冬凌草甲素联合地塞米松对多发性骨髓瘤U266细胞增殖及凋亡的影响

目的：探讨冬凌草甲素联合地塞米松对多发性骨髓瘤 U266细胞增殖与凋亡的影响及其相关分子机制。方法培养 U266细胞至对数生长期,分别采用高、中、低浓度的地塞米松、冬凌草甲素单独或联合处理 U266细胞,以二甲基亚砜（DMSO）处理为对照,于处理后24、48、72 h 利用 CCK-8法检测细胞增殖情况;应用 Annexin V-FITC／PI 标记及流式细胞术检测细胞凋亡;利用实时荧光定量 PCR 法检测细胞 Notch1、NF-κB／p65、bcl-2基因表达水平的变化;Westernblot 法分析 Notch1、cleavedNotch1、NF-κB／p65、bcl-2蛋白表达水平的变化。结果与对照组相比,不同剂量的药物处理组均能有效抑制 U266细胞的增殖并促进其凋亡（P＜0.05）,两种药物联合应用对 U266细胞的抑制增殖和诱导凋亡具有协同作用（P＜0.05）。 U266细胞经地塞米松处理后其 NF-κB／p65、bcl-2 mRNA 和蛋白表达水平均有所下调（P＜0.05）,冬凌草甲素处理组及联合处理组 U266细胞 Notch1、cleaved Notch1、NF-κB／p65、bcl-2蛋白表达水平均显著下调（P＜0.05）。结论冬凌草甲素联合地塞米松对 U266细胞增殖的抑制作用及凋亡诱导作用显著增强,其作用机制可能与抑制 Notch1信号通路有关。     

体外消炎痛抑制多发性骨髓瘤细胞增殖及其机制研究

消炎痛 ( Indomethacin,IN)是一种临床常用的非甾体类抗炎药 ,近年来研究发现 ,IN具有抑制结肠癌、白血病等肿瘤细胞的增殖作用 [1,2 ] ,具有较广谱的抗肿瘤效应。本研究观察了在不同浓度的 IN作用下 ,对多发性骨髓瘤 ( multiple myeloma,MM)细胞株和 MM患者瘤细胞体外增殖的影响 ,确定IN是否有抗骨髓瘤细胞的增殖作用 ,并通过测定瘤细胞自分泌的 IL- 6及瘤细胞表面 IL- 6受体的变化 ,探讨 IN对骨髓瘤细胞的作用机制。材料与方法一、试剂　 IN、二甲亚砜 ( DM30 )、四甲基偶氮唑蓝 ( MTT)分别为 Sigma公司、瑞士 Flnka公司产品 …     

亲环素A在恶性肿瘤中的研究进展

亲环素A(CypA)是亲环素家族的重要成员,具有肽基脯氨酰基顺反式异构酶(PPIase)活性和分子伴侣功能,在蛋白质折叠和转运过程中发挥着重要作用。环孢素A(CsA)是CypA的细胞内受体,两者结合形成CypA-CsA复合物,该复合物可以促进T细胞凋亡,抑制T细胞活化及阻断信号转导,从而参与免疫抑制。在炎性反应过程中,CypA与CD147结合,产生促炎信号,介导炎性反应。CypA在多种恶性肿瘤中高表达,使肿瘤细胞逃避免疫监视,促进肿瘤细胞增殖、侵袭、转移和耐药等。CypA与丝裂原激活蛋白激酶(MAPK)的活化有关,其中研究最多的是细胞外信号调节激酶(ERK)/MAPK和p38/MAPK。本文主要针对CypA在恶性肿瘤中促进肿瘤生长、侵袭、转移、多药耐药(MDR)和免疫逃逸的作用机制作一综述。     

激活素A在多发性骨髓瘤发病中的研究进展

摘 要：多发性骨髓瘤（MM）是恶性浆细胞肿瘤,引起血钙升高、肾损害贫血和骨损害（CRAB症状,症状性骨髓瘤）。在MM患者中,高水平的激活素A不仅与MM的病情进展有关,与MM的溶骨性骨质破坏、贫血也相关,且影响MM患者的预后。抗激活素A疗法的应用,不仅可治疗MM相关的骨质破坏、贫血,也能改善MM患者的预后,故激活素A可能是MM或肿瘤性骨病的有希望的治疗靶点,特别是合并肿瘤性贫血的患者     

