Ang-1在多发性骨髓瘤中的表达和青蒿琥酯、β-榄香烯抗骨髓瘤效应
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摘要
第一部分Ang-1在多发性骨髓瘤中的表达及临床意义
目的：骨髓血管新生在多发性骨髓瘤(multiple myeloma,MM)的发生、发展和预后中起着重要的作用,血管生成素-1(angiopoietin-1,Ang-1)在此过程中扮演了重要角色。本课题旨在研究Ang-1及其受体Tie-2在MM患者和骨髓瘤细胞株RPMI-8226中的表达情况,探讨其表达水平变化的临床意义。
方法：采用RT-PCR和Western blot法检测112例MM患者、24例非肿瘤患者(下称对照组)以及RPMI-8226细胞株中Ang-1及其受体Tie-2的表达水平,分析阳性率及表达量在MM患者与对照组、MM不同分期间的差异,探讨Ang-1表达与MM患者骨髓微血管密度、临床分期及预后的关系。
结果：MM组Ang-1表达的阳性率、表达量均显著高于对照组(P<0.05)；Ang-1在初治组和复发/难治MM组的表达阳性率无明显差异(P>0.05),但复发/难治MM组的Ang-1表达量高于初治组(P<0.05)；Tie-2 mRNA仅在12例MM患者中检出,对照组未检出。MM患者骨髓微血管密度明显高于对照组(25.21±0.8 vs 5.23±0.2,P<0.01)；Ang-1阳性患者的骨髓微血管密度高于Ang-1阴性患者(32.98±1.7 vs 16.55±1.3,P<0.05)；Ang-1蛋白在Ⅱ期和Ⅲ期MM患者的表达阳性率差异无统计学意义(52.1% vs 60.9%,P>0.05),但Ⅲ期MM患者Ang-1蛋白的表达水平明显高于Ⅱ期患者(0.4±0.07 vs 0.22±0.04,P<0.05)；初治患者中,治疗无效组Ang-1蛋白表达阳性率明显高于有效组(19.1% vs 70.0%,P<0.01)。
结论：Ang-1在MM中高表达；Ang-1表达水平与MM临床分期、判断预后及选择靶向治疗有关。
第二部分青蒿琥酯抑制人骨髓瘤细胞诱导的血管新生
目的：观察青蒿琥酯对人骨髓瘤细胞株RPMI-8226细胞诱导的血管新生的影响,并探讨其机制。
方法：采用MTT法观察青蒿琥酯对人骨髓瘤细胞株RPMI-8226细胞增殖的影响；采用HUVEC迁移实验、HUVEC小管成型实验、主动脉环血管生成实验和鸡胚绒毛尿囊膜(CAM)体内血管生长实验观察青蒿琥酯对RPMI-8226细胞诱导血管新生的影响；采用酶联免疫吸附法检测青蒿琥酯对RPMI-8226细胞分泌VEGF和Ang-1的影响；免疫蛋白印迹法检测青蒿琥酯对RPMI-8226细胞VEGF和Ang-1蛋白表达的影响,
结果：
1青蒿琥酯以时间、剂量依赖方式抑制人骨髓瘤细胞株RPMI-8226细胞增殖。药物作用24 h和.48 h后,其IC5o值分别为36.33±2.65和14.31±3.28μmol/l。
2青蒿琥酯在较低浓度(2.5μmol/l)即开始对血管内皮细胞迁移有抑制作用；12.5μmol/l时即可抑制内皮小管形成。
3主动脉环血管生成实验结果显示,与单纯RPMI-8226细胞组相比,青蒿琥酯预处理组新生血管形成明显减少,出现时间明显滞后。第14天,单纯RPMI8226细胞组新生血管数为114.5±1.87；3、6和12μmol/l青蒿琥酯预处理组新生血管数分别为60.33±1.86、34.83±2.14和10±1.41,差异具有统计学意义(P<0.05)
4鸡胚绒毛尿囊膜(CAM)体内血管生长实验结果显示,单纯RPMI-8226细胞组具有很强的刺激血管新生能力；而ART处理组的RPMI-8226细胞刺激血管新生能力明显减弱,3、6和12μmol/l青蒿琥酯处理RPMI-8226细胞组与单纯RPMI8226细胞组比较,微血管数目分别减少21.9%,38.24%和76.9%。
5青蒿琥酯抑制人骨髓瘤细胞株RPMI-8226细胞分泌VEGF和Ang-1。与单纯RPMI-8226细胞对照组上清液中VEGF、Ang-1含量(578±26、185.14±32 pg/ml)相比,经3、6和12μmol/l青蒿琥酯处理的RPMI8226细胞条件培养液中VEGF、Ang-1含量下降,分别为502±27、145±21；227±35、65±18; 121±14、36±6pg/ml。
6青蒿琥酯抑制人骨髓瘤细胞株RPMI-8226细胞VEGF和Ang-1蛋白表达。与单纯RPMI8226细胞对照组相比,经3、6和12μmol/l青蒿琥酯处理的RPMI8226细胞VEGF蛋白表达量分别下降28.2%、57.1%和73.6%；Ang-1蛋白表达量分别下降28.9%、44.4%和71.1%,呈剂量依赖性。
