沉默环磷腺苷效应元件结合蛋白1对乳腺癌细胞生物学行为的影响

目的：探究环磷腺苷效应元件结合蛋白（cAMP-response element binding protein, CREB1）基因沉默对乳腺癌MCF-7和MDA-MB-231细胞增殖、凋亡、迁移和侵袭的影响。方法：针对人CREB1的基因序列设计并构建2条短发夹RNA（short hairpin RNA，shRNA），采用慢病毒转染shRNA至人乳腺癌MCF-7和MDA-MB-231细胞系抑制其CREB1的表达。将实验组分为shCREB1#1组和shCREB1#2组，同时将shSCR空载质粒转染至上述细胞系作为阴性对照组。采用实时定量PCR和Western-blot法检测转染效率；CCK-8法检测细胞的增殖能力；集落形成实验检测细胞的集落形成能力；流式细胞术检测细胞周期和凋亡率；细胞划痕实验和Transwell实验检测细胞的迁移和侵袭能力；Western-blot法检测细胞周期及细胞凋亡相关蛋白的表达。结果： 在MCF-7和MDA-MB-231细胞中，相较于shSCR组，shCREB1#1和shCREB1#2组中CREB1基因的mRNA和蛋白表达水平均下降（P＜0.001）。沉默CREB1后，MCF-7和MDA-MB-231细胞的增殖能力、集落形成能力、迁移和侵袭能力减弱且细胞的凋亡率升高（P＜0.05）。此外，沉默CREB1可使细胞周期蛋白CDK2、CDK4、CDK6、Cyclin D1以及抗凋亡蛋白Bcl-2、Survivin的表达水平下调而促凋亡蛋白Caspase 3和Bax的表达水平上调。 结论：沉默CREB1可抑制乳腺癌细胞的增殖、迁移和侵袭能力并诱导细胞凋亡。     

青蒿素对MCF-7乳腺癌细胞Akt及survivin蛋白表达影响的研究

目的研究青蒿素对MCF-7乳腺癌细胞Akt及survivin蛋白表达的影响。方法体外培养人乳腺癌细胞系MCF-7细胞株,采用MTT法检测青蒿素对细胞的抑制作用,流式细胞仪检测细胞周期及凋亡的影响,Western blot分析Akt与survivin蛋白表达的改变,多组均数比较采用单因素方差分析,组间两两比较用LSD检验。结果青蒿素对MCF-7乳腺癌细胞体外生长具有明显的抑制作用,并呈时间和剂量依赖性;青蒿素对MCF-7细胞出现G0/G1期阻滞;与对照组比较,青蒿素明显抑制Akt与survivin蛋白的表达。结论青蒿素可以通过抑制Akt与survivin蛋白表达及对细胞周期的影响而诱导乳腺癌细胞的凋亡,从而发挥抗肿瘤作用。     

p27^kip1、Skp2与肿瘤

细胞周期的运行受多种因素所调控。有两个主要调控点 :一个处于G1/S转折点 ,是控制细胞进入S期的控制点(G1期检测点 ) ,另一个处于G2 /M转折点 ,是控制细胞进入M期的控制点 (G2 期检测点 )。参与细胞周期调控的主要因子有 :周期蛋白 (cyclin) ,周期蛋白依赖性激酶 (cyclindepen dentkinase,CDK ) ,周期蛋白依赖性激酶抑制剂 (CKI)。CDK与cyclin CDK复合物正性催化细胞周期过程 ,而CKI通过与cyclin ,CDK或cyclin CDK复合物结合抑制并阻断细胞周期进程。p2 7kip1就是一种新发现的CKI ,p2 7kip1活性的改变影响细胞周期进程 ,进而…     

补骨脂素对乳腺癌耐药细胞株周期和细胞凋亡的影响

目的研究补骨脂素对人乳腺癌多药耐药细胞凋亡和周期的影响。方法用MTT法检测不同浓度的补骨脂素对人乳腺癌MCF-7/ADR耐药细胞株的体外生长抑制作用,并运用流式细胞仪观察细胞凋亡和细胞周期的变化情况,采用RT-PCR检测基因caspase-3、Bcl-2和p53的表达。结果补骨脂素对人乳腺癌MCF-7/ADR细胞株有明显的抑制作用,其IC50值为(18.785±1.253)μg/ml;作用24 h后,流式细胞仪检测显示细胞周期S期细胞减少,G0/G1期细胞增加(P<0.05),但对早期凋亡细胞影响较小。并上调凋亡基因p53和凋亡执行基因caspase-3(P<0.05),下调抑凋亡基因Bcl-2(P<0.05)。结论补骨脂素对人乳腺癌MCF-7/ADR耐药细胞有明显体外抑制作用,其抑制机制与影响细胞周期及诱导细胞凋亡有关。     

