Therapy of human carcinoma xenografts with antibodies to EGFr and HER-2 conjugated to radionuclides emitting low-energy electrons

PURPOSE: Low-energy electrons (10-50 keV) can be effective and specific cytotoxic agents when delivered to the cell surface by antibodies, because their path length in tissue is comparable to a cell diameter. In this study, we have begun to evaluate the therapeutic potential of antibodies (Abs) conjugated to (111)In against carcinoma xenografts in nude mice. METHODS: Abs to EGFr or HER-2 were labeled with (111)In to a high specific activity of approximately 1.48 GBq/mg (40 mCi/mg). They were injected into nude mice 5-6 days after inoculation of human carcinoma cells, either A431 or SK-OV-3, and tumor growth was monitored. In preliminary in vitro experiments, we calculated the cumulative decays per cell, estimated the centigray dose delivered to the nucleus, and related this to the fraction surviving. RESULTS: Abs to both antigens provided significant protection in nude mouse xenograft models (p values ranging from <0.05 to <0.001). Some mice appeared to be cured, but most had delayed tumor growth. The specificity of the effect was demonstrated by testing non-reactive Abs labeled in the same way. The radioactivity was required, because unconjugated Abs had no therapeutic effect. The maximum tolerated dose was required in order for therapy to be effective, but most of the treated mice had no significant weight loss or other overt signs of toxicity. CONCLUSION: Abs labeled with nuclides emitting low-energy electrons, such as (111)In, can be effective therapeutic agents against microscopic s.c. tumors. This strategy should be considered for clinical applications.     

DNA凝胶电泳和放射自显影研究153Sm诱发骨肉瘤细胞凋亡

目的　研究受１５３ＳｍＥＤＴＭＰ内照射治疗时诱发骨肉瘤细胞凋亡的ＤＮＡ损伤特征,以及１５３Ｓｍ 核素在瘤细胞中的行径特性。方法　观察了１５３ＳｍＥＤＴＭＰ内照射不同时间,诱发骨肉瘤细胞凋亡的ＤＮＡ 凝胶电泳和微观放射自显影的示踪动态。结果　研究发现,在１５３ＳｍＥＤＴＭＰ内照射作用下,骨肉瘤细胞的ＤＮＡ 呈现出在核小体间断裂的阶梯状条带形成的凋亡特征,放射性核素１５３Ｓｍ 可迅速透过瘤细胞膜,也可被瘤细胞吞噬,呈亲膜性积聚和膜包裹着的凋亡小体呈现。结论　１５３ＳｍＥＤＴＭＰ内照射治疗时,可诱发骨肉瘤细胞凋亡发生,其诱发凋亡的程度随１５３Ｓｍ 内照射作用时间延长而增升。     

用99Tcm-rh-Annexin V检测放疗或化疗后肿瘤细胞凋亡

目的 验证99Tcm直接法标记的重组人膜联蛋白V(99Tcm-rh-AnnexinV)用于早期评价放化疗后肿瘤组织细胞凋亡状态的可行性.方法 将小鼠肝癌细胞(Hca-F25)接种于实验小鼠右腋下,8 d后当肿瘤生长至直径约1 cm时,将模型鼠分为A(化疗组,13只)、B(放疗组,10只)、C(荷瘤对照组,12只)3组.其中A、C组按注射示踪剂后不同时间(1、4、6 h)各分为3个亚组(A1组3只,A2组4只,A3组6只;C1组3只,C2组4只,C3组5只);A组接受环磷酰胺单次化疗(按体重150 mg/kg,腹腔内注射),C组腹腔内注射同等量生理盐水,20h后A、C组鼠尾静脉注射99Tcm-rh-Annexin V 3.7MBq(0.5μg)/只.B组按不同吸收剂量(2、5、10 Gy)分为3个亚组(B1组4只,B2组3只,B3组3只),放疗后1 h注射与A、C组相同剂量示踪剂,6 h后显像.小鼠模型显像后即刻处死,取组织(肿瘤、血、肌肉)称重后,测量放射性计数,并计算每克组织百分注射剂量率(%ID/g)及肿瘤(T)/肌肉(M)、T/血液(B)放射性比值,应用流式细胞仪检测细胞凋亡百分率.结果 A、B组小鼠肿瘤组织中凋亡细胞百分率[(11.16±3.83)%、(17.40±5.20)%]均明显高于C组[(2.11±1.51)%,F=56.414、94.748,P＜0.001];A2、A3及B组肿瘤组织%ID/g均明显高于C2及C3组(F值分别为14.307、7.074、28.672,P均＜0.05),且与凋亡细胞百分率呈明显正相关(A2、A3组r=0.813,P=0.002;B组r=0.780,P=0.004),而C组肿瘤组织%ID/g与凋亡细胞百分率无相关性(r=-0.238,P=0.229).结论 接受放疗或化疗后,小鼠肝癌组织对99Tcm-rh-AnnexinV的摄取明显增加,且其摄取程度与肿瘤组织内凋亡细胞量呈明显正相关;应用99Tcm-rh-AnnexinV显像可较准确地反映治疗后早期肿瘤组织细胞凋亡的状态.     

