Heparin regulates B6FS cell motility through a FAK/actin cytoskeleton axis

Voudouri,Kallirroi; Nikitovic,Dragana; Berdiaki,Aikaterini; Papachristou,Dionysios J.; Tsiaoussis,John; Spandidos,Demetrios A.; Tsatsakis,Aristides M.; Tzanakakis,George N.

doi:10.3892/or.2016.5057

Heparin regulates B6FS cell motility through a FAK/actin cytoskeleton axis

Authors:
- Kallirroi Voudouri
- Dragana Nikitovic
- Aikaterini Berdiaki
- Dionysios J. Papachristou
- John Tsiaoussis
- Demetrios A. Spandidos
- Aristides M. Tsatsakis
- George N. Tzanakakis
View Affiliations

Affiliations: Laboratory of Anatomy‑Histology‑Embryology, School of Medicine, University of Crete, Heraklion 71003, Greece, Laboratory of Anatomy‑Histology‑Embryology, School of Medicine, University of Patras, Patras 23001, Greece, Laboratory of Virology, School of Medicine, University of Crete, Heraklion 71003, Greece, Laboratory of Toxicology, School of Medicine, University of Crete, Heraklion 71003, Greece
Published online on: August 30, 2016 https://doi.org/10.3892/or.2016.5057
Pages: 2471-2480
Copyright: © Voudouri 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

Soft tissue sarcomas are rare, heterogeneous tumors of mesenchymal origin with an aggressive behavior. Heparin is a mixture of heavily sulfated, linear glycosaminoglycan (GAG) chains, which participate in the regulation of various cell biological functions. Heparin is considered to have significant anticancer capabilities, although the mechanisms involved have not been fully defined. In the present study, the effects of unfractionated heparin (UFH) and low‑molecular‑weight heparin (LMWH) on B6FS fibrosarcoma cell motility were examined. Both preparations of heparin were shown to both enhance B6FS cell adhesion (p<0.01 and p<0.05), and migration (p<0.05), the maximal effect being evident at the concentration of 10 µg/ml. The utilization of FAK‑deficient cells demonstrated that the participation of FAK was obligatory for heparin‑dependent fibrosarcoma cell adhesion (p<0.05). The results of confocal microscopy indicated that heparin was taken up by the B6FS cells, and that UFH and LMWH induced F‑actin polymerization. Heparitinase digestion demonstrated that the endogenous heparan sulfate (HS) chains did not affect the motility of the B6FS cells (p>0.05, not significant). In conclusion, both UFH and LMWH, through a FAK/actin cytoskeleton axis, promoted the adhesion and migration of B6FS fibrosarcoma cells. Thus, our findings indicate that the responsiveness of fibrosarcoma cells to the exogenous heparin/HS content of the cancer microenvironment may play a role in their ability to become mobile and metastasize.

