Low molecular weight heparin suppresses receptor for advanced glycation end products-mediated expression of malignant phenotype in human fibrosarcoma cells
The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor and its engagement by ligands such as high mobility group box 1 (HMGB1) is implicated in tumor growth and metastasis. Low molecular weight heparin (LMWH) has an antagonistic effect on the RAGE axis and is also reported to exert an antitumor effect beyond the known activity of anticoagulation. However, the link between the anti-RAGE and antitumor activities of LMWH has not yet to be fully elucidated. In
... elucidated. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, invasion, and metastasis by blocking the RAGE axis using in vitro and in vivo assay systems. Stably transformed HT1080 human fibrosarcoma cell lines were obtained, including human full-length RAGE-overexpressing (HT1080 RAGE ), RAGE dominant-negative, intracellular tail-deleted RAGE-overexpressing (HT1080 dnRAGE ), and mock-transfected control (HT1080 mock ) cells. Confocal microscopy showed the expression of HMGB1 and RAGE in HT1080 cells. The LMWH significantly inhibited HMGB1induced NFjB activation through RAGE using an NFjB-dependent luciferase reporter assay and the HT1080 cell lines. Overexpression of RAGE significantly accelerated, but dnRAGE expression attenuated HT1080 cell proliferation and invasion in vitro, along with similar effects on local tumor mass growth and lung metastasis in vivo. Treatment with LMWH significantly inhibited the migration, invasion, tumor formation, and lung metastasis of HT1080 RAGE cells, but not of HT1080 mock or HT1080 dnRAGE cells. In conclusion, this study revealed that RAGE exacerbated the malignant phenotype of human fibrosarcoma cells, and that this exacerbation could be ameliorated by LMWH. It is suggested that LMWH has therapeutic potential in patients with certain types of malignant tumors. (Cancer Sci 2013; 104: 740-749) R eceptor for advanced glycation end products is a multiligand pattern-recognition receptor, having an extracellular region with one V-type and two C-type immunoglobulin-like domains, one transmembrane region, and a short C-terminal intracellular stretch. (1, 2) The V domain of RAGE is critical for the binding of distinct ligands, such as AGE, (1,2) S100 proteins, (3, 4) b-amyloid, (5,6) Mac1 ⁄ CD11b, (7) lipopolysaccharides, (8) phosphatidylserine, (9) and HMGB1. (10) (11) (12) The interaction between RAGE and these ligands has been implicated in proinflammatory reactions, diabetic vascular complications, neurodegenerative disorders, and cancer. (1, 13, 14) The ligation of RAGE is reported to cause the activation of multiple intracellular signaling pathways, including Cdc42 ⁄ Rac1, p38 MAP kinase, (15) PKC, (16) and NFjB, (17) as well as the production of reactive oxygen species. The biological outcomes are also known to be divergent among cell types or under different conditions. Concerning malignant tumors, the HMGB1-RAGE axis is reported to be involved in tumor growth, invasion, and metastasis. (10,18) RAGE expression has been documented in human malignancies of the stomach, (19) colon and rectum, (20) prostate, (21) lung, (20) breast, (20) and bone. (22) Soft tissue sarcomas are rare malignant tumors arising from mesenchymal tissues. Multiagent chemotherapy has improved both the survival rate and limb function in patients with certain mesenchymal malignant tumors, such as osteosarcoma (23) and Ewing's sarcoma. (24) Despite advances in chemotherapy regimens, however, high-grade soft tissue sarcoma remains a fatal disease with frequent distant metastases. (25) (26) (27) To improve the prognosis of high-grade soft tissue sarcoma, new therapeutic strategies are needed for the suppression of cancer spread and metastasis. In 1930, Goerner et al. (28) first reported the inhibitory effect of heparin against the growth of transplanted tumor tissues. Subsequently, anticoagulant therapy with unfractionated heparin (29) (30) (31) or warfarin (32) (33) (34) has come to be indicated for several types of cancers. Numerous subsequent studies confirmed that anticoagulant agents improve the overall survival of cancer patients. (28) (29) (30) (31) (32) (33) Low molecular weight heparin is a fragmented and fractionated heparin consisting of short polysaccharide chains with anticoagulant activity. (35) The effect of LMWH on the improvement of overall survival has also been shown in various types of cancers. (36) (37) (38) Altinbas et al. (39) reported that treatment with LMWH improved chemotherapy responsiveness and overall survival in small cell lung cancer. Several mechanisms other than anticoagulation have been proposed to underlie the antitumor activity of LMWH. (39) Harvey et al. (40) suggested that LMWH may have an antagonistic effect on CXCR4 in breast cancer cells and thus inhibit tumor cell metastasis. Our group reported that LMWH, dalteparin obtained by deaminative cleavage of heparin with nitrous acid, had an antagonistic effect on RAGE. (41) Moreover, we reported its preventive and therapeutic effects on diabetic nephropathy in vivo. (41) On the basis of these findings, we hypothesized that the antitumor activity of LMWH, at least in part, depends on the competitive inhibition of RAGE-ligand interaction. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, migration, invasion, and distant metastasis by blocking the RAGE axis in vitro and in vivo using wild-type RAGE-and dominant negative RAGE-overexpressing human fibrosarcoma HT1080 cell lines.