硼替佐米联合阿霉素诱导T细胞淋巴瘤细胞凋亡的实验研究

目的 探讨蛋白酶抑制剂硼替佐米对淋巴瘤细胞株Jurkat细胞的凋亡诱导作用及其可能机制,同时观察联合硼替佐米与阿霉素对Jurkat细胞有无协同作用.方法 采用四甲基偶氮唑蓝(MTT)比色法检测硼替佐米、阿霉素对Jurkat细胞体外生长的抑制作用,油镜下观察细胞形态变化.采用DNA的碘化丙啶(PI)染色及Annexin V-PI双标记法检测细胞凋亡率,Western blot法检测硼替佐米、阿霉素对Jurkat细胞caspase-3、caspase-8和聚(腺苷二磷酸-核糖)多聚酶(PARP)蛋白表达水平的影响.结果 10～320 ng/ml硼替佐米处理Jurkat细胞24、48和72 h,均能明显抑制细胞增殖,且生长抑制率与药物作用浓度呈正相关,硼替佐米作用24、48和72 h的相关系数分别为0.900、0.849和0.679(均P<0.01),呈浓度依赖性.以10～320 ng/ml硼替佐米单药或联合低剂量阿霉素(125ng/ml)处理Jurkat细胞24 h,硼替佐米的IC_(50)从(137.64±6.82)ng/ml降为(20.44±2.85)ng/ml.细胞凋亡率与硼替佐米的作用浓度呈正相关(P<0.01).硼替佐米作用Jurkat细胞后,caspase-3、caspase-8和PARP蛋白均出现明显的剪切带.结论 硼替佐米具有诱导Jurkat细胞凋亡的作用,通过外源性途径诱导凋亡是其机制之一.硼替佐米与阿霉素有一定的协同作用,二者联合能增强Jurkat细胞对硼替佐米的敏感性.     

硼替佐米联合毛喉素诱导硼替佐米耐药骨髓瘤细胞凋亡的实验研究

背景与目的：硼替佐米作为蛋白酶体抑制剂已成为新诊断和复发多发性骨髓瘤患者临床治疗的主要药物,但仍有部分患者对硼替佐米产生耐药而影响其长期生存。近年来研究发现,提高细胞内环腺苷酸浓度可以诱导骨髓瘤细胞凋亡并延缓其生长,成为骨髓瘤治疗新途径。该研究通过观察硼替佐米联合腺苷酸环化酶激动剂毛喉素(forskolin)对硼替佐米耐药骨髓瘤细胞的作用,探究克服骨髓瘤耐药的可能途径及其机制。方法：以硼替佐米耐药骨髓瘤细胞株H929-R和原代细胞为模型,通过观察细胞生长、形态、凋亡相关基因的改变来研究硼替佐米、毛喉素单独或联合处理对硼替佐米耐药细胞增殖及凋亡的影响;并应用Rh123/PI双染法检测药物处理前后细胞内线粒体跨膜电位的变化;同时采用实时荧光定量聚合酶链反应(real-time lfuorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测线粒体凋亡相关基因转录水平及抗凋亡基因Bcl-2和Mcl-1的蛋白水平的改变。结果：硼替佐米(20 nmol/L)与毛喉素(50 nmol/L)能够协同诱导硼替佐米耐药细胞凋亡。进一步研究发现,毛喉素可协同硼替佐米促使耐药细胞内线粒体跨膜电位下降并下调其Bcl-2和Mcl-1蛋白的表达。结论：毛喉素联合硼替佐米能够诱导硼替佐米耐药骨髓瘤细胞凋亡。     

硼替佐米对K562细胞株DNMT1表达的影响

[目的]探讨蛋白酶体抑制剂硼替佐米(bortezomib)对K562细胞株DNA甲基转移酶(DNMTs)表达、细胞凋亡的影响.[方法]以不同浓度的硼替佐米(0、6、20、60nmol/L)处理白血病K562细胞株,Western blot法检测细胞内DNMT1的表达,流式细胞术检测细胞凋亡.[结果]与对照组相比,硼替佐米可显著抑制DNMT1表达;硼替佐米作用于细胞12、24、36h后细胞凋亡率逐渐增加,60nmol/L硼替佐米作用36h后细胞凋亡率最高,为61.68％+3.20％.[结论]硼替佐米抑制K562细胞株DNMT1表达,并诱导细胞凋亡,呈浓度依赖性.     