照射后不同p53基因状态胃癌细胞的G1期阻滞和凋亡

目的　评价抑癌基因ｐ５３（ 野生型ｐ５３） 对照射后人胃癌细胞系（ＢＧＣ８２３） 的Ｇ１ 期阻滞和凋亡的控制作用。方法　３ 种具有不同ｐ５３ 状态的人胃癌细胞系,即转染人野生型ｐ５３ 基因的ＢＧＣ８２３ｗｔｐ５３ 细胞、转染人突变型ｐ５３ 基因的ＢＧＣ８２３ｍｕｔｐ５３ 细胞和转染无ｐ５３ 基因的空载质粒的ＢＧＣ８２３ｖｅｃｔ 细胞,用流式细胞计分析细胞,４Ｇｙ 照射后０、８ 和２４ 小时后各细胞时相分布和凋亡的反应。结果　照射４Ｇｙ 后８ 小时和２４ 小时后的ＢＧＣ８２３ｗｔｐ５３ 细胞出现强烈的Ｇ１ 期阻滞（分别占原细胞总数的６７－９％ 和６１－１ ％） ,而ＢＧＣ８２３ｍｕｔｐ５３ 、ＢＧＣ８２３ｖｅｃｔ 细胞几乎没有Ｇ１ 期阻滞;照射４Ｇｙ 后８ 小时和２４ 小时后的ＢＧＣ８２３ｗｔｐ５３ 细胞出现明显的预示凋亡的亚Ｇ１ 峰,凋亡细胞比例分别达１３－０ ％ 和１５－３ ％ ;而ＢＧＣ８２３ｍｕｔｐ５３ 和ＢＧＣ８２３ｖｅｃｔ 细胞几乎没有出现亚Ｇ１ 峰和凋亡细胞比例都为零。结论　野生型ｐ５３ 基因具有促进照射后肿瘤细胞的Ｇ１ 期阻滞和凋亡作用,而ｐ５３ 变异和缺失则减低了肿瘤细胞对放射线的反应。     

Bystander effect in tumor cells produced by Iodine-125 labeled human lymphocytes

Abstract

Purpose: To investigate the ability of human lymphocytes labeled with DNA-incorporated ¹²⁵I to exert an inhibitory (antiproliferative) bystander effect on co-cultured human colon adenocarcinoma LS174T cells in vitro.

Materials and methods: Human peripheral blood lymphocytes were stimulated to synthesize DNA in the presence of phytohemagglutinin (PHA) and labeled with 5-[¹²⁵I]iodo-2′-deoxyuridine. Human colon adenocarcinoma LS174T cells were co-cultured with the ¹²⁵I-labeled lymphocytes in various ratios for 5 days and the proliferation of the LS174T cells was assessed. Further, the supernatant media from these co-cultures were: (i) Transferred to LS174T cells and their proliferation measured after 5 days, (ii) used to assess the clonogenic survival of LS174T cells, and (iii) screened for factors that suppress growth.

Results: A significant reduction in the proliferation of LS174T cells was observed when co-cultured either with ¹²⁵I-labeled lymphocytes (56?±?3.5%) or the supernatant media (52.5?±?1.3%) obtained from these co-cultures. Clonogenic survival of LS174T cells grown in the supernatant media corroborated the decrease in tumor cell growth.

