Possible contribution of aminopeptidase N (APN/CD13) to migration and invasion of human osteosarcoma cell lines

Liang,Wenqing; Gao,Bo; Xu,Guojian; Weng,Dong; Xie,Minghua; Qian,Yu

doi:10.3892/ijo.2014.2664

Possible contribution of aminopeptidase N (APN/CD13) to migration and invasion of human osteosarcoma cell lines

Authors:
- Wenqing Liang
- Bo Gao
- Guojian Xu
- Dong Weng
- Minghua Xie
- Yu Qian
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Affiliations: Department of Orthopedics, Shaoxing People's Hospital, Shaoxing, Zhejiang 312000, P.R. China, Department of Orthopedics, The 306th Hospital of PLA, Beijing 100101, P.R. China
Published online on: September 22, 2014 https://doi.org/10.3892/ijo.2014.2664
Pages: 2475-2485

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Abstract

Osteosarcoma is the most common primary malignancy of the bone. Aminopeptidase N (APN/CD13), a Zn+2‑dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion mechanism. This study explores the potential involvement of APN in migration and invasion of human osteosarcoma cells in vitro using inhibitors and activators of APN. Cells treated with APN inhibitor bestatin displayed decreased migration and invasion in a Boyden chamber Transwell assay. Western blotting revealed reduced levels of mitogen‑activated protein kinase (MAPK) and phosphatidylinositol 3‑kinase (PI3K) pathway proteins, reduced phosphorylation of p38, ERK1/2 and JNK and decreased levels of NF‑κB. Bestatin treatment also lowered APN, matrix metalloproteinase (MMP)‑2 and ‑9 enzymatic activity and their mRNA expression. Reduced MMP‑2 and ‑9 protein levels were also observed. By comparison, cells treated with cytokine interleukin‑6 (IL‑6), a stimulator of APN, displayed increased migration and invasion. Western blotting revealed increased levels of MAPK and PI3K pathway proteins, phosphorylated p38, ERK1/2 and JNK, and NF‑κB. IL‑6 treatment also increased APN and MMP‑2 and ‑9 enzymatic activity. An increase of APN, MMP‑2 and ‑9 mRNA levels, and MMP‑2 and ‑9 protein levels was also observed. Together these experiments reveal potential enzymatic and signalling roles for APN in osteosarcoma and establish a starting point for an in‑depth analysis of the role of APN in regulating invasiveness. A deeper knowledge about the regulatory mechanisms of APN may contribute to the development of anti‑metastatic therapies.