结论：青蒿琥酯抑制人骨髓瘤RPMI-8226细胞增殖；青蒿琥酯抑制人骨髓瘤RPMI-8226细胞诱导的血管新生；青蒿琥酯抗骨髓瘤细胞诱导血管新生的效应与其抑制RPMI-8226细胞的VEGF、Ang-1表达有关。
第三部分β-榄香烯对人骨髓瘤细胞增殖与凋亡的影响
目的：研究β-榄香烯对人骨髓瘤细胞系RPMI-8226增殖、凋亡的影响,探讨其作用的分子机制
方法：用MTT法检测β-榄香烯对人骨髓瘤细胞系RPMI-8226增殖的影响；Annexin V/PI双标流式术和Hoechst33342/PI荧光染色检测β-榄香烯对RPMI-8226骨髓瘤细胞凋亡的影响；Western blot法检测β-榄香烯对RPMI-8226骨髓瘤细胞Bcl-2、Caspase-3、DR-4和NF-κB p65蛋白表达影响。
结果：β-榄香烯对RPMI-8226骨髓瘤细胞生长具有明显抑制作用,且呈浓度和时间依赖性(r=0.723,P<0.05)；10-80μmol/L范围β-榄香烯作用48 h后可诱导骨髓瘤细胞凋亡。与对照组凋亡率(5.4%±1.1%)相比,β-榄香烯处理组凋亡率随药物浓度而递增,分别为22.1%±3.8%(10μmol/L)、39.5%±3.1%(20μmol/L)、58.4%±2.8%(40μmol/L)、65.4%±2.1%(80μmol/L)(P<0.05)；β-榄香烯以时间依赖方式上调Caspase-3、DR-4蛋白表达,并可下调Bcl-2、NF-κB p65蛋白表达。
结论：β-榄香烯可通过诱导细胞凋亡而有效抑制骨髓瘤细胞增殖；其诱导细胞凋亡与细胞内、外凋亡通路激活、抗凋亡机制被抑制有关。
Part one Expression of angiopoietin-1 in multiple myeloma and its clinical significance
Objective: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 cell
Objective: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 mechanism
Objective: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.
目的：骨髓血管新生在多发性骨髓瘤(multiple myeloma,MM)的发生、发展和预后中起着重要的作用,血管生成素-1(angiopoietin-1,Ang-1)在此过程中扮演了重要角色。本课题旨在研究Ang-1及其受体Tie-2在MM患者和骨髓瘤细胞株RPMI-8226中的表达情况,探讨其表达水平变化的临床意义。
方法：采用RT-PCR和Western blot法检测112例MM患者、24例非肿瘤患者(下称对照组)以及RPMI-8226细胞株中Ang-1及其受体Tie-2的表达水平,分析阳性率及表达量在MM患者与对照组、MM不同分期间的差异,探讨Ang-1表达与MM患者骨髓微血管密度、临床分期及预后的关系。
结果：MM组Ang-1表达的阳性率、表达量均显著高于对照组(P<0.05)；Ang-1在初治组和复发/难治MM组的表达阳性率无明显差异(P>0.05),但复发/难治MM组的Ang-1表达量高于初治组(P<0.05)；Tie-2 mRNA仅在12例MM患者中检出,对照组未检出。MM患者骨髓微血管密度明显高于对照组(25.21±0.8 vs 5.23±0.2,P<0.01)；Ang-1阳性患者的骨髓微血管密度高于Ang-1阴性患者(32.98±1.7 vs 16.55±1.3,P<0.05)；Ang-1蛋白在Ⅱ期和Ⅲ期MM患者的表达阳性率差异无统计学意义(52.1% vs 60.9%,P>0.05),但Ⅲ期MM患者Ang-1蛋白的表达水平明显高于Ⅱ期患者(0.4±0.07 vs 0.22±0.04,P<0.05)；初治患者中,治疗无效组Ang-1蛋白表达阳性率明显高于有效组(19.1% vs 70.0%,P<0.01)。
结论：Ang-1在MM中高表达；Ang-1表达水平与MM临床分期、判断预后及选择靶向治疗有关。
第二部分青蒿琥酯抑制人骨髓瘤细胞诱导的血管新生
目的：观察青蒿琥酯对人骨髓瘤细胞株RPMI-8226细胞诱导的血管新生的影响,并探讨其机制。
方法：采用MTT法观察青蒿琥酯对人骨髓瘤细胞株RPMI-8226细胞增殖的影响；采用HUVEC迁移实验、HUVEC小管成型实验、主动脉环血管生成实验和鸡胚绒毛尿囊膜(CAM)体内血管生长实验观察青蒿琥酯对RPMI-8226细胞诱导血管新生的影响；采用酶联免疫吸附法检测青蒿琥酯对RPMI-8226细胞分泌VEGF和Ang-1的影响；免疫蛋白印迹法检测青蒿琥酯对RPMI-8226细胞VEGF和Ang-1蛋白表达的影响,
