Discussion
Metastasis has been recognized as the main cause of fatal outcomes in lung cancer patients. It is of interest that low-grade intravascular coagulation has been observed in most patients with solid tumors (Rickles, Edwards, Barb & Cronlund, 1983). Malignant tumors often exhibit hypercoagulability, which is correlated with high levels of activated thrombin (Caine, Stonelake, Lip & Kehoe, 2002). Studies have shown that exogenous thrombin is capable of enhancing tumor adhesion to platelets (Nierodzik, Kajumo & Karpatkin, 1992), endothelial cells (Klepfish, Greco & Karpatkin, 1993), and fibronectin (Chen et al., 2014) in vitro, and revealed that exogenous thrombin promotes tumor growth (Nierodzik et al., 1998). In this study we demonstrated that thrombin expression in tumor tissues was significantly correlated with metastatic potential of NSCLC, postoperative tumor recurrence, and poor prognosis of NSCLC patients. This finding is supported by the fact that a high thrombin expression was significantly associated with the aggressive histopathological characteristics of NSCLC, such as high TNM stage. Thrombin deficiency impairs tumor progression. This indicates that thrombin may serve as an independent predictor for tumor recurrence and prognosis of NSCLC patients, which might be an explanation as to why malignant tumors often exhibit hypercoagulability.
To further explore the role of thrombin in the lung cancer, we used exogenous thrombin to treat NSCLC cell lines in vitro. we found that 10 nmol/L thrombin can promote NSCLC cell invasion, angiogenesis, and metastasis in vitro. r-hirudin and DTIP are new bivalent direct thrombin inhibitors that could effectively prevent the formation of thrombosis and embolism (Mo, Zhang, Chen, Wang & Song, 2009; Zhao et al., 2017). These results showed that r-hirudin and DTIP could inhibit thrombin-induced cell invasion, angiogenesis, and metastasis in vitro. r-hirudin and DTIP inhibit progression, dissemination and spontaneous metastasis in NSCLC mice models, thus prolonging the survival time of mice in lung cancer models. Furthermore, in our studies, we did not find increased bleeding after administration of DTIP, slight subcutaneous hemorrhage was observed after r-hirudin administration for three weeks continuously. These results show that DTIP could be extended to anti-cancer therapy.
The cleavage of the thrombin-receptor PAR-1 by thrombin exposes a new N terminus that binds to the receptor to induce trans-membrane signaling (McLaughlin, Shen, Holinstat, Brooks, Dibenedetto & Hamm, 2005). PAR-1 overexpression has been reported in malignant invasive tumor cell lines (Black et al., 2007; Queiroz et al., 2014). Our data suggested that PAR-1 is highly expressed in human NSCLC tissues, but showed no difference based on subtype and clinical stage. To further explore the role of thrombin in the PAR-1-mediated NSCLC metastasis, we used exogenous thrombin to treat PAR-1 deficient NSCLC cells in vitro, and found that the invasion and metastasis of PAR-1 deficient cell lines was inhibited in vitro and in vivo; thrombin had no effect on their invasive and metastatic.
In our study, we showed that thrombin promotes the activation of RhoA through the activation of PAR-1, thereby inducing cytoskeletal rearrangements, actin stress fiber formation, and activation of NF-κB. Activation of cytosolic NF-κB leads to expression of MMP9 and inflammatory marker IL6. r-hirudin and DTIP can bind to thrombin specifically and block the thrombin binding to PAR1, thereby inhibiting RhoA and NF-κB activation and reducing the expression of MMP9 and IL6 in NSCLC cells, which would be hugely beneficial for suppressing metastasis.
Chemotherapy used in NSCLC seem to cause primarily vascular complications, including venous or arterial thromboembolic events. In a nonrandomized study, the addition of low-molecular-weight heparin (LMWH) to standard gemcitabine/cisplatinum chemotherapy significantly improved survival in patients with locally advanced or metastatic pancreatic carcinoma (Icli et al., 2007). The total response rate for patients treated with LMWH was almost 60%, compared with only 12% for patients treated with chemotherapy only. In our studies, we found combination therapy of DTIP and chemotherapy results in improved anti-tumor efficacy, and DTIP could potentiate gemcitabine-induced inhibition of lung cancer and inhibit paclitaxel resistance in mice. This approach merits further evaluation in more extensive studies and in NSCLC patients.