ATP生物荧光肿瘤药敏检测技术在乳腺癌化疗中的应用研究

目的 探讨ATP生物荧光肿瘤药敏检测技术(ATP-TCA)在乳腺癌化疗中的应用价值。方法 取40例乳腺癌改良根治术、淋巴结活检和胸腔积液标本进行ATP-TCA药敏检测。结果 ATP-TCA肿瘤药敏检测技术在乳腺癌标本中的可评价率为90％(36/40);化疗药物对乳腺癌的杀伤作用具有较强的个体差异性;10种化疗药物的敏感率分别为:氟尿嘧啶(5-Fu)33.3％、顺铂(DDP)37.5％、环磷酰胺(CTX)29.2％、足叶乙苷(VP-16)16.7％、丝裂霉素(MMC)22.0％、表阿霉素(EPI)41.7％、诺维本(NVB)45.8％、阿霉素(ADM)41.70k、泰素(PTX)54.2％、羟基喜树碱(HCPT)25.0％。初步研究表明ATP-TCA体外检测结果与实际临床疗效具有良好的相关性。结论 ATP-TCA检测技术是一种准确、可靠的肿瘤药敏检测技术。该技术检测结果与临床实际疗效具有较好的相关性,可用于指导乳腺癌化疗和化疗药物的新适应证研究。     

ATP抗肿瘤药物敏感性检测技术与卵巢癌临床治疗的相关性研究

目的 采用三磷酸腺苷生物荧光法(ATP-TCA)对卵巢癌组织标本进行体外药敏试验,并分析其与临床疗效的相关性。方法 取卵巢癌患者术后新鲜组织和腹水标本共34例,应用ATP-TCA技术对9种10组化疗药物进行体外药敏试验。结果 ATP-TCA技术的可评价率为94．0％,敏感性为90．0％,特异性为91．7％,阳性预测值为94．7％,阴性预测值为84．6％．整体预测值为90．6％。药物体外药敏检测结果与临床资料相符率较高。结论 ATP-TCA体外药敏检测结果与临床治疗反应有很好的相关性,是开展肿瘤个体化化疗的一种重要的体外药物筛选方法。     

乳腺癌ATP生物荧光法肿瘤体外化疗药物敏感性检测的可行性

目的：探讨ATP生物荧光法肿瘤体外药敏感性检测（ATP—TCA）在乳腺癌化疗中的应用。方法：应用ATP生物荧光肿瘤体外药物敏感性检测法,检测50例乳腺癌标本对6种常用化疗药物和3种联合化疗方案的敏感性。结果：组织标本的可评估率为92％,乳腺癌细胞对阿霉素（ADM）＋紫杉醇（PTX）联合体外有效率最高为95．3％,其次为ADM＋氟尿嘧啶（5-Fu）90．7％,多西他赛（TXT）72．5％,PTX70．7％,ADM50．0％,甲氨蝶呤（MTX）＋5-Fu 19．5％,MTX9．8％。化疗药物对乳腺癌细胞的杀伤作用具有较强的个体差异。结论：ATP生物荧光肿瘤体外药物敏感性检测法敏感性高、稳定性好、简单、快速、检测结果可靠,可用于临床指导个体化的乳腺癌化疗方案。     

Evaluation of in vitro chemosensitivity of antitumor drugs using the MTT assay in fresh human breast cancer

Practical criteria were developed in this paper for the purpose of evaluating chemosensitivity of fresh human breast cancer by the MTT assay. The survival rates at maximum inhibition (Imax %) and the concentrations of drugs which caused fifty percent reduction in absorbance compared to baseline values (IC₅₀) of 175 samples of 10 anti-tumor drugs were evaluated by logistic analyses of the dose-response curves. Distributions of Imax% appeared as normal curves, while those of the IC₅₀ significantly deviated from normal distribution (p < 0.0001). We assessed the in vitro chemosensitivity by comparing the Imax % of each drug on individual samples with the mean Imax % + SD which was obtained from the Imax% of 175 samples. If the individual Imax % > mean Imax % + SD, we thought the tumor sample was resistant to this drug. If the Imax % mean Imax % + SD, we would compare its IC₅₀ with Q₅₀ which was used as a cutoff point for in vitro chemosensitivity of anti-tumor drugs. The in vitro chemosensitivity could be graded as sensitive (Q₁–Q₂₅), intermediate (Q₂₆–Q₇₅), and resistant (Q₇₆–Q₁₀₀) by means of percentile method. If the individual IC₅₀ Q₇₆, the tumor sample would be defined as resistant. If the individual IC₅₀ Q₂₅, it would be defined as sensitive. In the range of Q₂₆–Q₇₅, we used Q₅₀ as a cutoff point between relative sensitivity and relative resistance. Preliminary results showed that the in vitro chemosensitivity to different anti-tumor drugs determined by these criteria were consistent with the clinical response in 83 advanced breast cancer patients.     