烟酰胺磷酸核糖转移酶对人多发性骨髓瘤U266细胞增殖和凋亡的影响及其机制

目的探讨小干扰RNA(siRNA)沉默烟酰胺磷酸核糖转移酶(NAMPT)基因表达对人多发性骨髓瘤U266细胞增殖、凋亡的影响及其相关机制。方法在体外合成针对NAMPT基因的siRNA并转染U266细胞,实验分为si-NAMPT组(转染siRNA-NAMPT的U266细胞)、si-NC组(转染阴性对照siRNA的U266细胞),采用四甲基偶氮唑盐(MTT)法检测转染后U266细胞增殖情况,流式细胞术检测细胞凋亡情况,蛋白质印迹法检测转染后各组细胞NAMPT、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、糖原合成酶激酶3β(GSK-3β)、磷酸化GSK-3β(p-GSK-3β)、β-catenin表达水平。结果si-NAMPT组转染48、72 h后与si-NC组比较,对U266细胞增殖抑制作用(570 nm处吸光度值)增加(48 h:0.78±0.06比1.62±0.11;72 h:1.23±0.14比2.37±0.18),差异均有统计学意义(t=3.54,P=0.034;t=4.72,P<0.01)。si-NAMPT组与si-NC组相比,细胞早期凋亡率升高[(53.42±0.25)%比(25.98±3.18)%],差异有统计学意义(t=4.41,P<0.01)。与si-NC组相比,si-NAMPT组p-AKT、p-GSK-3β、NAMPT、β-catenin蛋白水平均降低(均P<0.05)。结论沉默NAMPT基因可明显抑制U266细胞增殖并诱导细胞凋亡,其可能通过抑制AKT-GSK-3β-β-catenin信号通路发挥作用。     

cAMP信号通路与多发性骨髓瘤

多发性骨髓瘤（MM）细胞克隆性增殖涉及细胞内多个信号通路.环腺苷酸（cAMP）信号通路作为一种重要的细胞内信息传递系统,与包括骨髓瘤细胞在内的多种恶性淋巴细胞增殖和凋亡的失调相关.靶向调变cAMP信号通路可以诱导包括骨髓瘤细胞在内的多种恶性淋巴细胞增殖阻滞和凋亡,其机制涉及线粒体介导细胞凋亡和cAMP调控多种细胞内递质.对cAMP介导骨髓瘤细胞凋亡深入研究分析,将为临床治疗MM提供可能途径和潜在靶点.     

冬凌草甲素抑制多发性骨髓瘤细胞H929增殖和诱导凋亡的分子机制

目的:研究不同浓度的冬凌草甲素对多发性骨髓瘤细胞H929增殖和凋亡的影响及其调控机制。方法:使用不同浓度的冬凌草甲素处理多发性骨髓瘤细胞H929；利用CCK-8法检测细胞增殖抑制率；利用Annexin-FITC/PI双染和流式细胞仪检测细胞凋亡能力；利用蛋白印迹实验检测凋亡通路中BCL-2家族蛋白及PI3K/Akt通路中相关蛋白的表达水平。结果:细胞增殖实验结果显示冬凌草甲素显著抑制了多发性骨髓瘤H929细胞的增殖，且呈浓度和时间依赖性；细胞凋亡实验结果显示冬凌草甲素显著促进了多发性骨髓瘤H929细胞的凋亡，呈浓度和时间依赖性递增；蛋白印迹实验结果显示，随着冬凌草甲素浓度的增加，多发性骨髓瘤H929细胞中BCL-2蛋白、p-PI3K蛋白和p-Akt蛋白表达量递减，Bax蛋白表达量递增。结论:冬凌草甲素能有效抑制骨髓瘤H929细胞增殖，诱导细胞发生凋亡，其机制可能与PI3K/Akt信号通路的受抑及凋亡通路的活化有关。     

