毛蚶抗癌蛋白对人乳腺癌细胞MCF-7的生长抑制作用

目的:从毛蚶软体部位中分离出具有一定抗癌活性的毛蚶蛋白组分,研究其对人乳腺癌细胞MCF-7的生长抑制作用。方法:以新鲜毛蚶软体部分为原材料,分离获得毛蚶抗癌蛋白组分(命名为NS);倒置相差显微镜观察不同浓度(50、100、200、400μg/mL)NS组分对MCF-7癌细胞生长的影响,将200μg/mL NS组分作用MCF-7细胞不同时间(12、24、48 h)后,Giemsa染色观察癌细胞及核形态的变化,流式细胞术检测NS组分对MCF-7细胞周期及细胞凋亡的影响,Western blot检测细胞周期及细胞凋亡相关蛋白的表达。结果:与阴性对照组比较,各浓度NS组分对人乳腺癌MCF-7细胞生长均具有明显的抑制作用(P<0.05或P<0.01);倒置相差显微镜下可见200μg/mL NS组分作用MCF-7细胞12 h后,部分细胞收缩变圆、脱落,24、48 h后细胞脱落现象更加明显;Giemsa染色发现,NS组分处理12 h后细胞出现有丝分裂相增加,继而出现多核或核碎裂等现象;流式细胞术检测发现NS组分主要引起细胞G2-M期阻滞;NS处理组凋亡率均较对照组明显升高(P均<0.01),且随着药物作用时间的延长,细胞凋亡比例逐渐增加。Western blot检测发现NS组分作用MCF-7细胞后p53及procaspase-3蛋白表达量均上调。结论:毛蚶抗癌蛋白NS组分体外对人乳腺癌MCF-7细胞的细胞毒作用主要通过引起细胞发生G2-M期阻滞、诱导细胞凋亡实现的,期间伴随p53及procaspase-3蛋白表达上调。     

G2—M期阻滞与肿瘤

Ｇ２－Ｍ期阻滞是细胞对射线等ＤＮＡ损伤剂的普遍反应,与基因组不稳定性,肿瘤发生和治疗密切相关。本文着重阐述了近年来报道的在Ｇ２－Ｍ期阻滞的分子机制,。与凋亡的关系以及生物不意义方面的研究进展。     

辐射诱发肿瘤细胞增殖抑制和凋亡的相关研究

目的 探讨不同辐射敏感性的肿瘤细胞受照后G2/M期阻滞和凋亡发生的关系。为提高肿瘤放射治疗效果提供理论依据。方法 以人早幼粒白血病细胞株HL-60、人T淋巴细胞性白血病细胞系CEM和人红白血病细胞系K562细胞为对象,采用形态学观察、DNA琼脂糖凝胶电泳和流式细胞仪等方法检测细胞周期的改变和凋亡的发生。结果 辐射敏感的HL-60和CEM细胞受照后首先发生G2/M期阻滞,然后在阻滞退出过程中或之后发生凋亡;辐射耐受的K562细胞受照后只发生G2/M期阻滞,不发生凋亡;照射并加入咖啡因（CAF）,抑制3种细胞的G2/M期阻滞,促进凋亡;照射并加入佛波酯（TPA）,增加HL-60和CEM细胞的G2/M期阻滞,抑制凋。结论 辐射诱发肿瘤细胞的凋亡与G2/M期阻滞有关,用药物调控G2/M期阻滞可影响辐射所致的凋亡。     

咖啡因促进受照肿瘤细胞凋亡的研究

目的：探讨肿瘤细胞受照后Ｇ２／Ｍ期阻滞和凋亡的关系及调控。方法：以Ｋ５６２细胞为对象,用流式细胞仪,形态学,ＤＮＡ电泳和凋亡蛋白２．７（ＡＰＯ２．７）等方法检测细胞周期和凋亡。结果：（１）２０Ｇｙγ射线照射Ｋ５６２细胞后引起Ｇ２／Ｍ期阻滞,４８ｈＧ２／Ｍ期比例为７１．２％,流式细胞仪,形态学和ＡＰＯ２．７检测凋亡比例分别为２．６％,２．０％±１．１Ａ％和２２．５％。（２）照前加入１０ｍｍｏｌ／Ｌ咖     