View Figures

View References

1	Meric F, Hess KR, Varma DG, Hunt KK, Pisters PW, Milas KM, Patel SR, Benjamin RS, Plager C, Papadopoulos NE, et al: Radiographic response to neoadjuvant chemotherapy is a predictor of local control and survival in soft tissue sarcomas. Cancer. 95:1120–1126. 2002. View Article : Google Scholar : PubMed/NCBI
2	Nedea EA and DeLaney TF: Sarcoma and skin radiation oncology. Hematol Oncol Clin North Am. 20:401–429. 2006. View Article : Google Scholar : PubMed/NCBI
3	Jemal A, Siegel R, Ward E, Murray T, Xu J and Thun MJ: Cancer statistics, 2007. CA Cancer J Clin. 57:43–66. 2007. View Article : Google Scholar : PubMed/NCBI
4	Linhardt RJ: 2003 Claude S. Hudson Award address in carbohydrate chemistry. Heparin: Structure and activity. J Med Chem. 46:2551–2564. 2003. View Article : Google Scholar : PubMed/NCBI
5	Engelberg H: Actions of heparin that may affect the malignant process. Cancer. 85:257–272. 1999. View Article : Google Scholar : PubMed/NCBI
6	Vlodavsky I and Friedmann Y: Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest. 108:341–347. 2001. View Article : Google Scholar : PubMed/NCBI
7	Tyagi SC, Kumar S and Katwa L: Differential regulation of extracellular matrix metalloproteinase and tissue inhibitor by heparin and cholesterol in fibroblast cells. J Mol Cell Cardiol. 29:391–404. 1997. View Article : Google Scholar : PubMed/NCBI
8	Smorenburg SM and Van Noorden CJ: The complex effects of heparins on cancer progression and metastasis in experimental studies. Pharmacol Rev. 53:93–105. 2001.PubMed/NCBI
9	Elias EG, Shukla SK and Mink IB: Heparin and chemotherapy in the management of inoperable lung carcinoma. Cancer. 36:129–136. 1975. View Article : Google Scholar : PubMed/NCBI
10	Kakkar AK and Macbeth F: Antithrombotic therapy and survival in patients with malignant disease. Br J Cancer. 102(Suppl 1): S24–S29. 2010. View Article : Google Scholar : PubMed/NCBI
11	Malavaki CJ, Theocharis AD, Lamari FN, Kanakis I, Tsegenidis T, Tzanakakis GN and Karamanos NK: Heparan sulfate: Biological significance, tools for biochemical analysis and structural characterization. Biomed Chromatogr. 25:11–20. 2011. View Article : Google Scholar : PubMed/NCBI
12	Karamanos NK and Tzanakakis GN: Glycosaminoglycans: From 'cellular glue' to novel therapeutical agents. Curr Opin Pharmacol. 12:220–222. 2012. View Article : Google Scholar : PubMed/NCBI
13	Halkin H, Goldberg J, Modan M and Modan B: Reduction of mortality in general medical in-patients by low-dose heparin prophylaxis. Ann Intern Med. 96:561–565. 1982. View Article : Google Scholar : PubMed/NCBI
14	Chahinian AP, Propert KJ, Ware JH, Zimmer B, Perry MC, Hirsh V, Skarin A, Kopel S, Holland JF, Comis RL, et al: A randomized trial of anticoagulation with warfarin and of alternating chemotherapy in extensive small-cell lung cancer by the Cancer and Leukemia group B. J Clin oncol. 7:993–1002. 1989.PubMed/NCBI
15	Hettiarachchi RJ, Smorenburg SM, Ginsberg J, Levine M, Prins MH and Büller HR: Do heparins do more than just treat thrombosis? The influence of heparins on cancer spread. Thromb Haemost. 82:947–952. 1999.PubMed/NCBI
16	von Tempelhoff GF, Harenberg J, Niemann F, Hommel G, Kirkpatrick CJ and Heilmann L: Effect of low molecular weight heparin (Certoparin) versus unfractionated heparin on cancer survival following breast and pelvic cancer surgery: A prospective randomized double-blind trial. Int J Oncol. 16:815–824. 2000.PubMed/NCBI
17	Mousa SA and Mohamed S: Anti-angiogenic mechanisms and efficacy of the low molecular weight heparin, tinzaparin: Anti-cancer efficacy. Oncol Rep. 12:683–688. 2004.PubMed/NCBI
18	Ettelaie C, Fountain D, Collier ME, Beeby E, Xiao YP and Maraveyas A: Low molecular weight heparin suppresses tissue factor-mediated cancer cell invasion and migration in vitro. Exp Ther Med. 2:363–367. 2011. View Article : Google Scholar : PubMed/NCBI
