Lidamycin induces apoptosis of B-cell lymphoma cells and inhibits xenograft growth in nude mice

OBJECTIVE To study the cytotoxicity of Lidamycin （LDM） and its induction of apoptosis in Raji and Daudi cells of B-cell lymphoma, and the inhibition of growth of the lymphoma Raji xenograft in nude mice. METHODS MTT assay was used to observe the inhibition by LDM on the proliferation of the Raji and Daudi ceils. Annexin V-FITC/PI double-stain, in combination with flow cytometry （FCM）, was used to determine the induction of apoptosis by LDM in Raji cells. The B-cell lymphoma Raji xenograft model in nude mice was set up to detect the in vivo antitumor activity of LDM. RESULTS LDM markedly inhibited the proliferation of the Raji and Daudi cells in vitro, with IC50 values of 7.13×10^-11 mol/L and 2.91×10^-10 mol/L, respectively. The apoptotic rates of Raji cells were respectively 77.98% and 67.63% at 0.5 nmol/L and 0.25 nmol/L of LDM, indicating an obvious induction of apoptosis in Raji cells. LDM inhibited the formation and growth of human B-cell lymphoma Raji xenograft in nude mice. The inhibition rates of tumor growth were respectively 74.9% and 65.2% in LDM at dosage group of 0.05 mg/kg and 0.025 mg/kg, suggesting an apparent prolongation of survival time in the nude mouse bearing lymphoma. CONCLUSION LDM can effectively induce apoptosis of the B-cell lymphoma cells and inhibit the xenograft growth in nude mice.     

MDA-7/IL-24对Burkitt淋巴瘤细胞的诱导分化作用

目的:研究黑色素瘤分化相关基因-7(melanoma differentiation associated gene 7,MDA-7)/IL-2对Burkitt淋巴瘤细胞的促分化作用并探讨其作用机制.方法:构建稳定过表达MDA-7/IL-24的人Burkitt淋巴瘤Raji和Daudi细胞株,MTS法检测稳定转染MDA-7/IL-24对Raji和Daudi细胞活力的影响;Transwell小室实验检测转染MDA-7/IL-24对Raji和Daudi细胞侵袭和迁移能力的影响;流式细胞术检测细胞凋亡水平及免疫表型;Western blotting技术检测转染MDA-7/IL-24对Raji和Daudi细胞表达分化相关蛋白Myb、BLIMP1及BCL-6的影响;建立裸鼠Raji细胞移植瘤模型,检测在体内环境中稳定转染MDA-7/IL-24对Raji细胞生物活性的影响.结果:过表达MDA-7/IL-24的Raji和Daudi细胞其增殖(P.＜0.05)、侵袭(P＜0.01)及迁移(P＜0.01)能力均明显下降,但凋亡细胞无明显增加(P＞0.05),表达CD45及CD138的水平均明显增加(P＜0.01),而表达CD10的水平明显下降(P＜0.01).过表达MDA-7/IL-24的Raji和Daudi细胞表达BLIMP1的水平明显增加(P＜0.01),而表达Myb及BCL-6的水平均明显减低(P＜0.01).MDA-7/IL-24过表达组裸鼠模型Raji细胞移植瘤质量明显低于对照组[(1.23±0.21)vs(1.96±0.24)g,P＜0.01].结论:转染MDA-7/IL-24可能通过诱导分化作用抑制Burkitt淋巴瘤细胞的生物活性.     