柴胡皂苷D通过诱导C2-M期阻滞抑制结直肠癌SW480细胞的增殖

目的:观察柴胡皂苷D（SSD）对于结直肠癌肿瘤细胞增殖的影响并初步探索其潜在的分子机制。方法:运用MTT、细胞计数试验及克隆形成试验观察SSD对细胞增殖的影响。流式细胞仪检测SSD对细胞凋亡及周期分布情况的影响。RT-PCR和Western-blot技术检测G2-M周期阻滞关键调控因子在SW480细胞中的表达。结果:MTT试验发现SSD能够显著抑制结直肠癌肿瘤细胞SW480的增殖,而对正常结直肠细胞FHC的增殖无明显影响。细胞计数试验和克隆形成试验进一步验证了SSD对于SW480细胞增殖的影响(P<0.05)。流式细胞术检测发现SSD不影响细胞的凋亡(P>0.05),但却显著诱导G2-M周期阻滞(P<0.05)。SSD在mRNA水平下调了G2-M周期调控因子CCNA1、CCNA2、CCNB1和CCNB2的表达(P<0.05);在蛋白水平,CCNA2、CCNB1和p34/cdc2明显被下调,p-H3S10上调,p21WAF1/CIP1被明显上调。结论:SSD可以通过上调p21WAF1/CIP1的表达诱导G2-M周期阻滞来抑制结直肠肿瘤细胞SW480的增殖。     

天佛参口服液对体外人癌细胞抑制效应及其对细胞动力学影响的实验研究

本实验采用人癌细胞体外培养的方法,证明了天佛参口服液(HRD)可抑制人癌细胞集落的形成,直接杀伤癌细胞,抑制人癌细胞的生长,降低其生长率,生长率和药物浓度呈负相关。对MGC_(803)、SMMC_(7721)、Hela细胞的半数抑制浓度(IC_(50))分别为10.42μl/ml 12.55μl/ml和9.30μl/ml。显微分光光度计检测MGC_(803)细胞小鼠S_(180)腹水细胞核DNA显示:低浓度时使细胞相对DNA含量直方图明显右移,说明低浓度时即可影响癌细胞的代谢,阻滞细胞增殖于G_2+M期,高浓度时虽有右移,但不明显说明高浓度时的主要机制是直接杀伤作用。     

新型拓扑异构酶抑制剂抗肿瘤活性及其机制研究

目的 拓扑异构酶是近年来发现的多种肿瘤化疗的重要靶点,与肿瘤细胞的发生、增殖和发展密切相关.本研究分析两种新型拓扑酶抑制剂的体外抗肿瘤活性,并初步探讨其作用机制.方法 采用MTT法检测两种受试物对人肿瘤细胞增殖的抑制作用;质粒pBR322解旋反应检测抑制剂对TopoⅠ催化活性的影响,蛋白质印迹法检测细胞中Topo Ⅰ和TopoⅡ的蛋白表达变化;Hoechst 33258荧光染色、AO荧光染色、彗星实验和流式细胞术等方法观察两种受试物对HEp-2细胞凋亡和细胞周期的影响.结果 MTT法的结果显示,受试物1和2对9种人肿瘤细胞株均有显著的抗肿瘤活性(P＜0.05),并呈现浓度依赖性;受试物1和2可抑制Topo Ⅰ介导的质粒DNA超螺旋的解旋,并且两种受试物都可下调细胞中Topo Ⅰ、Ⅱ的蛋白表达,P＜0.05;Hoechst 33258荧光染色表明受试物可诱导HEp-2细胞凋亡,AO染色也得到相同的结果;彗星实验检测出两种受试物可导致DNA损伤;流式细胞仪检测的结果表明,处理组的细胞凋亡率高于对照组(P＜0.05),并且,受试物1使HEp-2细胞周期在S期和G2/M期发生阻滞(P＜0.01),而受试物2使其阻滞在G1期(72 h),P＜0.05;蛋白质印迹法结果显示促凋亡蛋白Bid、Bax和JNK表达增加(P＜0.01),抗凋亡蛋白Bcl-2表达减少(P＜0.01),细胞周期蛋白Cyclin B1和Cyclin D1表达减少(P＜0.05).结论 受试物1和2可以抑制HEp-2细胞的生长,这可能与以下机制有关:通过抑制Topo Ⅰ的催化活性并下调细胞中TopoⅠ和TopoⅡ的表达;调控凋亡相关基因的表达,诱导其凋亡;造成肿瘤细胞周期紊乱,促进凋亡.     

