Expression of vascular endothelial growth factor and its receptors KDR and Flt-1 in gastric cancer cells

Abstract

Aim: The expression of vascular endothelial growth factor (VEGF) and its receptors KDR and Flt-1 by gastric carcinoma tissues and different gastric carcinoma cell lines was detected to elucidate the molecular mechanism of this growth factor in promoting tumor growth.

Methods: The expression of VEGF, Flt-1 and KDR was determined by reverse transcription-polymerase chain reaction (RT-PCR) in gastric cancer cell lines RF-1, RF-48, AGS-1, NCI-N87, NCI-SNU-1, NCI-SNU-5, NCI-SNU-16 and KATO-III. The expression of Flt-1 and KDR in paraffin-embedded specimens of gastric cancer was determined by immunohistochemistry. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the role of VEGF in tumor cell proliferation.

Results: All 8 gastric cancer cell lines analyzed expressed VEGF(121) and VEGF(165) and six of them expressed both Flt-1 and KDR, while cell line NCI-SNU-5 expressed Flt-1 only and cell line KATOIII expressed neither Flt-1 nor KDR. The gastric carcinoma tissues expressed Flt-1 and KDR widely, with the positive rate of expression of Flt-1 and KDR being 84.6 % and 70 % respectively. The exogenous VEGF stimulated the growth of KDR-positive cell lines NCI-N87 and AGS-1 in a dose-dependent manner but exhibited no effect on the growth of KDR-negative cell line NCI-N87.

Conclusion: VEGF and its receptors KDR and Flt-1 were expressed widely in gastric carcinoma cells and the VEGF stimulated KDR-positive tumor cell growth directly. These results suggest that VEGF may play a role in promoting tumor growth and metastasis by participating in both paracrine and autocrine pathways.

Figure 1

Detection of expression of VEGF and VEGFR in eight gastric carcinoma cell lines by RT-PCR. A. VEGF was ampli-fied using primers designed to detect all known splicing vari-ants and two isoforms 531 bp and 663 bp corresponding to VEGF₁₂₁ and VEGF₁₆₅ were obtained in all cell lines. B. Flt-1 of the expected size (1212 bp) was amplified in all cell lines except KATOIII. C. KDR of the expected size (927 bp) was amplified in all cell lines except SNU-5 and KATOIII. D. GAPDH was amplified in each cell line as a positive control for RT-PCR.

Figure 2

Immunohistochemical analysis of Flt-1 and KDR on gastric carcinoma specimens ( × 400). A. Expression of Flt-1. B. Expression of KDR. below sections are respectively nega-tive controls of the left-hand side sections of the same specimens.

Figure 3

Effects of recombinant human VEGF₁₆₅ on prolifera-tion of AGS-1, NCI-N87 and KATOIIIcells. The cells were treated with concentrations of VEGF₁₆₅ indicated, and their vi-ability was assessed using MTT and expressed as mean per-centage of the untreated controls ± SE (n = 4).