19	Alyahya R, Sudha T, Racz M, Stain SC and Mousa SA: Anti-metastasis efficacy and safety of non-anticoagulant heparin derivative versus low molecular weight heparin in surgical pancreatic cancer models. Int J Oncol. 46:1225–1231. 2015.
20	Tiozzo R, Cingi MR, Pietrangelo A, Albertazzi L, Calandra S and Milani MR: Effect of heparin-like compounds on the in vitro proliferation and protein synthesis of various cell types. Arzneimittelforschung. 39:15–20. 1989.PubMed/NCBI
21	Au YP, Kenagy RD, Clowes MM and Clowes AW: Mechanisms of inhibition by heparin of vascular smooth muscle cell proliferation and migration. Haemostasis. 23(Suppl 1): 177–182. 1993.PubMed/NCBI
22	Bennett MR, Evan GI and Newby AC: Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma, heparin, and cyclic nucleotide analogues and induces apoptosis. Circ Res. 74:525–536. 1994. View Article : Google Scholar : PubMed/NCBI
23	Miralem T, Wang A, Whiteside CI and Templeton DM: Heparin inhibits mitogen-activated protein kinase-dependent and -independent c-fos induction in mesangial cells. J Biol Chem. 271:17100–17106. 1996. View Article : Google Scholar : PubMed/NCBI
24	Nikitovic D, Assouti M, Sifaki M, Katonis P, Krasagakis K, Karamanos NK and Tzanakakis GN: Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines. Int J Biochem Cell Biol. 40:72–83. 2008. View Article : Google Scholar
25	Nikitovic D, Zafiropoulos A, Tzanakakis GN, Karamanos NK and Tsatsakis AM: Effects of glycosaminoglycans on cell proliferation of normal osteoblasts and human osteosarcoma cells depend on their type and fine chemical compositions. Anticancer Res. 25:2851–2856. 2005.PubMed/NCBI
26	Chatzinikolaou G, Nikitovic D, Asimakopoulou A, Tsatsakis A, Karamanos NK and Tzanakakis GN: Heparin - a unique stimulator of human colon cancer cells' growth. IUBMB Life. 60:333–340. 2008. View Article : Google Scholar : PubMed/NCBI
27	Chatzinikolaou G, Nikitovic D, Berdiaki A, Zafiropoulos A, Katonis P, Karamanos NK and Tzanakakis GN: Heparin regulates colon cancer cell growth through p38 mitogen-activated protein kinase signalling. Cell Prolif. 43:9–18. 2010. View Article : Google Scholar
28	Fthenou E, Zafiropoulos A, Tsatsakis A, Stathopoulos A, Karamanos NK and Tzanakakis GN: Chondroitin sulfate A chains enhance platelet derived growth factor-mediated signalling in fibrosarcoma cells. Int J Biochem Cell Biol. 38:2141–2150. 2006. View Article : Google Scholar : PubMed/NCBI
29	Boeryd B: Action of heparin and plasminogen inhibitor (EACA) on metastatic tumour spread in an isologous system. Acta Pathol Microbiol Scand. 65:395–404. 1965.PubMed/NCBI
30	Hagmar B and Norrby K: Evidence for effects of heparin on cell surfaces influencing experimental metastases. International journal of cancer Int J Cancer. 5:72–84. 1970. View Article : Google Scholar : PubMed/NCBI
31	Maat B: Extrapulmonary colony formation after intravenous injection of tumour cells into heparin-treated animals. Br J Cancer. 37:369–376. 1978. View Article : Google Scholar : PubMed/NCBI
32	Takeuchi A, Yamamoto Y, Munesue S, Harashima A, Watanabe T, Yonekura H, Yamamoto H and Tsuchiya H: Low molecular weight heparin suppresses receptor for advanced glycation end products-mediated expression of malignant phenotype in human fibrosarcoma cells. Cancer Sci. 104:740–749. 2013. View Article : Google Scholar : PubMed/NCBI
33	Chalkiadaki G, Nikitovic D, Berdiaki A, Katonis P, Karamanos NK and Tzanakakis GN: Heparin plays a key regulatory role via a p53/FAK-dependent signaling in melanoma cell adhesion and migration. IUBMB Life. 63:109–119. 2011.PubMed/NCBI
