rmhTNF-α联合顺铂杀伤肿瘤细胞作用研究

目的：观察顺铂联合重组改构人肿瘤坏死因子α（recombinant mutant human tumor necrosis factor-α, rmhTNF-α）对肿瘤细胞的增敏效果,并通过体外建立紫杉醇耐药细胞株研究rmhTNF-α有无逆多药耐药(multidrug resistance , MDR)的作用。方法采用结晶紫法检测不同浓度的rmhTNF-α及DDP对人肺腺癌细胞系A549、小鼠成纤维细胞系L929及经紫杉醇诱导耐药细胞系A549/T的生长抑制率,并用光学显微镜观察细胞形态。结果检测结果提示rmhTNF-α不仅具有抑制细胞生长的作用还具有增强DDP对肿瘤细胞杀伤的作用,光学显微镜下能明显观察到两药联合可促使肿瘤细胞呈凋亡形态。结论 rmhTNF-α联合DDP对人肺腺癌细胞系A549、小鼠成纤维细胞系L929及经紫杉醇诱导耐药细胞系A549/T具有一定的协同杀伤效应,且发现rmhTNF-α有逆MDR的作用。     

丹皮酚协同顺铂抗人宫颈鳞癌SiHa细胞及其机制

[目的]探讨丹皮酚（paeonol,Pae）和顺铂（CDDP）联合用药对人宫颈癌SiHa细胞的增殖抑制、凋亡诱导作用。[方法]应用MTT法检测不同浓度的Pae或CDDP单独及联合用药对宫颈癌SiHa细胞增殖的抑制作用,同时观察Pae与CDDP的协同抗肿瘤作用。流式细胞仪测定Pae联合CDDP用药对SiHa细胞凋亡的诱导作用。[结果]各浓度Pae（9.375～300mg/L）和CDDP（0.625～10mg/L）对人宫颈癌SiHa细胞均有明显增殖抑制作用,且呈时间—剂量依赖效应关系。18.75、37.5、75mg/LPae分别与2.5、5、75mg/LCDDP联用时具有协同作用,且以75mg/LPae与5mg/LCDDP联用时协同作用最显著（CDI=0.544）。75mg/LPae组细胞凋亡率为8.26%±1.12%,5mg/LCDDP组细胞凋亡率为29.62%±2.48%,与75mg/LPae联合5mg/LCDDP组细胞凋亡率（52.49%±4.39%）比较均有显著性差异（P〈0.01）。[结论]Pae与CDDP联合应用具有显著的协同抗人宫颈癌SiHa细胞增殖作用,其机制可能与两者协同诱导SiHa细胞凋亡有关。     

免疫毒素与化疗药物体外协同杀伤实验研究

近十年来,一些植物毒素,特别是蓖麻毒素(Ricin),由于具有很强的抑制蛋白质合成的作用被广泛用于与单克隆抗体等结合制成免疫毒素(Immunotoxins,简称ITS)的研究。但是,ITS体内应用效果很不理想,影响的因素很多,如肿瘤细胞表面抗原的变异、脱落、产生耐药株、循环中抗原的中和作用以及机体免疫系统对结合物的清除作用等。阿霉素、丝裂霉素C两者对多种肿瘤细胞均有很强的杀伤     

蟾毒灵与顺铂协同抑制乳腺癌MCF-7细胞增殖的机制探讨

目的 探讨蟾毒灵（Bufalin）联合顺铂对乳腺癌MCF-7细胞增殖及凋亡的影响,并探讨可能的协同机制。方法 采用MTT法检测空白对照组（仅培养液）、单纯细胞对照组和实验组的细胞增殖率,实验组加入不同浓度的顺铂或Bufalin单药或以固定比例（Bufalin∶顺铂=1 nmol／L∶1 μmol／L）联合作用24 h。分别采用20 nmol／L Bufalin、20 μmol／L顺铂单药或联合作用24 h,用流式细胞术检测细胞凋亡,并用Western Blotting检测活化的Met（p-Met）、Met、活化的Src（p-Src）、Src、PARP及其裂解cleaved PARP蛋白的表达。结果 MTT法检测显示,顺铂和Bufalin均以剂量依赖方式抑制乳腺癌MCF-7细胞增殖。在顺铂≥0.1 μmol／L和Bufalin≥0.1 nmol／L时两药联合作用可协同抑制MCF-7细胞增殖（0＜CI＜0.433）。流式细胞术检测显示,20 μmol／L顺铂作用24 h可诱导（10.7±4.8）％ 的MCF-7细胞凋亡,而20 nmol／L Bufalin作用24 h未明显诱导细胞凋亡,20 nmol／L Bufalin与20 μmol／L顺铂联合作用24 h后可诱导（40.8±8.5）％的MCF-7细胞凋亡。Western Blotting检测显示,顺铂作用后可导致Met和Src活化,Bufalin与顺铂联合作用后可抑制顺铂诱导的Met和Src活化,同时上调PARP裂解。结论 Bufalin可能通过抑制顺铂诱导的Met和Src活化与顺铂协同抑制MCF-7细胞增殖,增强顺铂诱导的细胞凋亡。     

