【作者】 甄永占；

【导师】 甄永苏； 李电东； 邵荣光；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2009， 博士

【摘要】 多发性骨髓瘤（multiple myeloma,MM）是骨髓浆细胞克隆性增殖的血液系统恶性肿瘤,发生率约占血液系统肿瘤的10%,全身恶性肿瘤的1%,是血液系统发病居第二位的常见恶性肿瘤。尽管MM的发病高峰为65至70岁,最近的统计结果显示MM的发病率正在提高,发病年龄出现年轻化趋势。MM特点是骨髓中浆细胞克隆性增殖,分泌M蛋白,同时伴有广泛的溶骨病变和骨质疏松。MM对化疗存在普遍抵抗性,目前还是一种难以治愈的致死性疾病。标准的化疗方案仅能使40-60%病人获得暂时缓解,中位生存期一般不超过3年。大剂量化疗联合造血干细胞移植可使缓解率明显提高。除了极少数病例,最终难免复发。正因为如此,人们对寻找新的抗癌药寄予厚望,以进一步提高MM患者的生存率和治愈率。近年来,随着对MM的发病机制和病理生理的深入研究,治疗MM的新思路、新方法相继出现。新药如免疫调节药物（Thalidomide analogues）和蛋白酶体抑制剂（bortezomib）的问世给MM的治疗带来革命性的改观,正逐步取代干细胞移植后的以蒽环类抗生素、长春新碱和地塞米松为主的常规化疗方案。力达霉素（Lidamycin,LDM）是本研究所从一株放线菌（Streptomycesglobisporus C-1027）代谢产物中获得的对多种肿瘤细胞有强烈杀伤作用的大分子肽类抗肿瘤抗生素,大量研究表明,LDM对多种肿瘤细胞及移植瘤具有极强的抗肿瘤活性,这都源于LDM对DNA的强烈地切割作用。最近研究显示LDM可引起肿瘤细胞染色体畸变及端粒功能异常。目前,LDM正作为一种新的化疗药物进行临床Ⅱ期试验研究。本课题选用一种小鼠骨髓瘤细胞株:SP2/0和2种人多发性骨髓瘤细胞株:U266和SKO-007,探讨LDM体内体外对鼠骨髓瘤和人多发性骨髓瘤的影响及其作用机制。此外本课题还初步研究了LDM联合bortezomib对人多发性骨髓瘤细胞U266和SKO-007的影响,从而为LDM联合bortezomib的临床应用提供基础。1.LDM抗鼠骨髓瘤作用的分子机制研究1.1 LDM作用于小鼠骨髓瘤细胞的体外实验研究1.1.1 MTS结果显示LDM显著抑制鼠骨髓瘤SP2/0细胞的增殖。与其它临床常用化疗药物相比,LDM抑制SP2/0细胞增殖的作用更强,其IC₅₀值远低于其它药物。也检测了不同剂量的LDM作用不同时间对SP2/0细胞的增殖抑制作用,结果显示,LDM抑制SP2/0细胞增殖的作用为浓度和时间依赖性。1.1.2 LDM对鼠骨髓瘤SP2/0细胞形态的影响。LDM作用SP2/0细胞12 h后,细胞形态发生明显改变,光学显微镜下可见典型表现为细胞体积变大,还观察到了胞浆空泡,在0.5 nM的浓度下空泡更明显。1.1.3 Hoechst33342荧光染色法和FITC-AnnexinⅤ/PI双染结合流式细胞仪检测LDM对SP2/0细胞凋亡的诱导作用。结果显示不同浓度的LDM作用细胞48 h后,细胞凋亡率呈浓度依赖性地增高。0.1 nM、0.5 nM、1 nM和2 nM LDM作用于SP2/0的细胞凋亡率分别为14.66%+1.31%、18.30%±2.34%、43.65%±1.12%和79.34%±1.19%,显著高于对照组的4.70%±0.46%。荧光显微镜下可见LDM作用48 h后的细胞出现典型的凋亡细胞形态学变化,染色质凝集、细胞核固缩、形成凋亡小体等多种改变。1.1.4 PI单染结合流式细胞仪检测LDM对SP2/0细胞周期分布的影响。结果显示不同浓度的LDM作用细胞48 h后均能诱导G2/M期阻滞,0.5 nM的LDM诱导的G2/M期阻滞最明显,随着浓度升高（1 nM和2 nM）,凋亡相关的亚G1峰比例显著增加,因此诱导的G2/M期阻滞作用反不如0.5 nM的LDM明显。1.1.5 Western blot法检测了LDM对细胞周期和凋亡相关蛋白的影响。结果显示LDM浓度及时间依赖性诱导SP2/0细胞caspase-3、caspase-7的活化及PARP（polyADP-ribose polymerase）的切割,浓度依赖性的下调凋亡抑制蛋白Bcl-2、Survivin的水平,上调p27蛋白的表达。1.1.6 Western blot法检测了LDM对对丝裂原活化蛋白激酶（Mitogen-activated protein kinases,MAPKs）信号通路分子的表达影响。结果显示磷酸化的（活化形式）JNK、ERK和p38 MAPK的表达随着LDM浓度升高而增加,MAPKs上游蛋白:K-ras和c-Raf的表达随着LDM浓度增加而下降,磷酸化的MEK的表达浓度依赖性升高。也检测了0.5 nM的LDM作用不同的时间对JNK的表达影响。结果显示磷酸化的JNK的表达随着作用时间的延长而增加。1.1.7分别应用JNK抑制剂（SP600125）、p38抑制剂（SB203580）和MEK抑制剂（U0126）预处理SP2/0细胞2h,再加入LDM（0.001 nM,0.01 nM,0.1 nM,和1 nM）继续作用48h,MTS结果显示单独的3种抑制剂对细胞的增殖抑制作用均不明显,但SP600125或SB203580分别与LDM合用后均降低了LDM对SP2/0的细胞毒作用,提示JNK和p38 MAPK的激活参与了LDM对SP2/0细胞的增殖抑制作用,而U0126与LDM合用后提高了LDM对SP2/0的细胞毒作用,提示ERK的激活有利于SP2/0细胞的生存。1.1.8 SP600125预处理SP2/0细胞2 h,再加入LDM（0.5 nM）继续作用48 h,Westernblot结果显示SP600125可消除LDM引起的JNK的活化,同时也消除了LDM引起的caspase-3和caspase-7的激活及PARP的切割。1.2 LDM作用于小鼠骨髓瘤的体内实验研究1.2.1 LDM作用鼠骨髓瘤细胞SP2/0的体内实验研究1.2.1.1 LDM作用于鼠骨髓瘤细胞SP2/0的体内实验研究（一）利用鼠骨髓瘤细胞SP2/0小鼠体内移植瘤模型,观察LDM对小鼠移植性骨髓瘤的生长抑制作用。1×10⁶ SP2/0细胞右侧腋窝皮下接种,在细胞接种后第1天和第8天给药,LDM按照0.1 mg/kg、0.05 mg/kg、0.025 mg/kg的剂量,尾静脉注射的方式给药。