BET抑制剂对弥漫性大B细胞淋巴瘤细胞生长及外周血Th17细胞数量和相关细胞因子表达的影响

目的:探讨溴结构域和超末端结构域（bromodomain and extra terminal domain，BET）抑制剂对弥漫性大B细胞淋巴瘤CRL-2630细胞生长的影响，以及对弥漫性大B细胞淋巴瘤BALB/c-nu裸鼠外周血中辅助性T细胞17（helper T cells,Th17）数量和相关细胞因子表达的影响。方法:培养弥漫性大B细胞淋巴瘤株CRL-2630，使用不同浓度BET抑制剂（2、4、8、16、32 nmol/L）处理48 h，32 nmol/L BET抑制剂处理不同时间（12、24、36、48 h），CCK-8法检测各处理细胞活性；集落形成实验检测不同BET抑制剂浓度处理后细胞集落形成能力；Annexin V-FITC/PI双染法检测不同BET抑制剂浓度处理后细胞凋亡情况；实时荧光定量PCR与Western blot检测32 nmol/L BET抑制剂处理CRL-2630细胞48 h后HMGA1 mRNA与蛋白的表达水平；构建HMGA1过表达载体并通过脂质体介导法转染CRL-2630细胞，并用32 nmol/L BET抑制剂处理48 h，检测细胞活性与凋亡情况；构建裸鼠弥漫性大B细胞淋巴瘤模型并采集外周血，流式细胞术检测Th17细胞比例，ELISA法检测相关细胞因子的含量。结果:在一定范围内，BET抑制剂呈剂量依赖性地抑制CRL-2630细胞的活性，32 nmol/L BET抑制剂以时间依赖性地抑制CRL-2630细胞的活性。随着BET抑制剂处理浓度的增高，CRL-2630细胞集落形成能力逐渐下降，凋亡率逐渐升高。32 nmol/L BET抑制剂处理CRL-2630细胞48 h后，细胞中HMGA1的mRNA和蛋白水平均明显下降。pcDNA3.4-HMGA1转染CRL-2630细胞再使用BET抑制剂处理后，细胞的活性升高而凋亡率明显下降。弥漫性大B细胞淋巴瘤裸鼠经BET抑制剂作用后，外周血Th17细胞比例和IL-6、IL-17、IL-23含量较生理盐水组明显下降。结论:BET抑制剂能有效抑制 CRL-2630细胞活性，并诱导其凋亡，在一定范围内呈时效和量效关系。BET抑制剂还可以抑制弥漫性大B细胞淋巴瘤裸鼠外周血Th17细胞数量和相关细胞炎性因子的分泌，其作用机制可能与HMGA1的表达下调有关。     

不同给药方式下三氧化二砷对T细胞淋巴瘤EL4细胞体内及体外抑制作用

背景与目的：体外实验证明,三氧化二砷（arsenictrioxide,As2O3）单药可抑制多种恶性淋巴瘤细胞的增殖,并呈时间依赖性和浓度依赖性。临床研究也提示,As2O3单药治疗多种病理亚型淋巴瘤有效。但目前As2O3的剂量、具体用法仍未确定。本实验研究不同给药方式As2O3对鼠源性T细胞淋巴瘤EIA细胞体内外的抗瘤作用,旨在了解As2O3不同给药方法对T细胞淋巴瘤疗效及不良反应。方法：MTT法检测8种浓度As2O3对EL4细胞的抑制作用,流式细胞仪分析、AnnexinV—FITC／PI双标记检测细胞凋亡,电镜观察凋亡形态变化。建立EIA细胞裸鼠移植瘤模型,观察不同方案腹腔注射As2O3对EIA裸鼠移植瘤的抑制作用及裸鼠的耐受情况。结果：与对照组相比,不同浓度As2O3对EL4细胞均存在直接抑制作用（P〈0．05）,作用72h时的IC50为1．28μmol／L。体内实验显示,4mg·（kg·d）^-1×7d与2mg·（kg·d）^-1×14d给药对裸鼠移植瘤的抑制作用相近,抑瘤率分别为58．8％和55．6％（P〈0．351）。移植瘤组织凋亡细胞增多并可见凋亡小体生成。毒性表现主要为急性肝损害的病理学变化。结论：As20,体外可抑制EIA细胞增殖并可以诱导细胞凋亡,在总剂量相同的情况下,4mg·（kg·d）^-1×7d与2mg·（kg·d）^-1×14d的给药方式对裸鼠移植瘤生长的抑制作用近似。     

