and further suggested that deregulation of Sema 3A pathway could be an important therapeutic target for prostate cancer progression [22]. Furthermore, overexpression of Sema 3A significantly suppresses in vivo breast tumor growth in mouse xenograft model [23]. However, the function of Sema 3A in regulation of melanoma progression is not well studied, and yet to be a field of intense investigation. Angiogenesis, or formation of new blood vessel from the existing one has been considered as the most important step during tumor progression [24]. Current advancement in cancer research has shown that targeting angiogenic pathways could be a more rational and promising anti-cancer therapeutic approach [25]. To date, vascular endothelial growth factor (VEGF) is considered as one of the most potent angiogenic factor that governs tumor angiogenesis [26]. Interaction between VEGF and one of its co-receptor neuropilin 1 (NRP1) is known to play an important role in tumor angiogenesis [27,28] and therefore blocking their interaction could be a rational anti-angiogenic therapeutic approach for cancer treatment [28,29]. Moreover, NRP1 has been identified as one of the co-receptors of Sema 3A [30,31]. Miao et al. have shown that Sema 3A and/or VEGF act as competitive ligand for binding to NRP1 and described that Sema 3A can attenuate VEGF-induced endothelial cell motility [32]. Thus, Sema 3A could act as a potent inhibitor of tumor angiogenesis by disrupting the interaction between VEGF and NRP1. Very recently, using transgenic mice model, Maione et al. have shown that Sema 3A acts as an endogenous angiogenesis inhibitor that blocks tumor growth by normalizing tumor vasculature [33]. Earlier data have shown that Sema 3F, another member of Sema 3 family, attenuates melanoma progression by inhibiting angiogenesis [34]. However, the correla- tion between Sema 3A expression and melanoma progression as well as angiogenesis is not clearly understood. Therefore, using multiple models, we have demonstrated the novel function of Sema 3A in suppression of melanoma progression and angiogenesis. Our study might be useful to develop more rational Sema 3A-mediated therapeutic strategy for the next generation of cancer management. Materials and Methods Cell lines, reagents and antibodies The murine melanoma cell lines (B16F1 and B16F10) and human melanoma cell lines (A375 and SK-Mel-28) were purchased from American Type Culture Collection (ATCC, Manassas, VA), cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine in a humidified atmosphere of 5% CO2 and 95% air at 37uC. Human umbilical vein endothelial cells (HUVEC) were cultured in EBM according to the manufacturer’s instructions (Lonza, Walkersville, MD). Human recombinant Sema 3A (Cat no. AF1250-S3-025) and mouse recombinant Sema 3A (Cat no. 5926-S3-025) were purchased from R&D Systems. Goat anti- Sema 3A (Cat no. SC 1148), rabbit anti-a-tubulin (SC-135659), rabbit anti-PARP (SC-25780) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-vWF antibody (Cat no. HPA002082), PI, DAPI, FITC-conjugated phalloidin, fibronectin, Dacarbazine (DTIC), curcumin were purchased from Sigma (St. Louis, MO). Boyden type cell migration chambers were obtained from Corning (Corning, NY). Growth factor depleted matrigel and matrigel coated invasion chambers were obtained from BD Bioscience (Bedford, MA). Mouse anti- Sema 3A (Cat no. MAB-1250) and anti-neuropilin1 (Cat no. AF566) antibodies were purchased from R&D System (Minne- apolis, MN). Rabbit anti-phospho p53 (Ser-15) (Cat no. 9284) antibody was obtained from Cell Signaling Technologies (Danvers, Semaphorin 3A Attenuates Melanoma Progression MA). The C57BL/6 mice were maintained at Experimental Animal Facility (EAF) of National Center for Cell Science (NCCS), Pune, India. The study was approved by Institutional Animal Care and Use Committee (IACUC), NCCS (Project Approval No. NCCS/ EAF/2007/B116). Sema 3A siRNA was purchased from Dharma- con International (Lafayette, CO). Human Sema 3A and GAPDH specific TaqMan gene expression quantitative real time PCR (Q- PCR) assay related reagents were procured from Applied Biosystems (Carlsbad, CA). BrdU labeling and detection kit was purchased from Roche. Anti-Goat Cy2 and anti-rabbit Cy3 were purchased from Chemicon International. RNA isolation, semi-quantitive RT–PCR and Q-PCR RNA isolation and reverse transcription-polymerase chain reaction (PCR) were performed as described earlier [35]. Total RNA was isolated from B16F1, B16F10, A375 and SK-Mel-28 cells by using Trizol reagent (Invitrogen) and reverse transcription- PCR was performed using following sets of primers: Sema 3A forward: 59-CAG CCA TGT ACA ACC CAG TG-39; Sema 3A reverse: 59-ACG GTT CCA ACA TCT GTT CC-39; GAPDH forward: 59-ACT CCA CTC ACG GCA AAT TC-39; GAPDH reverse: 59-CCT TCC ACA ATG CCA AAG TT-39. Q-PCR was performed with TaqMan gene expression assay (Applied Biosys- tems) according to manufacture’s instruction. GAPDH was used as internal control. The PCR products were analyzed by electro- phoresis using 1.0% agarose gel. Generation of Sema 3A stable clones Human Sema 3A cDNA in PCEP4 expression vector (H-Sema 3A-AP; a generous gift from Prof. Alex Kolodkin, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) was transfected in B16F10 cells using lipofectamine 2000 (Invitrogen; Carlsbad, CA). After transfection, cells were selected with 400 mg/ ml hygromycin and three positive clones were isolated and denoted as clone 1, 2 and 3. Melanoma clinical specimen analysis Normal skin biopsy and malignant melanoma specimens were collected from local hospitals with informed consent. Tissue sections were stained with hematoxylin and eosine and the occurrence of melanoma in these samples was analyzed with the help of expert onco-pathologist. The expression profiles of Sema 3A and phospho p53 (Ser-15) were analyzed by immunofluores- cence in tissue sections using their specific antibodies and visualized under confocal microscopy (Zeiss) as described earlier [35,36]. Western Blot Analysis The expression of Sema 3A in control or Sema 3A clones was determined by Western blot using anti-Sema 3A antibody as described [28]. The effect of Sema 3A on curcumin-induced PARP cleavage was also determined using anti-PARP antibody. Matrigel colony formation assay Matrigel colony formation assay was performed on growth factor depleted matrigel coated plate. Briefly, 250 ml cold matrigel was coated on 24 well plates and the plates were kept at 37uC for 1 h for polymerization. Equal number of cells (control B16F10 or clone 2) were grown on matrigel coated plates and incubated at 37uC for 7 days. After termination of experiments, colonies were visualized under microscope (Nikon) and photographed. PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33633