Conclusion: The observed reduction in the proliferation of LS174T cells in presence of ¹²⁵I-labeled lymphocytes or media obtained from such co-cultures can be attributed to an inhibitory (antiproliferative) bystander effect, probably mediated by factor(s) released from the dying ¹²⁵I-labeled lymphocytes.     

18F-FP-peptide用于化疗后肿瘤细胞凋亡显像

目的 研发含天冬氨酸-谷氨酸-缬氨酸-天冬氨酸(DEVD)核心的正电子核素标记的caspase-3多肽活性显像剂,以在体检测肿瘤细胞凋亡.方法 用国产合成模块通过点击化学合成( 18S,21S,24S,27S,30S)-27-(2-羧乙基)-21-(羧甲基)-30-( (2S,3R,4R,5R,6S)-6-((2-(4-(3-[18F]氟戊基)-1H-1,2,3三唑-1-yl)乙酰氨基)甲基)-3,4,5-三羟基四氢-2H-吡喃-2-羧酰氨)-24-异丙基-18-甲基-17,20,23,26,29-五羰基-4,7,10,13-四氧-16,19,22,25,28-五氨杂三十烷-1,32-二酸(18 F-FP-peptide).在体外18F-FP-peptide与经卡铂化疗的A549肺癌细胞混合60 min后,检测细胞放射性摄取率,摄取率=(放射性活度平均值×10×100％)/(细胞计数×细胞体积×放射性对照值).将18F-FP-peptide注射到经卡铂化疗的荷A549肿瘤小鼠体内,60 min后测其生物学分布,并行micro PET/CT显像.应用流式细胞仪检测细胞凋亡率,评价18 F-FP-peptide放射性摄取率与细胞凋亡率的相关性.数据处理使用SPSS 13.0统计软件,计量资料采用x-±s表示,组间比较采用单因素方差分析,相关性研究采用线性相关与回归分析.结果 18 F-FP-peptide的合成效率为(21.0±4.5)％(n=6,不校正),放化纯为99％,比活度为870 GBq/μmol.体外细胞实验研究表明,各组细胞凋亡率随卡铂化疗时间延长逐渐升高,经卡铂处理后0、1、2和24h后细胞凋亡率分别为(1.02±0.31)％、(4.85±1.26)％、(6.37±2.21)％和(10.23±2.43)％,对应的细胞摄取率分别为(0.06±0.01)％、(0.31±0.04)％、(0.44±0.02)％和(0.86±0.04)％,各组细胞凋亡率与放射性摄取率之间呈明显正相关(y=1.1244+11.0949x,r=0.9850,P＜0.01);荷A549肿瘤卡铂化疗后24h,肿瘤组织放射性摄取率为(0.33±0.02) ％ID/g,明显高于未化疗肿瘤组织的(0.08±0.01) ％ID/g(F=31.25,P＜0.01);而两者的细胞凋亡率分别为(15.50±1.47)％和(1.92±0.31)％,差异有统计学意义(F=33.54,P＜0.01);荷A549肿瘤化疗后细胞凋亡率和肿瘤对18 F-FP-peptide的放射性摄取率呈明显正相关(y=-1.9055+54.4341x,r=0.9907,P＜0.01);Micro PET显像示,未经化疗的肿瘤基本无放射性摄取,SUVmax与对侧比值为1.32±0.01,而经卡铂化疗的肿瘤明显摄取显像剂,SUVmax与对侧比值为3.46±0.02,两者比较差异有统计学意义(F=11.05,P＜0.01).结论 18F-FP-peptide是有临床潜在价值的caspase-3 活性显像剂.     

