The Effects Of HIF-1α& Downstream VEGF On The Proliferation Of CML Cells And The Synergistic Therapeutic Strategies Of T315I Mutant CML

Part I The Effect of HIF-1α on the Cell Proliferation of ChronicMyeloid Leukemia(CML) in Anaerobic ConditionsObjectives:(1) To detect the expression level of HIF-1αthrough the acquisition of bone marrow specimen of both CML patients and healthy volunteers.(2)To show the difference between the expression levels of HIF-1αm RNA in the two groups.(3)To conduct an analysis of the effect of the HIF-1αgene on the cell proliferation of CML and its possible mechanism.Method:To detect the difference of m RNA expression levels in the bone marrow between CML patients and normal patients through RT-PCR. To confirm the silencing of HIF-1αin the K562 cell line by Si RNA through RT-PCR. To determine the ability of proliferation and colony formation of K562 cell line after the silencing of HIF-1αthrough CCK-8.To determine the levels of p21,p53 and m RNA in the K562 cell line after the silencing of HIF-1α through RT-PCR. To determine the expression levels of p21 protein and p53 protein after the silencing of HIF-1αthrough the westernblot method.Results:1.The expression level of HIF-1α m RNA in the bone marrow of CML patients was significantly higher than that of control group.2.HIF-1α in silencing K562 cells inhibited the proliferation of the K562 cell line(P<0.05).3. HIF-1α in silencing K562 cells inhibited the colony formation of the K562 cell line(P<0.05).4. The expression of both m RNA and protein of p21 in the silencing K562 cell line was inhibited through its HIF-1αgene, while the expression of m RNA & protein of p53 was unaffected.Conclusion:(1) The expression level of HIF-1αin the bone marrow of patients with CML was significantly higher than that in normal people.(2) HIF-1α can promote the proliferation of CML cells by raising p21 expression. PART II The Effects of VEGF on the Cell Proliferation of ChronicMyeloid Leukemia(CLM) in Anaerobic ConditionsObjectives:To investigate expression of vascular endothelial growth factor(VEGF) in the serum of patients with chronic myeloid leukemia and its effects on cell proliferation.Method:Quantitative detection of serum VEGF of 12 CML patients(7 chronic-phase, 5 acute-phase) was conducted by enzyme-linked immunosorbent assay(ELISA). m RNA levels in VEGF were detected using the RT-PCR method after VEGF transfected K562 cells. The effects of transfection on K562 cell proliferation were observed.Results:1. There was a significant difference in the levels of serum VEGF in CML patients(115.8±32.72 ng/L) and the controls(391.95±91.21 ng/L)(P<0.05), showing that the expression of VEGF was higher in CML patients.2. Detection of serum VEGF levels in CML patients at different clinical stages showed that the level of serum VEGF in chronic phase patients(267.4±58.89 ng/L) was significantly lower than that in acute transformation phase patients(473±51.12 ng/L)(P<0.05).3. Compared to the control group, there was a significant decrease in the level of VEGF m RNA after the transfection of antisense nucleotide by K562 cells(P<0.01). As incubation continued, cell proliferation rate was significantly slower than the control group after the transfection of antisense nucleotide into K562 cells(P<0.05). Meanwhile, the transfection of sense nucleotide did not result in any significant difference in the rates of cell proliferation between CML and control groups, suggesting inhibition of K562 cell proliferation by low level expression of VEGF.4. The addition of exogenous VEGF165 to the cells transfected with antisense nucleotides could partially counteract the inhibition effect of K562 cell growth by antisense nucleotides(P<0.05), further suggesting the promotion effect of VEGF on cell proliferation.Conclusions:1. VEGF can promote the proliferation of CML cell line K562.2. VEGF can help monitor the efficacy of CML and predict its prognosis.Part III The Synergistic Strategies of T315 I Mutant CMLTherapyObjectives:To analyze the joint therapeutic effects of any two of the four drugs(LBH589+ATO, LBH589+17-AAG, 17-AAG+ATO and LBH589+Vx-680) on Ba/F3-P210-T315 I, as well as their synergistic effects compared to those of single drugs.Method:Examination by CCK-8 of the inhibition effects on Ba/F3-P210-T315 I and Ba/F3-P210 cell lines of single drugs of LBH589, ATO, 17-AAG and Vx-680 as well as those of combinations of LBH589+ATO, LBH589+17-AAG, 17-AAG+ATO and LBH589+Vx-680. The detection of the mono-therapeutic and combined therapeutic effects of the four drugs on the promotion of apoptosis of Ba/F3-P210-T315 I and Ba/F3-P210 cell lines, using annexin V PI kit and the flow cytometry technique. With the use of the Westernblot method detected the expression level of Aurora kinase after LBH589 acted on Ba/F3-P210-T315 I cell line.Results:1. The use of single drugs of LBH589,ATO,17-AAG and Vx-680 all inhibited the proliferation of Ba/F3-P210-T315 I cells. The effect was most obvious in Vx-680, whose inhibition rate grew exponentially as the drug concentration increased, followed by ATO. The four drugs’ IC50 concentration differed though: LBH589(24 nmol/L),ATO(480 nmol/L),17-AAG(60 nmol/L) and Vx-680(32 nmol/L).2. The four drugs also had inhibitory effects on Ba/F3-P210 cell proliferation, with the most obvious effect in Vx-680. IC50 concentration also differed among the four drugs: LBH589(24 nmol/L),ATO(480 nmol/L),17-AAG(60 nmol/L) and Vx-680(32 nmol/L).3. As IC50 concentration increased, the inhibition effects of the combined uses of LBH589+ATO, LBH589+17-AAG, 17-AAG+ATO and LBH589+Vx-680 differed on the proliferation of Ba/F3-P210-T315 I cell line, with the combination of LBH589+Vx-680 having the optimum inhibition effect.4. Using the annexin V PI kit and the flow cytometry analysis, it was found that the pro-apoptotic efficiency was most obvious with the combination of LBH589+Vx-680, followed by ATO+17-AAG and LBH589+17-AAG.5. The expression level of Aurora A kinase was significantly reduced after Ba/F3-P210-T315 I cells were affected by LBH589 with IC50 concentration, whereas the expression level of Aurora B kinase did not change significantly.Conclusions:1. The independent uses of LBH589, ATO, 17-AAG and Vx-680 all inhibited the proliferation of both Ba/F3-P210-T315 I and Ba/F3-P210 cells.2. The combined use of LBH589+Vx-680 had the best proliferation inhibition effect and pro-apoptotic effect on Ba/F3-P210-T315 I cells out of the four combinations of LBH589+ATO, LBH589+17-AAG, 17-AAG+ATO and LBH589+Vx-680.3. Such proliferation inhibition effect and pro-apoptotic effect may be achieved by the inhibition of Aurora A kinase.