结果显示LDM对SP2/0细胞移植瘤的生长有显著抑制作用,且呈剂量依赖性,0.1mg/kg、0.05 mg/kg、0.025 mg/kg剂量LDM组的抑瘤率分别为97.3%、86.3%和35.7%,与对照组相比具有显著性差异。接种后第10天LDM 0.1 mg/kg剂量组,有一只死亡。LDM 0.05mg/kg剂量组,动物状况良好。1.2.1.2 LDM作用于鼠骨髓瘤细胞SP2/0的体内实验研究（二）利用鼠骨髓瘤细胞SP2/0小鼠体内移植瘤模型,1×10⁶ SP2/0细胞右侧腋窝皮下接种,在细胞接种后第1天和第8天给药,调整LDM的给药剂量为0.06 mg/kg、0.04mg/kg、0.02 mg/kg,仍是尾静脉注射的方式给药。观察LDM对SP2/0细胞移植瘤的生长抑制作用。结果显示0.06 mg/kg、0.04 mg/kg、0.02 mg/kg的LDM对SP2/0细胞移植瘤的生长有显著抑制作用,且呈剂量依赖性,抑瘤率分别为92.1%、74.6.%和35.9%,与对照组相比具有显著性差异。2.LDM抗人多发性骨髓瘤细胞的作用及其分子机制研究2.1 LDM作用于人多发性骨髓瘤的体外实验研究2.1.1 MTS结果显示LDM显著抑制人MM细胞（U266和SKO-007）的增殖。LDM、VCR、5-FU、DEX、ADM对U266细胞的IC₅₀值分别为（5.751±0.153）×10^-11M、（7.083±0.106）×10^-7M、（3.246±0.084）×10^-7M、（1.778±0.113）×10^-4M、（2.514±0.127）×10^-8M。与其它临床常用化疗药物相比,LDM抑制U266细胞增殖的作用更强,其IC₅₀值远低于其它药物。在SKO-007细胞中观察到同样的结果。2.1.2 LDM对人MM细胞形态的影响。结果显示LDM作用U266和SKO-007细胞12 h后,细胞形态发生明显改变,光学显微镜下可见典型表现为细胞体积变大,还观察到了胞浆空泡。为了进一步观察细胞体积变大是否也影响了细胞核的大小,采用Wright-Giemsa染色,结果显示在LDM作用下U266和SKO-007的细胞核体积也变大。原因可能是LDM对整个细胞的骨架有影响,确切机制有待于进一步研究。2.1.3 Hoechst33342荧光染色法和FITC-AnnexinⅤ/PI双染结合流式细胞仪检测LDM对U266和SKO-007细胞凋亡的诱导作用。结果显示不同浓度的LDM作用细胞48 h后,细胞凋亡率呈浓度依赖性增高。0.1 nM、0.5 nM、1 nM和2 nM LDM作用于U266的细胞凋亡率分别为14.11%±2.31%、18.52%±3.65%、47.55%±4.56%和83.12%±5.33%,显著高于对照组的5.83%±1.45%。荧光显微镜下可见LDM作用后的细胞出现典型的凋亡细胞形态学变化,染色质凝集、细胞核固缩、形成凋亡小体等多种形态学改变。在SKO-007细胞中观察到同样的结果。2.1.4 PI单染结合流式细胞仪检测LDM对U266和SKO-007细胞周期分布的影响。结果显示不同浓度的LDM作用细胞48 h后均能诱导G2/M期阻滞,0.5 nM的LDM诱导的G2/M期阻滞最明显,随着浓度的增大（1 nM和2 nM）,凋亡相关的亚G1峰比例显著增加,因此诱导的G2/M期阻滞作用反不如0.5 nM的LDM明显。2.1.5利用Transwell实验观察LDM对U266和SKO-007细胞迁移能力的影响。结果显示LDM对这两种细胞的迁移能力均有显著的抑制作用,并呈明显的浓度依赖性。2.1.6 Western blot法测定了LDM对侵袭和转移相关靶点分子的影响。结果显示LDM可浓度依赖性地下调U266和SKO-007细胞与侵袭相关的VEGF、MMP-9的表达。2.1.7应用Western blot法测定了LDM对凋亡相关蛋白及细胞周期蛋白的表达的影响。结果显示LDM可浓度依赖性地诱导两种人多发性骨髓瘤细胞caspase-3、caspase-7的激活及PARP的切割,并显著下调凋亡抑制蛋白Bcl-2及Survivin的水平。同时LDM还可上调p21及p27蛋白的表达。2.1.8应用Western blot法测定了LDM对MAPKs信号通路分子的表达影响。结果显示LDM浓度依赖性地激活U266和SKO-007细胞JNK、ERK和p38 MAPK,MAPKs上游蛋白:K-ras、N-ras和c-Raf的表达因细胞类型不同而不同,在LDM的作用下,U266的K-ras、N-ras和c-Raf的表达浓度依赖性的降低,而SKO-007只有N-ras的表达浓度依赖性的降低,K-ras和c-Ra珀勺表达浓度依赖性的升高,在两个细胞中MEK的激活均为浓度依赖性。2.1.9为研究JNK、ERK和p38 MAPK的激活在LDM增殖抑制中的作用,分别应用SP600125、SB203580和U0126预处理U266细胞2 h,再加入LDM（0.001 nM,0.01 nM,0.1 nM,和1 nM LDM）继续作用48 h,MTS结果显示3种抑制剂单独对细胞的增殖抑制作用均不明显,但SP600125或SB203580分别与LDM合用后均降低了LDM对U266的细胞毒作用,而U0126与LDM合用后提高了LDM对U266的细胞毒作用。在SKO-007细胞观察到同样的结果。2.1.10为了探讨MAPKs的激活在LDM诱导凋亡中的作用,分别用SP600125、SB203580和U0126预处理U266细胞2h,再加入LDM（0.5 nM或1 nM LDM）继续作用48h,FITC-AnnexinⅤ/PI双染结合流式细胞仪检测结果显示单独的3种抑制剂对细胞的凋亡诱导作用均不明显,但SP600125或SB203580分别与LDM合用后均降低了LDM对U266的细胞凋亡诱导作用,而U0126与LDM合用后则提高了LDM对U266的细胞凋亡诱导作用。在SKO-007细胞观察到同样的结果。2.1.11为了探讨JNK、p38 MAPK的激活在LDM诱导凋亡中的作用机制,分别用SP600125和SB203580预处理U266细胞2h,再加入LDM（0.5 nM）继续作用48 h,Western blot检测结果显示单独的两种抑制剂对细胞的凋亡相关蛋白的表达影响不大,但SP600125或SB203580分别与LDM合用后均降低了LDM对PARP和aspase3/7的激活。在SKO-007细胞观察到同样的结果。