Effects Of Recombinant Immunotoxin Anti-c-met/pe38kdel On Proliferation And Apoptosis Of Gastric Cancer Cell Lines

GC shows multiple gene changes, including both oncogenes and tumor suppressor genes. Among them, the one most frequently implicated in GC is the abnormal expression and amplification of the c-met gene, which expressed in most GC cell lines[4]. The proto-oncogene c-met which has been identified as the receptor of hepatocyte growth factor (HGF, also known as scatter factor, which has been linked to the malignant potential of GC) encodes a 190 kDa heterodimeric transmembrane tyrosine kinase, binding with HGF triggers tyrosine autophosphorylation of the intracellular domain of met[5] and induces pleiotropic responses such as proliferation, motility, morphogenesis, and angiogenesis in many types of cells including various tumor cells. Some study has proved that amplification of the proto-oncogene c-met are found in nearly 74% of GC[6]. HGF with its receptor may also play an important role in the progression and metastasis of GC[7]. Thus, c-met is considered a good candidate for targeted therapeutic intervention in this type of tumor.Objective To investigate and evaluate the effects of recombinant immunotoxin (IT) anti-c-Met/PE38KDEL on proliferation and apoptosis of gastric cancer(GC) cell lines MKN-45, SGC7901 and the normal gastric mucosa cell line GES-1 and the mechanism of its action. Methods First, we detect the express of c-Met protein of cells by Western Blot. Cell lines MKN-45, SGC7901 and GES-1 were incubated with different concentrations of IT. Cell proliferation was determined by CCK-8 assay (cell counting kit-8); [3H]-leucine incorporation assay was used to evaluated the ability of inhibit protein synthesis of IT; Cell apoptosis was analysed by flow cytometric; Caspase activities were measured with the designated caspase colorimetric protease assay and by Western Blot. Result 1.The expression of c-met was assayed in the GC cell lines MKN-45 , SGC7901 and the normal gastric mucosa cells GES-1. cellular protein extracts were examined by Western blotting analysis. Substantially grown under similar conditions, more c-met was found to be expressed by SGC7901 and MKN-45 cells than by GES-1 celle , and moreover, we also see that SGC7901 cells expressed slightly more c-met than MKN-45 cells (0.942 vs 1.204), but P>0.05. 2. IT inhibited the growth of GC cells in a time- and dose- dependent manner, 1, 10 and 100μg/ml IT caused a dramatic growth inhibition in MKN-45 and SGC7901 cells(P<0.01). After 48hr, the cell inhibition rate in MKN-45 and SGC7901 cells is about 75% and 95%, but only 30% in GES-1 cells. The effect of increasing concentrations of anti-c-Met/PE38KDEL on protein synthesis in GES-1, MKN-45 and SGC7901 cells were detacted, compared with IC50 value of approximately 120 ng/ml in GES-1 cells, the IT induced a time- and dose-dependent inhibition of protein synthesis with an IC50 value of 5.34 ng/ml and 0.83 ng/ml after 24 hr in MKN-45 and SGC7901 cells. 3. flow cytometric analysis were performed to determine whether the antiproliferative effect of IT was due to cell cycle arrest and/or induce cells apoptosis. It revealed that IT induced apoptosis rather than the cell cycles arrest to play its anticancer effect. 4. MKN-45 and SGC7901 cells showed 3.70, and 5.02-fold increases in caspase-3 enzyme activity as compared to untreated controls at 24 hr of IT treatment(P<0.01), while GES-1 resulted in 2.03-fold increases in caspase-3 enzyme activity (P<0.05) . Caspase-8 enzyme activity in two GC cell lines also increased(P<0.05), but not increased as high as caspase-3 . we also investigated activity of caspase-3 by Western blotting analysis. After 24 hr of IT treatment, procaspase-3 was proteolytically cleaved in a dose-dependent manner, followed by the appearance of the active caspase-3 fragment . In untreated control cells(0 ng/ml), little caspase-3 was detected.