A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro

Zhao,Li-Ping; Xu,Tian-Min; Kan,Mu-Jie; Xiao,Ye-Chen; Cui,Man-Hua

doi:10.3892/ijmm.2016.2540

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro

Authors:
- Li-Ping Zhao
- Tian-Min Xu
- Mu-Jie Kan
- Ye-Chen Xiao
- Man-Hua Cui
View Affiliations

Affiliations: Department of Gynecology and Obstetrics, The Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China, Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Jilin University, Changchun, Jilin 130021, P.R. China
Published online on: March 24, 2016 https://doi.org/10.3892/ijmm.2016.2540
Pages: 1310-1316
Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

View Figures

View References

1	Stewart BW and Kleihues P: World Cancer Report. IARC press; Lyon: 2003
2	Mao Y, Xu J, Li Z, Zhang N, Yin H and Liu Z: The role of nuclear β-catenin accumulation in the Twist2-induced ovarian cancer EMT. PLoS One. 8:e782002013. View Article : Google Scholar
3	Fazioli F and Blasi F: Urokinase-type plasminogen activator and its receptor: new targets for anti-metastatic therapy? Trends Pharmacol Sci. 15:25–29. 1994. View Article : Google Scholar : PubMed/NCBI
4	Mekkawy AH, Pourgholami MH and Morris DL: Involvement of urokinase-type plasminogen activator system in cancer: an overview. Med Res Rev. 34:918–956. 2014. View Article : Google Scholar : PubMed/NCBI
5	Andreasen PA, Kjøller L, Christensen L and Duffy MJ: The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer. 72:1–22. 1997. View Article : Google Scholar : PubMed/NCBI
6	Kwaan HC: The plasminogen-plasmin system in malignancy. Cancer Metastasis Rev. 11:291–311. 1992. View Article : Google Scholar : PubMed/NCBI
7	Berkenblit A, Matulonis UA, Kroener JF, Dezube BJ, Lam GN, Cuasay LC, Brünner N, Jones TR, Silverman MH and Gold MA: A6, a urokinase plasminogen activator (uPA)-derived peptide in patients with advanced gynecologic cancer: a phase I trial. Gynecol Oncol. 99:50–57. 2005. View Article : Google Scholar : PubMed/NCBI
8	Ghamande SA, Silverman MH, Huh W, Behbakht K, Ball G, Cuasay L, Würtz SO, Brunner N and Gold MA: A phase 2, randomized, double-blind, placebo-controlled trial of clinical activity and safety of subcutaneous A6 in women with asymptomatic CA125 progression after first-line chemotherapy of epithelial ovarian cancer. Gynecol Oncol. 111:89–94. 2008. View Article : Google Scholar : PubMed/NCBI
9	Gold MA, Brady WE, Lankes HA, Rose PG, Kelley JL, De Geest K, Crispens MA, Resnick KE and Howell SB: A phase II study of a urokinase-derived peptide (A6) in the treatment of persistent or recurrent epithelial ovarian, fallopian tube, or primary peritoneal carcinoma: a Gynecologic Oncology Group study. Gynecol Oncol. 125:635–639. 2012. View Article : Google Scholar : PubMed/NCBI
10	Heinemann V, Ebert MP, Laubender RP, Bevan P, Mala C and Boeck S: Phase II randomised proof-of-concept study of the urokinase inhibitor upamostat (WX-671) in combination with gemcitabine compared with gemcitabine alone in patients with non-resectable, locally advanced pancreatic cancer. Br J Cancer. 108:766–770. 2013. View Article : Google Scholar : PubMed/NCBI
11	Chen H, Hao J, Wang L and Li Y: Coexpression of invasive markers (uPA, CD44) and multiple drug-resistance proteins (MDR1, MRP2) is correlated with epithelial ovarian cancer progression. Br J Cancer. 101:432–440. 2009. View Article : Google Scholar : PubMed/NCBI
