FK866对非小细胞肺癌细胞迁移的影响

doi:10.3785/j.issn.1008-9292.2018.02.01

浙江大学学报（医学版）

2018, Vol. 47

Issue (1): 1-9 DOI: 10.3785/j.issn.1008-9292.2018.02.01

专题报道

FK866对非小细胞肺癌细胞迁移的影响

谢娴1(

),徐小方2,3,王琪2,卢韵碧1,吴明2,张纬萍1,*(

)

1. 浙江大学医学院药理学系, 浙江杭州 310058
2. 浙江大学医学院附属第二医院胸外科, 浙江杭州 310009
3. 浙江省肿瘤医院胸外科, 浙江杭州 310022

Effects of FK866 on migration of A549 cells and related mechanism

XIE Xian1(

),XU Xiaofang2,3,WANG Qi2,LU Yunbi1,WU Ming2,ZHANG Weiping1,*(

)

1. Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
2. Department of Thoracic Surgery, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
3. Department of Thoracic Surgery, Zhejiang Cancer Hospital, Hangzhou 310022, China

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摘要：

目的: 探讨低浓度烟酰胺磷酸核糖基转移酶（NAMPT）抑制剂FK866对人非小细胞肺癌细胞株（A549细胞）迁移的影响及作用机制。方法: MTT法检测不同浓度FK866对A549细胞增殖的影响；划痕实验检测1.0 nmol/L和10.0 nmol/L FK866对A549细胞迁移的影响；实时定量RT-PCR检测上皮间质转化相关蛋白上皮钙黏素和波形蛋白mRNA的表达量；蛋白质印迹法检测细胞外调节蛋白激酶1/2（ERK1/2）和磷酸化ERK1/2蛋白的表达量。结果: FK866作用时间越长、浓度越高，对A549细胞增殖的抑制作用越明显。FK866作用72 h时的半抑制浓度（IC₅₀）为9.55 nmol/L。以1.0、10.0 nmol/L的FK866预处理细胞48 h，划痕后继续给药48 h，两种浓度的FK866均能抑制A549细胞迁移；如不进行预处理，仅10.0 nmol/L浓度的FK866对划痕愈合有一定的抑制作用；1.0 nmol/L的FK866处理细胞72 h可上调上皮钙黏素和波形蛋白mRNA表达，并激活ERK1/2；以1.0 mmol/L的烟酰胺单核苷酸（NMN）或10.0 μmol/L的ERK1/2抑制剂U0126预处理，能够逆转FK866引起的上皮钙黏素和波形蛋白表达上调。结论: 低浓度FK866能够通过减少细胞内烟酰胺腺嘌呤二核苷酸和激活ERK1/2，增加上皮钙黏素表达，进而抑制细胞迁移，对肿瘤转移可能有一定的抑制作用；但其同时促进了波形蛋白的表达，不利于肿瘤的治疗。

关键词： 烟酰胺磷酸核糖基转移酶/拮抗剂和抑制剂; 烟酰胺磷酸核糖基转移酶/药理学; 癌, 非小细胞肺/病理学; 钙黏着糖蛋白类/代谢; 波形蛋白/代谢; 细胞运动; 肿瘤细胞, 培养的/药物作用

Abstract:

Objective: To investigate the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 on the migration of human non-small cell cancer A549 cells and related mechanism. Methods: The inhibition effect of FK866 on A549 cells was tested by MTT assay. A549 cells were treated with 1.0 and 10.0 nmol/L FK866, and the cell migration was evaluated by modified wound scratch assay. The mRNA expression of E-cadherin and vimentin was detected by real-time RT-PCR, and the expression of ERK1/2 and pERK1/2 was determined by Western blotting. Results: FK866 inhibited the proliferation of A549 cells in a time- and concentration-dependent manner; after treatment for 72 h, the IC₅₀ of FK866 was 9.55 nmol/L. When 1.0 nmol/L or 10.0 nmol/L FK866 was continuously applied 48 h before and 48 h after a scratch was made in wound scratch assay, the migration of A549 cells was significantly inhibited. However, when the FK866 was applied only 48 h after the scratch, the migration of A549 cells was inhibited by 10.0 nmol/L but not by 1.0 nmol/L FK866. The mRNA expression of E-cadherin and vimentin, and the activated ERK1/2 were significantly increased after 1.0 nmol/L FK866 treatment for 72 h. The pretreatment with nicotinamide adenine dinucleotide (NAD) precursor nicotinamide mononucleotide(1.0 mmol/L) or ERK1/2 inhibitor U0126 (10.0 μmol/L) reversed the up-regulation of E-cadherin and vimentin expression induced by FK866. Conclusions: Low concentration of FK866 decreases the migration of A549 cells through the inhibition of NAD level, activation of ERK1/2 and up-regulation of E-cadherin expression. However, it also up-regulates the expression of vimentin, indicating that it may have dual effects on the migration of tumor cells.