DIM对雌激素非依赖性乳腺癌细胞增殖的影响

目的探讨3,3-二吲哚基甲烷(DIM)对雌激素非依赖性乳腺癌细胞MDA-MB-468体外增殖和体内成瘤能力的影响及其机制。方法 6×10~6个MDA-MB-468细胞接种BALB/c裸鼠,建立乳腺癌细胞异种肿瘤移植模型,5 mg/(kg·d)DIM喂食,检测DIM对MDA-MB-468肿瘤细胞致瘤及生长能力的影响。体外实验,不同浓度(0,30,60μmol/L)的DIM处理MDA-MB-468细胞,采用CCK-8法检测DIM对细胞增殖的影响;流式细胞仪测定细胞周期和细胞凋亡;Western印迹检测细胞周期调控蛋白(CyclinD 1),CDK4,CDC25A的表达水平。结果异种肿瘤移植模型中,DIM可显著抑制MDA-MB-468细胞的成瘤能力。体外实验,DIM对乳腺癌MDA-MB-468细胞增殖的抑制作用呈时间和剂量依赖性。流式细胞仪结果显示;DIM可改变MDA-MB-468细胞进程,将其阻滞于G1/S、G2/M期,并且60μmol/L DIM作用组有明显的诱导凋亡作用。DIM作用MDA-MB-468细胞48 h后,细胞周期因子CyclinD I,CDK4及CDC25A蛋白表达呈剂量依赖性下降(P0.05)。结论 DIM可有效抑制雌激素非依赖性乳腺癌细胞的体外增殖和体内成瘤,其机制可能与细胞周期重要调控蛋白表达下调有关。     

他克莫司对人肾小球系膜细胞细胞周期相关蛋白表达的影响

目的探讨他克莫司对体外培养的人肾小球系膜细胞细胞周期相关蛋白cyclin D1、cyclin E及p27kip1的影响及可能机制。方法设正常对照组和他克莫司处理组,他克莫司组设为三个组(浓度分别为10μmol/L、25μmol/L、50μmol/L)。四甲基偶氮唑盐法(MTT)检测他克莫司组处理12h后肾小球系膜细胞的增殖情况;流式细胞仪检测他克莫司组分别作用12、24、48h后系膜细胞的上述三种蛋白质及与其结合的CDK2和CDK4的表达情况;选择有明显抑制作用的时间点(12h),Westernblot检测上述三种蛋白质的表达情况。结果 (1)作用12h,他克莫司组均能抑制系膜细胞的增殖。(2)作用12、24、48h,与对照组相比,浓度为10μmol/L他克莫司对细胞周期蛋白cyclin D1、cyclin E及抑制因子p27kip1的表达无明显影响,而浓度为25、50μmol/L时,cyclin D1及cyclin E明显降低,p27kip1表达明显升高,呈一定的剂量效应关系;且相同浓度的他克莫司在作用三个时间点后,cyclin D1、cyclin E及p27kip1的表达无明显差异。(3)作用12h,与对照组相比,浓度为10μmol/L他克莫司对周期蛋白依赖性激酶CDK2、CDK4的表达无明显影响,而浓度为25、50μmol/L时,CDK2、CDK4的表达明显降低。结论一定浓度的他克莫司可抑制系膜细胞的增殖,其作用机制至少部分与其影响细胞周期相关蛋白的表达有关。     

丁酸钠联合全反式维甲酸对MDS细胞株SKM-1的诱导分化作用及其机制初步探讨

本研究探讨丁酸钠(NaB)抑制MDS细胞株SKM-1细胞生长、诱导其分化的分子机制，并研究其与全反式维甲酸(ATRA)的协同作用。用台盼蓝拒染实验观察药物对细胞生长曲线的影响；四氮唑盐还原试验和细胞表面分化抗原检测观察药物对细胞的分化作用；流式细胞术分析细胞周期；RT-PCR检察D型细胞周期蛋白、CDK和P21在mRNA水平的表达。结果表明：NaB和(或)ATRA均可抑制SKM-1细胞的生长，诱导细胞分化，将细胞周期阻滞于G0／G1期；ATRA下调CDK6、CDK4、cyclin D3和cyclin D1 mRNA的水平；NaB下调CDK2、cyclin D2和cyclin D1 mRNA的水平；两药联用下调CDK6、CDK4、CDK2、cyclin D1、cyclin D2和cyclin D3 mRNA的水平；ATRA和(或)NaB均上调P21 mRNA的水平。结论:NaB诱导SKM-1的分化可能是通过上调P21 mRNA的水平和抑制cycLin D-CDK复合体的形成完成的，NaB与ATRA对SKM-1细胞株的分化有协同作用。     