Inhibition of human B-cell lymphoma by an anti-CD20 antibody and its chimeric F(ab')2 fragment via induction of apoptosis

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully used in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. Despite all clinical success the exact mechanisms of action of various anti-CD20 antibodies remains mostly unclear. Several mechanisms have been proposed to be responsible for the therapeutic activity of anti-CD20 antibodies, including antibody-dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity, and direct inhibition of tumor growth via induction of apoptosis. We previously produced an anti-CD20 mAb, HI47, and showed that the antibody effectively blocked human B-cell proliferation in vitro and inhibited xenografted B-cell lymphoma in nude mice. In this study, we engineered the chimeric versions of both the Fab and F(ab)'2 fragments of HI47 and produced the fragments in E. coli. Both fragments competed efficiently with HI47 for binding to CD20+ B cells, and inhibited proliferation of B-lymphoma cells in a dose-dependent manner. Mechanistic studies revealed that both antibody fragments induced significant degree of B-cell apoptosis that is independent of any cross-linking agents. Further, both the F(ab)'2 and Fab fragments when administered in vivo significantly inhibited the growth of human B-cell lymphoma xenografts in nude mice. The bivalent F(ab)'2 fragment showed consistently better efficacy compared to its monovalent Fab counterpart in inducing apoptosis and inhibiting B-cell lymphoma growth both in vitro and in vivo. Taken together, these observations suggest that HI47 and its fragments most likely exert their antitumor activity through induction of cell apoptosis, and cross-linking/dimerization of CD20 molecules on B- cell surface is an important, but not essential, process for therapeutic efficacy of HI47 and its fragments.     

鱼藤素对淋巴瘤Daudi细胞株细胞增殖细胞凋亡的影响及其机制

目的观察鱼藤素对人Burkitt淋巴瘤Daudi细胞株细胞增殖、细胞凋亡和细胞周期的影响,并探讨其分子机制。方法四甲基偶氮唑蓝(MTY)法检测细胞增殖活性,Hoechst 33258染色和Annexin-V／PI双标法检测细胞凋亡,流式细胞仪检测细胞周期分布,Western blot检测细胞内cyclin D1和pRb的蛋白表达。结果鱼藤素对Daudi细胞具有明显的增殖抑制作用,而对正常人外周血单个核细胞(PBMC)抑制作用不明显。鱼藤素可以诱导Daudi细胞凋亡,Hoechst 33258染色可见典型凋亡小体。Annexin V／PI双标法显示,鱼藤素诱导细胞发生早期凋亡,并呈剂量依赖性,20、40、80 nmol／L鱼藤素作用24 h时,凋亡率分别为15．46％±0．62％、18．48％±2．98％和31．42％±1．43％。鱼藤素作用Daudi细胞后,主要使细胞周期聚积于G0／G1期,G0／G1期细胞比例随鱼藤素剂量增大而逐渐增高,40 nmol／L鱼藤素作用24 h达56．56％;相反,S期细胞比例随鱼藤素剂量增大而逐渐降低,对G2／M期细胞作用不明显。鱼藤素使cyclin D1及pRb蛋白表达降低,呈剂量依赖关系。结论鱼藤素抑制Daudi细胞增殖,使细胞阻滞于G0／G1期,并诱导细胞凋亡。其抗肿瘤机制可能与下调cyclin D1和pRb蛋白表达有关。     

