05; **, p < 0.01). Results Increased c-Met expression in MKN-45 and SGC7901 cells To determine the c-Met protein expression levels in GC, we used western blotting to examine c-Met protein in two GC cells (MKN-45 and SGC7901) and one
normal gastric mucosa cells GES-1 (Figure 1A). c-Met proteins is 3-4 fold higher in MKN-45 and SGC7901cells than GES-1 cells. SGC7901 cells express slightly more c-Met than MKN-45 cells (Figure 1B). The optical densities (OD’s) of the Western blot bands were measured using ImageJ. The OD for each band was normalized to β-actin. MKN-45 and SGC7901 had a 0.94 and 1.27 fold increase in the expression of c-Met AZD2281 molecular weight over the control, but only 0.34 fold increased in GES-1. Figure 1 Overexpression of c-Met in castric carcinoma cell lines. Lysates (80 μg/lane) from normal gastric mucosa cells GES-1 and GC cell lines MKN-45 and SGC7901 were analyzed for c-Met protein level by western blot using an anti-c-Met antibody and an anti- β-actin antibody (loading control) (Figure 1A). The optical densities (OD’s) of the Western blot
bands were measured using Image J (Figure 1B). IT anti-c-Met/PE38KDEL selleck inhibitor inhibited cell proliferation and protein synthesis GC cells have significantly higher c-Met protein levels than normal gastric mucosa cells, therefore we tried to determine if IT anti-c-Met/PE38KDEL has GC-specific effects. The anti-proliferative effect of IT anti-c-Met/PE38KDEL on GES-1, MKN-45 and SGC7901 cells was measured using CCK8 kit. Cells were harvested at 24 or 48 hr after IT
treatment. As shown in Figure 2, IT inhibited GC cell growth in a time- AZD8931 purchase and dose- dependent manner. 1, 10 and 100 ng/ml of IT caused a dramatic growth inhibition in MKN-45 and SGC7901 cells (P< 0.01). 48 hr of IT treatment (100 ng/ml) resulted in a growth inhibition of 30% in GES-1 cells (Figure 2A). However, inhibitions of 75% and 95% were observed in MKN-45 and SGC7901 cells (Figure 2B and 2C), respectively. Further, we found that there is a strong correlation between c-Met expression and in vitro immunotoxin efficacy. Figure 2 IT anti-c-Met/PE38KDEL induced inhibition of cell proliferation. Cell growth inhibition as a function of varying concentrations of IT (expressed as a percentage of untreated cells), RNA Synthesis inhibitor Normal cell GES-1 (A), GC cells MKN-45 (B) and SGC7901 (C) were treated with various concentrations of IT for 24 hr and 48 hr. Given the high c-MET levels in MKN-45 and SGC7910 cell lines, we hypothesize that anti-c-Met/PE38KDEL can attenuate cancer cell growth through inhibition of protein synthesis via c-Met inhibition. The effects of anti-c-Met/PE38KDEL on protein synthesis in GES-1, MKN-45 and SGC7901 cells are shown in Figure 3. The IT’s IC50 value on GES-1 cells was approximately 120 ng/ml. However, IT induced more potent inhibitions of protein synthesis in MKN-45 and SGC7901 cells, with IC50 values of 5.34 ng/ml and 0.83 ng/ml, respectively.