2.1.12应用Western blot法测定了LDM对NF-κB及IκBα表达的影响。结果显示LDM剂量依赖性地诱导两种人多发性骨髓瘤细胞IκBα的磷酸化,但对总IκBα的表达无影响,在LDM的作用下,U266的NF-κB的表达浓度依赖性的降低,而SKO-007浓度依赖性的升高。2.2 LDM对蛋白酶体活性的影响检测了不同剂量的LDM作用于高纯化的20S蛋白酶体对其胰凝乳蛋白酶样活性的影响,发现我们所用的剂量（0.1 nM、0.5 nM、1 nM和2 nM）LDM对胰凝乳蛋白酶样活性没有明显的影响,不同剂量的LDM作用于MM细胞48 h,提取26S蛋白酶体,定量后检测胰凝乳蛋白酶样活性结果同上。可见LDM发挥强大的凋亡诱导作用,并不是通过对蛋白酶体活性抑制实现的。同时发现大剂量的LDM（100nM、1000 nM和10,000 nM）具有激活胰凝乳蛋白酶样活性的作用。3.LDM联合bortezomib作用于人多发性骨髓瘤的的体外实验研究3.1 LDM联合bortezomib抗多发性骨髓瘤的协同作用3.1.1 MTS结果显示,两药联合作用时,能够明显抑制细胞增殖,其CDI＜1,具有协同抗肿瘤作用。3.1.2 FITC-AnnexinⅤ/PI双染结合流式细胞仪检测结果显示联合用药可明显增强单药的凋亡诱导率。低联合浓度（0.5 nM的LDM与5 nM的bortezomib）对U266细胞的凋亡诱导率为22.91%,明显高于两药单用诱导的凋亡率15.07%及3.90%,较高联合浓度（0.5 nM的LDM与10 nM的bortezomib）对U266细胞的凋亡诱导率为67.11%,明显高于两药单用诱导的凋亡率15.07%及5.91%,在SKO-007细胞观察到同样的结果。3.2 LDM联合bortezomib协同抗多发性骨髓瘤的作用机制3.2.1 Western blot结果显示LDM联合bortezomib后可使PARP和caspase-3的切割显著增强,进一步上调了p21的表达。联合用药还降低NF-κB的水平。3.2.2 Western blot法测定了单药及联合用药对MAPKs的表达影响,结果显示,bortezomib呈剂量依赖性地激活JNK和p38 MAPK。两药联合后可使JNK和p38MAPK的激活显著增强。相反,两药联合后可使ERK的激活显著降低。综上,研究表明LDM能够显著抑制体外培养的鼠骨髓瘤和人多发性骨髓瘤的细胞的增殖,比其他常规化疗药细胞毒作用更大。并通过激活JNK和p38 MAPK诱导细胞凋亡,降低肿瘤细胞VEGF和MMP-9的蛋白水平限制细胞迁移。Bortezomib联合LDM显示,bortezomib能够加强LDM的细胞毒作用和凋亡诱导作用。而且,LDM具有显著的体内抗鼠骨髓瘤的作用。更多还原

【Abstract】 Multiple myeloma（MM） is a type of malignant plasma cells which are immune system cells in bone marrow that produce antibodies.MM is part of the broad group of diseases called hematological malignancies that account for approximately 10%of all hematological malignancies.It represents approximately 1%of all cancers.Although the peak age of onset of multiple myeloma is 65 to 70 years of age,recent statistics indicate both increasing incidence and earlier age of onset.It is characterized by the clonal proliferation of plasma cells predominantly located in the bone marrow releasing M protein and accompanied by bone lesions and osteoporosis.Standard chemotherapy only leads to temporary remission in 40-60%patients.Median survival is less than 3 years. Although high-dose chemotherapy and stem cell transplantation have improved the rate of complete remission,some myeioma cells escape the treatment and almost all MM patients experienced relapse in the end.Therefore,new therapeutic agents are needed for this disease.Over the last several years,significant insight into the dysregulation of various signal transduction pathways of MM has led to the development of new agents. The new drugs have greatly changed the management of multiple myeloma.Thalidomide analogues and bortezomib are in the process of overthrowing the current paradigm of anthracycline,vinca alkaloid and dexamethasone-based chemotherapy followed by autologous bone marrow transplantation.Lidamycin（LDM,C-1027）,a macromolecular peptide anticancer antibiotic produced by Streptomyces