PI3Kδ 抑制剂CAL-101 对淋巴瘤细胞系Raji和SUDHL-10的作用及其机制研究*

目的:探讨PI 3Kδ抑制剂CAL-101 对弥漫大B 细胞淋巴瘤细胞系SUDHL- 10和Burkitt 淋巴瘤细胞系Raji 的作用及其相关机制,为这类疾病的治疗提供新的思路。方法:用不同浓度的CAL-101 处理Burkitt 淋巴瘤细胞系Raji 和弥漫大B 细胞淋巴瘤细胞系SUDHL- 10,以MTT 法检测CAL-101 对两种细胞系的增殖抑制作用;以AnnexinV/PI流式细胞术和DAPI染色法检测细胞的凋亡情况;迁移实验检测淋巴瘤细胞向淋巴瘤基质细胞系HK的迁移比例;Westernblot法检测CAL-101 处理后淋巴瘤细胞表达磷酸化ERK 的变化;MTT 法结合CalcuSynsoftware软件分析检测CAL-101 是否可协同硼替佐米抑制淋巴瘤细胞的增殖。结果:5 μmol/L 及更高浓度的CAL-101 对Raji 细胞和SUDHL- 10细胞的增殖有明显的抑制作用,且呈剂量依赖性。5、10、15、20μmol/LCAL- 101 作用于Raji 细胞48h,细胞增殖抑制率分别为（29.17± 1.23）% 、（38.15± 1.51）% 、（46.46± 1.78）% 、（55.8 ± 2.01）% ,空白对照组为（1.15± 0.02）%（P<0.05）。 作用于SUDHL- 1048h,细胞增殖抑制率也逐渐增加（P<0.05）。 CAL-101 可诱导淋巴瘤细胞的凋亡,10、20μmol/LCAL-101 作用于Raji 细胞24h,AnnexinV-FITC/PI 双标法显示其凋亡率分别为（22.69± 3.83）% 和（49.96±7.36）% ,均高于对照组（5.23± 2.04）%（P<0.05）;作用于SUDHL- 10细胞同样得到相似的结果（P<0.05）。 CAL-101 可显著降低淋巴瘤细胞向基质细胞的迁移,且对Raji 细胞和SUDHL- 10细胞的迁移率的抑制作用也呈浓度依赖性,差异均具有统计学意义（P<0.05）;Westernblot检测发现CAL-101 处理细胞后磷酸化ERK 的表达明显降低;CAL-101 与硼替佐具有协同作用,可显著抑制淋巴瘤细胞的增殖,CI<1。结论:PI 3Kδ抑制剂CAL-101 可抑制淋巴瘤细胞Raji 和SUDHL- 10的增殖,诱导凋亡,抑制其向淋巴瘤基质细胞的迁移,其机制可能通过阻断ERK 信号途径而实现;CAL-101 有望为侵袭性淋巴瘤的治疗带来希望。      

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.     

蛋白酶体抑制剂在肿瘤坏死因子相对凋亡诱导配体诱导恶性淋巴瘤细胞凋亡抵抗中的作用及其分子机制

 目的　探讨体外蛋白酶体抑制剂在肿瘤坏死因子相对凋亡诱导配体（TRAIL）诱导恶性淋巴瘤细胞凋亡抵抗中的作用及其分子机制。方法　TRAIL与蛋白酶体抑制剂硼替唑咪（PS-341）处理恶性淋巴瘤Raji细胞后,采用MTT法检测药物不同作用时间对细胞的增生影响;流式细胞术检测细胞周期;Western blotting法检测Bax蛋白质水平变化;实时荧光定量RT-PCR法检测Bax mRNA的表达情况。结果　TRAIL在质量浓度500 μg／L时可减少Raji细胞的增生,但低于正常淋巴细胞Hmy2.ciR;TRAIL阻滞细胞周期于G0／G1期,Western blotting法检测Raji细胞Bax蛋白表达水平下降,实时荧光定量RT-PCR法检测Bax mRNA的表达水平却未见差异性改变,联合使用浓度为10 nmol／L PS-341后可明显提高TRAIL的诱导凋亡作用,阻滞于G0／G1期的细胞数增加,Bax蛋白表达水平也逐渐增加。结论　TRAIL在诱导恶性淋巴瘤细胞凋亡过程中出现抵抗,可能与促凋亡蛋白Bax表达水平下降有关,而Bax蛋白表达水平的下降与mRNA表达水平无关,可能与泛素-蛋白酶体途径的降解有关。     

A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma.

