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.     

Dual inhibition of enhancer of zeste homolog 1/2 overactivates WNT signaling to deplete cancer stem cells in multiple myeloma

Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug‐resistant myeloma stem cells. Side population (SP) cells show cancer stem cell‐like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non‐SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR‐S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient‐derived xenograft model. Moreover, long‐term continuous dosing of OR‐S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR‐S1, leading to significant advances in the treatment of MM.     

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。      

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.     

Inhibition of thioredoxin 1 leads to apoptosis in drug-resistant multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by the aberrant accumulation of clonal plasma cells in the bone marrow. Despite recent advancement in anti-myeloma treatment, MM remains an incurable disease. This study showed higher intrinsic oxidative stress and higher Trx1 and TrxR1 protein levels in MM cells compared to normal cells. Drug-induced Trx1 (PX-12) and TrxR1 (Auranofin) inhibition disrupted redox homeostasis resulting in ROS-induced apoptosis in MM cells and a reduction in clonogenic activity. Knockdown of either Trx1 or TrxR1 reduced MM cell viability. Trx1 inhibition by PX-12 sensitized MM cells to undergo apoptosis in response to the NF-кβ inhibitors, BAY 11-7082 and curcumin. PX-12 treatment decreased the expression of the NF-кβ subunit p65 in MM cells. Bortezomib-resistant MM cells contained higher Trx1 protein levels compared to the parental cells and PX-12 treatment resulted in apoptosis. Thus, increased Trx1 enhances MM cell growth and survival and exerts resistance to NF-кβ inhibitors. Therefore inhibiting the thioredoxin system may be an effective therapeutic strategy to treat newly diagnosed as well as relapsed/refractory MM.     

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.     

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。     

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     

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。     

Act1分子对骨髓瘤细胞增殖的负调节作用研究

目的:探讨调节因子Act1对骨髓瘤细胞中BAFF(B细胞活化因子)通路的调节作用,进而了解其对骨髓瘤细胞的增殖存活所产生的作用.方法:利用荧光定量PCR的方法测定20例多发性骨髓瘤(MM)病人及20例正常对照者的外周血中的BAFF分子和Act1分子的表达水平;通过小干扰RNA(siRNA)和基因过表达技术构建缺乏和过表达Act1的U266骨髓瘤细胞,利用免疫印迹(Western blot,WB)法测定骨髓瘤细胞在BAFF分子刺激下的NF-κB信号通路活化情况,即磷酸化的IκB(p-IκB)的表达水平.结果:MM病人外周血BAFF和Act1分子的表达水平较对照组均增高(P<0.01),二者之间亦存在相关性;缺乏Act1分子的骨髓瘤细胞IκB磷酸化水平较高,而对照组的细胞IκB磷酸化程度较低,趋势不明显,过表达Act1分子的细胞基本没有磷酸化的发生.结论:Act1分子在多发性骨髓瘤的发生发展中发挥重要作用,其作用是通过对BAFF分子刺激下NF-κB信号通路的负调节作用而发挥的,使其可能成为抑制骨髓瘤细胞增殖的作用靶点.     

Selective inhibition of Rab prenylation by a phosphonocarboxylate analogue of risedronate induces apoptosis, but not S-phase arrest, in human myeloma cells

Bisphosphonates (BPs) are widely used in the treatment of osteolytic bone disease associated with multiple myeloma, and have been demonstrated to exert antitumor effects both in vitro and in vivo. However, the precise molecular mechanisms involved in the direct antitumor effects of BPs in vitro are not known. Nitrogen-containing BPs, such as risedronate (RIS), act by inhibiting protein prenylation. A phosphonocarboxylate analogue of RIS, 3-PEHPC, has previously been shown in osteoclasts and macrophages to specifically inhibit prenylation of Rab GTPases. The aim of this study was to identify the molecular targets of RIS and 3-PEHPC in human myeloma cells and to determine the cellular effects of selective inhibition of Rab prenylation by 3-PEHPC as compared to nonspecific inhibition of protein prenylation by RIS in human myeloma cells. RIS dose-dependently inhibited prenylation of both Rap1A and Rab6, whereas 3-PEHPC only inhibited Rab6 prenylation. Both RIS and 3-PEHPC dose-dependently increased apoptosis in human myeloma cells. RIS induced an accumulation of cells in the S-phase of the cell cycle, associated with inhibition of DNA replication. In contrast, 3-PEHPC did not cause cell-cycle arrest. Furthermore, geranylgeraniol could prevent inhibition of prenylation, induction of apoptosis, and cell-cycle arrest in response to RIS, but not inhibition of Rab prenylation and apoptosis induced by 3-PEHPC, consistent with specific inhibition of Rab geranylgeranyl transferase by 3-PEHPC. In conclusion, our studies demonstrate that selective inhibition of Rab prenylation induces apoptosis, but not S-phase arrest, thus identifying distinct molecular pathways that mediate the antimyeloma effect of nitrogen-containing BPs.     