34	Chalkiadaki G, Nikitovic D, Katonis P, Berdiaki A, Tsatsakis A, Kotsikogianni I, Karamanos NK and Tzanakakis GN: Low molecular weight heparin inhibits melanoma cell adhesion and migration through a PKCa/JNK signaling pathway inducing actin cytoskeleton changes. Cancer Lett. 312:235–244. 2011. View Article : Google Scholar : PubMed/NCBI
35	Pankov R and Yamada KM: Fibronectin at a glance. J Cell Sci. 115:3861–3863. 2002. View Article : Google Scholar : PubMed/NCBI
36	Clark EA, Golub TR, Lander ES and Hynes RO: Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 406:532–535. 2000. View Article : Google Scholar : PubMed/NCBI
37	Inoue T, Nabeshima K, Shimao Y, Kataoka H and Koono M: Modulation of fibronectin synthesis by cancer cell-fibroblast interaction. Int J Oncol. 9:721–730. 1996.PubMed/NCBI
38	Mytilinaiou M, Bano A, Nikitovic D, Berdiaki A, Voudouri K, Kalogeraki A, Karamanos NK and Tzanakakis GN: Syndecan-2 is a key regulator of transforming growth factor beta 2/Smad2-mediated adhesion in fibrosarcoma cells. IUBMB Life. 65:134–143. 2013. View Article : Google Scholar : PubMed/NCBI
39	Kouvidi K, Berdiaki A, Nikitovic D, Katonis P, Afratis N, Hascall VC, Karamanos NK and Tzanakakis GN: Role of receptor for hyaluronic acid-mediated motility (RHAMM) in low molecular weight hyaluronan (LMWHA)-mediated fibrosarcoma cell adhesion. J Biol Chem. 286:38509–38520. 2011. View Article : Google Scholar : PubMed/NCBI
40	Thurzo V, Popovic M, Matoska J, Blasko M, Grófová M, Lizonová A and Steno M: Human neoplastic cells in tissue culture: Two established cell lines derived from giant cell tumor and fibrosarcoma. Neoplasma. 23:577–587. 1976.PubMed/NCBI
41	Hong IK, Jin YJ, Byun HJ, Jeoung DI, Kim YM and Lee H: Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrix metalloproteinase-9 expression of human melanoma cells through adhesion-dependent c-Jun activation signaling pathways. J Biol Chem. 281:24279–24292. 2006. View Article : Google Scholar : PubMed/NCBI
42	Karamanos NK, Axelsson S, Vanky P, Tzanakakis GN and Hjerpe A: Determination of hyaluronan and galactosaminoglycan disaccharides by high-performance capillary electrophoresis at the attomole level. Applications to analyses of tissue and cell culture proteoglycans. J Chromatogr A. 696:295–305. 1995. View Article : Google Scholar : PubMed/NCBI
43	Karamanos NK, Vanky P, Tzanakakis GN, Tsegenidis T and Hjerpe A: Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate. J Chromatogr A. 765:169–179. 1997. View Article : Google Scholar : PubMed/NCBI
44	Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB and Parsons JT: pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci USA. 89:5192–5196. 1992. View Article : Google Scholar : PubMed/NCBI
45	Weiner TM, Liu ET, Craven RJ and Cance WG: Expression of focal adhesion kinase gene and invasive cancer. Lancet. 342:1024–1025. 1993. View Article : Google Scholar : PubMed/NCBI
46	Fritzsche J, Simonis D and Bendas G: Melanoma cell adhesion can be blocked by heparin in vitro: Suggestion of VLA-4 as a novel target for antimetastatic approaches. Thromb Haemost. 100:1166–1175. 2008.
47	Moyano JV, Maqueda A, Albar JP and Garcia-Pardo A: A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells. Biochem J. 371:565–571. 2003. View Article : Google Scholar : PubMed/NCBI
48	Dudás J, Ramadori G, Knittel T, Neubauer K, Raddatz D, Egedy K and Kovalszky I: Effect of heparin and liver heparan sulphate on interaction of HepG2-derived transcription factors and their cis-acting elements: Altered potential of hepatocellular carcinoma heparan sulphate. Biochem J. 350:245–251. 2000. View Article : Google Scholar : PubMed/NCBI
49	Geiger B and Yamada KM: Molecular architecture and function of matrix adhesions. Cold Spring Harb Perspect Biol. 3:32011. View Article : Google Scholar
50	Parsons JT, Martin KH, Slack JK, Taylor JM and Weed SA: Focal adhesion kinase: A regulator of focal adhesion dynamics and cell movement. Oncogene. 19:5606–5613. 2000. View Article : Google Scholar : PubMed/NCBI