大蒜素抑制多发性骨髓瘤侧群细胞增殖机制研究

目的:探讨大蒜素对多发性骨髓瘤侧群细胞(side population,SP)增殖的影响及其作用机制。方法:培养RPM I-8226和NCI-H929细胞,应用Hoechst33342染色分析其侧群细胞的含量;对培养的侧群细胞,应用10μg/ml大蒜素处理,采用CCK8法分析大蒜素对侧群细胞增殖的影响,应用平板集落形成法分析大蒜素对侧群细胞集落形成能力的影响,流式细胞术分析大蒜素对侧群细胞周期的影响,Western blot法分析大蒜素对周期相关蛋白cyclin D1、cyclin E、CDK2和CDK4的表达。结果:在RPM I 8226和NCI-H929中均存在有侧群细胞,其所占比例分别为(3.17±0.98)%和(2.65±0.61)%。大蒜素能够呈时间依赖性抑制侧群细胞的增殖并显著抑制集落形成;大蒜素能够抑制周期相关蛋白cyclin D1、cyclin E、CDK2和CDK4的表达并诱导细胞发生G1/S周期阻滞。结论:大蒜素可以抑制多发性骨髓瘤侧群细胞增殖和集落的形成,并可能通过下调细胞周期相关蛋白的表达诱导细胞发生G1/S阻滞。     

白藜芦醇诱导食管癌细胞凋亡及其相关基因表达的研究

目的研究白藜芦醇诱导人食管癌Eca109细胞凋亡的作用并探讨其对凋亡相关基因survivin、bax和bcl-2表达的影响。方法不同浓度白藜芦醇作用于Eca109细胞后,采用流式细胞仪检测细胞凋亡、细胞周期分布及survivin、bax和bcl-2蛋白的表达。结果白藜芦醇能诱导食管癌Eca109细胞凋亡,使其细胞周期阻滞在GO／G1期;白藜芦醇可以上调bax蛋白的表达,下调survivin蛋白并协同抑制bcl-2蛋白的表达。结论白藜芦醇能明显诱导Eca109细胞凋亡,其机制可能与促进bax表达、抑制survivin和bcl-2表达有关。     

曲古抑菌素A抑制乳腺癌MCF-7细胞增殖和诱导其凋亡的作用

目的：研究组蛋白去乙酰化酶抑制剂曲古抑菌素A对乳腺癌MCF-7细胞的增殖抑制作用和诱导凋亡作用。方法：乳腺癌MCF-7细胞经不同剂量曲古抑菌素作用后,用二甲氧唑黄比色法（XTT）法测定曲古抑菌素A对乳腺癌MCF-7细胞的增殖抑制率;用免疫细胞化学染色法观察其对凋亡相关基因p21wafl表达的影响。结果：不同浓度的曲古抑菌素均可抑制MCF-7细胞的增殖,且抑制率具有剂量和时间依赖性;凋亡相关基因p21wafl在曲古押菌素A作用细胞中的表达明显高于未进行处理的细胞。结论：曲古押菌素可抑制体外培养的人乳腺癌（MCF-7）细胞生长,诱导癌细胞发生凋亡。     

2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061), a novel microtubule inhibitor,evokes G2/M cell cycle arrest and apoptosis in human breast cancer cells