顺铂联合辐射对人盲肠未分化腺癌细胞系的杀伤效应

本文应用人肓肠未分化腺癌细胞系(HCe-8693),进行顺铂联合辐射对该细胞系杀伤效应的研究。从细胞生长曲线、分裂指数、~3H-TdR掺入试验和测定观察,两者合用互补了单纯放疗或药物的不足,细胞生长明显受抑,分裂受阻,掺入量减少。细胞培养液中β_2-MG、Fn含量及药物浓度,R Pt组和Pt R组均明显低于其它各组。     

顺铂加热后的抗癌疗效及其作用机制的研究

目的 探讨顺铂（DDP）加热后对肝癌细胞的毒性,温度与DDP杀伤力的关系及其作用机制。方法 采用MTT法、台蓝排染法以及流式细胞术分析法。结果 DDP对肿瘤细胞有明显的杀伤力,其IC50值为0.57μg/ml。当DDP（2.0μg/ml）温度为45℃时,药物作用24h后,细胞总数分别是对照组、45℃单热组、单药组的12.3％、24.5％和44.0％,提示一次性加热DDP至41℃以上可明显提高其抗癌能力。结论 加热增强了DDP的细胞毒性,两者之间有着协同增敏作用,阻滞细胞周期是其抗肿瘤作用的主要机制之一。     

顺铂诱导肝癌细胞凋亡的治疗

目的　探讨广谱抗癌药物顺铂的抗肿瘤作用机制。方法　采用Hoechst PI荧光染色、透射电镜和直接免疫荧光法流式细胞术分析法检测细胞凋亡。结果　顺铂可以诱导肿瘤细胞凋亡 ,从药物作用 12h即可出现 ,贯穿全程 ,且呈逐渐增强趋势 ,即给药作用 12h、2 4h、3 6h ,凋亡细胞数分别为 11.4%、19.1%、3 9.4% ,并出现核浓缩、染色质浓集于核膜处、凋亡小体形成等典型的细胞凋亡结构。结论　诱导细胞凋亡是顺铂抗肿瘤作用的主要机制之一。     

托瑞米芬协同顺铂对人肺癌细胞株A549的影响

目的：研究托瑞米芬（TOR）对人肺腺癌细胞系A549的毒性作用及其与顺铂（DDP）联用的协同效应,探讨肺癌综合治疗的方向。方法：用MTT显色法检测TOR及与DDP联用后对A549细胞的毒性作用,测定其吸光度（A）值。用流式细胞仪检测细胞DNA含量,Western blot法检测p21蛋白表达。结果：TOR能直接抑制A549细胞的生长,≥5μmol/L的TOR可明显增强DDP的细胞毒性作用。TOR可加强DDP对S期、G2期及M期细胞的作用,且DDP＋TOR后p21蛋白表达增加。结论：≥5μmol/L的TOR与DDP联用对A549细胞具有显著的协同抗肿瘤效应。     

