Figure 1.
Cysteamine inhibited cell migration and invasion of pancreatic cancer cell lines.
A. For the matrigel invasion assay, cells were incubated with increasing concentrations of cysteamine in the matrigel chamber for 24 hours. The number of invaded cells on the opposite side of the membrane was counted. B and C. Wound healing assay. Cells were cultured until confluent and scratched using a sterilized yellow tip. They were incubated for 24 hours with 0–5 mM of cysteamine and the area of the wound between cell layers was measured. The black horizontal lines in figure (vehicle) depict the area at time 0 when wound was made and indicate the area of the wound when cells were scratched (B). D. For cell viability assay, pancreatic cancer cells were incubated with various concentrations of cysteamine and live cell number was counted at 24 and 48 hours. Data are expressed as mean ± S.D. of triplicate determinations. Statistical significances are shown by †: P<0.01 and ‡: P<0.001.
Figure 2.
Cysteamine inhibited MMP activity in pancreatic cancer cells.
A. To measure total MMPs activity in pancreatic cancer cell lines, cells were incubated with 0–5 mM of cysteamine for 24 hours, total protein extracted and the activity measured using fluorogenic substrate. The effect of cysteamine on MMP-9 at protein level by ELISA (B), MMP-9, MMP-12 and MMP-14 mRNA levels (C) and gelatinase zymographic activities (D) was determined after incubating pancreatic cancer cells with cysteamine for 24 hours. MMP-9 activity was determined by ELISA and mRNA by qRT-PCR. β-actin was used for a reference gene in qRT-PCR. Data represent MMP-9 protein in pg/μg total cell lysate and RFU percent ratio of mRNA/ β -actin. Data are expressed as mean ± S.D. of triplicate determinations and experiments were repeated three times. Statistical significances are shown by *: P<0.05, †: p<0.01 and ‡: P<0.001.
Figure 3.
Cysteamine inhibited MMP enzyme activities in vitro.
IC50 of MMPs was determined by incubating each MMP enzyme with various concentrations of cysteamine and batimastat in a fluorimetric assay as described in materials and methods. IC50 is the concentration of inhibitor at which 50% inhibition of proteolysis occurs.
Figure 4.
Cysteamine inhibited primary tumors and metastatic lesions in an orthotopic pancreatic cancer in vivo.
HS766T and MIA-PaCa2 cells (2×106) were implanted directly into the pancreas of 5–6 week-old female nude nu/nu mice and then peritoneal cavity and skin were closed using clips. Increasing doses of cysteamine were injected s.c. as described in materials and methods. Primary tumors and metastatic lesions of ≥ 5 mm were counted in vehicle and cysteamine treated mice. a: P<0.05, b: P<0.01 and c: P<0.05, d: P<0.001. +: moderate ascites, ++: severe ascites, –: no ascites.
Figure 5.
Cysteamine decreased metastasis and prolonged survival in an orthotopic pancreatic cancer mouse model.
HS766T and MIA-PaCa2 cells (2×106) were implanted directly into the pancreas of 5–6 week-old female nude nu/nu mice and then peritoneal cavity and skin were closed using clips. From day 4 after tumor implantation, mice were treated twice a day with vehicle or 25, 100, 250 mg/kg/day of cysteamine until the end of the experiment. The mice with tumors were sacrificed 4 weeks after cell implantation. A. A representative mouse from each treatment group harboring MIA-PaCa2 tumor is shown. Yellow arrows indicate primary tumors in the pancreas and blue triangles indicate metastatic lesions. In another experiment, control and cysteamine treated mice were followed for survival. Kaplan-Meier survival curves of mice harboring HS766T (B) and MIA-PaCa2 (C) tumors.
Figure 6.
Measurement of body weight of control and cysteamine treated mice.
Mice with HS766T tumors were treated with increasing doses of cysteamine and body weights were measured at day 10 and day 25 after tumor implantation. There was no significant difference among treatment groups.
Figure 7.
Effect of cysteamine on serum enzymes and creatinine levels.
Tumor bearing mice were treated with increasing doses of cysteamine and serum was collected from tail veins twice, i.e., pre-cysteamine treatment (day 4) and post- treatment (day 18). Liver, muscle and kidney function biomarkers were evaluated after cysteamine treatment. Alanine aminotransferase (ALT), creatine kinase (CK), aldolase, and creatinine (Cr) were measured in each group. There were no significant differences among treatment groups.
Figure 8.
Cysteamine decreased MMP activity in primary orthotopic tumors in mice.
A. At day 30 after orthotopic implantation of pancreatic cancer cells, tumors were collected, total protein extracted and MMP activity determined by using fluorogenic MMP substrate. B. MMP-9 mRNA levels were determined in total RNA extracted from tumors by qRT-PCR. β-actin was used for a reference gene. C. MMP-9 protein level was determined by ELISA. D. Zymographic assay was performed in cysteamine treated tumors. Data are expressed as mean ± S.D. of triplicate to quadruplicate determinations. Experiment was repeated two times. Statistical significances are shown by *: P<0.05, †: p<0.01 and ‡: P<0.001.