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1	Ottaviani G and Jaffe N: The epidemiology of osteosarcoma. Cancer Treat Res. 152:3–13. 2009. View Article : Google Scholar
2	Sung L, Anderson JR, Donaldson SS, Spunt SL, Crist WM and Pappo AS; Soft Tissue Sarcoma Committee of the Children’s Oncology Group. Late events occurring five years or more after successful therapy for childhood rhabdomyosarcoma: a report from the Soft Tissue Sarcoma Committee of the Children’s Oncology Group. Eur J Cancer. 40:1878–1885. 2004.
3	Cheng YY, Huang L, Lee KM, Li K and Kumta SM: Alendronate regulates cell invasion and MMP-2 secretion in human osteosarcoma cell lines. Pediatr Blood Cancer. 42:410–415. 2004. View Article : Google Scholar : PubMed/NCBI
4	Cho HJ, Lee TS, Park JB, et al: Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression. J Biochem Mol Biol. 40:1069–1076. 2007. View Article : Google Scholar : PubMed/NCBI
5	Xin ZF, Kim YK and Jung ST: Risedronate inhibits human osteosarcoma cell invasion. J Exp Clin Cancer Res. 28:1052009. View Article : Google Scholar : PubMed/NCBI
6	Fan DG, Dai JY, Tang J, et al: Silencing of calpain expression reduces the metastatic potential of human osteosarcoma cells. Cell Biol Int. 33:1263–1267. 2009. View Article : Google Scholar : PubMed/NCBI
7	Dass CR, Nadesapillai AP, Robin D, et al: Downregulation of uPAR confirms link in growth and metastasis of osteosarcoma. Clin Exp Metastasis. 22:643–652. 2005. View Article : Google Scholar : PubMed/NCBI
8	Wan X, Kim SY, Guenther LM, et al: Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin. Oncogene. 28:3401–3411. 2009. View Article : Google Scholar : PubMed/NCBI
9	Haydon RC, Deyrup A, Ishikawa A, et al: Cytoplasmic and/or nuclear accumulation of the beta-catenin protein is a frequent event in human osteosarcoma. Int J Cancer. 102:338–342. 2002. View Article : Google Scholar : PubMed/NCBI
10	Iwaya K, Ogawa H, Kuroda M, Izumi M, Ishida T and Mukai K: Cytoplasmic and/or nuclear staining of beta-catenin is associated with lung metastasis. Clin Exp Metastasis. 20:525–529. 2003. View Article : Google Scholar : PubMed/NCBI
11	Engin F, Bertin T, Ma O, et al: Notch signaling contributes to the pathogenesis of human osteosarcomas. Hum Mol Genet. 18:1464–1470. 2009. View Article : Google Scholar : PubMed/NCBI
12	Hughes DP: How the NOTCH pathway contributes to the ability of osteosarcoma cells to metastasize. Cancer Treat Res. 152:479–496. 2009. View Article : Google Scholar : PubMed/NCBI
13	Leow PC, Tian Q, Ong ZY, Yang Z and Ee PL: Antitumor activity of natural compounds, curcumin and PKF118-310, as Wnt/β-catenin antagonists against human osteosarcoma cells. Invest New Drugs. 28:766–782. 2010.PubMed/NCBI
14	Li Y, Zhang J, Ma D, et al: Curcumin inhibits proliferation and invasion of osteosarcoma cells through inactivation of Notch-1 signaling. FEBS J. 279:2247–2259. 2012. View Article : Google Scholar : PubMed/NCBI
15	Lin YM, Chang ZL, Liao YY, Chou MC and Tang CH: IL-6 promotes ICAM-1 expression and cell motility in human osteosarcoma. Cancer Lett. 328:135–143. 2013. View Article : Google Scholar : PubMed/NCBI
16	Berdiaki A, Datsis GA, Nikitovic D, et al: Parathyroid hormone (PTH) peptides through the regulation of hyaluronan metabolism affect osteosarcoma cell migration. IUBMB Life. 62:377–386. 2010.PubMed/NCBI
17	Yang R, Hoang BH, Kubo T, et al: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma. Int J Cancer. 121:943–954. 2007. View Article : Google Scholar : PubMed/NCBI
18	Dobashi Y, Watanabe H, Matsubara M, et al: Autocrine motility factor/glucose-6-phosphate isomerase is a possible predictor of metastasis in bone and soft tissue tumours. J Pathol. 208:44–53. 2006. View Article : Google Scholar : PubMed/NCBI
19	Niinaka Y, Harada K, Fujimuro M, et al: Silencing of autocrine motility factor induces mesenchymal-to-epithelial transition and suppression of osteosarcoma pulmonary metastasis. Cancer Res. 70:9483–9493. 2010. View Article : Google Scholar : PubMed/NCBI
