A Preliminary Study Of Chemosensitivity Affected By Vascular Endothelial Growth Factor In Lung Cancer Cells

Objective To investigate the effect by exogenous vascular endothelial growth factor on proliferation, apoptosis, chemosensitivity in cultured A549 lung cancer cells and the involvement of VEGF-VEGFR2-PI3K/Akt pathway, hoping to provide the experimental data of biological effects of VEGF on lung cancer cells.Methods Culture A549 lung cancer cell with different concentrations of exogenous VEGF in the medium containing different concentrations of serum, then the cell growth was monitored by MTT assay. The cells were treated with anticancer drugs alone or in combination of exogenous VEGF. The proliferation and apoptosis was measured by MTT assay. The distribution of cell cycle or the apoptosis rate was analysed by flow cytometry. After inhibition VEGF-VEGFR2-PI3K/Akt signaling pathway, the chemosensitivity was evaluated by MTT assay. The phosphorylation of Akt was detected by Western blot.Results Culturing the cells with medium containing 10% or 0.5% serum, the exogenous VEGF did not change the growth rates significantly. When A549 cells in the serum free culture, applying exogenous VEGF, proliferation and survival could be stimulated, and the cell population in G1 phase was decreased while the cell population in S and G2 phase was increased. Compared to chemotherapy alone, the survival of the cells was improved when it treated with anticancer drugs together with exogenous VEGF, which was suppressed by VEGF-VEGFR-PI3K/Akt inhibitors. Flow cytometry analysis indicated the proportion of apoptotic cells was lesser in the VEGF plus chemotherapy group. Western blot revealed the level of phosphorylated Akt reached the highest after the cells were incubated with exogenous VEGF for 30 minutes or 2 hours.Conclusion 1. Exogenous VEGF could prolong survival of A549 human lung cancer cells cultured cells in serum-free medium.2. Exogenous VEGF could reduce chemotherapy-induced apoptosis of A549 human lung cancer cells.3. VEGF-VEGFR-PI3K/Akt signaling pathway was involved in the protection against chemotherapy toxicity by exogenous VEGF in A549 human lung cancer cells.Objective To study the effect of vector-mediated RNA interference targeting VEGF combined with chemotherapy on cell proliferation and survival in A549 human lung cancer cell line, providing some data of RNA interference therapy for lung cancer.Methods The VEGF-specific shRNA-expressing plasmid pRS-VEGF-shRNA was amplified in E. coli DH5α. The shRNA sequence targeting human VEGF was confirmed by DNA sequencing analysis. After the plasmid was transfected into A549 cells by liposome, VEGF mRNA expression and VEGF protein concentration was detected by RT-PCR and ELISA separately. The cytotoxity of RNAi plasmid alone or in combination with anticancer drugs was evaluated by MTT assay.Results The sequencing proved the targeting sequence was identical to part of VEGF cDNA. RT-PCR showed the down-regulation of VEGF mRNA expression. ELISA showed the concentration of VEGF protein in the supernatant of the medium was decreased. MTT assay indicated the growth of the plasmid transfected cells was inhibited. After the cells were treated with plasmid and anticancer drugs, compared to the control group, the number of apoptotic cells increased.Conclusion 1. Plasmid pRS-VEGF-shRNA could be successfully transfected into A549 cells, resulting in the inhibition of VEGF mRNA expression and VEGF protein secretion.2. After the expression of endogenous VEGF was suppressed, the growth of A549 cells was affected.3. The cytotoxicity of anticancer drugs was enhanced when the secretion of endogenous VEGF was down-regulated by plasmid-mediated RNAi in A549 lung cancer cells.