Reproducibility of linear tumor measurements using PACS: comparison of caliper method with edge-tracing method

The aim of this study was to evaluate inter- and intra-observer reproducibility when making electronic caliper linear tumor measurements on picture archiving and communications systems (PACS) and compare them with linear measurements obtained from circumferential tracing of tumor perimeter. Three radiologists measured 64 masses from 30 patients on body CT scans in two separate settings. Long axis and perpendicular short axis were measured using electronic calipers. The edge of each tumor was traced electronically and the long and short axes were calculated by computer software. The reproducibility of a measurement was evaluated by computing and comparing the absolute value of the mean difference between initial and subsequent measurements. The mean differences ±95% confidence interval (CI) between two measurements of the long by short axis were 3.8±2.6×3.1±1.8 mm when the caliper method was used and 3.5±2.0×3.2±1.5 mm when the tumor tracing method was used. There was no statistically significant difference in individual intra-observer reproducibility of tumor axes measurements. Neither long- nor short-axis single-dimension measurements resulted in significantly greater or lesser intra-observer reproducibility. When comparing caliper and tracing measurements, the overall mean difference (3.42±1.8 vs 3.38±1.4 mm) was not statistically significant. There was close correlation between the individual measurements made by each observer whether these were made by electronic calipers and when these were calculated from electronic tracings (Pearson correlations between 0.79 and 0.949). Current PACS systems allow reproducible linear, long or short axis, tumor measurements. There is no significant difference in reproducibility of measurements whether these are made directly with electronic calipers or calculated from tumor edge tracings.     

99m Tc-MIBI监测肿瘤照射后生物学效应的

目的　研究照射后肿瘤细胞摄取MIBI能力的变化及其与肿瘤细胞凋亡、坏死和增殖状态的关系。方法　将离体肿瘤细胞和实验动物按照射剂量 0、5、10、2 0Gy与照射后 2 4、48、72h进行分组比较。离体肿瘤细胞分别测定单位质量肿瘤细胞摄取99mTc MIBI的放射性计数值 (计数·min- 1 ·mg- 1 cellprotein) ,凋亡指数 (ApoptosisIndex ,AI) ,集落形成率 (Platingefficiency,PE)。实验动物计算肿瘤组织微分摄取率 (differentialuptakeratio,DUR)和AI值。结果　不同剂量照射后 ,肿瘤细胞的99mTc MIBI摄取值与AI值呈负相关 (r=- 0 93) ,与PE值呈正相关 (r =0 84)。 2 4hDUR值与AI负相关 (r=- 0 793)。结论　不同剂量照射后 ,肿瘤细胞活力变化可能用99mTc MIBI作为示踪剂得到反映     

(32)P-磷酸铬体外诱导人非小细胞肺癌A549细胞凋亡

目的观察^32P-磷酸铬(^32P胶体)体外诱导人非小细胞肺癌(NSCLC)A549细胞周期变化和发生细胞凋亡的现象，建立剂量-效应和时间-效应关系。方法将^32P胶体加入A549细胞培养体系进行内照射，初始放射性浓度分别为0，93，180，278，370和463MBq／L，采用Giemsa染色、透射电镜、末端原位标记(TUNEL)等方法进行凋亡细胞的形态学、超微病理、生化特征检测，应用流式细胞术定量检测细胞周期变化和细胞凋亡率。结果A549细胞受照射后S期 G2-M期细胞比率在96h以内呈上升趋势，之后逐渐下降。A54g细胞受照射后72h左右出现兴奋性表现，96h起各受照组均检出凋亡，各剂量组在照射后120h达到凋亡高峰。结论在较低剂量范围内，^32P胶体内照射可以体外诱导人NSCLC A54g发生72h以后的迟发相凋亡，细胞凋亡率与内照射初始放射性浓度呈正相关。     

白藜芦醇诱导人宫颈癌Hela细胞凋亡及其机制

目的探讨白藜芦醇诱导人宫颈癌Hela细胞的凋亡作用及其分子机制。方法应用不同浓度的白藜芦醇作用于人宫颈癌Hela细胞,采用四甲基偶氮唑蓝（MTT）法检测药物对细胞的增殖抑制率;流式细胞术（FCM）检测细胞凋亡、细胞周期分布：免疫组化法检测Survivin及Caspase-3的表达。结果白藜芦醇能明显抑制Hela细胞的增殖,并呈剂量和时间依赖性。经白藜芦醇处理Hela细胞后,FCM分析发现各实验组S期细胞比例增高,G2／M期细胞比例减少,凋亡率明显高于对照组,并呈剂量依赖性（P〈0．01）;各实验组Heal细胞Survivin的表达均低于对照组,而Caspase-3的表达均高于对照组（P〈0．01）。结论白藜芦醇能明显抑制人宫颈癌Hela细胞的增殖,诱导其凋亡,其机制可能与抑制Survivin的表达、上调Caspase-3的表达有关。     

