Recombinant hirudin suppresses the viability, adhesion, migration and invasion of Hep-2 human laryngeal cancer cells

Abstract

Abstract. Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC. IntroductionLaryngeal carcinoma (LC) is a common head and neck malignancy and accounts for ~2.4% of newly diagnosed malignancies worldwide every year (1,2). Surgery, chemotherapy and radiation therapy are the current treatment modules for laryngeal cancer (3-5). Although early-stage laryngeal cancer can be effectively treated with surgery or radiotherapy, the 5-year survival rate of patients with advanced LC is still below 60% even after systematic surgery and post-surgical adjuvant radiotherapy or chemotherapy (6). In addition, surgery may result in complete or partial loss of swallowing and vocal functions. Many patients have to maintain a tracheal cannula on a long-term basis due to laryngeal stenosis after surgery, which markedly impairs their quality of life (6,7). Therefore, there is urgent need to develop novel approaches and strategies for the treatment of these advanced LC patients.The development and progression of cancer are closely associated with thrombosis (8). Thrombin, the key terminal enzyme of coagulation, enhances angiogenesis, stimulates the adhesion of tumor cells to platelet ...

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review

“…Remarkably, rH significantly down-regulated the expression of proteins FAK and VEGF-R, suggesting that the anti-tumor effect of rH might be associated with the suppression of angiogenesis by down-regulating the VEGF/VEGF-R pathway. (Lu et al, 2015). These findings also suggested that targeting thrombin by rH is a potential strategy for treating LC.…”

Clinical significance of plasma D-dimer and fibrinogen in digestive cancer: A systematic review and meta-analysis

“…As non-invasive and inexpensive tests, they are suitable for initial screening or use in combination with other biomarkers for certain types of cancer diagnosis or prevention. Moreover, recombinant hirudin, an inhibitor of thrombin to prevent the formation of fibrinogen and d dimer, has been reported to inhibit the growth and metastasis in several types of cancer cells [62,63]. Based on the fact that the binding sites of fibrinogen with immune cells are different from its coagulation cascade recognition sites, it is an ideal target for pharmacological intervention without affecting its coagulation functions [64].…”

Antiplatelet agents for cancer treatment: a real perspective or just an echo from the past?

“…Hirudin inhibited TCIPA in non-small-cell lung cancer cell lines (NSCLC) and in SCLC, but in the last case, blocking TCIPA required addition of the ADP scavenger apyrase with hirudin [ 60 ]. Recombinant hirudin (rH) has also been shown to inhibit growth and exert anti-metastatic effects in several types of cancers through multiple mechanisms [ 185 , 188 ]. Experimental studies with laryngeal carcinoma cells (HEp-2 cells) demonstrated that rH inhibited the adhesion, migration, and invasion of the cancer cells.…”