亲环素A过表达对高温、紫外线和顺铂引起的H1299细胞凋亡的影响

目的:探讨人肺癌细胞株H1299过表达亲环素A(CyPA)后对高温、紫外线和顺铂作用引起的细胞凋亡的影响。方法:采用重组慢病毒介导构建CyPA过表达H1299细胞株H1299 CyPA OE,并用Western blot方法验证CyPA过表达细胞株是否成功构建。培养H1299和H1299 CyPA OE细胞株,分别进行高温(44 ℃处理60 min)、紫外线(90 μW/cm²处理60 min)和顺铂(37.5、75和150 μg/mL作用2 h)处理后,用高内涵分析平台检测细胞的晚期凋亡。结果:H1299 CyPA OE细胞能检测到绿色荧光,且其CyPA蛋白表达较H1299细胞明显增高,表明H1299 CyPA OE细胞株构建成功。在不同顺铂浓度下,H1299 CyPA OE细胞凋亡率较H1299细胞均明显降低(P均<0.05),而在高温和紫外线作用下,H1299 CyPA OE细胞凋亡率与H1299细胞相比差异均无统计学意义(P均>0.05)。结论:CyPA过表达对顺铂诱导的凋亡有抵抗作用,表明CyPA可作为潜在的联合化疗的基因靶点,有望为临床个体化治疗提供新的思路。     

MiR-410对多发性骨髓瘤细胞增殖和侵袭能力的影响

摘 要：［目的］ 检测miR-410与HMGB1在多发性骨髓瘤中的表达水平及其对细胞增殖与侵袭能力的影响,探讨miR-410在多发性骨髓瘤致病中的作用。［方法］ 实时荧光定量PCR检测多发性骨髓瘤骨髓及细胞中miR-410的表达水平;蛋白质印迹用于分析HMGB1蛋白表达水平;CCK8实验和Boyden 侵袭小室实验分别用于检测细胞增殖活性及侵袭能力;双荧光素酶报告实验用于检测miR-410与HMGB1之间的相互作用;通过重组质粒构建及细胞转染调节相关基因在细胞内的表达;通过建立裸鼠异位移植瘤模型,在体验证miR-410对肿瘤生长的影响。［结果］ MiR-410在多发性骨髓瘤骨髓及细胞中表达均下调（P<0.01）,而HMGB1表达均上调（P<0.01）。MiR-410抑制多发性骨髓瘤细胞的增殖和侵袭（P<0.01）。MiR-410通过与HMGB1的3’UTR作用,负向调控HMGB1的表达。MiR-410通过下调HMGB1抑制多发性骨髓瘤细胞增殖和侵袭（P<0.05）。MiR-410抑制多发性骨髓瘤移植瘤小鼠肿瘤生长（P<0.05）。［结论］ MiR-410通过负向调控HMGB1表达,抑制多发性骨髓瘤细胞增殖和侵袭,从而抑制肿瘤发生发展。     

亲环素A在肿瘤中的作用机制

亲环素A在多种肿瘤细胞中高表达,可通过诱导炎-癌转化,加快肿瘤细胞转录周期,促进肿瘤细胞侵袭转移,抑制肿瘤细胞凋亡,以及降低肿瘤细胞对化疗药物的敏感性等多种途径调控多种肿瘤的发生发展,提示亲环素A可能是一种促癌基因,有望成为肿瘤治疗新靶标.     

