Cyclopamine sensitizes multiple myeloma cells to circularly permuted TRAIL-induced apoptosis

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a promising anti-myeloma drug prototype. The aim of the present study was to investigate the synergistic effects of cyclopamine and circularly permuted TRAIL (CPT) on the proliferation and apoptosis of multiple myeloma cells. The results showed that the inhibitory effects of cyclopamine on the proliferation of human myeloma RPMI-8226 and SKO-007 cells were weak. RPMI-8226 cells were sensitive to CPT; however, the proliferation of SKO-007 cells was not effectively inhibited by CPT. SKO-007 cells were thus considered resistant to cyclopamine and CPT and used for subsequent experiments. Treatment with a combination of cyclopamine and CPT significantly inhibited cell proliferation. Moreover, the Q value showed that cyclopamine combined with CPT could synergistically inhibit the proliferation of SKO-007 cells. Cyclopamine increased CPT-induced apoptosis in the SKO-007 cells and exhibited a synergistic induction of apoptosis when combined with CPT. Moreover, the combination of cyclopamine and CPT decreased the ratio of myeloma stem cells. Quantitative PCR showed that cyclopamine decreased the mRNA expression levels of GLI1/GLI2/GLI3 and increased the expression levels of death receptor 4. In conclusion, the present study showed that a combination of cyclopamine and CPT exhibited synergistic effects on the inhibition of proliferation and induction of apoptosis in myeloma cells.     

PDK1 inhibition is a novel therapeutic target in multiple myeloma

Background:

Cancer cells utilise the glycolytic pathway even when adequate oxygen is present, a phenomenon known as the Warburg effect. We examined whether this system is operative in multiple myeloma (MM) cells and whether glycolysis inhibition is a potential therapeutic modality.

Methods:

The MM cells were purified from 59 patients using CD138-immunomagnetic beads. The expression levels of genes associated with glycolysis, c-MYC, GLUT1, LDHA, HIF1A and pyruvate dehydrogenase kinase-1 (PDK1) were determined by real-time PCR. Glucose consumption and lactate production by MM cell lines were analysed. Oxamate, an LDH inhibitor, and dichloroacetate (DCA), a PDK1 inhibitor, were employed. Inhibition of PDK1 expression was achieved using a siRNA.

Results:

High LDHA expression was found to be an indicator of poor prognosis. It was also positively correlated with the expression of PDK1, c-MYC and GLUT1. Greater glucose consumption and lactate production in MM cells was associated with higher LDHA expression. All the glycolysis inhibitors (oxamate, DCA and PDK1 siRNA) induced apoptosis in MM cells. DCA combined with bortezomib showed additive cytotoxic effects.

Conclusion:

The present data suggest that the Warburg effect is operative in MM cells. As PDK1 is not overexpressed in normal tissues, PDK1 inhibition could serve as a novel therapeutic approach.     

2-甲氧基雌二醇对多发性骨髓瘤患者骨髓细胞的影响

目的:探讨2-甲氧基雌二醇(2ME2)对多发性骨髓瘤患者骨髓瘤细胞及造血前体细胞凋亡的影响。方法:采用DNA片断原位末端标记(TUNEL法)检测多发性骨髓瘤患者骨髓瘤细胞及造血前体细胞凋亡。结果:14例多发性骨髓瘤患者骨髓瘤细胞经1、4、16μmol/L2-甲氧基雌二醇作用48小时后,凋亡率分别为(10.5±1.2)%、(21.2±2.3)%、(36.8±3.6)%,呈现剂量依赖性。经统计学分析,各组之间及与空白对照组相比,差异显著(P<0.05)。骨髓中粒系、红系、淋巴系细胞出现凋亡较少,经统计学分析,各组之间及与空白对照组相比,无明显差异(P>0.05)。相同的2ME2浓度条件下,骨髓中粒系、红系、淋巴系细胞凋亡率与骨髓瘤细胞组凋亡率相比,差异显著(P<0.05)。结论:2-甲氧基雌二醇可选择性诱导骨髓瘤患者骨髓中骨髓瘤细胞凋亡,对骨髓瘤患者骨髓中粒系、红系、淋巴系细胞影响很小。     

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.     