51	Castellot JJ Jr, Wong K, Herman B, Hoover RL, Albertini DF, Wright TC, Caleb BL and Karnovsky MJ: Binding and internalization of heparin by vascular smooth muscle cells. J Cell Physiol. 124:13–20. 1985. View Article : Google Scholar : PubMed/NCBI
52	Bârzu T, Pascal M, Maman M, Roque C, Lafont F and Rousselet A: Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: Comparison between heparin-sensitive and heparin-resistant cultures. J Cell Physiol. 167:8–21. 1996. View Article : Google Scholar : PubMed/NCBI
53	Casu B and Lindahl U: Structure and biological interactions of heparin and heparan sulfate. Adv Carbohydr Chem Biochem. 57:159–206. 2001. View Article : Google Scholar
54	Shrivastava S, Nayak SK, Nayak P and Sahu S: Fibrosarcoma of maxilla: A rare case report. J Oral Maxillofac Pathol. 20:1622016. View Article : Google Scholar : PubMed/NCBI
55	Thway K, Robertson D, Jones RL, Selfe J, Shipley J, Fisher C and Isacke CM: Endosialin expression in soft tissue sarcoma as a potential marker of undifferentiated mesenchymal cells. Br J Cancer. Jul 19–2016.Epub ahead of print. View Article : Google Scholar :
56	Noujaim J, Thway K, Sheri A, Keller C and Jones RL: Histology-Driven Therapy: The Importance of Diagnostic Accuracy in Guiding Systemic Therapy of Soft Tissue Tumors. Int J Surg Pathol. 24:5–15. 2016. View Article : Google Scholar
57	Blizniukov OP and Zamogil'naia IaA: Pleomorphic fibrosarcoma. Vopr Onkol. 58:54–60. 2012.In Russian.
58	Rabenstein DL: Heparin and heparan sulfate: Structure and function. Nat Prod Rep. 19:312–331. 2002. View Article : Google Scholar : PubMed/NCBI
59	Chen Q, Zhou Z, Shan L, Zeng H, Hua Y and Cai Z: The importance of Src signaling in sarcoma. Oncol Lett. 10:17–22. 2015.PubMed/NCBI
60	Hauck CR, Hsia DA and Schlaepfer DD: The focal adhesion kinase - a regulator of cell migration and invasion. IUBMB Life. 53:115–119. 2002. View Article : Google Scholar : PubMed/NCBI
61	Hall JE, Fu W and Schaller MD: Focal adhesion kinase: Exploring Fak structure to gain insight into function. Int rev Cell Mol Biol. 288:185–225. 2011. View Article : Google Scholar : PubMed/NCBI
62	Medeiros VP, Paredes-Gamero EJ, Monteiro HP, Rocha HA, Trindade ES and Nader HB: Heparin-integrin interaction in endothelial cells: Downstream signaling and heparan sulfate expression. J Cell Physiol. 227:2740–2749. 2012. View Article : Google Scholar
63	Moussa FM, Hisijara IA, Sondag GR, Scott EM, Frara N, Abdelmagid SM and Safadi FF: Osteoactivin promotes osteoblast adhesion through HSPG and αvβ1 integrin. J Cell Biochem. 115:1243–1253. 2014. View Article : Google Scholar : PubMed/NCBI
64	Fatma N, Singh DP, Shinohara T and Chylack LT Jr: Heparin's roles in stabilizing, potentiating, and transporting LEDGF into the nucleus. Invest Ophthalmol Vis Sci. 41:2648–2657. 2000.PubMed/NCBI
65	van de Water L III, Schroeder S, Crenshaw EB III and Hynes RO: Phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin. J Cell Biol. 90:32–39. 1981. View Article : Google Scholar : PubMed/NCBI
66	Kovalszky I, Dudás J, Oláh-Nagy J, Pogány G, Töváry J, Timár J, Kopper L, Jeney A and Iozzo RV: Inhibition of DNA topoisomerase I activity by heparan sulfate and modulation by basic fibroblast growth factor. Mol Cell Biochem. 183:11–23. 1998. View Article : Google Scholar : PubMed/NCBI
67	Timar J and Paterson H: Localization and production of proteoglycans by HT1080 cell lines with altered N-ras expression. Cancer Lett. 53:145–150. 1990. View Article : Google Scholar : PubMed/NCBI
68	Busch SJ, Martin GA, Barnhart RL, Mano M, Cardin AD and Jackson RL: Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription. J Cell Biol. 116:31–42. 1992. View Article : Google Scholar : PubMed/NCBI