| Background and objective:The importance of bone marrow angiogenesis in carcinogenesis and progression of acute leukemia has been widely recognized in recent years, but the mechanisms are still not completely clarified. BM microenvironment and leukemic blasts contribute equally to angiogenesis by secreting different angiogenic growth factors and mediators. Increased vascularization is not only seen in acute myeloid leukemia (AML) but also in acute lymphoblastic leukemia (ALL). Angiogenesis is a complexed physiopathologic process, not only hypoxia, oncogenes and anti-oncogenes, but also other cytokines are involved. As an important translation regulating factor, HIF-1α(hypoxia inducible factor-1α) activates the transcription of multiple genes encoding angiogenic growth factors and cytokines, which may be involved in the angiogenesis in acute leukemia.The past research indicates that the abnormal expression of HIF-1αwas found in the carcinogenesis and progression of many solid tumors, which is positively related to the angiogenesis and prognosis; and the inactivation of oncogenes, hypoxia and other cytokines contribute to it. The increased HIF-1αexpression is also found in acute leukemia, but its relationship with angiogenesis still lacks direct evidence. On the basis of our past research, this study aims to investigate the expression of HIF-1αand its downstream angiogenic factor-VEGF in acute leukemia, the relationship between HIF-1αand bone marrow MVD (micro vessel density) in acute leukemia, and the transduction pathways involved in acute leukemia bone marrow angiogenesis. What's more, we want to study the anti-angiogenesis effect of ginsenoside Rg3 and the involved signal transduction pathways of HIF-1α, so as to provide reliable experimental foundation for the role of HIF-1αin the bone marrow micro-environment.Methods:1. The relationship between angiogenesis and expression of HIF-1αin acute leukemia bone marrow and bone marrow stromal cells (BMSCs).The bone marrow biopsy specimen and BMSCs were obtained from 15 patients with acute myeloid leukemia (AML),10 patients with acute lymphocytic leukemia (ALL)and 10 normal people.(1) The expression of HIF-1α, VEGF and MVD in bone marrow biopsy specimen was detected by means of SP immunohistochemical method.(2) Isolate and cultivate the BMSCs of acute leukemia and normal people.(3) HIF-1αand VEGF proteins in acute leukemia BMSCs were detected by western blot.2. The anti-angiogenesis effect of ginsenoside Rg3 in acute leukemia bone marrow and related mechanisms.Thirty-one patients with acute leukemia were divided into AML and ALL groups, another twenty-eight healthy volunteers were served as control group.(1) Isolate and cultivate the BMSCs of acute leukemia and normal people(2) Groups:BMSCs were exposed to ginsenoside Rg3 at a concentration of 20,40,60, 80, 100mg/L respectively, and the processing time were 6h,12h,24h,48h respectively.(3) The appropriate time and concentration of ginsenoside Rg3 were assessed by MTT method.(4) The BMSCs were treated with ginsenoside Rg3, and mRNA of HIF-1αand VEGF were detected by RT-PCR.(5) The changes of the expression of HIF-1α, VEGF, p-Akt and p-ERK proteins were measured by western blot and immunofluorescence methods.Results:1. The relationship between angiogenesis and expression of HIF-1αin acute leukemia bone marrow and BMSCs.(1) The expression of HIF-1αin AML and ALL groups was significantly higher than that in normal group (P<0.05), there was no significant difference between AML and ALL groups (P>0.05). (2) VEGF expression in AML and ALL groups was significantly higher than that in normal group (P<0.05), there was no significant difference between AML and ALL groups (P>0.05). (3) The micro vessel density (MVD) in AML and ALL groups were significantly higher than those in normal group (P<0.05), there was no significant difference between AML and ALL groups (P>0.05). (4) In acute leukemia bone marrow biopsy specimen, HIF-la expression was positively correlated with the expression of VEGF and MVD (r=0.848, P<0.05; r=0.211, P<0.05), and the expression of VEGF was positively correlated with the expression of MVD (r=0.249, P< 0.05). (5) In acute leukemia BMSCs, HIF-1αprotein was positively correlated with the expression of VEGF and MVD (r=0.683, P<0.05; r=374, P<0.05), and the expression of VEGF was positively correlated with the expression of MVD (r=0.491, P<0.05).2. The anti-angiogenesis effect of ginsenoside Rg3 in acute leukemia bone marrow and related mechanism.After the addition of ginsenoside Rg3, MTT showed that Rg3 reduced the vitality of BMSCs in time-dependent and dose-dependent manner, and the appropriate concentration was 40ug/mL and the processing time was 24 h, respectively. Rg3 not only inhibited HIF-1αand VEGF mRNA expression but also protein expression(P<0.05). Rg3 could down-regulate phosphorylation of Akt and ERK, which were the key proteins of PI3K/Akt and MAPK signal transduction pathways respectively (P<0.05).Conclusion:1. The expression of MVD in acute leukemia was significantly higher than the normal people, it indicates angiogenesis plays an important role in the carcinogenesis and progression in acute leukemia.2. Overexpression of HIF-1αwas found in acute leukemia bone marrow biopsy specimen and BMSCs. HIF-1αmay induce angiogenesis in acute leukemia by up-regulating the transcription of VEGF gene. Over expression of HIF-1αin BMSCs may play an important role in the carcinogenesis and aggression in acute leukemia.3. The results indicate ginsenoside Rg3 may reduced VEGF expression in human bone marrow stromal cells through hypoxia-inducible factor la (HIF-la) via MAPK and PI3K/Akt signal pathways, which may be one of the mechanisms of anti-angiogenesis effect of Rg3. These results demonstrate a new function of ginsenoside Rg3 in inhibiting angiogenesis in acute leukemia.
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