A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same single-chain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19(+)CD22(+) Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC(50) = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.     

三氧化二砷对恶性淋巴瘤细胞株凋亡的研究

目的 研究不同浓度的三氧化二砷对恶性淋巴瘤细胞株的影响。方法 采用细胞染色方法观察细胞凋亡的形态,原位末端标记法（TUNEL)检测凋亡细胞,流式细胞仪检测细胞的DNA含量的形态,原位末端标记法（TUNEL）检测凋亡细胞,流式细胞仪检测细胞的DNA含量和bcl－2蛋白的表达。结果 0．5μmol/L-2.0μmol/L三氧化二砷抑制B淋巴瘤细胞株Raji细胞生长,出现细胞凋亡的形态学改变,流式细胞仪检测Raji细胞亚C1期细胞明显增多,bcl－2蛋白的表达明显降低,呈现剂量时间依赖效应。0．5μmol／L－4．0μmol／L三氧化二砷对T淋巴瘤细胞株Jurkat细胞无明显作用。结论 三氧化二砷抑制恶性B淋巴瘤细胞生长和促进细胞凋亡的作用。     

国产雷帕霉素对人淋巴瘤细胞Raji增殖的影响

目的探讨国产雷帕霉素（宜欣可）对人淋巴瘤细胞株Raji细胞体外生长及对mTOR/p 70S6K信号通路的影响。方法MTT法检测不同浓度（0、1、5、10、20、40、50、100 nmol/L）国产雷帕霉素作用不同时间（24、48、72 h）对Raji 细胞增殖的影响。光学显微镜观察Raji细胞形态学变化。流式细胞仪测定国产雷帕霉素对Raji细胞周期分布和凋亡的影响。Western blot 方法检测国产雷帕霉素处理前后对Raji 细胞mTOR、p70S6K、p-p70S6K蛋白的影响。结果国产雷帕霉素对Raji细胞增殖有明显的抑制作用（不同浓度P<0.01或P<0.05）,呈现明显的剂量-效应和时间-效应依赖关系。国产雷帕霉素明显抑制Raji细胞周期发展（P<0.05）,但没有发生明显的凋亡（P>0.05）。0、10、50、100 nmol/L国产雷帕霉作用于Raji细胞的mTOR、p-p70S6K,其蛋白量随药物浓度增大而降低（P<0.05）,p70S6K随药物浓度增大而升高（P<0.05）。结论人淋巴瘤细胞株Raji中存在mTOR/p70S6K信号通路激活状态,宜欣可可抑制该通路激活并通过阻滞细胞周期发展抑制Raji细胞增殖。     

雷帕霉素对人非霍奇金淋巴瘤Raji细胞生物学行为的影响及其机制研究

目的 探讨不同浓度雷帕霉素作用不同时间对人非霍奇金淋巴瘤Raji细胞生物学行为的影响及其相关机制.方法采用0、10、50、100、250、500 nmol/L雷帕霉素分别作用Raji细胞24、48、72 h,采用CCK-8法测定Raji细胞增殖抑制率;采用流式细胞术测定Raji细胞凋亡及细胞周期;采用Caspase-3、Caspase-9活性检测试剂盒检测Raji细胞中Caspase-3、Caspase-9的酶活性;采用蛋白印迹法及反转录聚合酶链反应(RT-PCR)检测Raji细胞中bcl-2、p53蛋白及其mRNA表达情况.结果作用24 h后,随着雷帕霉素浓度由0 nmol/L逐渐增加至500 nmol/L,Raji细胞增殖抑制率由(23.7±4.2)％升高至(51.7±3.7)％(P<0.01);细胞凋亡率由(4.9±1.9)％升高至(20.5±1.5)％(P<0.01);G0/G1期细胞比例由(40.8±1.4)％增加至(63.6±1.7)％(P<0.01);Caspase-3酶活性由0.16±0.05增加至1.08±0.04(P<0.01);Caspase-9酶活性由0.19±0.04增加至1.34±0.06(P<0.01);bcl-2 mRNA表达量由0.90±0.03减少至0.46±0.03,p53 mRNA表达量由2.51±0.41增加至5.85±0.21,并且bcl-2蛋白表达降低,p53蛋白表达增高.Raji细胞48 h和72 h实验结果与24 h实验结果趋势一致.结论雷帕霉素可能通过Caspase-3、Caspase-9、bcl-2、p53途径抑制Raji细胞增殖,并诱导Raji细胞凋亡.     