^131I-GMCSF诱导HL-60细胞凋亡与细胞周期阻滞的研究

目的观察和探讨131Ⅰ-GMCSF诱导HL-60细胞凋亡及其与细胞周期的关系.方法采用体外放射免疫治疗模型,通过MTT比色法、TUNEL来研究131Ⅰ-GMCSF辐射后HL-60细胞的存活率、凋亡率的改变;用流式细胞术及免疫细胞化学方法细胞周期的改变.结果放射性浓度≥18.5×108 Bq/L时HL-60细胞存活率显著下降.131Ⅰ-GMCSF能致HL-60细胞凋亡、细胞发生G2/M期阻滞.结论 GMCSF作为载体携带131Ⅰ能诱导HL-60细胞凋亡,凋亡与细胞发生G2/M期阻滞有关.     

RNA干扰抑制UHRF1基因表达对食管癌细胞放射增敏作用的研究

目的 研究RNA干扰抑制UHRF₁表达对食管癌细胞系(TE-1)放射敏感性的影响及作用机制。方法 以慢病毒感染方法将UHRF₁基因的短发夹状RNA (shRNA)转入TE-1细胞,并分为未转染组、转染NC-shRNA组、转染UHRF₁-shRNA组,前二者为对照组。采用 RT-PCR和蛋白印记法检测转染前后细胞UHRF₁的mRNA和蛋白表达,成克隆法、流式细胞术及蛋白印记法检测转染UHRF₁-shRNA联合X线照射对TE-1细胞放射敏感性、周期、凋亡及DNA损伤标识蛋白γ-H₂AX的影响。     

The antitumor activity of the fungicide ciclopirox

Ciclopirox olamine (CPX) is a synthetic antifungal agent clinically used to treat mycoses of the skin and nails. Here, we show that CPX inhibited tumor growth in human breast cancer MDA‐MB‐231 xenografts. To unveil the underlying mechanism, we further studied the antitumor activity of CPX in cell culture. The results indicate that CPX inhibited cell proliferation and induced apoptosis in human rhabdomyosarcoma (Rh30), breast carcinoma (MDA‐MB231) and colon adenocarcinoma (HT‐29) cells in a concentration‐dependent manner. By cell cycle analysis, CPX induced accumulation of cells in G₁/G₀ phase of the cell cycle. Concurrently, CPX downregulated cellular protein expression of cyclins (A, B1, D1 and E) and cyclin‐dependent kinases (CDK2 and CDK4) and upregulated expression of the CDK inhibitor p21^Cip1, leading to hypophosphorylation of retinoblastoma protein. CPX also downregulated protein expression of Bcl‐xL and survivin and enhanced cleavages of Bcl‐2. Z‐VAD‐FMK, a pan‐caspase inhibitor, partially prevented CPX‐induced cell death, suggesting that CPX‐induced apoptosis of cancer cells is mediated at least in part through caspase‐dependent mechanism. The results indicate that CPX is a potential antitumor agent.     

Apoptosis and cell growth inhibition as antitumor effector functions of interferons

Since their introduction to the clinic some 30 yr ago, interferons (IFNs) have become standard therapy for a range of disorders, including malignant and benign tumors as well as various viral diseases. Although IFNs will induce remissions in some patients with cancer, they are of no benefit or, at best, lead only to minor improvements in the great majority of patients with malignant disease. One of the great challenges of IFN research is to understand the multiple ways by which IFNs influence the behavior of tumor cells and to identify the factors that underlie the resistance of some tumors to IFNs. This reviews is written with a focus on two anticellular effects of IFN, inhibition of proliferation and induction of apoptosis, possible mechanisms underlying the antitumor action of IFN. In addition, possible reasons for IFN tumor cell resistance are also discussed.     

Protein kinase A inhibition facilitates the antitumor activity of xanthohumol,a valosin‐containing protein inhibitor

Xanthohumol (XN), a simple prenylated chalcone, can be isolated from hops and has the potential to be a cancer chemopreventive agent against several human tumor cell lines. We previously identified valosin‐containing protein (VCP) as a target of XN; VCP can also play crucial roles in cancer progression and prognosis. Therefore, we investigated the molecular mechanisms governing the contribution of VCP to the antitumor activity of XN. Several human tumor cell lines were treated with XN to investigate which human tumor cell lines are sensitive to XN. Several cell lines exhibited high sensitivity to XN both in vitro and in vivo. shRNA screening and bioinformatics analysis identified that the inhibition of the adenylate cyclase (AC) pathway synergistically facilitated apoptosis induced by VCP inhibition. These results suggest that there is crosstalk between the AC pathway and VCP function, and targeting both VCP and the AC pathway is a potential chemotherapeutic strategy for a subset of tumor cells.     