20	Fromigue O, Hamidouche Z, Vaudin P, et al: CYR61 downregulation reduces osteosarcoma cell invasion, migration, and metastasis. J Bone Miner Res. 26:1533–1542. 2011. View Article : Google Scholar : PubMed/NCBI
21	Bauvois B: Transmembrane proteases in cell growth and invasion: new contributors to angiogenesis? Oncogene. 23:317–329. 2004. View Article : Google Scholar : PubMed/NCBI
22	Carl-McGrath S, Lendeckel U, Ebert M and Röcken C: Ectopeptidases in tumour biology: a review. Histol Histopathol. 21:1339–1353. 2006.
23	Hitzerd SM, Verbrugge SE, Ossenkoppele G, Jansen G and Peters GJ: Positioning of aminopeptidase inhibitors in next generation cancer therapy. Amino Acids. 46:793–808. 2014. View Article : Google Scholar : PubMed/NCBI
24	Kido A, Krueger S, Haeckel C and Roessner A: Possible contribution of aminopeptidase N (APN/CD13) to invasive potential enhanced by interleukin-6 and soluble interleukin-6 receptor in human osteosarcoma cell lines. Clin Exp Metastasis. 17:857–863. 1999. View Article : Google Scholar : PubMed/NCBI
25	Fujii H, Nakajima M, Saiki I, Yoneda J, Azuma I and Tsuruo T: Human melanoma invasion and metastasis enhancement by high expression of aminopeptidase N/CD13. Clin Exp Metastasis. 13:337–344. 1995. View Article : Google Scholar : PubMed/NCBI
26	Kehlen A, Lendeckel U, Dralle H, Langner J and Hoang-Vu C: Biological significance of aminopeptidase N/CD13 in thyroid carcinomas. Cancer Res. 63:8500–8506. 2003.PubMed/NCBI
27	Petrovic N, Schacke W, Gahagan JR, et al: CD13/APN regulates endothelial invasion and filopodia formation. Blood. 110:142–150. 2007. View Article : Google Scholar : PubMed/NCBI
28	Hong DY, Lee BJ, Lee JC, Choi JS, Wang SG and Ro JH: Expression of VEGF, HGF, IL-6, IL-8, MMP-9, telomerase in peripheral blood of patients with head and neck squamous cell carcinoma. Clin Exp Otorhinolaryngol. 2:186–192. 2009. View Article : Google Scholar : PubMed/NCBI
29	Loo WT, Sasano H and Chow LW: Pro-inflammatory cytokine, matrix metalloproteinases and TIMP-1 are involved in wound healing after mastectomy in invasive breast cancer patients. Biomed Pharmacother. 61:548–552. 2007. View Article : Google Scholar : PubMed/NCBI
30	Güllü IH, Kurdoğlu M and Akalin I: The relation of gelatinase (MMP-2 and -9) expression with distant site metastasis and tumour aggressiveness in colorectal cancer. Br J Cancer. 82:2492000.PubMed/NCBI
31	Mizutani K, Kofuji K and Shirouzu K: The significance of MMP-1 and MMP-2 in peritoneal disseminated metastasis of gastric cancer. Surg Today. 30:614–621. 2000. View Article : Google Scholar : PubMed/NCBI
32	Korpi JT, Hagström J, Lehtonen N, et al: Expression of matrix metalloproteinases-2, −8, −13, −26, and tissue inhibitors of metalloproteinase-1 in human osteosarcoma. Surg Oncol. 20:e18–e22. 2011.
33	Ferrari C, Benassi S, Ponticelli F, et al: Role of MMP-9 and its tissue inhibitor TIMP-1 in human osteosarcoma: findings in 42 patients followed for 1–16 years. Acta Orthop Scand. 75:487–491. 2004.PubMed/NCBI
34	Foukas AF, Deshmukh NS, Grimer RJ, Mangham DC, Mangos EG and Taylor S: Stage-IIB osteosarcomas around the knee. A study of MMP-9 in surviving tumour cells. J Bone Joint Surg Br. 84:706–711. 2002. View Article : Google Scholar : PubMed/NCBI
35	Himelstein BP, Asada N, Carlton MR and Collins MH: Matrix metalloproteinase-9 (MMP-9) expression in childhood osseous osteosarcoma. Med Pediatr Oncol. 31:471–474. 1998. View Article : Google Scholar : PubMed/NCBI
36	Bellido T, O’Brien CA, Roberson PK and Manolagas SC: Transcriptional activation of the p21(WAF1,CIP1,SDI1) gene by interleukin-6 type cytokines. A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. J Biol Chem. 273:21137–21144. 1998. View Article : Google Scholar : PubMed/NCBI
37	Franchimont N, Gangji V, Durant D and Canalis E: Interleukin-6 with its soluble receptor enhances the expression of insulin-like growth factor-I in osteoblasts. Endocrinology. 138:5248–5255. 1997.PubMed/NCBI
38	Jilka RL, Weinstein RS, Bellido T, Parfitt AM and Manolagas SC: Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res. 13:793–802. 1998. View Article : Google Scholar : PubMed/NCBI