12	Wang L, Madigan MC, Chen H, Liu F, Patterson KI, Beretov J, O'Brien PM and Li Y: Expression of urokinase plasminogen activator and its receptor in advanced epithelial ovarian cancer patients. Gynecol Oncol. 114:265–272. 2009. View Article : Google Scholar : PubMed/NCBI
13	Dorn J, Harbeck N, Kates R, Gkazepis A, Scorilas A, Soos-aipillai A, Diamandis E, Kiechle M, Schmalfeldt B and Schmitt M: Impact of expression differences of kallikrein-related peptidases and of uPA and PAI-1 between primary tumor and omentum metastasis in advanced ovarian cancer. Ann Oncol. 22:877–883. 2011. View Article : Google Scholar
14	Salameh MA, Robinson JL, Navaneetham D, Sinha D, Madden BJ, Walsh PN and Radisky ES: The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin. J Biol Chem. 285:1939–1949. 2010. View Article : Google Scholar :
15	Zhao L and Yan W: Expression and identification of recombinant humanuPA17-KPI in Pichia pastoris. Chin J Lab Diagn. 14:1685–1688. 2010.
16	Lai KC, Huang AC, Hsu SC, Kuo CL, Yang JS, Wu SH and Chung JG: 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 : PubMed/NCBI
17	Lin Y, Cui M, Xu T, Yu W and Zhang L: Silencing of cyclooxy-genase-2 inhibits the growth, invasion and migration of ovarian cancer cells. Mol Med Rep. 9:2499–2504. 2014.PubMed/NCBI
18	Vergara D, Merlot B, Lucot JP, Collinet P, Vinatier D, Fournier I and Salzet M: Epithelial-mesenchymal transition in ovarian cancer. Cancer Lett. 291:59–66. 2010. View Article : Google Scholar
19	Evans DM and Lin PL: Suppression of pulmonary metastases of rat mammary cancer by recombinant urokinase plasminogen activator inhibitor. Am Surg. 61:692–696; discussion 696–697. 1995.PubMed/NCBI
20	Praus M, Wauterickx K, Collen D and Gerard RD: Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors. Gene Ther. 6:227–236. 1999. View Article : Google Scholar : PubMed/NCBI
21	Guo Y, Higazi AA, Arakelian A, Sachais BS, Cines D, Goldfarb RH, Jones TR, Kwaan H, Mazar AP and Rabbani SA: A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo. FASEB J. 14:1400–1410. 2000. View Article : Google Scholar : PubMed/NCBI
22	Guo Y, Mazar AP, Lebrun JJ and Rabbani SA: An antiangiogenic urokinase-derived peptide combined with tamoxifen decreases tumor growth and metastasis in a syngeneic model of breast cancer. Cancer Res. 62:4678–4684. 2002.PubMed/NCBI
23	Ploug M, Østergaard S, Gårdsvoll H, Kovalski K, Holst-Hansen C, Holm A, Ossowski L and Danø K: Peptide-derived antagonists of the urokinase receptor. Affinity maturation by combinatorial chemistry, identification of functional epitopes, and inhibitory effect on cancer cell intravasation. Biochemistry. 40:12157–12168. 2001. View Article : Google Scholar : PubMed/NCBI
24	Zaravinos A: The regulatory role of MicroRNAs in EMT and cancer. J Oncol. 2015(865816)2015. View Article : Google Scholar : PubMed/NCBI
25	Chandrasekar N, Mohanam S, Gujrati M, Olivero WC, Dinh DH and Rao JS: Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells. Oncogene. 22:392–400. 2003. View Article : Google Scholar : PubMed/NCBI
26	Wang Q, Wang Y, Zhang Y, Zhang Y and Xiao W: The role of uPAR in epithelial-mesenchymal transition in small airway epithelium of patients with chronic obstructive pulmonary disease. Respir Res. 14(67)2013. View Article : Google Scholar
27	Jankun J and Skrzypczak-Jankun E: Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride. Cancer Biochem Biophys. 17:109–123. 1999.