Key words: Nicotinamide phosphoribosyltransferase/antagonists & inhibitors Nicotinamide phosphoribosyltransferase/pharmacology Carcinoma, non-small-cell lung/pathology Cadherins/metabolism Vimentin/metabolism Cell movement Tumor cells, cultured/drug effects

收稿日期: 2017-12-22 出版日期: 2018-06-12

CLC:

R961

基金资助: 浙江省自然科学基金(LY16H010004, LY18H170001, LQ17H010002);国家自然科学基金(81573400);国家科技支撑计划(2015BAI13B02);浙江省公共技术应用研究(2016F82G2010036)

通讯作者: 张纬萍 E-mail: 21518401@zju.edu.cn;weiping601@zju.edu.cn

作者简介: 谢娴(1992-), 女, 硕士研究生, 主要从事老年性疾病研究; E-mail:21518401@zju.edu.cn; https://orcid.org/0000-0003-1642-1734

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引用本文:

谢娴,徐小方,王琪,卢韵碧,吴明,张纬萍. FK866对非小细胞肺癌细胞迁移的影响[J]. 浙江大学学报（医学版）, 2018, 47(1): 1-9.

XIE Xian,XU Xiaofang,WANG Qi,LU Yunbi,WU Ming,ZHANG Weiping. Effects of FK866 on migration of A549 cells and related mechanism. J Zhejiang Univ (Med Sci), 2018, 47(1): 1-9.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.02.01 或 http://www.zjujournals.com/med/CN/Y2018/V47/I1/1

