Cytostatic and cytotoxic response of Ehrlich ascites tumor cells in vivo on chronic treatment with cytarabine, bleomycin, and peplomycin

The cytokinetic response of Ehrlich ascites tumor (EAT) cells in vivo upon chronic treatment at low dosage levels with cytarabine (1-beta-D-arabinofuranosylcytosine, ara-c) bleomycin (BLM) and peplomycin (PEP) was estimated. Bivariate DNA histograms allow the simultaneous evaluation of the cell cycle status of living and killed cells. It could be confirmed that ara-C is cytostatic on cells in S phase. Pronounced cytotoxicity was observed in G1 and G2+M phase. BLM and PEP showed no (or neglectable ) accumulation of vital cells in any cycle phase. Both drugs, however, are cytotoxic on cells, regardless their position within the cell cycle. A successive application of ara-C and BLM (or PEP) in a cell kinetics-directed therapy schedule may be taken into account.     

Effect of cis-diammino-dichloro-platinum (II) on cell growth and cell cycle progression of a mouse ascites tumor growing in vivo

Cell kinetic studies of cis-platin (CDDP) have been few with divergent results. In this study, the effect of 12.5 and 25 micrograms CDDP/animal corresponding to approximately 0.4 and 0.8 mg CDDP/kg body weight was described when treating the Bp8 ascites sarcoma growing in mice. From sequential studies of the total number of cells together with the composition of cells in the cell cycle, the flow of cells through the cell cycle was calculated. A dose-dependent cell growth inhibition was found with almost immediate effect following the higher dose, while an inhibitory effect on cell growth with the lower dose was delayed 24 hours. The cell flow from the various phases of the cell cycle for the first 24.48 hours following administration of the lower dose, was increased from the G1-phase and mitosis, with normal flow from the S-phase and depressed outflow from the G2-phase. Thereafter the flow rates from all cell cycle phases were decreased. For the higher dose, the outflow rates from all phases of the cell cycle were depressed up to 72 hours. The maximum platination of DNA was found one hour after treatment. The results indicate that the mechanisms responsible for the passage through the G2 stage of the cell cycle are more sensitive to the treatment of CDDP than replicative DNA synthesis. This experimental system may be used as a basis for further mechanistic studies of the antitumor effect of anticancer drugs.     

3-取代芳基氧化吲哚(PH II-7)对肿瘤细胞周期的影响

目的观察3-取代芳基氧化吲哚(PH II-7)对肿瘤细胞周期分布的影响，明确PH II-7的抗敏感肿瘤和耐药肿瘤的共同机制。方法用流式细胞仪检测细胞周期分布，Western印迹分析细胞周期相关蛋白的表达，³H-TdR参入法检测细胞DNA合成，ELISA测定酪氨酸激酶的活性。结果PH II-7对多种肿瘤细胞（包括耐药细胞）的周期分布均有影响，阻滞细胞G₁期至S期的移行，细胞周期相关蛋白CDK2，Rb和c-myc的表达被抑制，Cyclin E的表达升高。PH II-7还可抑制³H-TdR的参入，抑制EGFR的酪氨酸激酶的活性。结论抗耐药肿瘤新药PH II-7是一种细胞周期阻滞剂，可能是通过抑制CDK2而使肿瘤细胞阻滞在G₁期，同时也说明细胞周期阻滞可能是抗耐药肿瘤的新方向。     

Comparative flow cytometric analysis of bleomycin toxicity on normal and tumour sheep cell kinetics in vitro.

The cytotoxic effects of bleomycin on cell proliferation, morphology and cycle progression were compared in a sheep tumour cell line and in normal sheep sinus cells. They were concentration and time dependent, irreversible, and were accompanied by increased cell diameter and cytosolic synthetic activity. Cell accumulation in the G2M phase, without much change in the cell cycle progression through the S phase, was observed using flow cytometry. Although the tumour cells showed a greater sensitivity to low bleomycin concentrations compared to the normal cells, both cell types exhibited equal sensitivity to higher drug concentrations. Provided that tumour cells are also preferentially affected by bleomycin in vivo, these findings raise the possibility that bleomycin cytotoxicity can be reduced or avoided in normal tissue.     