globisporus C-1027,displayed extremely potent cytotoxicity to culture cancer cells and markedly inhibited the growth of transplantable tumors in mice and human cancer xenografts in nude mice.The potent efficacy of LDM was ascribing to its DNA strand-scission activity.Recent study displayed the chromosomal aberrations and telomere dysfunction induced by LDM.LDM is currently being evaluated in phaseⅡclinical trials,as a potential chemotherapeutic agent in China.Studies on the molecular mechanisms of LDM alone and in combination with bortezomib against MM were investigated. 1 Studies on the molecular mechanisms of LDM against mouse myelom1.1 Effects of LDM on mouse myeloma SP2/0 cells in vitro1.1.1 SP2/0 cells were treated with various concentrations of LDM for various times. Antiproliferation activities were measured by MTS assay.Remarkedly growth inhibition effects of LDM were found in mouse myeloma SP2/0 cells.The IC₅₀ value of LDM for the SP2/0 cells was（5.623±0.051）×10^-10M,much lower than those of other drugs such as ADM,5-FU,VCR,and DEX.SP2/0 cells treated with the indicated concentration of LDM also showed a decreased cell proliferation in a time-dependent manner.1.1.2 After 12 h incubation with low dose LDM（0.1 nM,0.5 nM）,the treated SP2/0 cells exhibited morphological changes,including cell enlargement.Vacuoles appeared in cytoplasm,especially the cells exposed to LDM at the dose of 0.5 nM.1.1.3 Hoechst 33342 staining and flow cytometry combined with FITC-AnnexinⅤ/PI staining showed that LDM induced apoptosis in SP2/0 cells dose-dependently.The hoechst 33342 staining was used to assess the change in nuclear morphology after treatment with LDM.The nuclei of untreated cells were normal in appearance and exhibited diffused staining of the chromatin.After exposure to LDM for 48 h,most cells presented typical morphological changes of apoptosis such as chromatin condensation or a shrunken nucleus.FITC-AnnexinⅤ/PI staining showed LDM at 0.1 nM induced earlier apoptosis in SP2/0 cells.The ratio of apoptosis was significantly enhanced when cells were incubated with 1 or 2 nM LDM for 48 h.It suggests that apoptosis was the predominant mode of LDM-induced cell death.1.1.4 The flow cytometric cell cycle analysis showed that LDM induced cell cycle arrest at G2/M phase in SP2/0 cells.At 0.5 nM LDM,the number of G2/M phase cells reached the peak,however,the number of G2/M phase cells decreased due to apoptosis with further increasing concentrations.1.1.5 Western blot showed that LDM significantly increased caspase-3 and caspase-7 activities as well as PARP cleavage.The decreased Bcl-2 indicated that mitochondriacaspase cascade was responsible for LDM induced apoptosis.The level of Survivin