significantly inhibited PM-induced IL-6 release. Taken together, these results suggest that surfactin functions as a suppressor of PM-induced MMP2/9-dependent oral cancer cell migration and invasion by inhibiting the activation of phosphoinositide 3-kinase (PI3K)/Akt/mTOR and PI3K/Akt/nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)/IL-6 signaling pathways. Key words: Surfactin, Invasion, Particulate matter, Oral cancer, Matrix metalloproteinase 3 Introduction In many parts of the world, new cases of oral cancer and deaths are increasing. Known risk factors include smoking, drinking, human papillomavirus (HPV), and betel quid chewing [1]. It is also believed that exposure to heavy metals and emissions from petroleum and chemical plants is also related to the development of oral cancer, and it is well known that air pollution, especially ambient air particulate matter (PM), is harmful to the respiratory and cardiovascular system [2]. The combined effects of household and ambient air pollution cause approximately 7 million premature deaths every year, mainly due to heart disease, stroke, lung cancer, chronic obstructive pulmonary disease, and acute respiratory infections leading to increased mortality [3, 4]. The composition of PM is very complicated including nitrate, sulfate, ammonia, and so on. Compared to PM10, PM2.5 can cause greater harm to human health. PM2.5 generally penetrates the lung barrier and enters the blood system. Most studies in the past have explored the relationship between betel nuts, cigarettes, or alcohol and oral cancer, but few studies have studied the relationship between air pollution and oral cancer. The most common malignant tumor in the oral cavity is oral squamous cell carcinoma (OSCC). Moreover, cancer cell migration/invasion is the most important cause of cancer mortality [5]. Matrix metalloproteinases (MMPs) belong to a family of zinc-dependent endopeptidases [6]. Members of the MMP family include collagenase, gelatinase, stromalysin, stromelysin, and membrane-type MMP [6]. Moreover, MMP- 9 can mediate extracellular matrix (ECM) remodeling by cleaving many ECM proteins. MMP-2 or MMP-9 has been shown to play an important role in regulating metastasis including oral cancer [5, 7]. PM2.5 also has been shown to induce MMP-2 and MMP- 9 expression in keratinocytes [6]. Therefore, reducing the expression of MMP-2 and MMP-9 or its upstream regulatory signaling pathways is essential for the treatment of malignant tumors. Up-regulation of MMP-2 and MMP-9 expression was mediated through various signaling pathways [8, 9]. Here, we studied the novel pathways involved in PM-mediated MMP-2 and MMP-9 expression in the SCC4 and SCC25 human oral squamous cell carcinoma cell lines. Surfactin is a bacterial cyclic lipopeptide generated by Bacillus subtilis [10]. Surfactin has been shown to possess some properties including anti-cancer, anti- bacterial, and anti-viral activities [11]. Even though surfactin has been regarded as a potential anti-cancer agent, its specific effects on cancer cells and the detailed 4 mechanisms involved are still unknown. Park et al. indicated that surfactin reduced TPA-mediated breast cancer cell migration/invasion via the inhibition of MMP-9 levels [10]. In addition, Wang et al. also proved that surfactin could promote apoptosis of HepG2 cells via the reactive oxygen species (ROS) signaling [12]. Here, we studied the anti-cancer effects of surfactin on PM-induced human oral squamous cell carcinoma cell migration and invasion and the novel mechanisms underlying these processes. The present study proved that PM induces MMP-2/9-dependent cell migration and invasion via the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and PI3K/Akt/NF-κB and activator protein-1 (AP-1)/interleukin-6 (IL-6) signaling pathways. Moreover, surfactin can inhibit MMP-2/9 expression via inhibition of the activation of these two pathways induced by PM and then reduce cell migration and invasion. 5 Materials and methods Materials We purchased anti-Akt, anti-phospho-Akt, anti-mTOR, anti-phospho-mTOR, anti-MMP-2, anti-MMP-9, anti-GAPDH, anti-tissue inhibitor of matrix metalloproteinase (TIMP)-1, anti-TIMP-2, anti-phospho-c-Jun, and anti-phospho-p65 antibodies from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Surfactin and urban PM (SRM 1648a) were purchased from Sigma (St. Louis, MO, USA). Cell culture SCC4 and SCC25 human oral squamous cell carcinoma cell lines were kindly provided by Dr. J. F. Liu (School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan). SCC4 and SCC25 cells were grown in DMEM/F12 supplemented with 10% FBS, 2 mM glutamine and 0.4 μg/ml hydrocortisone. Cells were maintained as monolayer cultures in a humidified atmosphere of 5% CO2 at 37°C. Cell viability The cell viability of SCC4 and SCC25 cells in response to PM or surfactin was assessed using PrestoBlue Cell Viability Reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. Western blot Cells were incubated with PM under various experimental design conditions at 37°C. Western blot was then performed based on previously published literature in our laboratory [13]. At last, membranes were incubated with the anti-Akt, anti-phospho- Akt, anti-mTOR, anti-phospho-mTOR, anti-MMP-2, anti-MMP-9, anti-GAPDH, anti- TIMP-1, anti-TIMP-2, anti-phospho-c-Jun, or anti-phospho-p65 antibody for 24 h and then incubated with the anti-mouse or anti-rabbit horseradish peroxidase antibody for 1 h. We used enhanced chemiluminescence (ECL) reagents to observe immunoreactive bands. Real-Time PCR Total RNA was extracted by using TRIzol reagent. We further reverse-transcribed 6 mRNA into cDNA and analyzed by real-time PCR using SYBR Green PCR reagents (Applied Biosystems, Branchburg, NJ, USA) based on previously published literature in our laboratory [14]. Transient transfection with siRNAs Human scrambled, MMP-2, MMP-9, p65, p50, c-Jun, c-Fos, p85, p110, Akt, mTOR, and IL-6 siRNAs were from Sigma (St. Louis, MO). Transient transfection of siRNAs was performed using a Lipofectamine 2000 Transfection Reagent according to the manufacturer’s instructions. Analysis of luciferase reporter gene activity Human MMP-2, MMP-9, AP-1, and NF-κB promoter-luciferase constructs were kindly provided by Dr. C. W. Lee (Department of Nursing, Chang Gung University of Science and Technology, Puzi City, Chiayi County, Taiwan). The luciferase activity was quantitatively assessed as previously described [15] using a luciferase assay system (Promega, Madison, Wis.). Firefly luciferase activities were standardized for β-gal activity. Gelatin zymography Cells were seeded onto 6-well culture plates and made quiescent at confluence by incubation in serum-free DMEM/F12 for 24 h. Growth-arrested cells were treated with PM under various experimental design conditions at 37°C. The culture medium was collected and centrifuged at 10000 × g for 5 min at 4°C to remove cell debris.