ATP生物荧光法在乳腺癌化疗药物筛选中的应用

背景与目的：乳腺癌是女性最常见多发的恶性肿瘤,化疗在乳腺癌治疗中占有重要的地位,本文旨在探讨基于三磷酸腺苷（adenosine triphosphate,ATP）的肿瘤化疗药物敏感性检测方法在乳腺癌药物筛选中的应用。方法：应用ATP-TCA法,检测50例乳腺癌标本体外对6种常用化疗药物和3种联合化疗方案的敏感性。结果：组织标本的可评估率为92%,多柔比星（ADM）联合紫杉醇（PTX）对乳腺癌细胞在体外抑制率最高,为95.3%;其次为ADM联合氟尿嘧啶（5-FU）为90.7%,多西他赛（TXT）为72.5%,PTX为70.7%,ADM为50.0%;甲氨蝶呤（MTX）联合5-FU为19.5%,MTX为9.8%。此外,化疗药物对乳腺癌细胞的杀伤作用具有较强的个体差异性。结论：ATP-TCA法操作简便,敏感性好,可能对临床个体化疗具有指导价值。     

三磷酸腺苷生物荧光法体外药敏试验指导复发或难治性非霍奇金淋巴瘤化疗的临床研究

 目的　探讨三磷酸腺苷生物荧光法（ATP-TCA）体外药敏试验在复发或难治非霍奇金淋巴瘤（NHL）治疗中的临床应用价值。方法　应用ATP-TCA技术对复发或耐药淋巴瘤患者术后新鲜组织标本共34 例进行体外药敏试验,观察用敏感药物化疗疗效。结果　淋巴瘤标本的药物敏感性具有个体差异性,不同化疗药物的体外抑瘤活性不同。药敏试验组总有效率（RR）为82.4 %（28／34）,完全缓解（CR）率为52.9 %（18／34）;DICE化疗组RR为60.0 %（18／30）,CR率为33.3 %（10／30）;GDP化疗组RR为62.3 %（33／53）,CR率为26.4 %（14／53）。药敏试验组的RR与DICE组及GDP组相比,差异有统计学意义（χ2＝3.93,P＝0.047;χ2＝3.98,P＝0.046）。结论　ATP-TCA 体外药敏检测结果与临床治疗反应有很好的相关性,是开展肿瘤个体化化疗的一种重要的体外药物筛选方法。     

Weekly low-dose mitoxantrone plus doxorubicin as second-line chemotherapy for advanced breast cancer

Summary Weekly low dose mitoxantrone (3 mg/m²) plus doxorubicin (8 mg/m²) was administered as second-line chemotherapy to 33 patients with advanced breast cancer. Four out of 28 evaluable patients (14%) obtained a partial response with a median duration of 34 weeks (range 18–67⁺ weeks), while 8 patients (29%) showed stable disease with a median duration of 28 weeks (range 11⁺–60 weeks). Gastrointestinal toxicity and alopecia were mild. Grade II and III leukopenia occurred in 63% of the courses without serious infectious disease. Four patients experienced an asymptomatic drop of 16–20% in the left ventricular ejection fraction (LVEF) after relatively low cumulative doses of each drug, and one patient with a history of pericarditis carcinomatosa and mediastinal irradiation developed a heart failure. In conclusion, this second-line combination treatment had moderate activity in breast cancer and caused only few subjective side effects, especially with respect to gastrointestinal symptoms.     

人乳腺癌细胞原代培养体外药敏试验研究

目的:探讨用ATP生物荧光肿瘤体外药敏检测技术(ATP-TCA)研究乳腺癌药物敏感性的异质性以及个体化治疗的可行性。方法:用ATP-TCA检测114例乳腺癌标本对13种单药或联合用药的敏感性。结果:个体之间的药物敏感性存在着明显的异质性。单药中有效的药物为紫杉醇、长春瑞滨、表阿霉素、丝裂霉素和阿霉素,敏感率分别为73·3%、64·1%、62·3%、49·9%和40·0%。联合用药(紫杉醇 表阿霉素,环磷酰胺 表阿霉素 氟尿嘧啶)的敏感率高于单药。结论:乳腺癌对抗癌药物的敏感程度存在着异质性。ATP生物荧光肿瘤药敏检测技术可用于为乳腺癌选择合适的化疗药物。     