硼替佐米对胰腺癌细胞增殖、凋亡及PI3K/Akt通路的影响

目的:探讨硼替佐米（BTZ）对胰腺癌细胞增殖、凋亡及PI3K/Akt通路的影响。方法:体外培养人胰腺癌细胞株PANC-1细胞,分别给予硼替佐米（0,2,10,50,250 nmol/L）作用24 h,采用MTT法检测PANC-1细胞活力,采用Annexin V/PI双染法检测细胞凋亡情况,采用流式细胞术检测细胞周期,采用Western blot检测细胞凋亡及PI3K/Akt通路相关蛋白表达情况。结果:与0 nmol/L硼替佐米相比,不同浓度硼替佐米处理后,PANC-1细胞生长抑制率均显著升高（P＜0.05）,G0/G1期细胞比例均显著升高（P＜0.05）,细胞凋亡率显著升高（P＜0.05）,细胞中cleaved-caspase-3、Bax蛋白表达增高（P＜0.05）,Bcl-2、PI3K、p-Akt蛋白表达降低（P＜0.05）,且呈剂量依赖性;裸鼠成瘤实验中,不同浓度硼替佐米作用后,肿瘤瘤体质量显著降低（P＜0.05）,且呈剂量依赖性。结论:硼替佐米能抑制胰腺癌细胞PANC-1增殖,诱导其凋亡,推测硼替佐米可能通过调节PI3K/Akt通路,激活凋亡蛋白基因表达,抑制抗凋亡蛋白基因的表达,诱导PANC-1细胞凋亡。     

Apatinib联合5-Fu协同抑制乳腺癌MCF-7细胞的体外研究

目的 探讨阿帕替尼(Apatinib)联合5-氟尿嘧啶(5-Fu)对乳腺癌MCF-7细胞的抑制作用.方法 采用人乳腺癌细胞株MCF-7,首先采用MTr法及应用流式细胞仪测定不同浓度apatinib作用后对其细胞增殖和周期的影响,其次将MCF-7细胞分为4组,即对照组、Apatinib单药组、5-Fu单药组、Apanitib+ 5-Fu联合组.对各组细胞给予相应药物处理,48 h后分别应用流式细胞仪检测各组药物对MCF-7细胞株凋亡的作用.结果 Apatinib单药对MCF-7细胞有增殖抑制作用,且存在时间剂量依赖关系,而对其细胞周期影响不大.与对照组相比,Apatinib联合5-Fu作用后有协同诱导凋亡作用,经流式细胞仪检测,Apatinib组诱导的细胞凋亡率为12.05％,5-Fu组为25.76％.与单药组比,Apatinib+5-Fu联合组凋亡率升高更为明显,达34.90％ (P＜ 0.05).结论 Apatinib和5-Fu的联合应用在体外协同抑制乳腺癌MCF-7细胞并诱导凋亡,使抗肿瘤活性显著增强.     

蛋白酶体抑制剂治疗多发性骨髓瘤现状

 【摘要】　多发性骨髓瘤（MM）是骨髓浆细胞克隆性增殖的血液系统恶性肿瘤,其发病率约占血液系统肿瘤的10 %。蛋白酶体是细胞内的一种复合酶,它可以降解泛素标记的蛋白,调节细胞内蛋白水平,在保持细胞内环境的稳定中起重要作用。近年来,新型靶向药物蛋白酶体抑制剂的应用,使MM的治疗取得了很大的进展,进一步改善了骨髓瘤的治疗疗效。硼替佐米是首个获美国食品和药物管理局（FDA）批准用于MM临床治疗的蛋白酶体抑制剂,它能可逆地抑制26S蛋白酶体糜蛋白样活性。但是,大多数硼替佐米初治后缓解的患者因产生耐药而最终导致疾病复发,因此,人们开始寻找与硼替佐米作用机制不同的蛋白酶体抑制剂。目前研究发现了一些能特异地结合蛋白酶体活性位点并且不可逆的抑制其功能的抑制剂,这些药物正在进行相关的临床试验。对已用于治疗MM的蛋白酶体抑制剂硼替佐米进行讨论,并对正在进行前期临床试验的新型蛋白酶体抑制剂进行概述,旨在解决硼替佐米的耐药性问题。     