Breast cancer is the leading cause of cancer death in women worldwide, and novel chemotherapeutic drugs with high activity and no drug resistance for treating breast cancer are needed urgently. In this study, we investigated the antitumor effect of 2-methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061), which has a strong inhibition of cell growth in MCF-7 and MDA-MB-231 cells. We demonstrated that SQ0814061 (SQ) time-dependently induced cell cycle arrest at G2/M phase and subsequently progressed into apoptosis, which is associated with microtubule depolymerization. Western blot analysis revealed that up-regulation of cyclin B1 and Aurora A was related with G2/M phase arrest in MCF-7 and MDA-MB-231 cells treatment with SQ. However, the formation of multinucleated cells after a long time exposed to SQ of MCF-7 cells delayed the cell death. In addition, apoptosis induced by SQ is correlated with the down-regulation of the PI3K-Akt-MDM2 pathway in MCF-7 and MDA-MB-231 cells. Treatment with the PI3K specific inhibitor, LY294002, increased SQ-induced cell growth inhibitory rate and apoptosis rate of MCF-7 and MDA-MB-231 cells. Moreover, SQ induced MCF-7 and MDA-MB-231 cells to generate reactive oxygen species (ROS), and the SQ-induced cell death was ROS dependent. In conclusion, all the data demonstrated that SQ exhibited its antitumor activity through disrupting the microtubule assembly, inducing cell cycle arrest and eventually apoptosis which is associated with PI3K-Akt-MDM2 pathway in MCF-7 and MDA-MB-231 cells. Therefore, the novel compound SQ is a promising microtubule inhibitor that has tremendous potentials for therapeutic treatment of human mastocarcinoma.     

Benzyl isothiocyanate-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species and regulated by Bax and Bak

Epidemiologic studies have revealed an inverse correlation between dietary intake of cruciferous vegetables and the risk of breast cancer. We now show that cruciferous vegetable constituent benzyl isothiocyanate (BITC) effectively suppresses growth of cultured human breast cancer cells (MDA-MB-231 and MCF-7) by causing G(2)-M phase cell cycle arrest and apoptosis induction. On the other hand, a normal mammary epithelial cell line (MCF-10A) is significantly more resistant to growth arrest and apoptosis by BITC compared with breast cancer cells. The BITC-mediated cell cycle arrest was associated with a decrease in levels of proteins involved in regulation of G(2)-M transition, including cyclin B1, cyclin-dependent kinase 1, and cell division cycle 25C. The BITC-induced apoptosis correlated with induction of proapoptotic proteins Bax (MCF-7) and Bak (MDA-MB-231 and MCF-7) and down-regulation of antiapoptotic proteins Bcl-2 and Bcl-xL (MDA-MB-231). The SV40-immortalized mouse embryonic fibroblasts derived from Bax and Bak double knockout mice were significantly more resistant to BITC-induced DNA fragmentation compared with wild-type mouse embryonic fibroblasts. The BITC treatment caused rapid disruption of the mitochondrial membrane potential, leading to cytosolic release of apoptogenic molecules, which was accompanied by formation of autophagosome-like structures as revealed by transmission electron microscopy. The BITC-mediated apoptosis was associated with generation of reactive oxygen species and cleavage of caspase-9, caspase-8, and caspase-3. Apoptosis induction by BITC was significantly attenuated in the presence of a combined superoxide dismutase and catalase mimetic EUK134 as well as caspase inhibitors. In conclusion, the present study reveals a complex signaling leading to growth arrest and apoptosis induction by BITC.     

5,7,4⿲-Trihydroxy-6,8-diprenylisoflavone and lupalbigenin,active components of Derris scandens,induce cell death on breast cancer cell lines

BackgroundNatural products are a potential source for cancer chemotherapeutic development. This current study was performed to investigate the anti-tumor potential of 5,7,4⿲-trihydroxy-6,8-diprenylisoflavone (TD) and lupalbigenin (LB), plant flavonoids found in Derris scandens Benth (family: Leguminosae), in cancer and normal cell lines.MethodsThe human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468, the human colon cancer cell line SW-620, and the mouse fibroblast cell line L-929 were used to test their anti-cancer activity. Apoptotic cell levels were measured by staining with annexin-V and propidium iodide and Western blot analysis was performed to confirm the apoptotic mechanism.ResultsThe results revealed that TD and LB showed specific cytotoxicity against MDA-MB-231 and MCF-7 cells. To elucidate mode of cell death via cytotoxic activities, breast cancer cell lines were treated. TD and LB induced MDA-MB-231 and MCF-7 cells to apoptosis, with the highest number of apoptotic cells at 24 and 72 h, respectively. Furthermore, TD and LB inhibited cell cycle progression via up-regulation of p21. Both compounds stimulated apoptosis through down-regulation of bcl-2, up-regulation of bax and releasing of cytochrome C proteins.ConclusionsTD and LB have significant anti-cancer effects against human breast cancer cells via cell cycle arrest and the induction of apoptosis through mitochondria signaling pathways, and may be potential anti-cancer agents for the treatment of breast cancer.     