Role of Tyrosine Phosphorylation in Radiation-Induced Cell Cycle-Arrest of Leukemic B-Cell Precursors at the G2-M Transition Checkpoint

Here we provide experimental evidence that ionizing radiation induces inhibitory tyrosine phosphorylation of the p34^cdc2 kinase in human leukemic B-cell precursors. Herbimycin A markedly reduced tyrosine phosphorylation of p34^cdc2 in irradiated leukemic B-cell precursors, thereby preventing radiation-induced cell cycle arrest at the G2-M transition checkpoint. Thus, tyrosine phosphorylation is directly responsible for the inactivation of p34^cdc2 in irradiated human leukemic B-cell precursors and activation of protein tyrosine kinases is a proximal and mandatory step in radiation-induced G2-arrest arrest at the G2-M checkpoint. Human WEE1 kinase isolated from unirradiated or irradiated leukemic B-cell precursors had minimal tyrosine kinase activity towards p34^cdc2. We detected no increase of human WEE 1 kinase activity after radiation of leukemic B-cell precursors, as measured by (a) autophosphorylation, (b) tyrosine phosphorylation of a synthetic peptide derived from the p34^cdc2 amino-terminal region or (c) recombinant human p34^cdc2-cyclin B complex. Thus the signaling pathway leading to inhibitory tyrosine phosphorylation of p34^cdc2 and G2-arrest in irradiated human leukemic B-cell precursors functions independent of p49^WEE1Hu and enzymes which augment the tyrosine kinase activity of p49^WEE1Hu.     

Curcumin analog B14 has high bioavailability and enhances the effect of anti‐breast cancer cells in vitro and in vivo

Curcumin has a variety of anticancer properties, but low bioavailability prevents its use in chemotherapeutic applications. To address this problem, we tested the efficacy of the synthetic curcumin analog B14 in breast cancer cells and explored the mechanism by which B14 inhibits proliferation and metastasis of breast cancer cells. We used the breast cancer cell line MCF‐7, MDA‐MB‐231 to study the anticancer effects of B14 and assessed cell viability, cell migration and invasion, cell cycle, and apoptosis, in addition, the antitumor effect of B14 in vivo was examined in mice bearing MDA‐MB‐231 cells. We found that, as the concentration of B14 increased, cell viability decreased in a dose‐dependent manner. Compound B14 exerted the best antitumor activity and selectivity for MCF‐7 and MDA‐M‐231 cells (IC₅₀ = 8.84 μmol/L and 8.33 μmol/L, respectively), while its IC₅₀ value for MCF‐10A breast epithelial cells was 34.96 μmol/L. B14 has been shown to be a multi‐targeted drug that alters the expression of cyclin D1, cyclin E1, and cyclin‐dependent kinase 2 (CDK2), and ultimately induces G1 phase cell cycle arrest. At the same time, B14 activates the mitochondrial apoptosis pathway in breast cancer cells. Furthermore, B14 was more effective than curcumin in inhibiting cell migration, invasion, and colony formation. In tumor‐bearing mice, analog B14 significantly reduced tumor growth and inhibited cell proliferation and angiogenesis. The pharmacokinetic test found that B14 was more stable than curcumin in vivo. Our data reveal the therapeutic potential of the curcumin analog B14 and the underlying mechanisms to fight breast cancer cells.     

p53 protein regulates the effects of amifostine on apoptosis,cell cycle progression,and cytoprotection