白皮杉醇增强顺铂杀伤喉癌Hep-2细胞的作用及其机制

目的 探讨白皮杉醇增强顺铂对喉癌细胞的杀伤作用及其分子机制。方法 体外培养喉癌细胞Hep-2,联合使用白皮杉醇及顺铂,CCK8法检测喉癌Hep-2细胞的增殖能力,流式细胞术检测喉癌Hep-2细胞的凋亡率,Hoechst染色检测喉癌Hep-2细胞中细胞核固缩比例,蛋白印迹法检测BCL-2家族蛋白表达变化。结果 白皮杉醇在50 μmol/L时对喉癌Hep-2细胞的增殖凋亡并无显著影响。与顺铂组相比,白皮杉醇联合顺铂组细胞的增殖能力显著降低,48 h细胞吸光度值白皮杉醇联合顺铂组较顺铂组降低60%,流式细胞术检测发现48 h时,白皮杉醇联合顺铂组喉癌Hep-2细胞的凋亡率显著高于顺铂组（P<0.05）,Hoechst染色发现白皮杉醇联合顺铂组喉癌Hep-2细胞的细胞核固缩比例显著增高,差异有统计学意义。蛋白印迹检测发现,白皮杉醇可以显著下调BCL-2蛋白表达水平,上调BAX蛋白表达水平。结论 白皮杉醇可以有效增强顺铂对喉癌细胞的杀伤作用,其分子机制与白皮杉醇可以显著调控BCL-2家族蛋白表达有关。     

顺铂诱导肝癌细胞凋亡的研究

目的探讨广谱抗癌药物顺铂的抗肿瘤作用机制.方法采用Hoechst-PI荧光染色、透射电镜和直接免疫荧光法流式细胞术分析法检测细胞凋亡.结果顺铂可以诱导肿瘤细胞凋亡,从药物作用12h即可出现,贯穿全程,且呈逐渐增强趋势,即给药作用12h、24h、36h,凋亡细胞数分别为11.4%、19.1%、39.4%,并出现核浓缩、染色质浓集于核膜处、凋亡小体形成等典型的细胞凋亡结构.结论诱导细胞凋亡是顺铂抗肿瘤作用的主要机制之一.     

基因重组毒素HELβ1-PE38KDEL与顺铂杀伤乳腺癌细胞系的协同作用

目的：研究HELβ1－PE38KDEL与常规化疗药物面铂杀伤乳腺癌细胞系的协同作用。方法：采用细胞增殖、软琼脂集落形成等实验，测定HELβ1－PE38KDEL和顺铂对高表达erbB2、3、4的乳腺癌细胞MDA－MB－453，胃癌细胞N87的协同作用，并以低表达erbB2、3、4的乳腺癌细胞2LMP为对照。结果：HELβ1－PE38KDEL和顺铂联用对MDA－MB－453和N87具有协同杀伤作用（CI＜1）。对2LMP无协同作用（CI＞1）。结论：对高表达erbB-2、3、4的乳腺癌细胞，基因重组毒素HELβ1－PE38KDEL与顺铂联用具有协同杀伤作用。     

TRAIL与阿霉素联用协同杀伤人结肠癌细胞SW480

背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumornecrosisfactor-relatedapoptosisinducingligand,TRAIL)可选择性杀伤肿瘤细胞,而不影响正常细胞生长。当一部分肿瘤细胞对TRAIL不敏感时,特定的其它药物可增强其杀伤作用。本文旨在探讨结肠癌细胞SW480对TRAIL的敏感性,以及TRAIL与阿霉素联用对细胞的杀伤作用及可能作用机制。方法:常规培养结肠癌细胞SW480。利用MTT法检测细胞毒性作用,流式细胞术定量分析凋亡细胞比例,透射电镜在亚细胞结构形态上证实凋亡细胞,Westernblot分析p53及bcl-2蛋白表达变化。结果:(1)SW480细胞对TRAIL不敏感,100ng/mlTRAIL只能杀伤7.8%的细胞,IC50>1000ng/ml,且不存在浓度依赖性。(2)SW480细胞对阿霉素敏感,存在浓度依赖性作用,IC50=65μmol/L,0.86μmol/L的阿霉素对细胞不表现杀伤作用。(3)TRAIL与阿霉素合用表现出协同作用,亚毒性浓度TRAIL(100ng/ml)与亚毒性浓度阿霉素(0.86μmol/L)联用可杀伤80%SW480细胞。流式细胞学证实这种杀伤作用主要通过诱导细胞凋亡实现,透射电镜亦观察到大量凋亡细胞存在。药物作用前后,p53及bcl-2蛋白表达水平无明显改变。结论:结肠癌细胞株SW480对TRAIL不敏感,但TRAIL与亚毒性浓度阿霉素联用对癌细胞有协同杀伤作用,这种细胞毒性作用主要表现     