硼替佐米对老年白血病患者原代细胞增殖、凋亡及其bcl-2家族蛋白表达的影响

 目的 研究硼替佐米对老年白血病原代细胞增殖、凋亡及其bcl-2家族蛋白表达的影响。方法 MTT比色法检测细胞增殖活力;荧光显微镜形态观察、流式细胞仪检测细胞凋亡;Western-blot检测Bcl-2、Bax蛋白表达。结果 50～5000nM硼替佐米均可抑制细胞增殖并诱导凋亡,其中50nM和5000nM处理细胞24h,细胞增殖活力分别下降至90%和70%,细胞凋亡率分别为10.2±2.3%和13.3±3.3%;延长处理时间至48h,细胞活力进一步下降至86%和60%,细胞凋亡率增至18.4±3.9%和20.7±3.7%,与对照组相比差异均具有统计学意义（P值均<0.05）;50～5000nM硼替佐米处理细胞24-48h,Bcl-2蛋白表达逐渐下降,Bax蛋白表达逐渐增加。结论 硼替佐米对老年白血病原代细胞具有抑制增殖、诱导凋亡作用,其作用机制可能与Bcl-2家族蛋白的表达水平变化相关。     

TNF-ɑ调控LRG1促进乳腺癌MCF-7细胞增殖、侵袭和迁移

目的:研究TNF-α对乳腺癌MCF-7细胞中LRG1蛋白表达的调控及其对乳腺癌MCF-7细胞增殖、侵袭和迁移能力的影响及相关分子机制。方法:MTT实验检测不同浓度TNF-α处理后MCF-7细胞活力;EdU实验、Transwell实验和划痕实验分别检测抑制LRG1表达后细胞增殖、侵袭以及迁移能力;Western blot检测细胞内MAPK信号通路中p-p38蛋白表达。结果:低浓度TNF-α处理乳腺癌MCF-7细胞,细胞活力增强;抑制LRG1表达后细胞增殖能力下降,侵袭细胞数、细胞迁移率以及p-p38蛋白表达均下降。结论:TNF-ɑ通过调控LRG1的表达促进乳腺癌MCF-7细胞增殖、侵袭和迁移,这一过程可能通过激活p38MAPK信号通路来实现。     

硼替佐米增加耐药白血病细胞株K562/DNR对化疗药物的敏感性

目的:观察蛋白酶体抑制剂硼替佐米是否可以增加耐药白血病细胞株K562/DNR对化疗药物的敏感性.方法:MTT法计算硼替佐米逆转耐药倍数,流式细胞术(FCM)检测细胞调亡比及细胞内药物浓度.结果:表柔比星单独及联合硼替佐米作用于K562/DNR细胞株的IC50分别为417.0μg/ml和210.4 μg/ml,逆转倍数为1.98倍;柔红霉素单独及联合硼替佐米作用于K 562/DNR细胞株的IC50分别为457.7μg/ml和324.9 μg/ml,逆转倍数为1.4倍.单独应用表柔比星组细胞调亡比为(28.74±4.6)％,联合硼替佐米组细胞调亡比为(39.14±9.6)％.硼替佐米能使K562/DNR细胞内柔红霉素含量增加,而对K562/S细胞无类似影响.结论:蛋白酶体抑制剂硼替佐米能增加耐药白血病细胞株K562/ DNR的化疗药物的敏感性,逆转耐药性.     

