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. 2015 Oct;36(10):1266-76.
doi: 10.1038/aps.2015.25. Epub 2015 Jun 1.

DCLAK11, a multi-tyrosine kinase inhibitor, exhibits potent antitumor and antiangiogenic activity in vitro

Xiao-Bin Guo  1   2 , Xian-Jie Chen  3 , Lin-Jiang Tong  2 , Xia Peng  2 , Min Huang  2 , Hong-Chun Liu  2 , Hong Liu  3 , Jian Ding  2
Affiliations

DCLAK11, a multi-tyrosine kinase inhibitor, exhibits potent antitumor and antiangiogenic activity in vitro

Xiao-Bin Guo et al. Acta Pharmacol Sin. 2015 Oct.

Abstract

Aim: To investigate the molecular targets of DCLAK11, a novel compound discovered from a series of substituted pyridin-3-amine derivatives, and to characterize its anti-tumor properties in vitro.

Methods: Kinase inhibition was measured by an ELISA assay. Cell viability was assessed with an SRB or a CCK8 assay. The alterations induced by kinase signaling proteins in cancer cells were detected by Western blot. Apoptosis was determined by an Annexin V-PI assay. The following assays were used to evaluate the impact on angiogenesis: wound-healing, Transwell, tube formation and microvessel outgrowth from rat aortic rings.

Results: DCLAK11 was a multi-targeted kinase inhibitor that primarily inhibited the EGFR, HER2, and VEGFR2 tyrosine kinases with IC50 value of 6.5, 18, and 31 nmol/L, respectively. DCLAK11 potently inhibited the proliferation of EGFR- and HER2-driven cancer cells: its IC50 value was 12 and 22 nmol/L, respectively, in HCC827 and HCC4006 cells with EGFR exon deletions, and 19 and 81 nmol/L, respectively, in NCI-N87 and BT474 cells with HER2 amplification. Consistently, DCLAK11 blocked the EGFR and HER2 signaling in cancer cells with either an EGFR or a HER2 aberration. Furthermore, DCLAK11 effectively induced EGFR/HER2-driven cell apoptosis. Moreover, DCLAK11 exhibited anti-angiogenic activity, as shown by its inhibitory effect on the proliferation, migration and tube formation of human umbilical vascular endothelial cells and the microvessel outgrowth of rat aortic rings.

Conclusions: DCLAK11 is a multi-targeted kinase inhibitor with remarkable potency against tyrosine kinases EGFR, HER2 and VEGFR2, which confirms its potent anti-cancer activity in EGFR- and HER2-addicted cancers and its anti-angiogenic activity.

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Figures

Figure 1
Figure 1
Chemical structures of DCLAK11.
Figure 2
Figure 2
DCLAK11 blocks EGFR phosphorylation and downstream signaling in HCC827 (A) and HCC4006 (B) cells with concentration-dependent and time-dependent manner. Cells were cultured in the presence of different concentrations of DCLAK11 for 2 h or treated with indicated concentrations of DCLAK11 for increasing durations (0.25–6 h), then whole-cell lysates were assayed by Western blots.
Figure 3
Figure 3
HCC827 (A) and HCC4006 (B) cells were treated with increasing doses of DCLAK11 for 48 h and apoptotic rate was detected by flow cytometry with Annexin V-PI staining. Data are shown as mean±SD from three independent experiments. cP<0.01 vs control. Western blots were performed to observe the cleaved caspase-3 (Asp175), caspase-3, cleaved PARP and full-length PARP protein expression in HCC827 (C) and HCC4006 (D) cells respectively. The more cleaved caspase-3 and cleaved PARP expression represents for the higher level of apoptosis. Representative data are shown.
Figure 4
Figure 4
DCLAK11 inhibits HER2 signaling and induces apoptosis in cancer cells with HER2 amplification. (A, B) Concentration- and time-dependent inhibitive activity of DCLAK11 on HER2, AKT, and Erk1/2 phosphorylation in NCI-N87 (A) and BT474 (B) cells. Cells treated with increasing concentrations of DCLAK11 for 2 h or treated with indicated concentrations of DCLAK11 for increasing durations (0.25–6 h) were lysated and subjected to Western blot analysis.
Figure 5
Figure 5
DCLAK11 induces apoptosis in cancer cells with HER2 amplification. (A, B) N87 (A) and BT474 (B) cells were treated with increasing concentrations of DCLAK11 and apoptotic rate was detected by flow cytometry with Annexin V-PI staining. Data are shown as mean±SD from three independent experiments. bP<0.05, cP<0.01 vs control. (C, D) Western blots were performed to observe the cleaved caspase-3 (Asp175), caspase-3, cleaved PARP and full-length PARP protein expression in N87 (C) and BT474 (D) cells, respectively. The more cleaved caspase-3 and cleaved PARP expression represents for the higher level of apoptosis. Representative data are shown.
Figure 6
Figure 6
DCLAK11 exhibits antiangiogenic activities. (A) DCLAK11 inhibits the VEGF-stimulated VEGFR2 phosphorylation and signal transduction. HUVECs were starved, then incubated with indicated concentrations of DCLAK11 for 6 h, and VEGF165 (50 ng/mL) was added to the cultures during the last 10 min. Protein samples were subjected to Western blot analysis. Representative data are shown. (B) Effects of DCLAK11 on the migratory ability of HUVECs (wound-healing test). HUVECs were grown to confluence in complete media, wound were made using 96 well WoundMaker and culture in absence or presence of the different concentrations of DCLAK11. (C) DCLAK11 inhibits HUVEC migration in a transwell migration assay. HUVECs treated with various concentrations of DCLAK11 were seeded in both chambers. The upper chamber was filled with serum-free medium, and the bottom chamber was filled with the complete medium containing 20% FBS. (D) DCLAK11 inhibits tube formation of HUVECs. Cells were placed in 96-well plates coated with Matrigel. The tubular structures were photographed after 8 h treatment of DCLAK11. (E) Effect of DCLAK11 on sprouting from rat aortic segments. Rat aortic segments were cultured on Matrigel and treated various concentrations of DCLAK11 for 7 d.
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