Bruceantin inhibits multiple myeloma cancer stem cell proliferation

Multiple myeloma (MM) continues to claim the lives of a majority of patients. MM cancer stem cells (CSCs) have been demonstrated to sustain tumor growth. Due to their ability to self-renew and to express detoxifying enzymes and efflux transporters, MM-CSCs are rendered highly resistant to conventional therapies. Therefore, managing MM-CSCs characteristics could have profound clinical implications. Bruceantin (BCT) is a natural product previously demonstrated to inhibit the growth of MM in RPMI 8226 cells-inoculated mouse xenograft models, and to cause regression in already established tumors. The objectives of the present study were to test the inhibitory effects of BCT on MM-CSCs growth derived from a human primary tumor, and to explore a mechanism of action underlying these effects. BCT exhibited potent antiproliferative activity in MM-CSCs starting at 25 nM. BCT induced cell cycle arrest, cell death and apoptosis in MM-CSCs as well as inhibited cell migration and angiogenesis in vitro. Using a qPCR screen, it was found that the gene expression of a number of Notch pathway members was altered. Pretreatment of MM-CSCs with the γ-secretase inhibitor RO4929097, a Notch pathway inhibitor, reversed BCT-induced effects on MM-CSCs proliferation. In this study, BCT was shown to be an effective agent in controlling the proliferation, viability and migration of MM-CSCs as well as angiogenesis in vitro. The effect on MM-CSCs proliferation may be mediated by the Notch pathway. These results warrant further investigation of BCT in a broader set of human-derived MM-CSCs and with in vivo models representative of MM.     

The novel histone deacetylase inhibitor,AR‐42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells

Multiple myeloma (MM) remains incurable with current therapy, indicating the need for continued development of novel therapeutic agents. We evaluated the activity of a novel phenylbutyrate‐derived histone deacetylase inhibitor, AR‐42, in primary human myeloma cells and cell lines. AR‐42 was cytotoxic to MM cells at a mean LC₅₀ of 0.18 ± 0.06 μmol/l at 48 hr and induced apoptosis with cleavage of caspases 8, 9 and 3, with cell death largely prevented by caspase inhibition. AR‐42 downregulated the expression of gp130 and inhibited activation of STAT3, with minimal effects on the PI3K/Akt and MAPK pathways, indicating a predominant effect on the gp130/STAT‐3 pathway. AR‐42 also inhibited interleukin (IL)‐6‐induced STAT3 activation, which could not be overcome by exogenous IL‐6. AR‐42 also downregulated the expression of STAT3‐regulated targets, including Bcl‐xL and cyclin D1. Overexpression of Bcl‐xL by a lentivirus construct partly protected against cell death induced by AR‐42. The cyclin dependent kinase inhibitors, p16 and p21, were also significantly induced by AR‐42, which together with a decrease in cyclin D1, resulted in G₁ and G₂ cell cycle arrest. In conclusion, AR‐42 has potent cytotoxicity against MM cells mainly through gp130/STAT‐3 pathway. The results provide rationale for clinical investigation of AR‐42 in MM.     

Targeting SIRT1 to inhibit the proliferation of multiple myeloma cells

Multiple myeloma (MM) is the second most common hematopoietic malignancy and remains an incurable disease. Thus, novel drugs and therapeutic methods are required for patients with MM. The present study aimed to investigate the effect of sirtuin 1 (SIRT1) inhibitor cambinol on the proliferation and apoptosis of myeloma cell lines, RPMI8226 and U266. Moreover, the present study evaluated the underlying molecular mechanisms of proliferation inhibition and apoptosis induced by cambinol. A Cell Counting Kit-8 assay was used to measure the viability of RPMI8226 and U266 cells treated with cambinol. Apoptosis and the cell cycle were analyzed via flow cytometry. The expression levels of caspase-3, poly(ADP-ribose) polymerase 1 (PARP), p53, acetylated p53 (Ac-p53), Bcl-2, cyclin D1 and p21 were detected in cells treated with cambinol using western blot analysis. The results demonstrated that cambinol inhibited the proliferation of RPMI8226 and U266 cells in a time- and dose-dependent manner. Increased apoptosis and G₁ cell cycle arrest, together with enhanced procaspase-3 degradation and PARP cleavage were identified in cambinol-treated cells compared with controls. Western blotting results also revealed the upregulation of p53 acetylation and p21, as well as the downregulation of Bcl-2 and cyclin D1 in cells treated with cambinol. In conclusion, the present results suggest that cambinol inhibits the proliferation and induces apoptosis in RPMI8226 and U266 cells by regulating acetylation of p53 via the targeting of SIRT1.     