表 1 实时定量RT-PCR引物序列

图 1 不同浓度FK866作用24、48和72 h对细胞增殖的抑制效果比较

图 2 各组细胞划痕实验结果

图 3 各组细胞迁移率比较

图 4 FK866对细胞中上皮钙黏素和波形蛋白mRNA表达的影响

图 5 NMN抑制FK866引起的上皮钙黏素和波形蛋白mRNA表达上调

图 6 FK866上调细胞中ERK1/2磷酸化水平

图 7 U0126抑制FK866引起的上皮钙黏素和波形蛋白mRNA表达上调

1	RONGVAUX A , SHEA R J , MULKS M H et al. Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis[J]. Eur J Immunol, 2002, 32 (11): 3225- 3234 doi: 10.1002/1521-4141(200211)32:11<3225::AID-IMMU3225>3.0.CO;2-L
2	VAN BEIJNUM J R , MOERKERK P T , GERBERS A J et al. Target validation for genomics using peptide-specific phage antibodies:a study of five gene products overexpressed in colorectal cancer[J]. Int J Cancer, 2002, 101 (2): 118- 127 doi: 10.1002/ijc.v101:2
3	CHIARUGI A , D?LLE C , FELICI R et al. The NAD metabolome-a key determinant of cancer cell biology[J]. Nat Rev Cancer, 2012, 12 (11): 741- 752 doi: 10.1038/nrc3340
4	XIAO Y , ELKINS K , DURIEUX J K et al. Dependence of tumor cell lines and patient-derived tumors on the NAD salvage pathway renders them sensitive to NAMPT inhibition with GNE-618[J]. Neoplasia, 2013, 15 (10): 1151- 1160 doi: 10.1593/neo.131304
5	WILSON C , NICHOLES K , BUSTOS D et al. Overcoming EMT-associated resistance to anti-cancer drugs via Src/FAK pathway inhibition[J]. Oncotarget, 2014, 5 (17): 7328- 7341
6	ESPINDOLA-NETTO J M , CCS C , TARRAGó M et al. Preclinical efficacy of the novel competitive NAMPT inhibitor STF-118804 in pancreatic cancer[J]. Oncotarget, 2017, 8 (49): 85054- 85067
7	FOLGUEIRA M A , CARRARO D M , BRENTANI H et al. Gene expression profile associated with response to doxorubicin-based therapy in breast cancer[J]. Clin Cancer Res, 2005, 11 (20): 7434- 7443 doi: 10.1158/1078-0432.CCR-04-0548
8	SHACKELFORD R E , MAYHALL K , MAXWELL N M et al. Nicotinamide phosphoribosyltransferase in malignancy:a review[J]. Genes Cancer, 2013, 4 (11-12): 447- 456 doi: 10.1177/1947601913507576
9	CHEN H , WANG S , ZHANG H et al. Nicotinamide phosphoribosyltransferase (NAMPT) in carcinogenesis:new clinical opportunities[J]. Expert Rev Anticancer Ther, 2016, 16 (8): 827- 838 doi: 10.1080/14737140.2016.1190649
10	FRANZEN C A , BLACKWELL R H , TODOROVIC V et al. Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes[J]. Oncogenesis, 2015, 4 e163 doi: 10.1038/oncsis.2015.21
11	MIN H , SUN X , YANG X et al. Exosomes derived from irradiated esophageal carcinoma-infiltrating T cells promote metastasis by inducing the epithelial-mesenchymal transition in esophageal cancer cells[J]. Pathol Oncol Res, 2018, 24 (1): 11- 18 doi: 10.1007/s12253-016-0185-z
12	SUNDARAM M V. RTK/Ras/MAPK signaling[M/OL]. (2018-01-02)[2016-02-11]. http://www.wormbook.org/chapters/www_RTKRasMAPKsignal-ing/RTKRasMAPKsignaling.html.
13	UZGARE A R , KAPLAN P J , GREENBERG N M . Differential expression and/or activation of P38MAPK, erk1/2, and jnk during the initiation and progression of prostate cancer[J]. Prostate, 2003, 55 (2): 128- 139 doi: 10.1002/(ISSN)1097-0045
14	BRANCA M , CIOTTI M , SANTINI D et al. Activation of the ERK/MAP kinase pathway in cervical intraepithelial neoplasia is related to grade of the lesion but not to high-risk human papillomavirus, virus clearance, or prognosis in cervical cancer[J]. Am J Clin Pathol, 2004, 122 (6): 902- 911 doi: 10.1309/VQXFT880JXC7QD2W
15	CHEN X , YE S , XIAO W et al. ERK1/2 pathway mediates epithelial-mesenchymal transition by cross-interacting with TGFβ/Smad and Jagged/Notch signaling pathways in lens epithelial cells[J]. Int J Mol Med, 2014, 33 (6): 1664- 1670 doi: 10.3892/ijmm.2014.1723
16	GALLI U , TRAVELLI C , MASSAROTTI A et al. Medicinal chemistry of nicotinamide phosphoribosyl-transferase (NAMPT) inhibitors[J]. J Med Chem, 2013, 56 (16): 6279- 6296 doi: 10.1021/jm4001049
17	HOLEN K , SALTZ L B , HOLLYWOOD E et al. The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor[J]. Invest New Drugs, 2008, 26 (1): 45- 51 doi: 10.1007/s10637-007-9083-2
18	TRAVELLI C , DRAGO V , MALDI E et al. Reciprocal potentiation of the antitumoral activities of FK866, an inhibitor of nicotinamide phosphoribosyl-transferase, and etoposide or cisplatin in neuroblastoma cells[J]. J Pharmacol Exp Ther, 2011, 338 (3): 829- 840 doi: 10.1124/jpet.111.184630
19	VON H A , BERGLUND A , LARSSON R et al. Safety and efficacy of NAD depleting cancer drugs:results of a phase Ⅰ clinical trial of CHS 828 and overview of published data[J]. Cancer Chemother Pharmacol, 2010, 65 (6): 1165- 1172 doi: 10.1007/s00280-009-1125-3
20	YUAN Y M , FANG S H , QIAN X D et al. Leukotriene D4 stimulates the migration but not proliferation of endothelial cells mediated by the cysteinyl leukotriene cyslt(1) receptor via the extracellular signal-regulated kinase pathway[J]. J Pharmacol Sci, 2009, 109 (2): 285- 292 doi: 10.1254/jphs.08321FP
21	ZHANG L Y , LIU L Y , QIE L L et al. Anti-proliferation effect of APO866 on C6 glioblastoma cells by inhibiting nicotinamide phosphoribosyltransfe-rase[J]. Eur J Pharmacol, 2012, 674 (2-3): 163- 170 doi: 10.1016/j.ejphar.2011.11.017
22	ALAEE M , KHAGHANI S , BEHROOZFAR K et al. Inhibition of nicotinamide phosphoribosyltransf-erase induces apoptosis in estrogen receptor-positive MCF-7 breast cancer cells[J]. J Breast Cancer, 2017, 20 (1): 20- 26 doi: 10.4048/jbc.2017.20.1.20
23	YANG P , ZHANG L , SHI Q J et al. Nicotinamide phosphoribosyltransferase inhibitor APO866 induces C6 glioblastoma cell death via autophagy[J]. Pharmazie, 2015, 70 (10): 650- 655
24	LEIBOVICH-RIVKIN T , LIUBOMIRSKI Y , BERNSTEIN B et al. Inflammatory factors of the tumor microenvironment induce plasticity in nontransformed breast epithelial cells:EMT, invasion, and collapse of normally organized breast textures[J]. Neoplasia, 2013, 15 (12): 1330- 1346 doi: 10.1593/neo.131688

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