survivin基因沉默对“三阴”乳腺癌细胞株中caspase3和caspase7基因的影响

目的 探讨“三阴”乳腺癌细胞中沉默survivin基因对抑癌基因caspase3和caspase7的影响.方法 针对survivin基因设计干扰片段并与真核表达载体pGCsilencerTMU6/Neo/GFP进行连接,之后转染“三阴”乳腺癌细胞MDA-MB-231;通过RT-PCR鉴定survivin基因mRNA水平沉默效果;用RT-PCR检测沉默survivin基因对caspase3和caspase7表达及细胞凋亡的影响.结果 survivin基因沉默可引起MDA-MB-231细胞凋亡;caspase3和caspase7表达升高,灰度分析结果显示差异具有统计学意义（P =0.008）.结论 （1）RNAi技术沉默MDA-MB-231细胞中survivin基因,达到了对其表达的抑制效果;（2）survivin基因沉默可上调caspase3和caspase7的表达;（3） survivin基因沉默可引起细胞凋亡;（4） survivin基因可作为“三阴”乳腺癌基因治疗的新靶点.     

Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells

Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2-CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics.     

Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines

Sulforaphane, an isothiocyanate found in cruciferous vegetables, has been shown to induce phase 2 detoxication enzymes and inhibit the growth of chemically induced mammary tumors in rats, although the exact mechanisms of action of sulforaphane are not understood. In this study, we evaluated the effects of sulforaphane on cell growth and death in several human breast cancer cell lines and examined the hypothesis that sulforaphane acts as a histone deacetylase (HDAC) inhibitor in these cell lines. Sulforaphane treatment inhibited cell growth, induced a G(2)-M cell cycle block, increased expression of cyclin B1, and induced oligonucleosomal DNA fragmentation in the four human breast cancer cell lines examined, MDA-MB-231, MDA-MB-468, MCF-7, and T47D cells. Activation of apoptosis by sulforaphane in MDA-MB-231 cells seemed to be initiated through induction of Fas ligand, which resulted in activation of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, whereas apoptosis in the other breast cancer cell lines was initiated by decreased Bcl-2 expression, release of cytochrome c into the cytosol, activation of caspase-9 and caspase-3, but not caspase-8, and poly(ADP-ribose) polymerase cleavage. Sulforaphane inhibited HDAC activity and decreased the expression of estrogen receptor-alpha, epidermal growth factor receptor, and human epidermal growth factor receptor-2 in each cell line, although no change in the acetylation of H3 or H4 was seen. These data suggest that sulforaphane inhibits cell growth, activates apoptosis, inhibits HDAC activity, and decreases the expression of key proteins involved in breast cancer proliferation in human breast cancer cells. These results support testing sulforaphane in vivo and warrant future studies examining the clinical potential of sulforaphane in human breast cancer.     

Stathmin表达水平与乳腺癌细胞侵袭能力相关性研究

目的 探讨乳腺癌细胞MDA-MB-231和MCF-7中Stathmin基因表达水平与细胞生长、黏附、侵袭等生物学行为之间的关系,为进一步研究乳腺癌转移机制奠定实验基础。方法 应用RT-PCR和Western Blot方法检测MDA-MB-231和MCF-7细胞中Stathmin基因的表达水平,同时利用细胞增殖试验、细胞黏附试验和细胞侵袭试验检测MDA-MB-231和MCF-7细胞的生长、黏附、侵袭能力,分析Stathmin表达与细胞的生长、黏附、侵袭能力之间的关系。结果 RT-PCR和Western Blot检测结果显示,Stathmin基因在MDA-MB-231和MCF-7细胞中表达均高于正常对照细胞(F=10.173,P<0.05),且MDA-MB-231细胞中的表达水平明显高于MCF-7细胞中的表达水平(t=4.562,P<0.05)。而MDA-MB-231细胞在生长、黏附和侵袭能力方面均强于MCF-7细胞(P<0.05)。结论 Stathmin表达水平高的乳腺癌细胞相应的生长、黏附、侵袭能力较强,Stathmin表达水平与细胞侵袭能力密切相关。     

Inhibition of growth and induction of apoptosis in human cancer cell lines by an ethyl acetate fraction from shiitake mushrooms