also decreased after LDM treatment.The up-regulation of p27 was the evidence for G2/M phase arrest.1.1.6 Western blot showed that LDM significantly activates MAPKs family.LDM strongly induced phosphorylation of the three members of MAPKs family including JNK, p38 MAPK,and ERK in SP2/0 cells in a dose-dependent fashion.After LDM treatment, the up-stream proteins of MAPKs:K-ras and c-Raf decreased,whereas the level of phosphorylation of MEK increased.By 0.5 nM LDM treatment for various times, Western blot showed that LDM strongly induced phosphorylation of JNK in SP2/0 cells in a time-dependent fashion.1.1.7 To determine nthe role of MAPKs activation in cytotoxicity of LDM.SP2/0 cells were treated with LDM at different concentration in the presence or absence of the JNK inhibitor SP60012,the p38 MAPK inhibitor SB203580,and the MEK inhibitor U0126, respectively.In the absence of LDM,the inhibitors of JNK,p38 MAPK and MEK alone did not significantly show cytotoxicity in SP2/0 cells.However,both inhibitors, SP600125 and SB203580,（markedly depressed LDM-induced cytotoxicity,whereas U0126 significantly enhanced LDM-induced cytotoxicity.1.1.8 Western blot examined the mechanism of JNK activation in apoptosis induced by LDM.Consistent with the reduction of LDM-induced cytotoxicity,the phosphorylation of JNK and cleavage of caspase-3,caspase-7,and PARP triggered by LDM was blocked by SP600125.1.2 Effects of LDM on mouse myeloma SP2/0 cells in vivo1.2.1 In vivo antitumor activity of LDM（1）:Twenty-Four female BALB/c mice（20±2 g）, obtained from the Institute for Experimental Animals,Chinese Academy of Medical Sciences,Beijing） at the age of 4-6 weeks were used for SP2/0 mouse myeloma isografts. Animals were randomly divided into groups（n=6） in a manner that minimized the difference in body weight between the groups.SP2/0 cells（1×10⁶） resuspended in 200μl PBS were inoculated subcutaneously into BALB/c mice.On day +1 after inoculation, mice were receiving LDM or PBS（vehicle control） treatment,given intravenously once weekly via tail vein for a total of two injections.The doses of LDM were 0.1 mg/kg,0.05 mg/kg,and 0.025 mg/kg,respectively.Mice were weighted and tumor sizes were measured with a caliper and recorded every other day.The tumor volume was determined using the formula:（length×width²）×0.5.Growth of the established s.c.tumors in mice was decreased significantly when treated with LDM compared with control animals. Treatment with LDM at the dose of 0.1 mg/kg;0.05 mg/kg,and 0.025 mg/kg inhibited the growth of mouse myeloma SP2/0 isografts by 97.3%,86.3%,and 35.7%,respectively. On day +10 after inoculation a mouse in 0.1 mg/kg group died,indicating that 0.05 mg/kg is the tolerated dose for the