| BACKGROUNDRecent observations have suggested that angiogenesis and immunosuppression are the main biological mechanisms responsible for leukemia progression. Elevated angiogenesis has been proven to be associated with malignant growth, progress and poor prognosis in human leukemia. Moreover, the neovasculogenesis could resist to chemotherapy and provide some paracrine cytokines to maintain residual leukemic cell growth. Vascular endothelial growth factor (VEGF) is the most important pro-angiogenic factor, produced by many human hematologic malignancies. Recently, it has become clear that angiogenesis mediated by VEGF plays an important role in the pathophysiology of leukemia. VEGF mRNA and protein expressions often correlate positively with tumor angiogenesis, so VEGF maybe a potential target for anti-angiogenic therapy of leukemia. It has been reported that immune deficiency or impairment is a common phenomenon in patients with cancer, and tumor cells have developed a variety of cellular and molecular mechanisms to escape from antitumor immune responses. Tumor-induced immune defects were known to occur in all major branches of the immune system. Dendritic cells (DC) play a central role in antitumor immune responses. Abnormal differentiation of DC and its inability to stimulate T cells are the important factors in tumor escaping from immune-system supervision. It has been shown that the function of DC is impaired and the quantity of DC is decreased in a leukemia-bearing host. Recent research has shown that VEGF may also play an immunosuppressant role by inhibiting the maturation of DC from hematopoietic progenitors in addition to its angiogenic activity. OBJECTIVESBased on VEGF dual roles of angiogenic activity and immune inhibition, we expected therapeutic blockade of VEGF action might inhibit leukemic angiogenesis as well as improve prospects for immunotherapy. In this study, we observed the effect of VEGF antisense phosphorothioated oligodeoxynucleotide (AS-ODN) on the expression of VEGF in human leukemic cell lines (HL-60 and K562 cells), investigated whether in vitro transfection of VEGF AS-ODN could improve the functional maturation of DC derived from K562 leukemic cell lines. Furthermore we explored the role of NF-kappa B in the activation of DC mediated anticancer immunity through reducing VEGF. These studies would provide the experimental foundation for combination of antiangiogenic and immunotherapy in leukemia with VEGF AS-ODN. METHODS1. VEGF AS-ODN inhibited VEGF expression in leukemic cells1.1 Leukemic cell lines: HL-60 and K5621.2 Methods: The mRNA leveles of VEGF and its receptors were measured by RT-PCR, the protein expressiones of VEGF and its receptors were observed by immunohistochemistry. ELISA assay was used to detect the VEGF protein secretion of leukemic cells. VEGF AS-ODN transfected leukemic cells:The proliferation of leukemic cells was observed in various AS-ODN concentration and different culture time. Based on these results, the optimal ODN concentration and culture time were selected for following experiment. Groups including AS-ODN group, Missense-ODN(MS-ODN)group, control group without ODN were set. All cells were cultured in RPMI 1640 culture media at 37 ℃ and 5% CO2 conditions. Detected the VEGF level of leukemic cells: VEGF mRNA and protein expression of leukemic cells transfected with VEGF AS-ODN were measured by RT-PCR, immunohistochemistry and ELISA, and analyzed their changes.2. Investigated the effect of VEGF AS-ODN on the leukemic DC function and its possible mechanism2.1 Observed the change of VEGF expression in K562 cells after transfected with VEGF AS-ODN:After transfected with VEGF AS-ODN (5 μmol/L) for 24 h, VEGF mRNA and protein of k562 cells were detected by RT-PCR and ELISA, and analyzed their changes.2.2 Induction of DC generation from K562 transfected with VEGF AS-ODNAfter transfected with VEGF AS-ODN for 24 h, K562 cells were inoculated in RPMI 1...
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