B16F10 SDS-PAGE. Annexin V-FITC /PI

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1 NRP1 B16F10 * neuropilin1 NRP1 NRP1 mab B16F10 SDS-PAGE 1 1 B16F10 1 Annexin V-FITC /PI B16F B16F10 P < B16F10 P < B16F10 Akt ERK1 /2 Bcl-2 Caspase 3 Bax 1 B16F10 1 PI3K /Akt MEK /ERK1 /2 Bcl-2 /Bax /Caspase3 1 doi /cpj R965 A Effects of Anti-NRP1 Monoclonal Antibody on Cell Proliferation Migration and Apoptosis of Melanoma B16F10 Cells L Sha LI Zhe WANG Xian-jiang WANG Sheng-yu YANG Yun LUO Fang-hong WU Ting YAN Jianghua * Cancer Research Center of Medical College Xiamen Unversity Xiamen China ABSTRACT OBJECTIVE To investigate the effect of anti NRP1 monoclonal antibody NRP1 mab on melanoma cell B16F10 proliferation migration and apoptosis and explore its mechanism. METHODS Western blot immunofluorescent and immunocytochemistry staining were applied to determine the expression of NRP1 on B16F10 cells and its xenotransplanted tumors by NRP1 mab. The proliferation migration and apoptosis of melanoma cells were detected by MTT assay the scratch test and Annexin V-FITC /PI double labeled flow cytometry after administration with different concentrations of NRP1 mab. The expression level of some signal molecules was measured by Western blot. RESULTS The purified NRP1 mab has high purity and specificity. B16F10 cells and its xenotransplanted tumors highly expressed NRP1. NRP1 mab inhibit the proliferation and migration in a time-and dose-dependent manner P < Moreover NRP1 mab induce apoptosis of B16F10 in a dose-dependent manner P < The results of western blot revealed down-regulated expression of p-akt p-erk1 /2 Bcl-2 and caspase-3 and up-regulated Bax expression in B16F10 cells treated by NRP1 mab. CONCLUSION The purified NRP1 mab can effectively inhibit the proliferation and migration of B16F10 and induce apoptosis. Meanwhile the changed expression of the proteins suggests that NRP1 mab may inhibit cell proliferation and migration by ERK1 /2 and PI3K /Akt pathway and induce cell apoptosis by Bcl-2 /Bax /caspase-3 pathway consequencely playing anti-tumor role. KEY WORDS NRP1 mab melanoma proliferation migration apoptosis malignant melanoma R J0138 * Tel /Fax jhyan@ xmu. edu. cn Chin Pharm J 2014 October Vol. 49 No

2 8 ~ ml 1 ~ ~ 10 d 80% 37 1 h r min neuropilin 1 NRP1 semaphorin Sema 3A vascular endothelial growth factor rprotein A Sepharose TM β-actin MTT TRITC- receptor VEGF min sodium dodecyl sulfate-polyacrylamide gel electrophore NRP1 sis SDS-PAGE Sigma DMEM 1. 5 B16F10 NRP1 Gibico Annexin V /PI BD Akt p-akt ERK1 /2 p-erk1 /2 Cell Signal Bax Bcl-2 Caspase-3 Santa Cruz HRP ml ~ 6 d Hoechst cm 0. 5 cm 0. 3 cm RIPA 10% 5 μm 1. 2 NRP1 mab 10 min rprotein A PBS μl NRP1 B16F % CO 2 24 h a1a2 b1b2 c b1b2 NRP1 mab 48 h PBS 6 30% 4% 30 min 10% PBS min NRP1 mab PBS VEGF165 HGF TGF-β PDGF μl RIPA 1% b1b2 NRP1 mmol L - 1 PMSF 4 15 min r min % SDS- PAGE NRP1 mab NRP1 b1b Bcl-2 Bax Caspase Akt p-akt ERK1 /2 p-erk1 / TBST Tris-Buffered Saline and neuropilin1 NRP1 NRP1 mab h TBST 3 ECL Tween 20 3 HRP 1 10 NRP B16F10 NRP1 B16F10 3 TRITC 1 64 NRP1 mab 37 1 h PBS 3 Hoechst min PBS 3 Olympus 0. 25% r min min MTT Balb /c h 1808 Chin Pharm J 2014 October Vol. 49 No

3 NRP1 mab μg ml - 1 PBS 6 B16F h MTT 5 mg ml μl 4 h NRP1 2B C 150 μl dimethyl sulfoxide DMSO 5 min 570 nm A 2A B16F NRP1 mab B16F10 % = - NRP1 mab / 100% μl 1 mm μg ml - 1 NRP1 mab PBS 2% h Image- Pro Plus % = - / 100% 1 SDS-PAGE rprotein A M NRP1 mab NRP1 mab 100 Fig. 1 SDS-PAGE Analysis of the mabs after purification μg ml - 1 M - protein marker 1 - BSA 2 - purified NRP1 mab 3 - ascites PBS 48 h 100 μl Annexin V-FITC / PI 15 min 1 h 1. 9 SPSS13. 0 t x 珋 ± s 0 r 1-1 r 0 P < NRP1 mab rprotein A SDS- PAGE 95% A B C NRP B16F10 NRP1 Fig. 2 Western blot A immunocytochemistry staining B NRP1 mab and confocal immunofluorescent analysis C for the binding specificity of the mabs with NRP1 NRP1 b1b2 B16F10 NRP Chin Pharm J 2014 October Vol. 49 No