电化学治疗诱发大鼠种植型肝癌细胞凋亡的实验研究

目的　比较电化学治疗对大鼠种植型肝癌细胞凋亡的影响。方法　制作大鼠肝癌动物模型 ,应用电化学疗法进行治疗 ,7d后影像学、病理光镜观察肿瘤大小、坏死变化 ,计算肝肿瘤细胞凋亡指数 ,与对照组比较。结果　电化学治疗前 ,大鼠肝肿瘤平均体积为 (10 0± 6 )mm3 ,电化学治疗后 7d ,大鼠肝肿瘤平均体积为 (12 5± 10 )mm3 ,而荷瘤对照组肝肿瘤平均体积为 (190± 11)mm3 (P <0 .0 5 ) ;治疗组肝肿瘤细胞凋亡指数为 9.6 2 5 %± 1.172 % ,荷瘤对照组细胞凋亡指数为 3.5 2 3%± 0 .894 % (P <0 .0 1) ;治疗组病理检查 ,光镜下肿瘤细胞明显坏死。结论　电化学疗法能诱发细胞凋亡 ,促进肿瘤坏死 ,抑制肿瘤生长     

ANTP-SmacN7对肿瘤细胞的辐射增敏作用及其机制

目的 探讨ANTP-SmacN7融合蛋白对肿瘤细胞的辐射增敏作用及其机制。方法 合成ANTP-SmacN7融合蛋白并观察其细胞渗透能力,Western blot法检测辐射对XIAP表达量的影响,克隆形成实验观察ANTP-SmacN7融合蛋白的辐射增敏作用。Annexin V-FITC/PI双染法测定药物及射线对肿瘤细胞凋亡的影响,Western blot法检测ANTP-SmacN7阻断XIAP前后Caspase-8、Caspase-9和Caspase-3的变化。结果 ANTP-SmacN7融合蛋白可以进入细胞内,并在细胞内蓄积;辐射诱导肿瘤细胞体外XIAP表达量与肿瘤辐射敏感性呈负相关（r=0.82）;ANTP-SmacN7对肿瘤细胞有辐射增敏作用,增敏比为1.41;ANTP-SmacN7融合蛋白可促进4、8和10 Gy γ射线诱导的XIAP高表达肿瘤细胞的凋亡（t=-14.924、-7.294和-15.866,P<0.05）。辐射可诱导Caspase-3表达水平的升高,ANTP-SmacN7对Caspase-3蛋白表达水平不产生影响,但可增加活化Caspase-3的裂解片段的数量。结论 ANTP-SmacN7融合蛋白可通过诱导Caspase-3的活化降低肿瘤细胞的辐射抗性。     

电离辐射对EL-4细胞凋亡与坏死的影响

目的 研究不同剂量电离辐射对EL－4细胞凋亡和细胞坏死的影响。方法 采用PI和Hoechst 33342双染、流式细胞术检测。结果 实验结果表明：给予50－200mGy低剂量照射后6h,EL－4细胞凋亡和坏死较对照组没有升高,而给予1．0－8．0Gy大剂量X射线照射后6h,细胞凋亡较对照组有明显升高,4．0Gy以上剂量,细胞坏死显著升高。结论 一定剂量的电离辐射不仅可以诱导凋亡的产生,而且可直接导致细胞坏死,即细胞的死亡模式与受照剂量有关。     

绞股蓝皂甙诱导人口腔鳞癌颈淋巴结转移癌细胞凋亡的研究

 目的 探讨绞股蓝皂甙诱导人口腔鳞癌颈淋巴结转移癌GNM细胞凋亡的发生机制.方法 采用活细胞观察、超微结构观察、流式细胞仪及DNA琼脂糖凝胶电泳进行观察和检测.结果 50μg/ml的绞股蓝皂甙作用于GNM细胞后,细胞出现典型的核染色质凝集、核固缩、破裂;用FCM检测到G1峰前出现亚二倍体核型峰,DNA琼脂糖凝胶电泳带呈现DNA断裂损伤.结论 绞股蓝皂甙可诱导人口腔鳞癌颈淋巴结转移癌细胞发生凋亡.诱导细胞凋亡可能是其抑癌机理之一.     