28	Matthews H, Ranson M and Kelso MJ: Anti-tumour/metastasis effects of the potassium-sparing diuretic amiloride: an orally active anti-cancer drug waiting for its call-of-duty? Int J Cancer. 129:2051–2061. 2011. View Article : Google Scholar : PubMed/NCBI
29	Lester RD, Jo M, Montel V, Takimoto S and Gonias SL: uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells. J Cell Biol. 178:425–436. 2007. View Article : Google Scholar : PubMed/NCBI
30	Liu SQ, Huang JA, Qin MB, Su YJ, Lai MY, Jiang HX and Tang GD: Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways. Int J Colorectal Dis. 27:1569–1578. 2012. View Article : Google Scholar : PubMed/NCBI
31	Chou RH, Hsieh SC, Yu YL, Huang MH, Huang YC and Hsieh YH: Fisetin inhibits migration and invasion of human cervical cancer cells by down-regulating urokinase plasminogen activator expression through suppressing the p38 MAPK-dependent NF-κB signaling pathway. PLoS One. 8:e719832013. View Article : Google Scholar
32	Jo M, Lester RD, Montel V, Eastman B, Takimoto S and Gonias SL: Reversibility of epithelial-mesenchymal transition (EMT) induced in breast cancer cells by activation of urokinase receptor-dependent cell signaling. J Biol Chem. 284:22825–22833. 2009. View Article : Google Scholar : PubMed/NCBI
33	Blasi F and Sidenius N: The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling. FEBS Lett. 584:1923–1930. 2010. View Article : Google Scholar
34	Eastman BM, Jo M, Webb DL, Takimoto S and Gonias SL: A transformation in the mechanism by which the urokinase receptor signals provides a selection advantage for estrogen receptor-expressing breast cancer cells in the absence of estrogen. Cell Signal. 24:1847–1855. 2012. View Article : Google Scholar : PubMed/NCBI
35	Nowicki TS, Zhao H, Darzynkiewicz Z, Moscatello A, Shin E, Schantz S, Tiwari RK and Geliebter J: Downregulation of uPAR inhibits migration, invasion, proliferation, FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells. Cell Cycle. 10:100–107. 2011. View Article : Google Scholar :
36	Gogineni VR, Gupta R, Nalla AK, Velpula KK and Rao JS: uPAR and cathepsin B shRNA impedes TGF-β1-driven proliferation and invasion of meningioma cells in a XIAP-dependent pathway. Cell Death Dis. 3:e4392012. View Article : Google Scholar
37	Shetty S, Rao GN, Cines DB and Bdeir K: Urokinase induces activation of STAT3 in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol. 291:L772–L780. 2006. View Article : Google Scholar : PubMed/NCBI
38	Stewart AG, Xia YC, Harris T, Royce S, Hamilton JA and Schuliga M: Plasminogen-stimulated airway smooth muscle cell proliferation is mediated by urokinase and annexin A2, involving plasmin-activated cell signalling. Br J Pharmacol. 170:1421–1435. 2013. View Article : Google Scholar : PubMed/NCBI
39	Ulisse S, Baldini E, Sorrenti S and D'Armiento M: The urokinase plasminogen activator system: a target for anti-cancer therapy. Curr Cancer Drug Targets. 9:32–71. 2009. View Article : Google Scholar : PubMed/NCBI
40	Santarpia L, Lippman SM and El-Naggar AK: Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin Ther Targets. 16:103–119. 2012. View Article : Google Scholar : PubMed/NCBI
41	Romano G: The role of the dysfunctional akt-related pathway in cancer: establishment and maintenance of a malignant cell phenotype, resistance to therapy, and future strategies for drug development. Scientifica (Cairo). 2013:3171862013.