MiR-21下调SPRY2表达对多发性骨髓瘤细胞增殖和侵袭能力的影响及其机制研究

目的:探讨miR-21下调SPRY2基因表达在多发性骨髓瘤(multiple myeloma,MM)发生发展以及转移过程中的作用。方法:构建miR-21表达载体LV-anti-miR-21及脂质体转染法筛选稳定沉默SPRY2的MM细胞系。应用实时定量PCR和Western blot检测miR-21表达和SPRY2蛋白表达水平。四甲基偶氮唑蓝法(MTT法)检测细胞增殖能力;流式细胞仪分析细胞周期;划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力。结果:实时定量PCR及Western blot检测结果表明:转染U266细胞后,U266/LV-anti-miR-21慢病毒MOI 20组和MOI 40组miR-21表达量较U266/un组差异明显下降(P<0.05);转染miR-21 mimics组的U266细胞SPRY2的蛋白表达量较无转染组(untreated)和阴性对照组(siRNA)明显降低,差异均具有统计学意义(P<0.01)。与无转染组、阴性对照组相比:MTT法显示转染组48、72、96h细胞的生长速度明显减慢,增殖率明显下降(P<0.01);流式细胞术表明miR-21 mimics转染U266细胞48、72h后,细胞凋亡率分别为U266组[(24.7±1.97)%、(38.6±1.56)%]、siRNA组[(27.3±1.72)%、(37.3±1.59)%]和U266/miR-21组[(12.7±1.27)%、(22.1±1.63)%],与两对照组比较,U266/miR-21组细胞凋亡率明显降低,G0/G1期细胞明显减少,差异有统计学意义(P<0.05)。划痕实验显示转染组24、48h细胞迁移的能力明显减弱(P<0.05);Transwell实验证实转染组48、72h细胞穿过聚磷酸酯膜的U266细胞数明显减少,细胞穿膜能力明显降低(P<0.05)。结论:MiR-21下调SPRY2基因表达能够在体外条件下促进MM细胞的增殖和侵袭作用,揭示其在MM的发生、发展及转移过程中的作用及可能的机制,为确立新分子靶向治疗提供可靠的研究依据。     

艾拉莫德通过活化AMPK对骨髓瘤细胞株U266增殖的影响

目的:探讨艾拉莫德对多发性骨髓瘤（multiple myeloma,MM）细胞株U266细胞增殖的影响。方法:艾拉莫德（10、20和30 μg/ml）作用于骨髓瘤细胞株U266细胞后,采用四甲基偶氮唑蓝（MTT）法检测U266细胞活力;细胞周期染色试剂盒检测U266细胞周期分布情况;Western blot检测细胞周期相关蛋白CDK2、AMPK及p-AMPK的表达水平。结果:10、20和30 μg/ml的艾拉莫德处理24 h后,U266细胞活力呈剂量依赖性的降低。艾拉莫德（10、20和30 μg/ml）处理U266细胞24 h后,G0/G1期细胞数明显增加,S期细胞数明显降低。本研究还进一步发现10、20和30 μg/ml的艾拉莫德处理24 h后可显著下调U266细胞的CDK2蛋白表达水平。此外,艾拉莫德人处理骨髓瘤细胞株U266细胞24 h后,U266细胞的p-AMPK/AMPK水平明显增加。结论:艾拉莫德可抑制骨髓瘤细胞株U266细胞增殖以及细胞周期,其作用机制可能涉及促进AMPK的活化。     