To determine the role of p53 protein on the cellular effects of amifostine, we used molecularly engineered HCT116 colon cancer cells in which the p53 gene was inactivated by targeted homologous recombination or p53 protein was degraded by high-level expression of papillomavirus E6 protein. Amifostine induced a G1 arrest and protected against paclitaxel toxicity in p53-proficient but not in p53-deficient cells. In the absence of p53 protein, amifostine enhanced the cytotoxicity of paclitaxel. In addition, treatment of HCT116 cells with amifostine alone resulted in apoptotic cell death. Compared with p53-deficient cells, p53-proficient cells exhibited low-level resistance to amifostine-induced apoptosis. Amifostine induced the expression of p53 protein in p53-proficient cells and the expression of p21 protein in both p53-proficient and -deficient cells. These findings indicate that amifostine-induced G1 arrest and cytoprotection are mediated via a pathway that is dependent on p53 protein and that amifostine-induced expression of p21 protein is not sufficient to sustain a G1 arrest or to mediate cytoprotection. In addition, these findings identify p53 protein as a mechanism of resistance to amifostine-induced apoptosis.British     

Involvement of Cdc25c in Cell Cycle Alteration of a Radioresistant Lung Cancer Cell Line Established with Fractionated Ionizing Radiation

Cancer patients often suffer from local tumor recurrence after radiation therapy. Cell cycling, an intricatesequence of events which guarantees high genomic fidelity, has been suggested to affect DNA damage responsesand eventual radioresistant characteristics of cancer cells. Here, we established a radioresistant lung cancercell line, A549R , by exposing the parental A549 cells to repeated γ-ray irradiation with a total dose of 60 Gy.The radiosensitivity of A549 and A549R was confirmed using colony formation assays. We then focused onexamination of the cell cycle distribution between A549 and A549R and found that the proportion of cells inthe radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased. When A549 andA549R cells were exposed to 4 Gy irradiation the total differences in cell cycle redistribution suggested thatG2-M cell cycle arrest plays a predominant role in mediating radioresistance. In order to further explore thepossible mechanisms behind the cell cycle related radioresistance, we examined the expression of Cdc25 proteinswhich orchestrate cell cycle transitions. The results showed that expression of Cdc25c increased accompanied bythe decrease of Cdc25a and we proposed that the quantity of Cdc25c, rather than activated Cdc25c or Cdc25a,determines the radioresistance of cells.     

淫羊藿苷体内抑瘤作用及其机制

目的：探讨淫羊藿苷（icariin，ICA）对肝癌细胞H22荷瘤小鼠体内抑瘤作用及其机制。方法：肝癌细胞株H22右腋皮下接种C57BIM6小鼠，建立小鼠体内荷移植瘤模型。接种H22细胞后24h，用高、中、低不同剂量（9、4．5、2．25mg／ml）的ICA和环磷酰胺（cyclophosphamide，Cy）（30mg／kg）进行局部注射治疗，待荷瘤对照组肿瘤长至1g左右，各组小鼠称重，处死。留自凝血，取血清检测TNF—α；依次取脾脏及肿瘤组织称重；取1／2脾脏和肿瘤组织制成单细胞悬液，剩余的用10％中性甲醛溶液固定，做病理切片和H—E染色以及TUNEL实验；脾单细胞悬液用流式细胞术（FACS）检测CD3、CD4、CD8、DX5等抗原分子的表达；肿瘤单细胞悬液用AnnexinV／PI双染检测肿瘤细胞的早期与晚期凋亡情况，同时取适量的瘤细胞用70％乙醇固定以FACS检测肿瘤细胞周期各时相的分布情况。结果：Cy治疗组抑瘤率为67．16％，ICA高、中、低剂量治疗组抑瘤率分别为24．63％、32．84％、5．22％。AnnexinV／PI双染结果表明，Cy治疗组与ICA治疗组早期凋亡细胞率均高于荷瘤对照组[Cy组为（9．11±1．584）％，ICA组为（7．14±1．376）％，荷瘤对照组为（3．6±2．38）％]。TUNEL结果显示，ICA治疗组凋亡指数为（5．35±0．73）％，显著高于荷瘤对照组中的凋亡指数[（1．03±1．17）％]（P〈0．01）。正常小鼠血清中未检测到TNF—α的存在，而ICA治疗组及Cy治疗组中检测到微量TNF-α。病理检测表明，ICA中、高剂量组肿瘤组织中出现较多的灶状及小片状坏死，包膜处及肿瘤边缘可见瘤细胞坏死，包膜及肿瘤组织中可见少量的炎细胞（淋巴细胞与单核细胞）浸润。结论：ICA有显著的抑瘤作用，可能通过促进实体瘤细胞早期凋亡引起肿瘤组织坏死而起作用。