Figure 1.
The chemical structure of Sorafenib and HLC-080.
Table 1.
The inhibition of HLC-080 and Sorafenib on the proliferation of various cancer cells.
Figure 2.
The anti-proliferation effect of HLC-080 in colon cancer cells
. The time-effect relationship of 2.5 µM HLC-080 and 2.5 µM Sorafenib on proliferation in HT-29 cell line (A).The effect of HLC-080 and Sorafenib on colony formation in human colon cancer cell lines. HT-29 (B), HCT-8 (C) and HCT-116 (D) cells were seeded in a 6-well plate. After 24 h, cells were treated with various concentrations of HLC-080 or Sorafenib. After 14 days of incubation, observe colony size and numbers by Giemsa stain and photograph each well. * P<0.05, ** P<0.01, *** P<0.001, compared with control group.
Table 2.
The effect of HLC-080 and Sorafenib in H22 xenograft model in vivo.
Figure 3.
Sorafenib and HLC-080 induced cell cycle arrest at G1 phase.
Cells were incubated with various concentrations of Sorafenib and HLC-080 for 96 h, cells were then harvested and stained with DAPI and analyzed by flow cytometry. Error bars indicate standard deviation. *p<0.05, **p<0.01, ***p<0.001, compared with control group.
Figure 4.
The effect of HLC-080 on EA.hy926 vascular tube formation.
EA.hy926 were seeded on matrigel in 96-well plates at 3×104 cells per well. The cells were mixed with different concentrations of HLC-080 and photographed at various time points, as described in the methods. (A) Control at 6 h showing EA.hy926 without additional stimuli. (B) HLC-080 1.0 µM added to EA.hy926, showing vascular tubes at 6 h. (C) HLC-080 5.0 µM added to EA.hy926, showing vascular tubes at 6 h. (D) Control at 24 h showing EA.hy926 without additional stimuli. (E) HLC-080 1.0 µM added to EA.hy926 formed only a small number of short, incomplete tubes at 24 h (F) HLC-080 5.0 µM added to EA.hy926, exhibited more significant effects at 24 h.
Figure 5.
The effect of HLC-080 on cell invasion in HT-29 cells.
Cells were seeded in a transwell chamber and allowed to migrate across the chamber toward cell-specific conditioned medium for 24 h. Photomicrographies of stained migrating cells were taken under brightfield illumination. Representative images are shown for HT-29 control (A) and HT-29 disposed by HLC-080 1.0, 5.0 µM (B, C). Quantification of the invasion is expressed as the number of invasive cells in five random microscopic fields per well (mean ± SD; ***p<0.001 versus control group cells) (D). Results were obtained from three separate experiments.
Figure 6.
The effect of inhibition of Raf/MEK/ERK pathway in HT-29 cells in vitro.
(A) HT-29 cells were incubated with increasing concentrations of HLC-080 for 2 h. (B) HT-29 cells were incubated with increasing concentrations of HLC-080 for 72 h. Whole-cell lysates were subjected to western blot analysis. For western blot analysis, samples were transferred to a polyvinyldine diflouride membrane by semi-wet electrophoresis and incubated with indicated primary antibody p-c-Raf (Ser259), c-Raf, p-MEK1/2 (Ser217/221), MEK1/2, p-ERK1/2 (Thr202/Tyr204), ERK1/2). Actin was used as loading and transfer control. The experiment was repeated twice and similar results were obtained.
Table 3.
HLC-080 and Sorfenib inhibits p-c-Raf (Ser259) activities in vitro.