曲古菌素A和硼替佐米诱导卵巢癌细胞凋亡的协同作用

目的：探讨组蛋白去乙酰化酶抑制剂曲古菌素A与蛋白酶体抑制剂硼替佐米单独及联合应用对人卵巢癌细胞株SKOV3存活率和凋亡率的影响。初步探讨两种药物联合应用对诱导SKOV3细胞凋亡具有的协同作用。 方法曲古菌素A、硼替佐米单独或者联合应用于卵巢癌细胞后,用四甲基偶氮唑蓝（MTT）比色法测定细胞增殖活性,并计算细胞存活率,Annexin-V/PI法流式细胞仪检测细胞凋亡率,蛋白质免疫印迹法（Western blot）检测相关蛋白表达水平。通过检测Caspases-3的活性及其底物多聚ADP核糖聚合酶（PARP）的表达水平进一步说明不同用药组诱导细胞凋亡的情况。 结果联合用药组诱导的细胞凋亡率和单独用药组比较,差异有统计学意义（P<0.05）。几种凋亡相关蛋白Bcl-2、Mcl-1和Bcl-X_L, 在联合用药组的表达显著低于单独用药组。 在相同的时间点,联合用药组Caspase-3的活性和单独用药组比较,差异有统计学意义（P<0.001）。结论：低剂量的曲古菌素A和硼替佐米联合作用人卵巢癌细胞系能诱导凋亡,并且这种作用远远强于相同剂量单独用药引起的凋亡。这两种药物的联合应用可能成为人卵巢癌化疗中的新方案。     

Repression of breast cancer cell growth by proteasome inhibitors in vitro: impact of mitogen-activated protein kinase phosphatase 1

Mitogen activated protein kinase phosphatase-1 (MKP-1) has emerged as an important protein mediating breast cancer oncogenesis and chemoresistance to cancer chemotherapies, especially proteasome inhibitors. In this in vitro study, we utilized the breast cancer epithelial cell lines MCF-7 and MDA-MB-231, in comparison to MCF-10A control cells, to examine the impact of MKP-1 on breast cancer cell growth and repression by proteasome inhibitors. We confirm that proteasome inhibitors MG-132 and bortezomib induce MKP-1 protein upregulation and we show that one of the ways in which bortezomib increases MKP-1 in breast cancer cells, in addition to inhibition of ubiquitin-proteasome system, is via upregulation of MKP-1 mRNA expression in p38 MAPK-mediated manner. Notably, these effects are specific to cancer cells, as bortezomib activated p38 MAPK and induced MKP-1 in MCF-7 and MDA-MB-231 breast cancer cells, but not in control cells (MCF-10A). We took a dual approach toward targeting MKP-1 to show that bortezomib-induced effects are enhanced. Firstly, treatment with the non-specific MKP-1 inhibitor triptolide reduces breast cancer cell growth and augments proteasome inhibitor-induced effects. Secondly, specific knock-down of MKP-1 with siRNA significantly repressed cell viability by reduced cyclin D1 expression, and enhanced repression of cancer cell growth by proteasome inhibitors. Taken together, these results indicate that removing the unwanted (MKP-1-inducing) effects of bortezomib significantly improves the efficacy of proteasome inhibition in breast cancer cells. Thus, future development of drugs targeting MKP-1 offer promise of combination therapies with reduced toxicity and enhanced cell death in breast cancer.     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的：研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法：采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果：甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达（68.40±4.87）%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论：甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的:研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法:采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果:甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达(68.40±4.87)%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论:甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。     

CIP2A is a target of bortezomib in human triple negative breast cancer cells

Introduction

Triple negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets, such as hormone receptors or human epidermal growth factor receptor type 2 (HER2); therefore, prognosis is poor. Bortezomib, a proteasome inhibitor, may exert efficacy in TNBC through its multiple cellular effects. Here, we tested the efficacy of bortezomib and examined the drug mechanism in breast cancer cells.

Methods

Five breast cancer cell lines: TNBC HCC-1937, MDA-MB-231, and MDA-MB-468; HER2-overexpressing MDA-MB-453; and estrogen receptor positive MCF-7 were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western Blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interfering RNA (siRNA). In vivo efficacy of bortezomib was tested in nude mice with breast cancer xenografts. Immunohistochemical study was performed on tumor tissues from patients with TNBC.