冬凌草甲素诱导人多发性骨髓瘤ARH-77细胞凋亡及其可能机制

摘 要 目的：探讨冬凌草甲素（oridonin,Ori）对人多发性骨髓瘤ARH-77细胞的致凋亡作用及其可能的机制。方法：不同浓度Ori（2.5、5、10 μmol/L）作用于ARH-77细胞,MTT法检测ARH-77细胞的增殖,相差显微镜和Hoechst 33258染色观察细胞凋亡的形态学改变,流式细胞术检测ARH-77细胞的早期凋亡及线粒体膜电位（Δψm）的变化,caspase 8凋亡试剂盒检测ARH-77细胞caspase 8的活化。结果：Ori对ARH-77细胞的生长有明显的抑制作用,且呈时间和剂量依赖性。10 μmol/L Ori作用于ARH-77细胞24 h后可见细胞变小、胞质中出现空泡,并可见凋亡小体。流式细胞术结果显示,经不同浓度Ori（2.5、5、10 μmol/L）处理后,细胞早期凋亡率分别为（15.07±0.78）%、（21.00±1.49）%和（27.45±2.47）%,均高于对照组的（5.27±1.46）%（P<0.01）。不同浓度Ori（2.5、5、10 μmol/L）处理后,反映Δψm的绿色荧光细胞百分率分别为（26.80±0.75）%、（36.81±2.27）%和（49.48±1.10）%,均高于对照组的（16.96±0.50）%（P<0.01）,提示ARH-77细胞凋亡有显著增加。同时,Ori处理可上调ARH-77细胞caspase 8的活性（P<0.01）。结论：冬凌草甲素能明显诱导多发性骨髓瘤ARH-77细胞的凋亡,其机制可能与激活内、外源凋亡通路有关。     

Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma

Background:

Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas.

Results:

At IC₅₀ concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3–7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (P<0.0001) reduced the survival of primary myeloma cells.

Conclusions:

Cathepsin B has a prominent function in mediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal–mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.     

蛇床子素诱导人多发性骨髓瘤 RPMI －8226细胞凋亡及其机制研究

目的：研究蛇床子素（Osthole）的抗骨髓瘤作用,并探讨其抗骨髓瘤的作用机制。方法：处于对数增殖期人多发性骨髓瘤细胞 RPMI －8226随机分为空白对照组及10、20、40、80、120、160和200μmol／L Osthole组,用 MTT 法检测细胞增殖率,采用 Western blot 法检测凋亡相关蛋白表达量。结果：细胞培养24、48和72h后,不同浓度 Osthole 组细胞增殖数显著低于空白对照组,且作用呈剂量依赖性。不同浓度 Osthole 组细胞凋亡相关蛋白 PARP 被切割明显高于对照组,p53蛋白的表达量明显高于对照组,抗凋亡蛋白 Mcl －1表达明显下降。结论：Osthole 能明显抑制人多发性骨髓瘤细胞 RPMI －8226细胞增殖,分子机制可能是诱导其细胞凋亡,其诱导凋亡的机制有待于进一步深入研究。     

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。     

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.     

多发性骨髓瘤综合治疗现状与进展

多发性骨髓瘤（MM）是浆细胞克隆增生性恶性肿瘤,其治疗效果较差,迄今仍视为不可治愈的疾病。近年来着眼于MM细胞内信号通路、骨髓微环境及二者的相互作用,MM的治疗有了很大进展。该文综述了MM的化学治疗、造血干细胞移植、生物治疗、支持治疗及免疫治疗现状及其进展。     

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...     

The cap-translation inhibitor 4EGI-1 induces apoptosis in multiple myeloma through Noxa induction

Background:

Cancer cells are frequently addicted to deregulated oncogenic protein translation. The small molecule 4EG-I selectively inhibits the cap-dependent translation of mRNAs. As multiple myeloma is an incurable disease that requires new therapeutic approaches, we investigated whether targeting the translation initiation pathway could be a target for myeloma therapy.

Methods:

Six myeloma cell lines and primary samples were included in this study. The 4EGI-1 effect was determined by AnnexinV staining and caspase activation. Modification of Bcl-2 protein expression was analysed, and the significance of modified proteins was analysed by knock-down experiments.

Results:

We demonstrated that 4EGI-1 impaired the assembly of the eIF4F complex and decreased the expression of the eIF4E-regulated proteins in myeloma cells. Furthermore, we showed that 4EGI-1 induced strong apoptosis in five out of six myeloma cell lines. Apoptosis is associated with the activation of the intrinsic mitochondrial pathway. The 4EGI-1 triggered Noxa induction only in cells undergoing apoptosis through endoplasmic reticulum (ER) stress. Furthermore, Noxa silencing prevented myeloma cells from 4EGI-1-induced apoptosis. Finally, Noxa induction led to a disruption of Mcl-1/Bim complexes in parallel to the generation of ‘Mcl-1-free Noxa''.

Conclusion:

Our results suggested that the use of inhibitors that directly target the translation initiation complex eIF4F could represent a potential novel approach for multiple myeloma therapy.     

多发性骨髓瘤的治疗

Multiple Myeloma(MM)is characterised by the accumulation of malignanat plasma cells in the bone marrow producing a monocolonal immungoglobulin.The standard conventiona therapy in the combination of melphalan and prednisone resulting in a response rate of 40%-60% and in a median survival time of approximately 3 years.In order to improve the therapeutic efficacy va rious combination regimens have been tested.Most randomized trials have frailed to show a significant improvement in survival time when combination chemotherapy is used instead of melphalan with or without prednisone.The benefit of maintenance therapy with interferon-alpha has been demonstrated.The toxicity of interferon-alpha,which may reduce the quality of life,should be considered.Recently,myeloma-treatment has been modified.High-dose chemotherapy accompaniced by hematopoietic stem-cell support via autologous transplant is recommended up to the age of 65-70 years.First results from a French study comparing single versus double autologous transplantaiton have shown a benefit in terms of event-free survival for the sequential approach.Vaccinations as an adoptive immuntherapy to treat minimal residual dsease are umder way.The mortality rate of allogeneic transplantation of hemaatopoietic stem cells has been reduced in the last 5 years.The use of reduced conditioning regimens or the partial depletion of T cells in peripheral blood stem cell transplants in an effort to decrease transplant related mortality are promising approaches.Thalidomide and its derivates are a new class of agents with independent anti-tumour activity in MM.Encouraging results with this antiangiogenic therapy in phase Ⅱ trials have been reported.Supportive therapies,such as the treatment of anaemia with erythropoietin,the management of renal failure and the use of bisphosphonates,improve the life quality of MM patients.     