激发型CD40单克隆抗体联合特异性细胞毒T淋巴细胞对B细胞淋巴瘤的免疫治疗作用

目的研究激发型CD40单克隆抗体5C11联合特异性细胞毒T淋巴细胞(CTL)对B细胞淋巴瘤的免疫治疗作用,方法人B细胞淋巴瘤Daudi细胞株经5C11刺激24或48 h,Annexin V/PI结合实验测定凋亡率,流式细胞术检测Fas表达率外周血单个核细胞来源的树突状细胞(DC),经凋亡Daudi细胞负载、5C11诱导成熟后,与自体T细胞混合培养,激发特异性CTL,JAM法检测5C11、CTL或5C11联合CTL对Daudi细胞的杀伤效应。皮下注射Daudi细胞,建立人源化SCID小鼠B细胞淋巴瘤模型;接种肿瘤细胞1周、3周后,分别经腹腔注射5C11、CTL、5C11 CTL进行治疗,通过肿瘤生长曲线、荷瘤小鼠生存期评价疗效。结果5C11作用后,Daudi细胞表面Fas的表达率显著上调,但细胞凋亡率没有明显改变。特异性CTL能有效杀伤靶细胞,且与5C11联合应用后,Daudi细胞DNA片段形成率显著增高,8 h为71.9%±4.0%,12 h达82.6%±4.4%经5C11、CTL、5C11联合CTL治疗后,荷瘤小鼠体内肿瘤生长速度均显著减缓,而且在低肿瘤负荷小鼠中,CTL、5C11联合CTL治疗分别取得了30.0%和70.0%的完全缓解率,与对照组相比,各治疗组小鼠生存期显著延长。结论激发型CD40单抗5C11可显著上调细胞表面Fas的表达,增强Daudi细胞对诱导凋亡的敏感性,从而促进肿瘤特异性CTL的体外杀伤和体内治疗效应。     

Oligonucleotides complementary to c-myb messenger RNA inhibit growth and induce apoptosis in human Burkitt lymphoma cells

A 24-mer (antisense) phosphorothioate oligonucleotide (ODN) corresponding to the codons 2-9 of the c-myb gene was evaluated for its effects on the growth of a human Burkitt lymphoma cell line (Raji) in vitro. Raji cells incubated with different concentrations of c-myb antisense ODN (5-15 mu g/ml) for 24-72 h showed a significant dose-dependent decrease in growth. The same concentrations of control (sense) or scrambled c-myb phosphorothioate ODNs did not inhibit Raji cell growth. The c-myb antisense ODN, but not the control ODNs, significantly decreased c-myb mRNA levels in treated cells as determined by RT-PCR. Additionally, the c-myb antisense ODN induced apoptosis of Raji cells as demonstrated by i) flow cytometry to enumerate the A(o) (apoptotic cell population) population of propidium iodide stained cells; ii) electron microscopy to evaluate the cell morphology; and iii) DNA fragmentation pattern. Thus, an antisense c-myb ODN causes significant growth inhibition of Burkitt lymphoma cells, and one mechanism of growth inhibition is the induction of apoptosis of the lymphoma cells. In addition, antisense c-myb ODN did not reduce CFU-GM or BFU-e colony-forming ability of normal hematopoietic stem/progenitor cells. Because the inhibition is sequence-specific and Burkitt lymphoma cell selective, evaluation of the therapeutic effects of c-myb antisense ODN against Burkitt lymphoma is warranted.     