OBJECTIVE: Shiitake (Lentinus edodes) mushrooms have been reported to have cancer-preventing properties. However, little research has been conducted verifying the antitumor activities of "mycochemicals" in shiitake mushrooms. In this study, potential roles of an ethyl acetate fraction from shiitake mushrooms were investigated by in vitro bioassays. DESIGN: The activities of an ethyl acetate fraction were evaluated by [3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide] (MTT), apoptosis bioassay, cell cycle analysis, and Western blot analysis using two human breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human nonmalignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9). RESULTS: Concentration-dependent antiproliferative effects of the fraction were observed in all cell lines using the MTT assay. Approximately 50 mg/L concentration of the fraction induced apoptosis in 50% of the population of four human tumor cell lines and the fraction-induced apoptosis may have been mediated through the pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell cycle arrest by significant decrease of S phase, which was associated with the induction of cdk inhibitors p21 and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumor cells, nonmalignant cells were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and cdk4 expression. A 51% antiproliferative effect occurred at the highest concentration of the fraction (800 mg/L). CONCLUSIONS: These data suggest that inhibition of growth in tumor cells by "mycochemicals" in shiitake mushrooms may result from induction of apoptosis.     

Hypoxia decreased chemosensitivity of breast cancer cell line MCF-7 to paclitaxel through cyclin B1

Hypoxia, frequently found in the center of solid tumors, may lead to enhance the production of key factor in cell survival, invasion, angiogenesis and loss of apoptosis. The low oxygen tension in hypoxic tumors is also known to interfere with the efficacy of chemotherapy, but the underlying mechanisms are not very clear. Paclitaxel (PTX) is an active agent used in breast cancer chemotherapy, which disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. In the present study, we try to determine whether hypoxia can decrease the chemosensitivity of human breast carcinoma cells to PTX and elucidate the underlying mechanism. We found that hypoxia could decrease PTX-induced cell death and G₂/M arrest. Furthermore, our results showed that hypoxia inhibit PTX-induced soluble tubulin polymerized. In addition, we also found hypoxia could suppress PTX-induced cell cycle protein—cyclin B1 expression in MCF-7 cells. To further investigate whether the inhibitory effect of hypoxia on PTX-induced cell death is mediated by decreasing levels of cyclin B1, cyclin B1-transfected MCF-7 cells were used under hypoxic condition. The data showed that the hypoxia-based decreasing chemosensitivity of breast cancer cells to PTX was reversed by cyclin B1. We also found that overexpression of cyclin B1 could significantly increase the sensitivity of MCF-7 cells to PTX by stimulating soluble polymerized tubulin. Overall, hypoxia decreases cyclin B1, which could in turn reverse hypoxia-induced decreasing chemosensitivity to PTX in breast cancer cell line MCF-7.     

Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner

BACKGROUND: Preclinical and clinical studies have shown that the proteasome inhibitor bortezomib (PS341, Velcade) is highly effective when combined with chemotherapeutic agents. The value of trastuzumab (Herceptin) in HER-2-positive (3+ score by immunohistochemistry or fluorescence in situ hybridization positive) breast cancer is also known; however, the response rate is <40% for metastatic breast cancer. These two pharmacologic agents prevent nuclear factor-kappaB (NF-kappaB) activation and induce nuclear accumulation of the cyclin-dependent kinase inhibitor p27(kip1), suggesting that combining bortezomib with trastuzumab could increase trastuzumab efficacy. METHODS: Drug cytotoxicity, both individually and together, and drug effects on p27 localization and NF-kappaB activation were investigated on four breast cancer cell lines: SKBR-3 (HER-2+++), MDA-MB-453 (HER-2++), HER-2-transfected MCF-7 (HER-2+++), and MCF-7 (HER-2-). RESULTS: Bortezomib induced apoptosis in HER-2-positive and HER-2-negative breast cancer cells in a dose- and time-dependent manner. Together, these drugs induced apoptosis of HER-2++/+++ cells at low concentrations, which had no effect when used alone, indicating there was a synergistic effect. Sequential treatment (trastuzumab then bortezomib) induced either necrosis or apoptosis, depending on the trastuzumab preincubation time. Susceptibility to bortezomib alone and the drug combination correlated with NF-kappaB activity and p27 localization. CONCLUSIONS: The addition of bortezomib to trastuzumab increases the effect of trastuzumab in HER-2+++/++ cell lines in a synergistic way. This effect likely results from the ability of these two drugs to target the NF-kappaB and p27 pathways. The potential clinical application of this drug combination is under current evaluation by our group in a phase 1 clinical trial.