Results

Bortezomib induced significant apoptosis, which was independent of its proteasome inhibition, in the three TNBC cell lines, but not in MDA-MB-453 or MCF-7 cells. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of bortezomib. We showed that bortezomib inhibited CIP2A in association with p-Akt downregulation in a dose- and time-dependent manner in all sensitive TNBC cells, whereas no alterations in CIP2A expression and p-Akt were noted in bortezomib-resistant cells. Overexpression of CIP2A upregulated p-Akt and protected MDA-MB-231 and MDA-MB-468 cells from bortezomib-induced apoptosis, whereas silencing CIP2A by siRNA overcame the resistance to bortezomib-induced apoptosis in MCF-7 cells. In addition, bortezomib downregulated CIP2A mRNA but did not affect the degradation of CIP2A protein. Furthermore, bortezomib exerted in vivo antitumor activity in HCC-1937 xenografted tumors, but not in MCF-7 tumors. Bortezomib downregulated CIP2A expression in the HCC-1937 tumors but not in the MCF-7 tumors. Importantly, CIP2A expression is readily detectable in tumor samples from TNBC patients.

Conclusions

CIP2A is a major determinant mediating bortezomib-induced apoptosis in TNBC cells. CIP2A may thus be a potential therapeutic target in TNBC.     

内质网应激对乳腺癌MCF-7细胞CCL5表达的影响

目的探讨内质网应激水平与人乳腺癌MCF-7细胞CCL5表达之间的关系,明确CCL5与人乳腺癌MCF-7细胞增殖侵袭转移能力之间的关系。方法使用内质网应激诱导剂(Tuniamycin）和内质网应激抑制剂(4-PBA）分别处理人乳腺癌MCF-7细胞,24小时后提取细胞总蛋白。Western blot检测MCF-7细胞内质网应激水平及CCL5表达情况。MTT比色法,Transwell小室法检测细胞增殖和侵袭转移能力。结果内质网应激诱导剂处理组细胞内质网应激处于高水平,同时CCL5表达量增高;抑制剂组细胞内质网应激水平低,CCL5表达量也随之降低。并且诱导剂组肿瘤细胞增殖较抑制剂组活跃。升高的CCL5部分分泌到培养液中,影响细胞侵袭转移能力。结论内质网应激能够诱导人乳腺癌MCF-7细胞CCL5的表达;内源性CCL5能促进MCF-7细胞的增殖;外源性CCL5能促进MCF-7细胞侵袭转移。     

Cotylenin A, a new differentiation inducer, and rapamycin cooperatively inhibit growth of cancer cells through induction of cyclin G2

Cotylenin A, a plant growth regulator, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), are potent inducers of differentiation of myeloid leukemia cells. Recently, we found that cotylenin A and rapamycin effectively inhibited the proliferation of several human breast cancer cell lines including MCF-7. Herein, we demonstrate that cotylenin A and rapamycin rapidly and markedly induced the cyclin G2 gene expression in several cancer cells including MCF-7 cells. The growth arrest of the MCF-7 cells at the G1 phase, induced by the treatment with cotylenin A and rapamycin or the culture in low serum medium, markedly induced the cyclin G2 gene expression. Anticancer drugs including doxorubicin, etoposide and 5-fluorouracil also induced cyclin G2 expression during induction of growth arrest of the MCF-7 cell at the G1 phase or G2/M phase. Ectopically inducible cyclin G2 expression potently inhibited the proliferation of MCF-7 cells. Furthermore, cyclin G2 knockdown induced by cyclin G2 small interfering RNA markedly reduced the potency of cotylenin A plus rapamycin to induce growth inhibition. Taken together, our results suggest that cotylenin A and rapamycin induce inhibition of cancer cell growth through the induction of cyclin G2. ( Cancer Sci 2008; 99: 1693–1698)     

Effects of the proteasome inhibitor bortezomib alone and in combination with chemotherapeutic agents in gastric cancer cell lines