mice.1.2.2 In vivo antitumor activity of LDM（2）:Based on the above-mentioned In vivo experiment,we modulated the dose of LDM.The used doses are 0.06 mg/kg,0.04 mg/kg and 0.02 mg/kg respectively.Administration of LDM significantly reduced myeloma SP2/0 tumor growth,compared with control animals.Comparisons of tumor volumes on day 20 following tumor implantation revealed significantly lower tumor volumes in the LDM-treated groups versus the control group.Treatment with LDM at the doses of 0.06, 0.04,and 0.02 mg/kg inhibited the growth of SP2/0 myeloma by 92.1%,74.6%,and 35.9%,respectively.On day 35 after tumor implantation BALB/c mice were killed by cervical dislocation,then tumors were carefully taken and weighed.Tumor weights in the LDM-treated groups were significantly lower than that of the control group.There was no significant body weight loss in the treated groups compared with controls.2 Studies on the molecular mechanisms of LDM against human multiple myeloma （MM）2.1 Effects of LDM on MM cell lines:U266 and SKO-007 cells in vitro2.1.1 U266 and SKO-007 cells were treated with various concentrations of LDM for various times.Antiproliferation effects were measured by MTS assay.Remarkedly growth inhibition effects of LDM were found in MM cells.The IC₅₀ values of LDM for the U266 and SKO-007 cells were（5.751±0.153）×10^-11 M and（1.585±0.178）×10^-10 M, respectively,much lower than those of other drugs（ADM,5-FU,VCR,and DEX）.2.1.2 After 12 h incubation with low dose LDM（0.1 nM,0.5 nM）,the treated U266 and SKO-007 cells exhibited morphological changes,including cell enlargement and appearance of vacuoles in cytoplasm.2.1.3 Hoechst 33342 staining and flow cytometry combined with FITC-AnnexinⅤ/PI staining showed that LDM induced apoptosis in U266 and SKO-007 cells dose-dependently.The hoechst 33342 staining was used to assess the change in nuclear morphology.The nuclei of untreated cells were normal in appearance and exhibited diffused staining of the chromatin.After exposure to LDM for 48 h,most cells presented typical morphological changes of apoptosis such as chromatin condensation or a shrunken nucleus.FITC-AnnexinⅤ/PI staining showed LDM at 0.1 nM induced earlier apoptosis in U266 and SKO-007 cells.The ratio of apoptosis was significantly enhanced when cells were incubated with 1 or 2 nM LDM for 48 h.It suggests that apoptosis was the predominant mode of LDM-induced cell death.2.1.4 Flow cytometric cell cycle analysis showed that LDM induced cell cycle arrest at G2/M phase in U266 and SKO-007 cells.At 0.5 nM LDM,the number of G2/M phase cells reached the peak,however,the number G2/M phase cells decreased due to apoptosis with further increasing concentrations.2.1.5 The effects of LDM on the