188Re-DTPA-DG致MCF-7乳腺癌细胞凋亡实验研究

目的研究不同放射性浓度^188Re-DTPA-脱氧葡萄糖（DG）对人MCF-7乳腺癌细胞凋亡的影响及其作用机制.方法用流式细胞仪检测^188Re-DTPA-DG处理后MCF-7乳腺癌细胞的凋亡.将人MCF-7乳腺癌细胞分成3组：^188Re-DTPA-DG实验组、^188ReO-4对照组和生理盐水组.其中前2组分3个不同放射性浓度组：37、55.5、74kBq/ml.结果^188Re-DTPA-DG、^188ReO-4均能导致肿瘤细胞发生凋亡,实验组与^188ReO-4对照组及生理盐水组在不同放射性浓度下差异均有显著性（P＜0.01）,随着放射性浓度增加,凋亡程度增大;^188Re-DTPA-DG较^188ReO-4更易诱导凋亡发生.结论^188Re-DTPA-DG能导致肿瘤细胞凋亡,并且与放射性浓度相关;其机制可能是辐射导致DNA严重损伤,使细胞周期发生阻滞.     

Short fluorodeoxyuridine exposure of different human glioblastoma lines induces high-level accumulation of S-phase cells that avidly incorporate 125I-iododeoxyuridine

Purpose Radio-iododeoxyuridine (IdUrd) is a potential Auger radiation therapy agent incorporated into DNA during the synthesis phase. In this study we sought to optimise S-phase targeting by modulating cellular cycling and radio-IdUrd DNA incorporation using short non-toxic fluorodeoxyuridine (FdUrd) incubations. Methods Three human glioblastoma cell lines with different p53 expression were pre-treated with various FdUrd conditions. After different intervals, ¹²⁵I-IdUrd DNA incorporation was measured. Fluorescence-activated cell sorter cell cycle analysis was performed after identical intervals post FdUrd pre-treatment. Results The highest increase in ¹²⁵I-IdUrd DNA incorporation was induced by 1-h incubation with 1 μM FdUrd. Increase in radio-IdUrd DNA incorporation was greatest 16–24 h after FdUrd, reaching factors of ≥7.5 over baseline incorporation in the three cell lines. Furthermore, cell synchronisation in S phase was observed with a peak of ≥69.5% in the three cell lines at 16 and 24 h post FdUrd, corresponding to an increase of 2.5–4.1 over baseline. Conclusion FdUrd-induced thymidine synthesis inhibition led to S-phase accumulation that was maximal after an interval of 16–24 h and time-correlated with the highest radio-IdUrd DNA incorporation. These observations might allow the rational design of an Auger radiation therapy targeting a maximal number of S-phase cells in single treatment cycles.     

Viability of human cancer cells in culture after exposure to diagnostic ultrasound

Reports have appeared in the literature regarding biological damage to human cells following exposure to diagnostic ultrasound. We have examined the effects of diagnostic ultrasound (Sonel 400, C.G.R.) on human cell lines established in our laboratory. We report here that exposure to diagnostic ultrasound, at maximum exposure intensity and at exposure time as long as 60 minutes, produces no cell lysis as determined by vital dye exclusion ability, and as confirmed by electron microscopy. However, the exposure to ultrasound produced by an apparatus delivering an acoustic power higher than the diagnostic levels (Sonoscope-Alcatel) can cause the complete lysis of the same cells.     

肿瘤细胞凋亡核素显像分子探针研究进展

测定肿瘤细胞凋亡是早期评价肿瘤疗效的指标之一, 放射性核素凋亡显像是目前研究最为广泛、技术最为成熟的体内肿瘤细胞凋亡分子影像学检测技术, 能在活体内动态、无创地检测抗肿瘤治疗引起的细胞凋亡, 有助于肿瘤疗效的早期评判和预后分析, 其中特异性分子探针技术的研究开发是放射性核素凋亡显像的关键技术之一。笔者将目前有代表性的肿瘤细胞凋亡核素显像分子探针进行了总结。