The Promotion Effects And Mechanisms Of Thrombin And Its Receptor-par1 On Lung Cancer

Thrombin, an important coagulation factor, is located in the common pathway of the intrinsic and extrinsic coagulation pathways. It is crucial for the activition of coagulation system. Studies have demonstrated that thrombin is one of the key factors for cancer developing. PAR1 (protease-activated receptor 1) is the most important receptor of thrombin, and it mediates the enhancement effects of thrombin in angiogenesis, invasion and metastasis, growth of cancer. This research is aimed to demonstrate the effects and mechanisms of thrombin and its receptor-PAR1 in the tumorigenesis and metastasis of lung cancer cells.In vivo, thrombin promoted the growth of Lewis lung cancer in mice. Compared with NS control, the lung weight of two thrombin groups (0.5U/animal, 1U/animal) increased 37% and 30%, respectively.Effect of endogenous thrombin on the growth and metastasis of Lewis lung cancer in mice were investigated with hirudin and a novel thrombin inhibitor. The results showed that hirudin and the novel thrombin inhibitor inhibited the tumor growth, reduced the speed of tumor growth. Compared with NS control, hirudin and the novel thrombin inhibitor significantly reduced the cancer migration to lung. The average lung weight and lung nodule number of the novel thrombin inhibitor-excited group were 0.143g and 9.2, respectively, which were both inferior than those of NS control group (0.185g, 17.5), the p value is smaller than 0.05. The results of tumor and lung derived tissue sections stained with H&E showed that the novel thrombin inhibitor could reduce the invasion and metastasis of Lewis lung tumor in vivo. At the end point of experiment, most mice in hirudin group died, 4 of 7 mice died in 2.5mg/kg hirudin group, and only 1 mouse survived in hirudin 5mg/kg group. The autopsy results of the died animals in two hirudin groups showed that heavy bleeding were found in abdominal cavity, which maybe the main cause of the death. On the contrary, only 1 mouse died in the novel thrombin inhibitor group. These results indicate that the bleeding side-effect of the novel thrombin inhibitor reduced dramatically. Thrombin promoted the malignant progession of Glc-82 tumor. Thrombin significantly reduced the body weight, enhanced the ratio of tumor weight to body weight, and shortened life span of tumor-bearing nude mice, and increased the lung nodule mass in certain thrombin-treated groups. The phosphorylation of ERK and the expression of FAK in the thrombin-excited mice tumor tissue were dramatically increased, while the expression of PTEN was remarkably reduced.In vitro, thrombin in solution didn't promote Glc-82 cells and PLA-801D cells adhere to ECM, but it activated integrin. Its mechanisms require more experiment data to explain. Coated thrombin promoted the adhesion property of Glc-82 cells and PLA-801D cells. The adhesion promotion effects were inhibited when thrombin was incubated with its native inhibitor-hirudin which inhibits thrombin's catalytic domain before coated. However, hirudin did not inhibit the adhesion promotion effects when thrombin had been coated in the culture plate. The results indicate that the conformation of thrombin changed after it is coated, and the adhesion promotion effects of coated thrombin don't depend on the catalytic domain. The promotion adhesion effects of coated thrombin were not inhibited by anti-PAR1 and anti-αⅤ(an integrin receptor subtype) antibodies, but GRGDS ( the competitive peptide of integrin, contains the sequence of RGD),significantly inhibited the adhesion promotion effects of coated thombin, indicating that integrin except integrinαⅤparticipated in the adhesion promotion effects of coated thrombin. The results of Western blot showed coated thrombin could activate integein, and this activation effects was blocked by GRGDS. These results indicate that coated thrombin may active integrin by RGD sequence which is exposed from the catalytic domain of thrombin by the conformation change. SFLLRN (PAR1 activating peptide) could increase the adhesion of lung cancer cells to ECM and activate integrin. This activation effects of SFLLRN was reduced by anti-αⅤand GRGDS. The results of confocal microscopy showed that PAR1 didn't colocalize with integrinαⅤneither in the presence of thrombin nor in the presence of SFLLRN.Aiming to demonstrate the effects of thrombin on the cancer stem cells, we investigated the relationship of the PAR1 expression and cancer stem cells. Compared with PLA-801C cells, PLA-801D cells showed significantly stronger adhesion to both rat collagen and fibronectin (p <0.01). The migration property of PLA-801D cells is higher than that of PLA-801C cells (p <0.01). PLA-801D cells could form four kinds of single cell clones, one of them could differentiate into the same four clones in sub-clones. However, only three kinds of clones were found in PLA-801C cell, and none of them could differentiate into the initial three clones in their sub-clones. PLA-801D cells showed better collagen gel contraction than PLA-801C cells (p <0.05).To investigate the effects of thrombin and PAR1 in the promotion of cancer, the eukaryotic cell expression vector of PAR1 was constructed and the recombinant adenovirus-PAR1 was established. We also constructed the expression vector containing PAR1 RNA interference sequence. It's the foundation of the next study work.Conclusion: Thrombin enhances the growth of Lewis lung cancer and promotes the development of lung cancer cell Glc-82 in vivo. The novel thrombin inhibitor and hirudin can inhibit the growth of Lewis lung cancer, and significantly suppress the invasion and metastasis of Lewis lung cancer in vivo. Furthermore, compared with hirudin, the bleeding side-effect of the new thrombin inhibitor reduces dramatically. Coated thrombin can expose the RGD sequence by conformation change. As a result, it can activate integrin, and promote the adhesion property of Glc-82 cells and PLA-801D. Thrombin in solution can activate integrin, but can't promote lung cancer cells adhere to ECM. Its mechanisms require more experiment data to explain.SFFLLRN can promote lung cancer cells adhere to ECM, it probably activates integrin by"inside-out"mechanisms, andαⅤintegrin may involve in these effects.PLA-801D cells express more PAR1 than PLA-801C cells, and show higher metastasis property. A grouplet of cells in PLA-801D cells show self-renewal and multi-directional differentiation characteristic of cancer stem cells. Moreover, PLA-801D cells display good biomechanical remodeling potential, indicating its good remodeling capacity for extracellular matrix. But the relationship of the express of PAR1 and cancer stem cells needs further exploration.The eukaryotic cell expression vector of PAR1 has been cloned and the recombinant adenovirus containing PAR1 gene has been established. The expression vector containing PAR1 RNA interference sequence has also been constructed, and stable cell line that PAR1 down-regulated has been created.