Cytostatic synergism between bromodeoxyuridine, bleomycin, cisplatin and chlorambucil demonstrated by a sensitive cell kinetic assay.

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse the interference of common cytostatic agents with cell activation and cell cycle progression of human B-cell lines. Bleomycin impaired both cell activation and G2 transit, the latter effect being oxygen dependent. The DNA alkylating agents cyclophosphamide, chlorambucil and mitomycin C caused G2 arrest, whereas cisplatin arrested cells in both the S and G2 phase of the cell cycle. Vinblastin interfered with mitosis, but in addition arrested cells in all phases of the cell cycle. The growth inhibitory action of bleomycin, cisplatin and chlorambucil was dependent upon the bromodeoxyuridine (BrdU) concentration in the culture medium. No interaction was found between BrdU and cyclophosphamide, mitomycin C and vinblastin. The cell cycle kinetic mechanism of the interaction between BrdU and bleomycin, cisplatin and chlorambucil was a potentiation of the G2 arrest. In conclusion, BrdU may be useful in clinical chemotherapy as a chemosensitizer for selected cytostatic agents.     

Inhibitory effect of tumor cell proliferation and induction of G2/M cell cycle arrest by panaxytriol

Panaxytriol, a polyacetylenic compound, isolated from red ginseng (Panax ginseng C.A. Meyer), was studied to determine its effects on the growth and cell cycle of tumor cell lines. The compound showed both significant cytotoxicity and inhibition of DNA syntheses in various tumor cells tested. For P388D1, a mouse lymphoma cell line, IC50 values for cytotoxicity and inhibition of DNA synthesis were 3.1 and 0.7 microg/ml, respectively. The cytotoxic effect of panaxytriol was both time- and dose-dependent. It also induced the cell cycle arrest of P388D1 at the G2/M phase, which was measured through flow cytometry. Particularly, the proportion of cells in the G2/M phase of the cell cycle increased from 9 % to 26 and 48 %, respectively, after 24 and 36 h exposure to panaxytriol at 5 microg/ml. There were corresponding decreases in the proportion of cells at the G0/G1 phase. The S phase also decreased during the 36-h treatment.     

三氧化二砷抑制小鼠B16黑色素瘤生长作用及其机制

目的　探讨三氧化二砷 (As2 O3 )对小鼠B16黑色素瘤的生长及其血管生成的抑制作用 ;同时观察其对B16细胞增殖活性、细胞形态、细胞周期及凋亡的影响。方法　选用小鼠黑色素瘤B16细胞接种C5 7BL/ 6J小鼠 ,观察腹腔注射As2 O3 对实体瘤的重量及成瘤率的影响 ;应用HE染色、Ⅷ RAg免疫组化染色观测瘤组织内新生血管密度 ;采用CellTiter 96AqueousOne试剂检测B16细胞增殖活力 ;Giem sa染色、Feulgen染色观察细胞形态学变化 ;流式细胞术分析细胞周期及细胞凋亡。结果　As2 O3 能显著抑制小鼠B16黑色素瘤的生长 ,治疗组成瘤率为 37 5 % ,抑瘤率达81 6 1% ,并能显著抑制瘤组织内血管生成 ;体外实验观察到As2 O3 能抑制B16细胞增殖 ,并存在浓度依赖效应 ,IC50 为32 99μmol·L-1;细胞形态学观察结果显示As2 O3 使B16细收稿日期 :2 0 0 4-0 2 -17,修回日期 :2 0 0 4-0 3 -2 8基金项目 :安徽省教育厅资助项目 ,No 2 0 0 4kJ2 79作者简介 :夏　俊 ( 1965 -) ,女 ,硕士 ,副教授 ,硕士生导师 ,研究方向 :肿瘤分子生物学 ,Tel:0 5 5 2 3 0 664 12 2 0 97,E mail:xia jun1965 @yahoo .com .cn崔秀云 ( 1941-) ,女 ,教授 ,博士生导师 ,研究方向 :癌基因与抑癌基因 ,Tel:0 411 472 0 64 8,E mail:cuixy @dlmedu .edu .     