mDRA-6与尼美舒利对人肝癌细胞系SMMC-7721细胞的协同杀伤效应

背景与目的:mDRA-6为本实验室制备的具有肿瘤细胞杀伤作用的抗人死亡受体5(death receptor5,DR5)的单克隆抗体,尼美舒利作为特异性环氧合酶-2(cyclooxygenase-2,COX-2)抑制剂,近年来发现其对某些肿瘤细胞系的细胞具有促凋亡作用,本研究探讨mDRA-6与尼美舒利对肝癌细胞系SMMC-7721的杀伤作用,及二者有无协同效应。方法:流式细胞术检测细胞表面DR5的表达率;分别用一定浓度的mDRA-6、尼美舒利、mDRA-6联合200μmol/L尼美舒利处理SMMC-7721细胞,MTT法检测细胞毒性作用,Hoechst33258染色观察SMMC-7721细胞核形态变化,流式细胞术定量分析凋亡细胞率。结果:SMMC-7721细胞表面DR5的表达率为95.0%,mDRA-6能够诱导SMMC-7721细胞凋亡,存在浓度依赖性(r=0.984,P=0.002),25ng/mL作用12h可杀伤10.5%的细胞,1600ng/mL作用12h可杀伤35.0%的细胞。尼美舒利能够诱导SMMC-7721细胞凋亡,200μmol/L作用12h可使5.0%的细胞凋亡,800μmol/L作用12h可杀伤34.0%的细胞,存在浓度依赖性(r=0.929,P=0.002)。尼美舒利与mDRA-6联合对SMMC-7721细胞具有协同杀伤作用(q=1.23),200μmol/L的尼美舒利协同25ng/mL与1600ng/mL的mDRA-6作用12h可分别杀伤31.2%与91.1%的SMMC-7721细胞,Hoechst33258染色和Annexin V/PI染色证实杀伤作用是通过诱导细胞凋亡实现的。结论:mDRA-6与尼美舒利均有杀伤SMMC-7721细胞的作用,二者具有协同作用,该作用是通过诱导凋亡实现的。     

Recombinant immunotoxin anti-c-Met/PE38KDEL inhibits proliferation and promotes apoptosis of gastric cancer cells

Background

Our study aims to evaluate the anti-growth effects of recombinant immunotoxin (IT) anti-c-Met/PE38KDEL on gastric cancer cells, and its mechnisms.

Methods

Gastric cancer cells were treated with increasing doses of IT and c-Met protein was quantified by Western blotting. Cell proliferation was determined by Cell Counting Kit-8 assay (CCK). [³H]-leucine incorporation assay was used to evaluate IT inhibition of protein synthesis. Cell apoptosis was quantified by flow cytometry. Caspase activities were measured using colorimetric protease assays.

Results

Cell growth and protein synthesis of the gastric cancer cell lines were suppressed by IT in a dose- and time-dependent manner. IT also induced apoptosis in a dose-dependent manner. The apoptosis rates of gastric cancer cell lines MKN-45 and SGC7901 were 19.19% and 27.37%, respectively when treated with 50 ng/ml of IT. There were significant increase ofcaspase-3 activity at 24 hr of IT treatment (100 ng/ml) (P < 0.01) in these gastric cancer cell lines.

Conclusions

IT anti-c-Met/PE38KDEL has anti-growth effects on the gastric cancer cell lines in vitro, and it provides an experimental basis for c-Met-targeted therapy towards in vivo testing.     

循环肿瘤细胞的检测及其临床应用

循环肿瘤细胞(CTC)可反映肿瘤的侵袭性.尽管许多检测及计数CTC的方法如磁性激活的细胞筛选、免疫细胞化学法、反转录聚合酶链式反应、流式细胞术开展了很久,直到最近这些方法才具有可行性.临床检测结果强烈提示,在某些肿瘤中,CTC的检测和计数可以用来预测预后、选择个体化治疗并可能作为评估治疗反应性的早期指标.     

Synergistic cytotoxic effect of sulindac and pyrrolidine dithiocarbamate against ovarian cancer cells

Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac) in vitro compounds affected the growth of tumor cells to a similar extent and synergized in cytotoxicity with PDTC. Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.     

