Table 1. Association of thrombin expression with the clinicopathologic characteristics of 132 NSCLC patients
Figure 1 The expression of thrombin in lung cancer is closely related to clinicopathological features and the prognosis of patients.(a-c) Thrombin expression in NSCLC patients. (a)Paraffin sections obtained from patients with resectable NSCLC tissues were stained for thrombin. (b) Score of thrombin expression in adjacent non-tumor lung tissue and in NSCLCs. (c) Thrombin expression in different types of NSCLC. (d) The overall survival rates of thrombin-positive patients and thrombin-negative patients. (e)The mRNA level of thrombin in BEAS-2B, A549, 95D, and PC9 was determined by Q-PCR. (f) The expression of thrombin in BEAS-2B, A549, 95D, and PC9 was determined by western blotting analysis. Right, Summary data of western blotting were given. All the results were expressed as mean ± SD. *p  < 0.05, **p  < 0.01, ***p  < 0.001, NS, not significant.
Figure 2Thrombin plays an important role in the progression of lung cancer. (a) Western blot depicting depletion of thrombin in A549 cells transfected with human thrombin gRNA compared with negative control. (b) Representative pictures of A549 cells migrated through the transwell are shown, Right, quantitative analysis of invasive cells. (c) Western blot depicting depletion of thrombin in Lewis cells transfected with mouse thrombin gRNA compared with negative control. (d) Representative pictures of Lewis cells migrated through the transwell are shown, Right, quantitative analysis of invasive cells. (e-i)Thrombin deficient LLC cells and control cells were injected subcutaneously into the right flank of mice. (e) Serial calipation of tumor volume after transplantation cells into mice. (f) Mice were humanely euthanized, and the tumors were resected 35 days after cell injection.(g) The weight of resected tumors was determined. (h)H&E stained sections of lung tissue. (i) The number of metastatic nodes per lung was determined. (j-l) In orthotopic lung tumor model, 1 × 106 cells in 50 µL RPMI-1640 medium and 50 µL Matrigel were injected into the left lung parenchyma through the left rib cage. (j)Survival rate of mice in different groups. (k) Representative photograph of lungs bearing tumors (left); Gross appearance of representative lung harvested viewed under a fluorescence stereoscope (middle); H&E stained sections of lung tissue (right). (l) Average tumor area in the lungs was measured. (m)Number of mice with tumor cells in liver. All the results were expressed as mean ± SD. ANOVA followed by Dunnett’s test was applied for multiple comparison. *p  < 0.05, **p  < 0.01, ***p  < 0.001, NS, not significant.
Figure 3 r-hirudin and DTIP inhibit thrombin-promoted cell migration, invasion and angiogenesis in vitro. (a)A549 and Lewis cells were pretreated with PBS, 10 nmol/L thrombin, 25 nmol/L r-hirudin, 50 nmol/L DTIP, 10 nmol/L thrombin + 25 nmol/L r-hirudin, 10 nmol/L thrombin + 50 nmol/L DTIP for 24 hours. The migration of r-hirudin and DTIP treated A549 and Lewis cells were assessed using wound healing assay. Right, quantitative analysis of wound area. (b) Transwell assay was performed to assess cell migration of A549 and Lewis cells. Right, quantitative analysis of invasive cells. (c) Endogenous GTP-bound form of RhoA was enriched by a pull-down assay and detected by Western blotting. Total RhoA was detected using anti-RhoA antibody. Bottom, summary data of western blotting were given. (d)Representative images of each group stained with phalloidin in A549 cells. (e)Quantification of the F-actin fluorescence intensity. (f)Percentages of ruffle-positive cells in different groups were calculated based on the immunofluorescence. (g) Expression of IL6 and MMP9 in different groups. (h) Effect of r-hirudin and DTIP against HUVEC tube formation on Matrigel. Left, representative photographs of five independent experiments were shown. Right, quantification of the inhibition activity of aspirin on tube formation. All the results were expressed as mean ± SD. ANOVA followed by Dunnett’s test was applied for multiple comparison. *p  < 0.05, **p  < 0.01, ***p  < 0.001, NS, not significant.