Molecular aspects of multiple myeloma

Multiple myeloma (MM) is a B-cell neoplasm characterized by bone marrowinfiltration with malignant plasma cells, which synthesize and secretemonoclonal immunoglobulin (Ig) fragments. Despite the considerable progressin the understanding of MM biology, the molecular basis of the disease remainselusive. The initial transformation is thought to occur in a post-germinalcenter B-lineage cell, carrying a somatically hypermutated Ig heavy chain(IGH) gene. This plasmablastic precursor cell colonizes the bone marrow,propagates clonally and differentiates into a slowly proliferating myelomacell population, all under the influence of specific cell adhesion moleculesand cytokines. Production of interleukin-6 by stromal cells, osteoblasts and,in some cases, neoplastic cells is an essential element of myeloma cellgrowth, with the cytokine stimulus being delivered intracellularly via theJack-STAT and ras signaling pathways. While karyotypic changes havebeen identified in up to 50% of MM patients, recent molecularcytogenetic techniques have revealed chromosomal abnormalities in the vastmajority of examined cases. Translocations mostly involve illegal switchrearrangements of the IGH locus with various partner genes (CCND1, FGFR3,c-maf). Such events have been assigned a critical role in MMdevelopment. Mutations in coding and regulatory regions, as well as aberrantexpression patterns of several oncogenes (c-myc, ras) andtumor suppressor genes (p16, p15) have been reported. Keyregulators of programmed cell death (BCL-2, Fas), tumor expansion(metalloproteinases) and drug responsiveness (topoisomerase II alpha) havealso been implicated in the pathogenesis of this hematologic malignancy. Atumorigenic role for human herpesvirus 8 (HHV8) was postulated recently,following the detection of viral sequences in bone marrow dendritic cells ofMM patients. However, since several research groups were unable to confirmthis observation, the role of HHV8 remains unclear. Translation of theadvances in MM molecular biology into novel therapeutic strategies isessential in order to improve disease prognosis.     

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.     

Getting to the root of the problem: the causes of relapse in multiple myeloma

Multiple myeloma (MM) remains incurable, and ultimately, patients exhibit disease progression under current treatment regimens. Proteasome inhibitors have emerged as frontline treatment of relapsed and refractory MM however, resistance to these drugs occur through poorly defined mechanisms. Numerous studies have identified different acquired resistance models such as β5 proteasome subunit mutations and stabilization of tumor suppressors and apoptotic proteins. In addition, recent findings have identified a progenitor organization in MM whereby early progenitor tumor cells show resistance to proteasome inhibitor therapy and cause progressive disease with maturation arrest. This editorial highlights the potential causes of MM relapse in the context of these tumor progenitor cells and the role these cells play in treatment failure.     

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。     

SIRT1 promotes proliferation and inhibits the senescence-like phenotype in human melanoma cells

SIRT1 operates as both a tumor suppressor and oncogenic factor depending on the cell context. Whether SIRT1 plays a role in melanoma biology remained poorly elucidated. Here, we demonstrate that SIRT1 is a critical regulator of melanoma cell proliferation. SIRT1 suppression by genetic or pharmacological approaches induces cell cycle arrest and a senescence-like phenotype. Gain and loss of function experiments show that M-MITF regulates SIRT1 expression, thereby revealing a melanocyte-specific control of SIRT1. SIRT1 over-expression relieves the senescence-like phenotype and the proliferation arrest caused by MITF suppression, demonstrating that SIRT1 is an effector of MITF-induced proliferation in melanoma cells. Interestingly, SIRT1 level and activity are enhanced in the PLX4032-resistant BRAF^V600E-mutated melanoma cells compared with their sensitive counterpart. SIRT1 inhibition decreases melanoma cell growth and rescues the sensibility to PLX4032 of PLX4032-resistant BRAF^V600E-mutated melanoma cells. In conclusion, we provide the first evidence that inhibition of SIRT1 warrants consideration as an anti-melanoma therapeutic option.     

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp.     

Association of interleukin-1A, interleukin-1B and interleukin-1 receptor antagonist gene polymorphisms with multiple myeloma

Interleukin-1 (IL-1) is a cytokine involved in the maturation and proliferation of B cells and plays a significant role in the development of lytic bone lesions, a major clinical feature of multiple myeloma (MM) patients. Genes that regulate products involved in the immune system are highly polymorphic and contribute to inter-individual differences that can influence the genetic predisposition and progression of particular diseases and cancers. In this study, we investigated the correlation between the single nucleotide polymorphisms IL1A -889, IL1B -511, IL1B +3954, IL1RN Mspa1 +11100 and susceptibility to MM in 74 patients and 160 controls. We found that individuals possessing IL1A -889 CT polymorphism had a higher risk in developing MM. Moreover, genotypes IL1B -511 CC, IL1B +3954 CC, IL-1RN Mspa1 +11100 CC and the combination of IL 1B +3954 CC with IL1B -511 CC or IL-1RN Mspa1 +11100 CC exerted a protective effect in individuals possessing them.