Expression Of Angiopoietin-1 In Multiple Myeloma And The Anti-myeloma Effect Of Artesunate And β-elemene

Part one Expression of angiopoietin-1 in multiple myeloma and its clinical significanceObjective:Bone marrow angiogenesis plays an important role in the development and prognosis of multiple myeloma (MM). Angiopoietin-1 (Ang-1) is the most potent stimulatory factor in angiogenesis. The present study was designed to examine the expression of Ang-1 mRNA and its receptor, Tie-2, mRNA in MM patients and RPMI-8226 cells, and to analyze the clinical significance of Ang-1 expression and its correlation with the tumorigenes and development of MM.Methods:Reverse transcription polymerase chain reaction (RT-PCR) and Western blot was performed to detect the expression level of Ang-1 and Tie-2 mRNA in RPMI-8226 cell line cells and in bone marrow samples from 112 MM patients and 24 control people. The relationships of Ang-1 expression level with microvessel density in the bone marrow of MM patients was studied.The different expression levels of Ang-1 in different groups and stages were analyzed.Results:The positive rate and expression level of Ang-1 were significantly higher in MM patients than that in control people (P<0.05). The positive rate of Ang-1 was not significantly different between newly diagnosed and relapsed/refractory MM patients (P>0.05), but the expression level of Ang-1 was significantly higher in relapsed/refractory MM patients than that in newly diagnosed MM patients (P<0.05). Tie-2 was detected only in 12 MM patients and was not detected neither in control group nor in RPMI-8226 cells. Microvessel density in bone marrow samples were significantly higher in MM patients than that in control people (25.21±0.8 vs 5.23±0.2,P<0.01). Microvessel density in bone marrow samples were higher in Ang-1-positive MM patients than that in Ang-1-negative MM patients (32.98±1.7 vs 16.55±1.3, P<0.05). The positive rate of Ang-1 protein was not significantly different between patients in stage II MM and patients stage III MM (52.1% vs 60.9%, P>0.05), but the expression level of Ang-1 protein was significantly higher in the patients in stageⅢthan that in stageⅡMM (0.4±0.07 vs 0.22±0.04, P<0.05). In the newly diagnosed MM patients, the positive rate of Ang-1 protein in PD patients was significantly higher than in PR and MR patients (19.1%vs 70.0%, P<0.01).Conclusion:High expression of Ang-1 was detected in MM patients and RPMI-8226 cells. The Ang-1 expression was associated with the staging and prognosis of MM, and Ang-1 might be a target for the therapy of MM.Part two Artesunate inhibiting the angiogenesis induced by myeloma cellObjective:To study the effect of artesunate (ART) on angiogenesis induced by human myeloma cell line PRMI- 8226 cells and to investigate its mechanism.Methods:MTT colorimetric assay was performed to observe the effect of artesunate on the proliferation of RPMI-8226 cells. The inhibition effect of artesunate on angiogenesis was studied by using the model of migration and tube-formation of HUVEC, angiogenesis in chick chorioallantoic membrane as well as aortic sprouting assay in fibrin gel. The levels of vascular endothelial growth factor (VEGF) and angiopoietin-1(Ang-1) in the culture supernatant of RPMI-8226 cells were measured by enzyme-linked immunosorbent assay (ELlSA). The expression of VEGF and Ang-1 protein in RPMI-8226 cells was measured by western blotting.Results:1. Artesunate inhibited the proliferation of RPMI-8226 cells in dose- and time-dependent manners. After 24 and 48 hours'treatment with ART, the IC50value of ART for RPMI-8226 cells was 36.33±2.65 and 14.31±3.28μmol/l, respectively, at each time point.2. In the lower concentration group (2.5μmol/1), artesunate inhibited migration of HUVEC. When the concentration of ART was 12.5μmol/1, artesunate inhibited tube-formation of HUVEC. 3. The aortic sprouting in fibrin gel experiment showed that the number of microvessel in ART groups decreased significantly and the forming time was delayed, compared with the control group.14 days after ART treatment, the number of neogenetic microvessels was 114.5±1.87 in control group,60.33±1.86 in 3μmol/l ART group,34.83±2.14 in 6μmol/l ART group and 10±1.41 in 12μmol/l ART group, respectively (P <0.05)4. The chick embryo choriallantoic membrane (CAM) experiment showed that the RPMI-8226 cells strongly stimulated angiogenesis, and this ability became weaker after the cells were pretreated with ART. After the pretreatment with 3,6, and 12μmol/l ART, the number of neogenetic microvessels decreased by 21.9%,38.24% and 76.9%, compared with that in RPMI-8226 cells group.5. The secretion of VEGF and Ang-1 from RPMI-8226 cells was inhibited by ART. In the culture supernatant of RPMI-8226 cells with no treatment, the levels of VEGF and Ang-1 were 578±26 pg/ml and 185.14±32 pg/ml. With the treatment of 3,6, and 12μmol/l ART, the VEGF levels in the supernatant were 502±27,227±35 and 121±14 pg/ml, and the Ang-1 levels were 145±21,65±18 and 36±6 pg/ml.6. The result of Western blotting showed that the expression levels of VEGF and Ang-1 protein in the ART treatment groups decreased gradually with the ART dose increasing. In 3,6, and 12μmol/l ART treatment groups, the expression of VEGF protein decreased by 28.2%, 57.1% and 73.6%, and the expression of Ang-1 protein decreased by 28.9%,44.4% and 71.1%, compared with that in no treatmetn RPMI-8226 cells group.Conclusion:ART could significantly inhibit the proliferation of RPMI-8226 cells and the angiogenesis induced by RPMI-8226 cells, which may be related to the decreasion in the expression level of VEGF and Ang-1 after ART treatment. Part three The effect ofβ-elemene on proliferation and apoptosis of RPMI-8226 cells and its mechanismObjective:To investigate the effect ofβ-elemene on proliferation and apoptosis of human multiple myeloma cell line RPMI-8226 cells and its mechanism.Methods:The effect of P-elemene on the growth inhibition of human multiple myeloma cell line RPMI-8226 cells was studied by MTT assay. The effect of P-elemene on the apoptosis induction of RPMI-8226 cells was studied by combined AnnexinV/PI protein iodide staining and combined Hoechst33342-PI staining. The effect ofβ-elemene on the expression of Bcl-2, Caspase-3, DR-4 and NF-κB p65 protein was studied by Western blotting analysis.Results:β-elemene inhibited the proliferation of RPMI-8226 cells in a time- and dose-dependent manner (r=0.723, P<0.05). Treatment with 10-80μmol/L P-elemene for 48 h induced apoptosis of RPMI-8226 cells. Compared to the apoptosis rate of the control group (5.4%±1.1%), the apoptosis rates of RPMI-8226 cells in P-elemene treatment groups were 22.1%±3.8%(10μmol/l),39.5%±3.1%(20μmol/l),58.4%±2.8%(40μmol/l), and 65.4%±2.1%(80μmol/l) (P<0.05). The expression of caspase-3 and DR-4 protein in RPMI-8226 cells treated withβ-elemene increased in a time-dependent manner, while Bcl-2 and NF-κB p65 protein decreased.Conclusion:P-elemene inhibited the proliferation of RPMI-8226 cells via inducing the cell apoptosis. Activating the mitochondrial and death receptor pathways of apoptosis and inhibiting the anti-apoptosis pathway may be one mechanism involved inβ-elemene-induced apoptosis.