Figure 4 r-hirudin and DTIP exert the ability of Anti-growth and anti-metastasis in orthotopic lung tumor model and lung cancer metastasis model.(a-c)In orthotopic lung tumor model, 1 × 106 cells were injected into the left lung through the left rib cage, and 1.0 mg/kg DTIP or 0.5 mg/kg r-hirudin was administered for 7 consecutive days after 3 days of the injection of LLC cells. (a)Survival of mice treated with saline, r-hirudin and DTIP after injection of LLC cells into the left lung. (b)Representative photograph of lungs (left); Gross appearance of representative lung harvested viewed under a fluorescence stereoscope (middle); H&E stained sections of lung tissue (right). (c) Number of NS, r-hirudin, and DTIP treated mice with liver metastasis. (d-j)Tail-vein injection of 1×106 LLC cells was performed in mice. The LLC were engineered to express red fluorescent protein to simplify detection of tumor foci in vivo. Mice that were injected with LLC were immediately treated with saline, 1.0 mg/kg DTIP or 0.5 mg/kg r-hirudin once a day for one week. We randomly divided the mice into two groups, one was sacrificed on the 14th day to observe the metastasis of tumors, and the other group was used to analyze the survival rate of mice. (d)Effect of r-hirudin and DTIP on the formation of metastatic nodes on day 14. Representative photograph of metastatic nodules on lungs (upper). Gross appearance of representative lung harvested from different groups viewed under a fluorescence stereoscope (bottom). (e)The number of metastatic nodes per lung was determined in different groups. (f) Representative micro-PET images of mice 14 days after injection. (g)H&E stained sections of lung tissue. (h)Survival of mice treated with saline, r-hirudin and DTIP after injection of LLC cells via tail-vein. (i)H&E stained sections of liver tissue. (j) Number of NS, r-hirudin, and DTIP treated mice with tumor cells in liver. All the results were expressed as mean ± SD. Compared with NS group by one-way ANOVA. *p  < 0.05.
Figure 5 r-hirudin and DTIP could inhibit the progression of tumor. LLC cells at a density of 1×106 in 0.1mL serum-free media were injected subcutaneously into the right flank of mice. (a-d) Mice that were injected with LLC cells were immediately treated with normal saline, 0.5 mg/kg r-hirudin or 1.0 mg/kg DTIP for one week. (a)The curve of tumor growth after injection of LLC cells. (b) In vivo imaging of each group at 4 weeks after cell injection. (c) H&E stained sections of tumor. (d) Number of NS, r-hirudin and DTIP treated mice with panniculus invasion. (e-n) After one week of the injection of LLC cells, mice were administered with normal saline, 1.0 mg/kg DTIP or 0.5 mg/kg r-hirudin for 21 consecutive days. (e) The curve of tumor growth after injection of LLC cells. (f) Weight of resected tumors driven from normal saline-, r-hirudin-, and DTIP-treated groups. (g) In vivo imaging of each group at 45 days after cell injection. (h)Representative photograph of metastatic nodules on lungs (upper). Gross appearance of representative lung harvested from different groups viewed under a fluorescence stereoscope (bottom) (i) Number of NS, r-hirudin and DTIP treated mice with lung metastasis. (j) Representative photograph of metastatic nodules on livers (upper). Gross appearance of representative single liver lobes harvested from different groups viewed under a fluorescence stereoscope (bottom). (k) Number of NS, r-hirudin and DTIP treated mice with liver metastasis. (l) Effect of r-hirudin and DTIP against primary tumor angiogenesis. Left, a typical photograph of immunohistochemical staining of CD31. Right, the histogram represents the number of microvessels. (m) Immunohistochemical analysis was performed on tumor samples to determine the expression of MMP9 and IL6 on tumors from NS, r-hirudin and DTIP treated mice. (n)Inhibition of phosphorylation of p65, Erk, STAT3, and Akt in tumors by r-hirudin and DTIP. Right, summary data of western blotting. All the results were expressed as mean ± SD. Compared with NS group by one-way ANOVA. *p  < 0.05, **p  < 0.01, ***p  < 0.001.
