Construction of oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor gene and its lethal effect to gastric cancer cells

Abstract

Objective To construct an double-regulated oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor (HGFK1) gene which exerts anti-tumor and anti-angiogenesis effect and observe its lethal effect to gastric cancer cells. Methods Adenoviral E1a and E1b genes were controlled by Human telomerase reverse transcriptase promoter (hTERT) and hypoxia regulatory element (HRE). TH-Ad-HGFK1-EGFP or TH-Ad-EGFP was constructed by inserting HGFK1 gene into the viral genome or not. The adenovirus was divided into three groups: the experimental group A (TH-Ad-HGFK1-EGFP), the experimental group B (TH-Ad-EGFP) and the control group C (Ad-EGFP). Gastric cancer cells SGC7901 and the normal human fibroblasts cell HF were infected by adenovirus, then the proliferation of adenovirus was counted by the half-cell culture infection dose (TCID50) method. Methyl thiazolyl tetrazolium (MTT) method was used to detect the inhibitory effect. 4, 6-Diamidino-2 -Phenylindole, Dihydrochloride (DAPI) method and Terminal -deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) method was used to observe the cell apoptosis. Results TH-Ad-HGFK1-EGFP and TH-Ad-EGFP was confirmed constructed successfully by DNA sequencing and PCR analyses. All the titer of the adenovirus was 2×1010 pfu/ml. The TCID50 results showed the proliferation of group A and group B in SGC7901 cells was 1.47×104 and 1.60×104 fold, which were significantly higher than that in group C. However in HF cells, the fold change of replication of group A and group B was only 110 and 124, which was significantly lower than that in group C. Group A showed potent inhibitory effect on the growth of SC7901 cells, but Group B and group C showed no obviously inhibitory effect. The inhibition ratio of group A (MOI=100, 72 h and 96 h) on SGC7901 cells was (71.34±8.27)%, (74.56±1.78)%, which had significant difference compared with those in group B (23.40±2.29)%, (31.94±6.62) % (P=0.002, P=0.003) or group C (21.18±2.32%, 21.18±2.32%)(P=0.003, P=0.000). The inhibition ratio of group A and group B (MOI=100, 96 h) on HF cells was 18.94±0.88% and 22.84±3.34%, which were significant lower than that in group C [(53.25±3.50)%, P=0.003, P=0.001]. Group A also showed cell apoptosis on SGC7901 cells (18.66±4.04%), which had significant difference in other groups. Conclusion The oncolytic adenovirus TH-Ad-HGFK1-EGFP showed remarkably replication capability, promoted apoptosis and inhibited proliferation of SGC7901. Key words: Gastric cancer; Kringle 1 domain of hepatocyte growth factor; Oncolytic adenovirus; SGC7901