39	Nishimura R, Moriyama K, Yasukawa K, Mundy GR and Yoneda T: Combination of interleukin-6 and soluble interleukin-6 receptors induces differentiation and activation of JAK-STAT and MAP kinase pathways in MG-63 human osteoblastic cells. J Bone Miner Res. 13:777–785. 1998. View Article : Google Scholar : PubMed/NCBI
40	Kehlen A, Göhring B, Langner J and Riemann D: Regulation of the expression of aminopeptidase A, aminopeptidase N/CD13 and dipeptidylpeptidase IV/CD26 in renal carcinoma cells and renal tubular epithelial cells by cytokines and cAMP-increasing mediators. Clin Exp Immunol. 111:435–441. 1998. View Article : Google Scholar
41	Chen YY, Chiang SY, Lin JG, et al: Emodin, aloe-emodin and rhein inhibit migration and invasion in human tongue cancer SCC-4 cells through the inhibition of gene expression of matrix metalloproteinase-9. Int J Oncol. 36:1113–1120. 2010.PubMed/NCBI
42	Lin CC, Chen JT, Yang JS, et al: Danthron inhibits the migration and invasion of human brain glioblastoma multiforme cells through the inhibition of mRNA expression of focal adhesion kinase, Rho kinases-1 and metalloproteinase-9. Oncol Rep. 22:1033–1037. 2009.
43	Lai KC, Huang AC, Hsu SC, et al: Benzyl isothiocyanate (BITC) inhibits migration and invasion of human colon cancer HT29 cells by inhibiting matrix metalloproteinase-2/-9 and urokinase plasminogen (uPA) through PKC and MAPK signaling pathway. J Agric Food Chem. 58:2935–2942. 2010. View Article : Google Scholar
44	Liu KC, Huang AC, Wu PP, et al: Gallic acid suppresses the migration and invasion of PC-3 human prostate cancer cells via inhibition of matrix metalloproteinase-2 and -9 signaling pathways. Oncol Rep. 26:177–184. 2011.PubMed/NCBI
45	Chiang JH, Yang JS, Ma CY, et al: Danthron, an anthraquinone derivative, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways. Chem Res Toxicol. 24:20–29. 2011. View Article : Google Scholar
46	Wen YF, Yang JS, Kuo SC, et al: Investigation of anti-leukemia molecular mechanism of ITR-284, a carboxamide analog, in leukemia cells and its effects in WEHI-3 leukemia mice. Biochem Pharmacol. 79:389–398. 2010. View Article : Google Scholar : PubMed/NCBI
47	Hennessy BT, Smith DL, Ram PT, Lu Y and Mills GB: Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov. 4:988–1004. 2005. View Article : Google Scholar : PubMed/NCBI
48	Qi M and Elion EA: MAP kinase pathways. J Cell Sci. 118:3569–3572. 2005. View Article : Google Scholar
49	Bond M, Chase AJ, Baker AH and Newby AC: Inhibition of transcription factor NF-kappaB reduces matrix metalloproteinase-1, -3 and -9 production by vascular smooth muscle cells. Cardiovasc Res. 50:556–565. 2001. View Article : Google Scholar : PubMed/NCBI
50	Bauvois B and Dauzonne D: Aminopeptidase-N/CD13 (EC 3.4.11.2) inhibitors: chemistry, biological evaluations, and therapeutic prospects. Med Res Rev. 26:88–130. 2006. View Article : Google Scholar : PubMed/NCBI
51	Balkwill F, Charles KA and Mantovani A: Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 7:211–217. 2005. View Article : Google Scholar : PubMed/NCBI
52	Lo CW, Chen MW, Hsiao M, et al: IL-6 trans-signaling in formation and progression of malignant ascites in ovarian cancer. Cancer Res. 71:424–434. 2011. View Article : Google Scholar : PubMed/NCBI
53	Tang CH, Chen CF, Chen WM and Fong YC: IL-6 increases MMP-13 expression and motility in human chondrosarcoma cells. J Biol Chem. 286:11056–11066. 2011. View Article : Google Scholar : PubMed/NCBI
54	Tu B, Du L, Fan QM, Tang Z and Tang TT: STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma. Cancer Lett. 325:80–88. 2012. View Article : Google Scholar : PubMed/NCBI
55	Mina-Osorio P: The moonlighting enzyme CD13: old and new functions to target. Trends Mol Med. 14:361–371. 2008. View Article : Google Scholar : PubMed/NCBI
56	Liao CL, Lai KC, Huang AC, et al: Gallic acid inhibits migration and invasion in human osteosarcoma U-2 OS cells through suppressing the matrix metalloproteinase-2/-9, protein kinase B (PKB) and PKC signaling pathways. Food Chem Toxicol. 50:1734–1740. 2012. View Article : Google Scholar : PubMed/NCBI