23-羟基桦木酸对B_(16)细胞系的诱导分化作用

目的评价 2 3 羟基桦木酸对黑色素瘤B16细胞的抑瘤作用。方法以MTT法测定细胞增殖的抑瘤率 ,并以B16细胞形态、黑色素含量、细胞周期变化及体内致瘤能力的测定作为观察指标。结果用 10～ 2 0 μg/ml的2 3 羟基桦木酸作用肿瘤细胞 ,见有不同程度的抑瘤作用 (P <0 .0 0 1)。表现为黑色素生成能力增加 ,细胞生长缓慢。可使B16细胞阻断在G1期 ,肿瘤体积明显缩小。结论 2 3 羟基桦木酸低剂量 (10～ 2 0 μg/ml)对B16细胞有明显的分化诱导作用 ,而对体内外黑色素瘤增殖有明显的抑制作用     

AC480, formerly BMS-599626, a pan Her inhibitor,enhances radiosensitivity and radioresponse of head and neck squamous cell carcinoma cells in vitro and in vivo

Purpose: The present study investigated the effect of AC480, a small molecule pan-HER tyrosine kinase inhibitor, on in vitro radiosensitivity and in vivo radioresponse of a human head and neck squamous cell carcinoma cell line. Methods: HN-5 cells were exposed to γ-radiation with and without AC480 and assayed for proliferation, clonogenic survival, apoptosis, cell cycle distribution, and DNA damage. The cells were analyzed by immunoprecipitation and western blotting for proteins involved in apoptosis, cell cycle regulation, and the EGFR pathway. The effect of AC480 on tumor radioresponse was assessed by tumor growth delay assay using HN5 tumor xenografts generated in nude mice. Results: At the molecular level, in HN-5 cells the agent inhibited the expression of pEGFR, pHER2, cyclins D and E, pRb, pAkt, pMAPK, pCDK1 and 2, CDK 6, and Ku70 proteins. The drug also induced accumulation of cells in the G1 cell cycle phase, inhibited cell growth, enhanced radiosensitivity, and prolonged the presence of γ-H₂AX foci up to 24 h after radiation. AC480 did not increase the percentage of cells undergoing radiation-induced apoptosis. The drug given before and during irradiation improved the radioresponse of HN5 tumors in vivo. Conclusion: AC480 significantly enhanced the radiosensitivity of HN-5 cells, expressing both EGFR and Her2. The mechanisms involved in the enhancement included cell cycle redistribution and inhibition of DNA repair. Both in vitro and in vivo data from our study suggest that AC480 has potential to increase tumor response to radiotherapy.     

Apoptosis induction and cell cycle perturbation in human hepatoma hep G2 cells by 10-hydroxycamptothecin

10-Hydroxycamptothecin (HCPT), a DNA topoisomerase I inhibitor, is an antitumor alkaloid isolated from a Chinese tree, Camptotheca acuminata, and exhibits a remarkable antihepatoma effect. We studied HCPT to determine whether or not its anti-hepatoma activity occurs through apoptosis induction and cell cycle disturbance using the MTT method, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. The results showed that HCPT inhibited proliferation of human hepatoma Hep G2, Bel-7402 and Bel-7404 cells at an optimal concentration of 0.1 microg/ml. This growth inhibition was dose and time dependent, and was accompanied by evidence of apoptotic changes and cell cycle perturbation in Hep G2 cells. Chromatin condensation and nuclear fragmentation were observed in Hep G2 cells by fluorescence microscopy. Agarose gel electrophoresis showed internucleosomal DNA fragmentation ('ladder pattern') of Hep G2 cells following treatment with HCPT, in a concentration- and time-dependent manner. Flow cytometry showed that HCPT induced a massive hypodiploid cell population and arrested cells in G2/M phase (at low dose) or in S phase (at high dose) in Hep G2 cells. The results of this study suggest that the anti-hepatoma effect of HCPT may result from apoptosis induction and cell cycle disturbance.     