依托泊苷对人骨髓瘤细胞 RPMI8226增殖与凋亡的影响

目的：探讨依托泊苷对人骨髓瘤细胞 RPMI8226的增殖抑制作用及其分子机制。方法：将依托泊苷作用于骨髓瘤细胞,用 MTT 法检测细胞增殖抑制率,光学显微镜和电子显微镜观察细胞形态及超微结构变化,流式细胞术检测细胞凋亡率和细胞周期分布,半定量 RT －PCR 检测 Bax 及 Caspase －3 mRNA 表达量的变化,Western blot 检测 Bax 及 Caspase －3蛋白表达量变化。结果：依托泊苷可抑制 RPMI8226细胞增殖,抑制率呈时间（r ＝0．926）和浓度（r ＝0．938）依赖性增强。24h 后在光学显微镜下可见依托泊苷组细胞数量减少,排列紊乱,细胞形态变得不规则,可见凋亡细胞及坏死细胞。48h 电子显微镜下可见细胞典型凋亡改变,凋亡小体形成。流式细胞术检测结果显示,依托泊苷组 RPMI8226细胞凋亡率明显增高（P ＜0．05）;依托泊苷作用48h 后将 RPMI8226细胞阻滞于 S 期（P ＜0．05）;依托泊苷作用48h,Bax、Caspase －3 mRNA 及蛋白表达量增加（P ＜0．05）。结论：依托泊苷可诱导 RPMI8226细胞凋亡,可能与细胞周期阻滞,激活细胞内、外源性凋亡通路有关。     

沉默COX-2抑制A549细胞的恶性增殖

Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lun...     

硼替佐米、三氧化二砷对多发性骨髓瘤细胞株KM_3细胞的抑制作用(英文)

Objective:The aim of our study was to investigate the effect of bortezomib in combination with arsenic trioxide(As2O3) on the proliferation and apoptosis in the human multiple myeloma cell line KM3.Methods:KM3 cells were cultured with different concentrations of bortezomib, As2O3 alone or in combination for different times.Cell proliferation was analyzed by MTT assay, and the IC50 was calculated.Cell morphology was observed under the light microscope(using Wright-Giemsa stain) and electric microscope.Agaros...     

Homing behaviour of the malignant cell clone in multiple myeloma

Multiple myeloma (MM) represents a B cell malignancy characterised by the presence of a monoclonal population of end-stage B cells in the bone marrow. Although fully matured bone marrow plasma cells are the predominant cell type in MM, there is much evidence that also more immature B cells are included in the malignant cell clone which are considered to be the myeloma precursor cells. The fact that these cells are detectable in the blood circulation and that their number increases with disease progression, makes it very likely that they represent the component of the tumour clone that mediates disease dissemination. This implies that these cells must have the potential to extravasate and home to the bone marrow environment. Like the migration mechanisms used by normal leukocytes and/or metastatic tumour cells of non-haematopoietic origin, it can be assumed that this bone marrow homing process is mediated by adhesive interactions and chemotactic signals provided by the microenvironment of the tumour. Once in the bone marrow compartment, myeloma cells will receive the appropriate signals to grow and survive. This aspect of tumour-homing is found to be the result of a functional interplay between the myeloma cells and the surrounding microenvironment, involving the action of several cytokines and adhesion molecules. In the end phase of the disease, myeloma cells can lose their stroma-dependency resulting in extramedullary tumour growth. We review normal B cell homing and discuss molecular mechanisms that determine the homing behaviour of the malignant cell clone in MM.     

The neural cell adhesion molecule NCAM in multiple myeloma

The Neural Cell Adhesion Molecule NCAM is a membrane glycoprotein and belongs to the immunoglobulin superfamily. It is expressed on neural cells as well as on various neuroendocrine tumors and can be detected in sera of patients with small cell lung cancer. Its role is attributed to tumor invasion and formation of metastases. Malignant plasma cells and a subset of plasma cells from patients with monoclonal gammopathy exhibit surface expression of NCAM whereas normal plasma cells do not express NCAM. Expression as measured by flow cytometry using anti-CD56 antibodies does not seem to correlate with clinical course, however leukemic myelomas and myeloma cell lines tend to loose NCAM surface expression.

An isoform of NCAM which is rich in polysialic acids and characteristic for embryonal NCAM (eNCAM) has been shown to be elevated in sera of patients with multiple myeloma using a chemiluminescence immunoassay. Patients with progressive myeloma tend to have high serum NCAM levels above the normal range of 20 U/ml. Analysis of 125 myeloma patients suggest that serum NCAM is a valuable parameter for tumor progression rather than tumor mass. Increase in serum NCAM may be associated with loss of adhesive function.