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of tumor cells. Bortezomib is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against several solid tumors. We examined the anti-proliferative and apoptotic effects of bortezomib in three gastric cancer cell lines (SNU638, MUGC-3 and MKN-28), along with its antitumor combination effects with other chemotherapeutic agents. Tumor cell growth inhibition and apoptosis was measured by MTT assay and FACS analysis, respectively. Apoptosis- and cell cycle-associated protein expression levels were measured by Western blot assay. Bortezomib induced the suppression of tumor cell growth and apoptosis in a dose-dependent manner with an inhibitory dose (ID)50 of approximately 0.5 microg/ml in all gastric cancer cell lines tested. Further combination treatment with cisplatin and docetaxel, in particular with docetaxel displayed dramatically increased tumor cell growth suppression in all three gastric cancer cell lines, as compared to single drug treatment alone. This was concomitant with the induction patterns of apoptotic cells. Bortezomib treatment increased the Bax protein expression. Moreover, combination treatment of bortezomib plus docetaxel resulted in a dramatic increase in the Bax expression. In contrast, Bcl-2 expression was decreased by combination treatment with bortezomib plus docetaxel in SNU638 cells. Finally, bortezomib, docetaxel and to a greater degree bortezomib plus docetaxel increased the expression levels of p27 proteins even without influencing p53 expression levels. Bortezomib has profound effects on tumor cell growth inhibition and induction of apoptosis in human gastric cancer cells, suggesting that bortezomib may be an effective therapeutic drug for patients with gastric cancer. Further combination studies with other chemotherapeutic drugs, in particular docetaxel showing more tumor cell growth inhibition and apoptosis suggest that combining bortezomib with docetaxel might be more effective for displaying tumor cell growth inhibitory effects in gastric cancer cells through regulation of Bcl-2, Bax and p27 proteins in vitro.     

Sesamin, a lignan of sesame, down-regulates cyclin D1 protein expression in human tumor cells

Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol-lowering, lipid-lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF-7. Furthermore, sesamin dephosphorylates tumor-suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF-7 cell proliferation by sesamin is correlated with down-regulated cyclin D1 protein expression, a proto-oncogene that is overexpressed in many human cancer cells. It was found that sesamin-induced down-regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down-regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down-regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent.     

GANT61通过自噬性细胞死亡抑制人乳腺癌MCF-7细胞增殖

 目的 观察Hedgehog通路抑制剂GANT61对人乳腺癌MCF-7细胞的抑制作用。方法 GANT61作用MCF-7细胞后,通过流式细胞术检测细胞死亡;提取处理细胞总RNA和蛋白。分别采用Real-timePCR和Western blot检测Hedgehog通路SHH、Gli1、Gli2及自噬标志物LC3-Ⅱ表达;处理细胞固定后通过电子显微镜观察GANT61诱导的自噬体形态;自噬抑制剂3-MA处理细胞或自噬相关基因atg5 siRNA转染细胞后再用GANT61作用,流式细胞术检测细胞死亡数量。结果 GANT61能明显诱导MCF-7死亡,降低Hedgehog通路SHH、Gli1、Gli2表达水平,提高LC3-Ⅱ蛋白表达,并可诱导MCF-7细胞产生自噬体。3-MA及atg5 siRNA可减弱GANT61诱导的细胞死亡。结论 GANT61通过诱导自噬性细胞死亡机制发挥抗乳腺癌细胞活性。     

5,4'-二-正辛烷氧基-7-二氟亚甲基异黄酮体外抗人类乳腺癌细胞作用

目的 探讨金雀异黄素(Gen)衍生物5,4'-二-正辛烷氧基-7-二氟亚甲基异黄酮(DOdFMG)体外抗人类乳腺癌细胞的作用.方法 平皿克隆形成法检测DOdFMG对人类乳腺癌MCF-7细胞抑制生长和增生作用,琼脂糖凝胶电泳法观测DOdFMG诱导MCF-7细胞凋亡作用,Western blotting 法测定DOdFMG对MCF-7细胞PTEN、pAkt、caspase-3蛋白的表达及活性.结果 DOdFMG抑制MCF-7细胞生长,呈剂量依赖性,比Gen具有更强的抗肿瘤活性,DOdFMG能诱导MCF-7细胞凋亡,DOdFMG处理的MCF-7细胞PTEN蛋白及caspase-3蛋白表达上调,pAkt蛋白表达下调,呈时间剂量依赖性.结论 DOdFMG具有抗人类乳腺癌MCF-7细胞的作用,其机制可能与抑制PKB/Akt通路的信号传导、激活PTEN及caspase-3有关,是一种新型治疗乳腺癌的候选药物.