糙苏素的抗肿瘤作用及机制研究

目的:研究糙苏素的抗肿瘤作用及其对端粒酶活性和细胞周期的影响。方法:采用血清药理学与体内、外抑瘤实验相结合的方法,MTT法检测糙苏素含药血清对体外培养的人白血病K562细胞增殖的抑制作用,聚合酶链反应-酶联免疫吸附法检测K562细胞在糙苏素作用后端粒酶活性的变化,流式细胞仪分析细胞周期的改变;体内实验采用小鼠肝癌H22细胞株接种小鼠,连续给药14d,计算抑瘤率。结果:糙苏素含药血清在10%～50%剂量范围内对K562细胞有抑制作用,且呈剂量依赖关系(P<0.05);糙苏素作用后端粒酶活性出现抑制,且各给药组随糙苏素浓度增加抑制作用显著增强(P<0.01),同一浓度随作用时间延长,抑制作用逐渐增强(P<0.05);K562细胞的DNA合成后期/分裂期(G2/M)细胞含量显著增高(P<0.01);糙苏素(2.5、5、10mg·kg-1)灌胃能显著抑制小鼠移植性肝癌H22细胞的生长。结论:糙苏素具有抗肿瘤作用,其作用是通过抑制肿瘤细胞的端粒酶活性而实现的。     

人8羟-基鸟嘌呤DNA糖苷酶1基因低表达增加肺腺癌细胞对博来霉素的敏感性

目的研究DNA碱基切除修复基因人8羟-基鸟嘌呤DNA糖苷酶1(hOGG1)低表达增加肺腺癌细胞对博来霉素(BLM)的敏感性的作用,为化疗增敏提供更多的实验依据。方法以肺腺癌A549细胞和通过稳定转染hOGG1核酶而获得的hOGG1低表达的A549-R细胞为研究对象,用MTT试验和集落形成抑制试验测定不同浓度BLM处理后两种细胞的存活率和形成集落的能力;体外微核试验及单细胞凝胶电泳检测两种细胞微核率及DNA损伤与修复的差异。结果BLM作用下A549-R细胞的IC50及集落形成率显著低于A549细胞;BLM可诱导两种细胞的微核率增高,而在相同浓度下A549-R细胞微核率较A549细胞更高;单细胞凝胶电泳结果显示,BLM作用下两种细胞均有不同程度DNA损伤,A549-R细胞的拖尾率和DNA迁移长度显著大于A549细胞;损伤后A549细胞修复发生较A549-R早,与A549细胞相比A549-R细胞更不易修复。结论hOGG1低表达使肺腺癌细胞DNA修复能力降低,从而使其对BLM的敏感性增强。     

Enhancement of antitumor activity of bleomycin by benzamide in vitro and in vivo

The cytotoxic effects of bleomycin on HeLa cells in culture were enhanced by incubation of the cells with benzamide, a potent inhibitor of poly(ADP-ribose) polymerase, at concentrations at which benzamide alone did not show any cytotoxicity. Benzamide plus bleomycin display enhanced therapeutic effects against Ehrlich ascites tumor cells in vivo. On daily treatment with various doses of bleomycin plus benzamide for 10 days, mice with Ehrlich ascites tumors survived longer than mice on treatment with bleomycin alone.     

阿司匹林诱导人肺腺癌细胞株SPCA-1凋亡研究

目的　研究非甾体类抗炎药阿司匹林对肺腺癌SP CA 1体外增殖抑制作用及凋亡的影响。方法　用噻唑蓝(MTT)法测定细胞存活率 ;用流式细胞术测定细胞周期时相分布 ,瑞氏染色、Hoechest/PI双染、扫描电镜观察细胞表面形态改变 ,琼脂糖电泳法观察细胞凋亡。结果　阿司匹林对肺腺癌SPCA 1细胞有较强的抑制作用 ,有明显的时间和剂量依赖性 (1 0～ 12 5mmol·L-1) ;可使SPCA 1细胞G0 /G1期、G2 /M期比例明显升高 ,S期比例下降 ;凋亡细胞增多。结论　阿司匹林可能通过影响细胞周期时相分布、诱导凋亡以及引起细胞膜形态而抑制肺腺癌SPCA 1细胞增殖。     