migration of MM cells were examined with transwell chamber assay.A dose dependent reduction in migration of U266 and SKO-007 cells was found after exposure to various concentrations of LDM.2.1.6 Western blot analysis showed that LDM downregulated the levels of VEGF and MMP-9 in a dose-dependent way.2.1.7 Western blot showed that LDM significantly increased caspase-3 and caspase-7 activities as well as PARP cleavage in MM cells.The decreased Bcl-2 indicated that mitochondria- caspase cascade was responsible for LDM induced apoptosis.The level of Survivin also decreased after LDM treatment.The up-regulation of p21 and p27 was the evidence for G2/M phase arrest.2.1.8 Western blot showed that LDM significantly activates MAPKs family.LDM strongly induced phosphorylation of the three members of MAPKs family including JNK, p38 MAPK,and ERK in both MM cells in a dose-dependent fashion.The up-stream protein of MAPKs:K-ras,N-ras,and c-Raf decreased in U266 cells after LDM treatment, whereas only N-ras decreased in SKO-007 cells.The level of phosphorylation of MEK increased in both cell lines.2.1.9 To determine nthe role of MAPKs activation in LDM-induced cytotoxicity.MM cells were treated with LDM at different concentrations in the presence or absence of the JNK inhibitor SP60012,the p38 MAPK inhibitor SB203580,and the MEK inhibitor U0126,respectively.In the absence of LDM,the inhibitors of JNK,p38 MAPK and MEK alone did not show significantly cytotoxicity in MM cells;however,both inhibitors, SP600125 and SB203580,markedly depressed LDM-induced cytotoxicity,whereas U0126 significantly enhanced LDM-induced cytotoxicity in MM cells.2.1.10 By FITC-AnnexinⅤ/PI staining,SP600125 and SB203580 inhibited LDM-induced apoptosis in U266 cells.Compared with LDM treatment alone, pretreatment with SP600125（14μM） for 2 h followed by LDM（0.5 nM,or 1 nM） treatment significantly decreased the rates of apoptosis,16.81%,43.67%versus 10.14%, and 28.94%,respectively.Pretreatment with SB203580（12.5μM） decreased the rates of apoptosis,from 18.72%,and 49.07%versus 10.71%,29.01%,respectively.However, U0126 augmented LDM-induced apoptosis.The rates of apoptosis by LDM（0.5 nM,1 nM） alone and LDM（0.5 nM,1 nM） plus U0126（10μM） were 18.02%,41.32%, 30.32%,and 66.38%,respectively.Similar results were observed in SKO-007 cell line.2.1.11 U266 cells were treated for 48 h with 0.5 nM LDM,SP600125,SB203580 or the combination,respectively.Western blot showed the cleavage of caspase-3,caspase-7,and PARP triggered by LDM was blocked by SP600125 and SB203580.These results suggest that JNK and p38 MAPK might play a role,at least in part,mediating U266 cell apoptosis triggered by LDM,confirming that the activation of JNK,p38 MAPK, cleavage