Effect on NB₄ Cells Proliferation, Differentiation and Resistance to Chemotherapy Apoptosis by VEGF-C cDNA Transfection.

VEGF-C secreted by kinds of tumor cells play the key role in lymphangiogenesis via activating tyrosine kinase of VEGFR-3, which is one of most important pathway of infiltration and metastasis of tumor cells. In recent years, the effects of VEGF-C on hematological malignant cells are paid attention. In the current study, the recombinant eukaryotic expression plasmid (pcDNA3.1-VEGF-C) and the vacant pcDNA3.1 vector were introduced into the acute promyelocytic leukemia cell line- NB₄ cells (VEGFR-3⁺/VEGFR-2⁻) by lipofectamine mediation and positive clones were screened by G418. The stable expression of VEGF-C was detected by reverse transcriptase-PCR and Western blotting. The proliferation ability of NB₄/VEGF-C cells is analysed by MTT assay. After NB₄/VEGF-C cells were induced by ATRA (1μmol/L), the expression levels of C/EBP_α gene which can promote myelocytes differentiation and maturation, CD11b on cells surface and morphological variation were analysed by real-time quantitative PCR (RT-QPCR), flow cytometry analysis (FCM), Wright-Giemsa staining respectively. Furthermore, after the NB₄/VEGF-C cells were cultured together with Etoposide (200ng/ml) for 48 hours, the numbers of AnnexinV⁺/PI⁻cells (apoptotic cells) were determined by FCM to investigate the effect of VEGF-C on the cells apoptosis and expression levels of antiapoptotic bcl-2 gene in these cells were analysed by RT-QPCR. The NB₄/pcDNA3.1 cells was used as control during the experiments. From these experiments, NB₄/VEGF-C cells can stably secret active protein VEGF-C in the cell supernatants. The cell growth curve shows that the proliferation ability of NB₄/VEGF-C cells is stronger compared with NB₄/pcDNA3.1cells. The expression levels of C/EBP_α gene of NB₄/VEGF-C cells after induced by ATRA is only 1/32 that of NB₄/pcDNA3.1 cells. Morphological analysis shows that the degree of promyelocytes gradually maturing into myelocytes among NB₄/VEGF-C cells is weaker compared with the controls after induced by ATRA. After induced by Etoposide, the percent of apoptotic cells of NB₄/VEGF-C cells (7.20±2.52%) is significantly lower than that of NB₄/pcDNA3.1 cells (16.07±3.58%)( P=0.005) and the bcl-2 gene expression level of NB₄/VEGF-C cells is 2.28 times more than that of NB₄/pcDNA3.1 cells. The results implied that the VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of NB₄ cells by autocrine pathway and inhibit the NB₄cells differentiation and maturation and chemotherapy-induced apoptosis via upregulating bcl-2 gene expression. Thus, VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.