抑制组蛋白去乙酰化酶与DNA甲基转移酶活性对胶质瘤恶性表型影响的研究

【摘要】 目的：探讨阻断组蛋白去乙酰化酶与DNA甲基转移酶的活性后对胶质瘤恶性表型的抑制作用。方法：选择丙戊酸（VPA）为组蛋白去乙酰化酶抑制剂,5-氮杂-2′-脱氧胞苷（Aza）为DNA甲基转移酶抑制剂。将U251胶质瘤细胞分为对照组、VPA治疗组、Aza治疗组和VPA+Aza联合治疗组。采用四唑盐(MTT)比色法分析肿瘤细胞增殖活性,流式细胞术分析细胞周期,Annexin V-FITC染色检测细胞凋亡,2D Matrigel、3D Matrigel、Transwell法检测胶质瘤细胞侵袭能力,并建立U251细胞裸鼠皮下移植瘤模型,进行肿瘤局部多点注射上述药物治疗,测量肿瘤体积。结果：与对照组比较,各治疗组肿瘤细胞的增殖在治疗2 d后均出现明显抑制,S期和G2M期细胞比例减少,诱导细胞周期阻滞于G0/G1期,治疗组细胞凋亡率明显下降,侵袭和运动迁移能力均明显下降,各治疗组鼠移植瘤体积增长较对照组明显减缓,与对照组相比差异均有统计学意义（P<0.05）。以上结果均以VPA+Aza联合治疗组最为显著。结论：联合阻断DNA甲基转移酶和组蛋白去乙酰化酶活性可抑制胶质瘤表观遗传学性征,抑制胶质瘤恶性表型。     

茵陈素对肺癌细胞增殖和细胞周期的影响

目的 :探讨茵陈素对肺癌细胞增殖和细胞周期的影响。方法 :应用光镜、四氮甲唑蓝 (MTT)方法和流式细胞术分析茵陈素对肺癌细胞形态学、生长和细胞周期的变化。结果 :茵陈素能抑制肺癌细胞的增殖 ,呈剂量依赖性 ,以160μg/ml抑制作用最明显 ,抑制率达52 4 %。80μg/ml时 ,S期和G2/M期细胞比例开始下降 ,细胞被阻滞于G0/G1 期 ,不能进入S期及G2/M期 ,增殖指数明显下降。结论 :茵陈素在体外对肺癌细胞具有抑制作用 ,通过抑制DNA合成 ,将细胞阻滞于G0/G1 期来抑制细胞增殖。     

Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri-n-butyltin chloride

Induction of apoptosis by tri-n-butyltin (TBT) in gill tissue of the mussel Mytilus galloprovincialis was investigated. The terminal dUTP nick-end labeling technique (TUNEL) was used to detect cells displaying DNA fragmentation within gill structures. Genomic DNA fragmentation was detected as characteristically ladder-like pattern of DNA fragments induced by single injection of different doses of TBT (1-5 microg/g) below the mantle, directly into the pallial fluid, after 24 h of incubation. DNA degradation of higher order DNA structure, as well as reduced G(0)/G(1) cell cycle region (the sub-G(1) region) was detectable after 1.5 h of TBT incubation. Presence of apoptotic cells in mussels' gills was indicated by the selective loss of G(2)/M cells concomitant with the appearance of cells with decreased DNA content in S and G(0)/G(1) cell cycle regions. The effect of the TBT on cell cycle in a mussel gill was a dose related and exposure time depending. The possible mechanism of induction of apoptosis in vivo in gill tissue of mussel treated with TBT is discussed.     