4 3A MTT P < h P < NRP1 mab h r NRP1 mab NRP1 mab 3B 2. 4 NRP1 mab B16F10 NRP1 mab 4A NRP1 mab P < NRP1 mab 4 B16F10 A μg ml - 1 B. n = 6 x 珋 ± s P < 1 P < P < NRP1 mab h Fig. 4 The migratory morphological changes A 200 and migratory index B of B16F10 cells detected by the scratch NRP1 mab test. n = 6 x 珋 ± s P < P < vs controls P < P < NRP1 mab B16F10 4B 48 h 3 NRP1 mab B16F10 A 200 NRP1 mab B16F10 B. n = 6 x 珋 ± s 1 P < P < Fig. 3 Morphological changes A 200 and proliferation B of B16F10 cells treated with NRP1 mab. n = 6 珋 x ± s 1 P < P < vs controls 1810 Chin Pharm J 2014 October Vol. 49 No. 20 NRP1 mab B16F μg ml μg ml - 1 P < MTT 5A 1 Hoechst NRP1 mab 100 μg ml - 1 B16F μg ml - 1 5B 2. 6 NRP1 mab B16F10 p-akt p-erk1 /2 Bcl-2 Bax caspase-3 p-akt p-erk1 /2 Bcl-2 Bax caspase-3 NRP1 mab p- Akt p-erk1 /2 Bcl-2 Bax caspase-3 β-actin

5 5 Fig. 5 B 400 B16F10 A B 400 Morphological and apoptotic index changes of B16F10 cells by flow cytometry analysis A and nuclear fluorescent staining 1 Tab. 1 NRP1 MAb. % n = 3 x 珋 ± s The percentage of cell apoptosis of B16F10 treated with NRP-1 mab and the correlation analysis. % n = 3 x 珋 ± s Groups Control 100 μg ml μg ml μg ml - 1 r Early apoptosis Q ± ± ± ± Late apoptosis Q ± ± ± ± P < P < Note 1 P < P < vs controls 6 Western blot p-akt p-erk1 /2 Bcl-2 Bax caspase-3 Fig. 6 Expression of p-akt p-erk1 /2 Bcl-2 Bax and caspase-3 protein detected by Western blot 3 B16F10 NRP1 VEGF Sema EGF PDGF FN 3 5 sirna NRP1 11 IgG 15 U87 U251 MCF7 NRP-1 mab NRP-1 b1b2 NRP1 mab B16F10 NRP1 mab B16F10 NRP1 mab Chin Pharm J 2014 October Vol. 49 No

6 NRP1 mab B16F10 Akt ERK1 /2 Bcl-2 Bax caspase-3 6 NRP-1 MAb NRP-1 NRP1 VEGFR TGFR PDGFR NRP1 6 BAGRI A TESSIER-LAVIGNE M WATTS R J. Neuropilins in. Clin Cancer Res GUTTMANN-RAVIV N KESSLER O SHRAGA-HELED N et al. The neuropilins and their role in tumorigenesis and tumor pro-. Cancer Lett BIELENBERG D R PETTAWAY C A TAKASHIMA S et al. Neuropilins in neoplasms Expression regulation and function. Exp Cell Res BAGCI T WU J K PFANNL R et al. Autocrine semaphorin 3A signaling promotes glioblastoma dispersal J. Oncogene CAO Y WANG L NANDY D et al. Neuropilin-1 upholds dedifferentiation and propagation phenotypes of renal cell carcinoma cells by activating Akt and sonic hedgehog axes J. Cancer Res MAZUREK A M OLBRYT M. The influence of neuropilin-1 silencing on semaphorin 3A and 3C activity in B16 F10 murine melanoma cells J. Neoplasma PEI X K CAI M ZHOU H M et al. Effect of Neuropilin-1 in. Mod Oncol PI3K MEK1 /2 Akt ERK1 / Akt ERK1 /2 tumor biology J gression J BAD Bax J Bcl-2 Bcl-2 Bax caspase NRP1 mab B16F10 PI3K /Akt MEK /ERK1 /2 Bcl-2 /Bax /caspase-3 proliferation of mice melanoma cells J NRP tumours J. Nature REFERENCES 1 L J J TAO X J. The progress on treatment of malignant melanoma J. China Cancer KLAGSBRUN M TAKASHIMA S MAMLUK R. The role of neuropilin in vascular and tumor biology J. Adv Exp Med Biol SOKER S MIAO H Q NOMI M et al. VEGF165 Mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding J. Cell Biochem BECKER P M WALTENBERGER J YACHECHKO R et al. Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability J. Circ Res KITSUKAWA T SHINIZU M SANBO M et al. Neuropilinsemaphorin III / D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice J. Neuron BECK B DRIESSENS G GOOSSENS S et al. A vascular niche and a VEGF-NRP1 loop regulate the initiation and stemness of skin 14 NI E R ZENG F W WANG S Y et al. Characterization and application of monoclonal antibodies against human NRP1 J. Immunol J LI X LUO F H WANG S Y et al. Monoclonal antibody against NRP1 b1b2 J. Hybridoma Chin Pharm J 2014 October Vol. 49 No