Effect of angiotensin II on immunotoxin uptake in tumor and normal tissue

 Purpose: To investigate the effect of the sarcosine analog of human angiotensin II ({sar}ATII) on the uptake and spatial distribution of immunotoxins (MW 210000 Da) in RD rhabdomyosarcoma xenografts in mice. This analog has a pressor activity similar to native angiotensin II (ATII) but a longer duration of action. Method: A period of elevated blood pressure of approximately 80 min, measured by noninvasive photoplethysmography, was achieved by a 40-min continuous i.p. infusion of {sar}ATII at 0.07 μg/min. Tumor-bearing animals were injected i.v. with ¹²⁵I-labeled specific and ¹³¹I-labeled nonspecific immunotoxins and made hypertensive by i.p. infusion of {sar}ATII. Radioactivity was measured in plasma, tumor, liver, kidney and muscle at 2, 6 and 24 h. Plasma radioactivity was subtracted from tissue values to calculate tissue uptake. To assess the spatial distribution of immunotoxin in the solid tumor, ¹²⁵I-labeled specific immunotoxin was injected i.v. into tumor-bearing animals, and quantitative autoradiography was performed on tumor sections. Results: The uptake of specific or nonspecific immunotoxins in tumor and normal tissues was not significantly different in {sar}ATII-hypertensive animals compared with saline-treated controls. In control animals, the spatial distribution of ¹²⁵I-labeled specific immunotoxins was very heterogeneous and contained punctate accumulations throughout the tumor. Treatment with {sar}ATII did not affect this distribution qualitatively or quantitatively. To examine a possible reason for the lack of {sar}ATII effect, we measured the interstitial pressure of the RD tumor using a fluid-filled micropipette connected to a servo-null pressure transducer. The interstitial pressure in this solid tumor was unexpectedly low, only 0.6±0.9 mm Hg. Conclusions: The sustained period of{sar}ATII-induced hypertension had no effect on RD tumor or normal tissue uptake or tumor spatial distribution of immunotoxin. In saline-treated controls, the heterogeneity of immunotoxin distribution does not arise from an elevated interstitial pressure. Further studies are needed to determine whether a correlation exists between responsiveness to ATII-induced hypertensive chemotherapy using macromolecular drugs and tumor type and/or physiological properties. Received: 31 October 1995 / Accepted: 25 March 1996     

Molecular targeting of malignant glioma cells with an EphA2-specific immunotoxin delivered by human bone marrow-derived mesenchymal stem cells

Immunotoxins have shown great promise as an alternative treatment for brain malignancies such as gliomas, but their failure to penetrate into the tumor mass remains a major problem. Mesenchymal stem cells exhibit tropism to tumor tissue and may serve as a cellular vehicle for the delivery and local production of antitumor agents. In this study, we used human bone marrow-derived mesenchymal stem cells (hMSCs) as a vehicle for the targeted delivery of EphrinA1-PE38, a very specific immunotoxin against the EphA2 receptor that is overexpressed in gliomas. hMSCs were transduced with adenovirus to express secretable EphrinA1-PE38. Our invitro assays confirmed the expression, release and selective killing effect of the immunotoxin produced by hMSCs. Furthermore, the intratumoral injection of engineered hMSCs was effective at inhibiting tumor growth in a malignant glioma tumor model. These results indicate that gene therapy utilizing EphrinA1-PE38-secreting hMSCs may provide a novel approach for the local treatment of malignant gliomas.     

托瑞米芬协同顺铂抑制A549细胞生长

目的：研究托瑞米芬（TOR）对人肺腺癌细胞系A549的毒性作用及其与顺铂（DDP）联用的协同效应，以期为肺癌的综合治疗提供新的方向。方法：用MTT显色法检测TOR及与DDP联用后对A549细胞的毒性作用，测定其A值。用流式细胞仪检测细胞DNA含量，Western Blotting法检测p53及p21蛋白表达，明确可能的机制。结果：TOR能直接抑制A549细胞的生长，＞或等于5μmol/L的ＴＯＲ浓度可明显增强DDP的细胞毒性作用，TOR可加强DDP对S期，G2及M期细胞的作用，且DDP加TOR后p21蛋白表达增加。结论：＞5μmol/L的TOR与DDP联用对A549细胞具有显著的协同抗肿瘤效应。其机制可能与DDP对细胞周期阻断的时相改变有关，且可能与p21蛋白诱导相关及可能和非p53依赖蛋白机制有关。