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.