Alterations in growth rate and cell cycle kinetics of rat liver tumor cells cultured in ethanol-containing medium. In vitro model of proliferative restriction in response to ethanol exposure

Mechanisms related to the growth suppressive effect of acute ethanol exposure on liver cells were investigated using an established line of ethanol-sensitive rat hepatic tumor cells (32IIIA) and recently developed cytochemical methods for analysis of hepatocyte cell cycle kinetics. Exposure of exponentially growing 32IIIA cells to ethyl alcohol (range 10-100 mM in the growth medium) for a period of 3 days resulted in concentration-dependent decreases (4-25%) in final population density and increases (18-35%) in mean population doubling time compared to untreated cells. Viability was unaffected by ethanol exposure in the concentrations indicated and for the duration period utilized, approximating 94% under all experimental conditions. Multiparametric flow cytometric analysis revealed significant ethanol-associated differences in specific growth parameters and growth state compartments of 32IIIA hepatic tumor cell populations. Most prominent was an ethanol-associated and concentration-dependent (a) increase in the fraction of cells in the G1 phase of the cell cycle, (b) increase in the coefficient of variation in the G1 DNA content measurement, and (c) accumulation (in the G1 phase) of cells with a very low mean RNA content. Increases in each of these cytochemically-defined parameters reflected increasing levels of ethanol in the growth medium. This study indicates that the effects of ethanol on cultured cells of hepatic origin are quite complex. It is concluded that the inhibition of proliferation observed during acute ethanol exposure of liver-derived 32IIIA cells in vitro is due to an accumulation of cells in the G1 compartment.     

Inhibition of bleomycin-induced cellular DNA strand scission by 1,10-phenanthroline.

Inhibition by 1,10-phenanthroline of cellular DNA strand scission induced by the antitumor antibiotic bleomycin in Ehrlich ascites tumor cells was studied. DNA alkaline elution was performed on cells after 1-hr bleomycin treatments. Pretreatment for 24 hr with initial 1,10-phenanthroline concentrations of 0.2 nmol/10(5) cells, which depletes cells of ferritin iron by 80%, had no consistent effect on bleomycin strand breakage. However, simultaneous treatment with 3.1 nmol of 1,10-phenanthroline/10(5) cells and with bleomycin concentrations from 5 to 25 microM decreased both apparent double-stranded breaks and random breakage. When cells were treated with both 3.1 nmol of 1,10-phenanthroline/10(5) cells and 25 microM bleomycin, washed free of both drugs, and incubated at 35 degrees for 1 hr, the resulting breakage was equivalent to that found in cells treated with bleomycin only. When the combination treatment was extended to 4 hr, cell washing and reincubation resulted in increased strand scission, as compared with strand scission in cells treated with bleomycin only. Growth inhibition by bleomycin was not affected appreciably by temporary suppression of DNA strand breakage activity.     

Sequence-dependent cytotoxicity of combination chemotherapy using paclitaxel, carboplatin and bleomycin in human lung and ovarian cancer

Combination chemotherapy for non-small cell lung cancer (NSCLC) and ovarian cancer typically consists of a regimen of a taxane such as paclitaxel and a platinum-containing agent. Bleomycin, which halts cell cycle progression at G2 phase, is an agent which might thereby increase taxane cytotoxicity. The goal of this study was to evaluate the effect of different paclitaxel-platinum or paclitaxel-bleomycin schedules on cytotoxicity in human NSCLC and ovarian cancer cells. The simultaneous combination of paclitaxel and carboplatin exhibited simple additivity in vitro, while sequential exposure studies indicated that carboplatin followed by paclitaxel produced greater than additive cytotoxicity using the isobologram analysis of combinatorial effects. In contrast, the simultaneous combination of paclitaxel and bleomycin consistently exhibited greater than additive effects indicating a potentially synergistic combination. Sequential exposure studies of bleomycin followed by paclitaxel produced similar synergistic findings. Experiments in SCID mice evaluating the combinations of paclitaxel and bleomycin supported the in vitro results, as significantly enhanced A549 lung tumor growth inhibition was observed when paclitaxel was administered 1 h after bleomycin. The synergistic activity shown by the combination of bleomycin and paclitaxel indicates a potentially beneficial novel combination for treatment of NSCLC and ovarian cancer.