二烯丙基二硫对人胃癌细胞裸鼠移植瘤的抗肿瘤作用

背景与目的:既往研究发现二烯丙基二硫(diallyldisulfide,DADS)在体外可抑制多种肿瘤细胞生长,但在体内抗肿瘤作用的研究报道较少。本实验旨在探讨DADS对人胃癌细胞移植瘤在BALB/C裸鼠体内生长的影响。方法:未经药物处理和经30mg/LDADS处理1天的胃癌细胞MGC803接种于裸鼠皮下;观察体外DADS处理MGC803细胞裸鼠移植瘤的成瘤情况和未处理MGC803细胞移植瘤成瘤后腹腔注射DADS对胃癌移植瘤在BALB/c裸鼠体内生长情况的影响。Westernblot检测瘤组织中增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)的表达情况。结果:30mg/LDADS处理的MGC803细胞移植裸鼠体内无一成瘤。荷瘤裸鼠腹腔注射DADS剂量为50、100和200mg/kg时的抑瘤率分别为27.8%、66.1%和73.0%,同时可抑制移植瘤癌细胞PCNA的表达。结论:DADS可明显降低胃癌细胞裸鼠移植瘤的成瘤性,并对移植瘤生长有明显抑制作用。     

Aspirin enhances doxorubicin-induced apoptosis and reduces tumor growth in human hepatocellular carcinoma cells in vitro and in vivo

Combined therapy with multiple drugs is a common practice in the treatment of cancer, which can achieve better therapeutic effects than a single drug, and can reduce the side effects as well as drug resistance. This study aimed to determine whether aspirin (ASA) shows synergism with doxorubicin (DOX) in HepG2 human hepatocellular carcinoma cells in vitro and in a HepG2 cell xenograft model in BALB/c nude mice. When treated in combination, DOX (0.25 nmol/ml) and ASA (5 μmol/ml) produced strong synergy in growth inhibition, cell cycle arrest and importantly, apoptosis in vitro in comparison to single treatments. Moreover, ASA (100 mg/kg/day orally) and DOX (1.2 mg/kg biweekly ip) induced synergistic antitumor activity in the HepG2 cell xenograft model in nude mice. Therefore, the combination of ASA and DOX could be used as a novel combination regimen which provides a strong anticancer synergy in the treatment of hepatocellular carcinoma.     

单价抗CD20抗体诱导人B细胞淋巴瘤Raji细胞的凋亡

背景与目的:抗CD20抗体和片段已应用于非霍奇金淋巴瘤的临床治疗,但仍需要开发新的抗CD20抗体和片段(未修饰的或放射性标记的),以治疗对美罗华(利妥昔单抗)无反应的患者。鼠源性抗CD20抗体HI47的嵌合抗体片段Fab和F(ab)'2已被构建。本研究目的是观察HI47(鼠抗-CD20抗体)和其嵌合抗CD20抗体片段抑制肿瘤细胞生长和诱导肿瘤细胞凋亡的作用。方法:用免疫荧光法测定抗CD20抗体与CD20+人B细胞淋巴瘤Raji细胞的结合能力;MTT法测定抗CD20抗体片段对Raji细胞生长的影响;用膜联蛋白Ⅴ染色和DNA琼脂糖凝胶电泳检测和验证抗CD20抗体片段诱导Raji细胞凋亡。结果:HI47和其嵌合的抗CD20抗体片段均可与CD20+Raji细胞结合,结合率可达90%以上;HI47不能与美罗华竞争结合Raji细胞;HI47和其嵌合的抗CD20抗体片段浓度为100μg/ml对Raji细胞的抑制率分别为:(57.0±1.5)%、(65.2±2.5)%、(77.2±3.2)%;单价的抗CD20抗体片段Fab(20μg/ml)能够诱导Raji细胞的凋亡,早期凋亡率为17%。结论:HI47的嵌合抗体片段对Raji细胞有抑制作用,能诱导Raji细胞的凋亡。