Figure 6 PAR-1 is a major determinant in thrombin-promoted progression of lung cancer.(a)Western blot depicting depletion of PAR-1 in A549 cells transfected with human PAR-1 gRNA compared with negative control. (b)Representative pictures of A549 cells migrated through the transwell are shown, Right, quantitative analysis of invasive cells. (c)Western blot depicting depletion of PAR-1 in Lewis cells transfected with mouse PAR-1 gRNA compared with negative control. (d)Representative pictures of Lewis cells migrated through the transwell are shown, Right, quantitative analysis of invasive cells. (e)GTP-bound form of RhoA was enriched by a pull-down assay and detected by Western blotting. Total RhoA was detected using anti-RhoA antibody. (f) Representative images of each group stained with phalloidin. Right, Measurement of ruffles-positive cells infected with the indicated lentivirus. (g-i) 1 × 106PAR-1 deficient LLC cells and control cells were injected into the left lung through the left rib cage. (g) Survival of mice in different groups. (h) Representative photograph and H&E stained sections of lung tissues. (i) Average tumor area in total lungs. (j-l) Tumor cells were injected subcutaneously into the right flank of mice. (j)Serial calipation of tumor volume after transplantation of LLCPar-1-/- and LLCNC cells into mice. (k) In vivo imaging of each group at 5 weeks after cell injection. (l)Representative images of anti-CD31 staining in tumor tissues. Right, the histogram represents the number of microvessels. All the results were expressed as mean ± SD. ANOVA followed by Dunnett’s test was applied for multiple comparison. *p  < 0.05, **p  < 0.01, ***p  < 0.001, NS, not significant.
Figure 7Combination therapy of DTIP and chemotherapy results in improved anti-tumor efficacy.(a-f)1×106LLC cells were subcutaneously injected in the right dorsal region of mice. One week after LLC inoculation, the mice were randomly divided into four groups, normal saline, DTIP (1 mg/kg per day, s.c.), gemcitabine (120 mg/kg once a week; i.p.), and combination DTIP with gemcitabine treatment groups, and then the mice were administered normal saline or DTIP for 21 consecutive days, gemcitabine for 4 consecutive weeks. (a) The curve of tumor growth after injection of LLC cells. (b)Mice were humanely euthanized and the tumors were resected at 45 days after cell injection. (c) Weight of resected tumors was determined in different groups. (d) Representative photograph of metastatic nodules on lungs (upper). H&E stained sections of representative single lung lobes harvested (bottom). (e)Number of mice with liver metastasis. (f) Survival rate of mice in different groups. (g-k)1×106LLC cells were subcutaneously injected in the right dorsal region of mice. One week after LLC inoculation, the mice were randomly divided into four groups, normal saline, DTIP (1 mg/kg per day, s.c.), paclitaxel (20 mg/kg every two days; i.p.), and combination DTIP with paclitaxel treatment groups, and then the mice were administered normal saline or DTIP for 21 consecutive days, paclitaxel for 4 consecutive weeks. (g) The curve of tumor growth after injection of LLC cells. (h) Mice were humanely euthanized and the tumors were resected at 42 days after cell injection. (i) Weight of resected tumors was determined in different groups.(j)H&E stained sections of representative single lung lobes harvested.(k) The number of pulmonary foci in lungs. All the results were expressed as mean ± SD. ANOVA followed by Dunnett’s test was applied for multiple comparison. *p  < 0.05, **p  < 0.01, ***p  < 0.001, NS, not significant. Gemcitabine: Gem; paclitaxel: PTX