of caspase-3,caspase-7,and PARP is associated with apoptosis induced by LDM.Similar results were observed in SKO-007 cells.2.1.12 Western blot showed that LDM induced phosphorylation of IκBαin MM cells in a dose-dependent fashion,but has no effects on the expression of the total IκBα.The level of NF-κB expression is decreased in U266 cells,whereas increased in SKO-007 cells after LDM treatment.2.2 Studies on the activity of proteasome after LDM treatmentThe chymotrypsin-like activity was examined after different doses of LDM treatment. LDM at the doses of 0.1 nM,0.5 nM,1 nM,and 2 nM showed no effects on the chymotrypsin-like activity of 20S proteasome,whereas high doses of LDM including 100 nM,1000 nM,and 10,000 nM activated that activity.3 Studies on the effects of LDM combination with bortezomib on human multiple myeloma cells in vitro3.1 Synergistic effects of LDM combined with bortezomib against human multiple myeloma3.1.1 Examined by MTS assay,the combination of LDM with bortezomib exerted much stronger greatly inhibition of the growth of U266 and SKO-007 cells than either agent alone.The CDI value less than 1.0 indicates synergism.3.1.2 By FITC-AnnexinⅤ/PI staining,the combination of LDM with bortezomib greatly enhanced the apoptotic rates in MM cells,as compared with that of either agent alone.3.2.The mechanism of LDM combination with bortezomib against human multiple myeloma.3.2.1 By Western blot,the LDM combined with bortezomib greatly enhanced the cleavage of PARP and the activation of caspase-3.The expression of p21 was significantly upregulated,whereas the level of NF-κB decreased.3.2.2 By Western blot,LDM combined with bortezomib greatly enhanced the activation of JNK and p38 MAPK,whereas it decreased the activation of ERK significantly.Conclusion:Taken together,our observations highlight the extremely potent cytotoxicity of LDM to mouse myeloma SP2/0 cells and human multiple myeloma（MM） cell lines including U266 and SKO-007.Moreover,MM cells show much higher sensitivity to LDM than 2BS cells.Cells treated with LDM display an increasing apoptotic ratio in a dose-dependent fashion.This is related to the changes of certain molecular events involving the activation of JNK and p38 MAPK.LDM can inhibit cell migration through down-regulating the expression of VEGF and MMP-9.Notably,bortezomib enhances the cytotoxicity of LDM and the induced-apoptosis by LDM in MM cells.Moreover,LDM is very effective in vivo in murine SP2/0 myeloma model,evidenced by significant inhibition of SP2/0 tumor growth in mice.These results provide the framework for further studies of the efficacy of LDM on human MM,with the potential to be developed as a chemotherapeutic agent for multiple myeloma.更多还原