Yonsei Med J.  2017 May;58(3):581-591. 10.3349/ymj.2017.58.3.581.

Potential Antitumor Activity of SIM-89 in Non-Small Cell Lung Cancer Cells

Affiliations
  • 1Department of Pulmonary, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China. hanbh520@163.com
  • 2Department of Basic Research, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China.
  • 3Department of Biology, East China Normal University, Shanghai, China.

Abstract

PURPOSE
c-Met and its ligand, hepatocyte growth factor (HGF), play a critical role in oncogenesis and metastatic progression. The aim of this study was to identify inhibited enzymogram and to test the antitumor activity of SIM-89 (a c-Met receptor tyrosine kinase inhibitor) in non-small cell lung cancer.
MATERIALS AND METHODS
Z"²-LYTE kinase assay was employed to screen the kinase enzymogram, and mechanism of action (MOA) analysis was used to identify the inhibited kinases. Cell proliferation was then analyzed by CCK8 assay, and cell migration was determined by transwell assay. The gene expression and the phosphorylation of c-Met were examined by realtime-PCR and western blotting, respectively. Finally, the secretion of HGF was detected by ELISA assay.
RESULTS
c-Met, activated protein kinase (AMPK), and tyrosine kinase A (TRKA) were inhibited by SIM-89 with the ICâ‚…â‚€ values of 297 nmol/L, 1.31 µmol/L, and 150.2 nmol/L, respectively. SIM-89 exerted adenosine triphosphate (ATP) competitive inhibition on c-Met. Moreover, the expressions of STAT1, JAK1, and c-Met in H460 cells were decreased by SIM-89 treatment, and c-Met phosphorylation was suppressed in A549, H441, H1299, and B16F10 cells by the treatment. In addition, SIM-89 treatment significantly decreased the level of HGF, which accounted for the activation of c-Met receptor tyrosine kinase. Finally, we showed cell proliferation inhibition and cell migration suppression in H460 and H1299 cells after SIM-89 treatment.
CONCLUSION
In conclusion, SIM-89 inhibits tumor cell proliferation, migration and HGF autocrine, suggesting it's potential antitumor activity.

Keyword

c-Met; antitumor; cell proliferation; cell migration; lung cancer

MeSH Terms

Antineoplastic Agents/*pharmacology
Blotting, Western
Carcinoma, Non-Small-Cell Lung/*drug therapy/enzymology/pathology
*Cell Line, Tumor
Cell Movement/drug effects
Cell Proliferation/*drug effects
Enzyme-Linked Immunosorbent Assay
Hepatocyte Growth Factor/metabolism
Humans
Lung Neoplasms/*drug therapy/enzymology/pathology
Phosphorylation
Protein Kinase Inhibitors/*pharmacology
Proto-Oncogene Proteins c-met/*antagonists & inhibitors/*genetics/metabolism
Signal Transduction/drug effects
Xenograft Model Antitumor Assays
Antineoplastic Agents
Protein Kinase Inhibitors
Hepatocyte Growth Factor
Proto-Oncogene Proteins c-met

Figure

  • Fig. 1 Inhibition of kinase activity by Staurosporine and SIM-89. (A) IC50 test of c-Met. (B) IC50 test of AMPK. (C) IC50 test of TRKA. Compound A in figure represents SIM-89. AMPK, activated protein kinase; TRKA, tyrosine kinase A.

  • Fig. 2 Lineweaver-Burk plots and IC50 test under different concentration of ATP by SIM-89. (A) Inhibition of c-Met. (B) IC50 test of c-Met. (C) Inhibition of AMPK. (D) IC50 test of AMPK. (E) Inhibition of TRKA. (F) IC50 test of TRKA. AMPK, activated protein kinase; TRKA, tyrosine kinase A.

  • Fig. 3 Cell proliferation inhibition by SIM-89 at different time points. ANOVA analysis were perform at 24, 48, and 72 h among groups (A) B16F10. (B) A549. (C) H441. (D) H460. (E) H1299. (F) H1993.

  • Fig. 4 The effect of SIM-89 on gene expression. (A) The relative expression of Met in different cell lines after the treatment of HGF and SIM-89. (B) The expression of STAT1 in H460 after the treatment of HGF and SIM-89. (C) The expression of Met in H460 after the treatment of HGF and SIM-89. (D) The expression of JAK1 in H460 after the treatment of HGF and SIM89. Inh represents SIM-89. HGF, hepatocyte growth factor.

  • Fig. 5 The secrection of hepatocyte growth factor (HGF) in different cell lines at different time points. (A) A549. (B) H441. (C) H460. (D) H1299. (E) H1993.

  • Fig. 6 The phosphorylation of Met in different cell lines. (A) B16F10. (B) A549. (C) H441. (D) H460, (E) H1299. All the four cell lines were stimulated with HGF (40 ng/mL) and SIM-89 (5 µM). HGF, hepatocyte growth factor.

  • Fig. 7 The cell migration after the treatment of SIM-89. (A and B) A549 cells before and after SIM-89 treatment. (C and D) H441 cells before and after SIM-89 treatment. (E and F) H1993 cells before and after SIM-89 treatment. (G and H) H1299 cells before and after SIM-89 treatment.


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