结果：
1青蒿琥酯以时间、剂量依赖方式抑制人骨髓瘤细胞株RPMI-8226细胞增殖。药物作用24 h和.48 h后,其IC5o值分别为36.33±2.65和14.31±3.28μmol/l。
2青蒿琥酯在较低浓度(2.5μmol/l)即开始对血管内皮细胞迁移有抑制作用；12.5μmol/l时即可抑制内皮小管形成。
3主动脉环血管生成实验结果显示,与单纯RPMI-8226细胞组相比,青蒿琥酯预处理组新生血管形成明显减少,出现时间明显滞后。第14天,单纯RPMI8226细胞组新生血管数为114.5±1.87；3、6和12μmol/l青蒿琥酯预处理组新生血管数分别为60.33±1.86、34.83±2.14和10±1.41,差异具有统计学意义(P<0.05)
4鸡胚绒毛尿囊膜(CAM)体内血管生长实验结果显示,单纯RPMI-8226细胞组具有很强的刺激血管新生能力；而ART处理组的RPMI-8226细胞刺激血管新生能力明显减弱,3、6和12μmol/l青蒿琥酯处理RPMI-8226细胞组与单纯RPMI8226细胞组比较,微血管数目分别减少21.9%,38.24%和76.9%。
5青蒿琥酯抑制人骨髓瘤细胞株RPMI-8226细胞分泌VEGF和Ang-1。与单纯RPMI-8226细胞对照组上清液中VEGF、Ang-1含量(578±26、185.14±32 pg/ml)相比,经3、6和12μmol/l青蒿琥酯处理的RPMI8226细胞条件培养液中VEGF、Ang-1含量下降,分别为502±27、145±21；227±35、65±18; 121±14、36±6pg/ml。
6青蒿琥酯抑制人骨髓瘤细胞株RPMI-8226细胞VEGF和Ang-1蛋白表达。与单纯RPMI8226细胞对照组相比,经3、6和12μmol/l青蒿琥酯处理的RPMI8226细胞VEGF蛋白表达量分别下降28.2%、57.1%和73.6%；Ang-1蛋白表达量分别下降28.9%、44.4%和71.1%,呈剂量依赖性。
结论：青蒿琥酯抑制人骨髓瘤RPMI-8226细胞增殖；青蒿琥酯抑制人骨髓瘤RPMI-8226细胞诱导的血管新生；青蒿琥酯抗骨髓瘤细胞诱导血管新生的效应与其抑制RPMI-8226细胞的VEGF、Ang-1表达有关。
第三部分β-榄香烯对人骨髓瘤细胞增殖与凋亡的影响
目的：研究β-榄香烯对人骨髓瘤细胞系RPMI-8226增殖、凋亡的影响,探讨其作用的分子机制
方法：用MTT法检测β-榄香烯对人骨髓瘤细胞系RPMI-8226增殖的影响；Annexin V/PI双标流式术和Hoechst33342/PI荧光染色检测β-榄香烯对RPMI-8226骨髓瘤细胞凋亡的影响；Western blot法检测β-榄香烯对RPMI-8226骨髓瘤细胞Bcl-2、Caspase-3、DR-4和NF-κB p65蛋白表达影响。
结果：β-榄香烯对RPMI-8226骨髓瘤细胞生长具有明显抑制作用,且呈浓度和时间依赖性(r=0.723,P<0.05)；10-80μmol/L范围β-榄香烯作用48 h后可诱导骨髓瘤细胞凋亡。与对照组凋亡率(5.4%±1.1%)相比,β-榄香烯处理组凋亡率随药物浓度而递增,分别为22.1%±3.8%(10μmol/L)、39.5%±3.1%(20μmol/L)、58.4%±2.8%(40μmol/L)、65.4%±2.1%(80μmol/L)(P<0.05)；β-榄香烯以时间依赖方式上调Caspase-3、DR-4蛋白表达,并可下调Bcl-2、NF-κB p65蛋白表达。
结论：β-榄香烯可通过诱导细胞凋亡而有效抑制骨髓瘤细胞增殖；其诱导细胞凋亡与细胞内、外凋亡通路激活、抗凋亡机制被抑制有关。
Part one Expression of angiopoietin-1 in multiple myeloma and its clinical significance
Objective: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 cell
Objective: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 mechanism
Objective: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.
引文
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