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