Artesunate overcomes drug resistance in multiple myeloma by inducing mitochondrial stress and non-caspase apoptosis

Although novel drugs have contributed immensely to improving outcomes of patients with multiple myeloma (MM), many patients develop drug resistance and ultimately succumb to MM. Here, we show that artesunate, an anti-malarial drug, reliably induces cell death in vitro in naïve as well as drug-resistant MM cells at concentrations shown to be safe in humans. Artesunate induced apoptosis predominantly through the non-caspase mediated pathway by primarily targeting mitochondria and causing outer mitochondrial membrane permeabilization that led to cytosolic and subsequent nuclear translocation of mitochondrial proteins apoptosis inducing factor (AIF) and endonuclease G (EndoG). Nuclear translocation of AIF and EndoG was accompanied by low levels of reactive oxygen species (ROS) and increased mitochondrial production of superoxide. These effects were present before apoptosis was evident and were related to intracellular levels of bivalent iron (Fe⁺²). Artesunate''s unique mechanism probably was at least partially responsible for, its ability to act synergistically with multiple anti-myeloma agents. Our findings suggest that artesunate acts through iron to affect the mitochondria and induce low ROS and non-caspase–mediated apoptosis. Its potency, toxicity profile, and synergism with other drugs make it an intriguing new candidate for MM treatment.     

三氧化二砷对多发性骨髓瘤细胞的影响

目的：了解多发性骨髓瘤细胞对Ａｓ２Ｏ３的反应及其可能机制。方法：使用多发性骨髓瘤细胞系ＲＰ－ＭＩ８２２６和Ｕ２６６细胞作为体外模型。细胞凋亡经细胞形态学、流式细胞仪和ＤＮＡ凝胶电泳判定通过测定细胞内荧光染料Ｒｈｏｄａｍｉｎｅ１２３的染色强度分析线粒体跨膜电位（△Ψｍ），并采用蛋白印迹分析蛋白剪切情况。结果：不同浓度的Ａｓ２Ｏ３对ＲＰＭＩ８２２６和Ｕ２２６细胞产生不同的效应：０．１～０．５ｍｏｌ／Ｌ的Ａｓ２Ｏ３抑制其增殖，２．０ｕｍｏｌ／Ｌ的Ａｓ２Ｏ３浓度的Ａｓ２Ｏ３诱导其凋亡，而１．０ｕｍｏｌ／Ｌ的Ａｓ２Ｏ３在抑制细胞增殖的同时轻度诱导细胞凋亡。Ａｓ２Ｏ３诱导的ＭＭ细胞凋亡伴随线粒体△Ψｍ下降的ｃｓａｐａｓｅ－３的活化。结论：Ａｓ２Ｏ３具有一个相对较广的诱导凋亡效应谱，而线粒体△Ψｍ破坏是Ａｓ２Ｏ３诱导细胞凋亡     

环孢素A 抑制亲环素A 对多发性骨髓瘤细胞增殖的影响

  目的  亲环素A（cyclophilinA,CyPA）在多种肿瘤组织中高表达,在肿瘤形成发展中扮演重要的角色。本研究对CyPA在多发性骨髓瘤（multiple myeloma,MM）患者骨髓标本中的表达及其对MM细胞增殖、凋亡能力的影响进行研究,探讨其与MM发生、进展的相关性。  方法  采用ELISA法检测骨髓标本及细胞培养上清中CyPA水平。不同浓度环孢素A（cyclosporin A,CsA）刺激MM细胞,CCK-8法检测细胞增殖能力、Western blot法检测PARP蛋白裂解评价细胞凋亡水平。  结果  单克隆免疫球蛋白血症、冒烟型骨髓瘤、MM患者骨髓中CyPA浓度逐渐升高,至MM达最高值,患者治疗后CyPA浓度明显下降。CsA处理MM细胞后,CyPA分泌减少,随时间延长细胞增殖能力呈浓度依赖性逐渐减弱,PARP蛋白裂解增加、细胞凋亡增多。  结论  随MM恶性程度增加,骨髓中CyPA浓度显著升高,经治疗缓解后CyPA浓度显著下降,CyPA介导的细胞增殖在MM致癌机制中可能起关键作用。CsA作为CyPA作用的抑制剂,具有促进细胞凋亡、阻碍细胞增殖的作用,无免疫抑制作用的CsA衍生物可能是潜在的MM有效治疗手段。