Uptake and distribution of doxorubicin in hormone-manipulated human breast cancer cells in vitro

Background: Kinetic resistance to cytotoxic drugs may account for the moderate responsiveness of breast cancer to chemotherapy. In the present study the in vitro effects of estradiol-mediated DNA stimulation on the cellular uptake of the DNA intercalating drug doxorubicin (DOX) were examined in MCF-7 human breast cancer cells. Methods: Using the fluorescent properties of the drug, the cellular uptake was investigated by high performance liquid chromatography (HPLC), and by flow cytometry. Results: The uptake of DOX (0.01–2 g/ml) by MCF-7 cells in suspension, incubated for 1 and 6 h, showed a strong correlation between the incubation concentration of DOX and the cellular uptake of the drug as measured by HPLC and flow cytometry. Simultaneous exposure of MCF-7 cells, in monolayer culture, to DOX (0.04–0.2 g/ml) and estradiol (1 nM) for 1–24 h showed no significant difference in uptake of the drug compared to control cultures exposed to DOX in the absence of estradiol. Neither was there a significant difference in uptake of DOX when MCF-7 cells were pretreated with estradiol (1 nM) for 16–24 h followed by a 0.5, 1, 6, and 21/23 h incubation with DOX (0.01–2 g/ml). Pretreatment with estradiol did not affect the retention of DOX as measured 24 h after a 0.5 h incubation with DOX (2 g/ml). Furthermore, fluorescence microscopy revealed no difference in the cellular DOX distribution pattern of estradiol-stimulated MCF-7 cultures compared to unstimulated cultures. Conclusion: From this study we can conclude that, for the human MCF-7 breast cancer cells in vitro, the uptake, retention, and cellular distribution of DOX are not influenced by estrogenic manipulation.     

人膀胱癌原代培养体外药敏试验的研究

目的:探讨用ATP生物荧光肿瘤体外药敏检测技术(ATP-TCA)研究膀胱癌药敏的异质性和个体化疗的可行性。方法:用ATP-TCA检测来自45例初发和6例复发膀胱癌患者的手术标本对4种化疗药物的敏感程度。结果:51例标本中有49例获得药敏结果,可评价率为96·0%。最有活性的药物是吡柔比星,有43·8%(21/48)的标本对它表现强敏感;28·6%(14/49)的标本无强敏感药物;69·4%(34/49)的标本无耐药药物。结论:膀胱癌对抗癌药物的敏感程度存在着异质性。ATP生物荧光肿瘤药敏检测技术可用于为膀胱癌选择合适的化疗药物。     

非小细胞肺癌体外化疗敏感试验影响因素的研究

目的：研究非小细胞肺癌对化疗药物原发敏感情况及药敏试验相关影响因素。方法：采用MTT比色法测定31例非小细胞肺癌标本对9种化疗药物的敏感性，每种药物设3个浓度等级，根据每种药物的抑制率进行分析。结果：31例肺癌标本对9种化疗药物的敏感程度依次为：异长春花硷83．87%、长春新碱77．42%、丝裂霉索74．19％、阿霉素69．72%、卡铂69．72％、顺铂61．29％、足叶乙甙58．06%、氨甲喋呤22．58%、5-氟脲嘧啶19．35％；足叶乙甙和阿霉素对肺鳞癌抑制率明显高于肺腺癌，足叶乙甙和顺铂对低分化肺癌抑制率明显高于中高分化肺癌。结论：体外药敏试验受到多因素影响，药敏结果能够较好指导临床化疗。     

Med19基因敲减增加乳腺癌细胞化疗敏感性及其可能的机制

目的:研究中介体(mediator,Med) 19对乳腺癌化疗敏感性的影响并分析其分子机制.方法:选用多柔比星(adriamycin,ADM)耐药的人乳腺癌细胞MCF-7/ADM和亲本细胞MCF-7(NC组),采用慢病毒载体介导RNA干扰方法构建Med9稳定低表达的MCF-7/ADM与MCF-7细胞株(KD组),并用Real-time PCR和Western blotting方法验证干扰效果.CCK-8法检测慢病毒介导的Med19敲减前后两种细胞对ADM、顺铂(cisplatin,DDP)和紫杉醇(taxinol,TAX)药物敏感性的变化.Real-time PCR和Western blotting检测Med19敲减对多药耐药基因1(multidrug resistance 1,MDR1)和细胞凋亡基因Bcl2、Bax及Caspase-3、active Caspase-3的表达的影响.流式细胞术检测敲减Med19及ADM处理对细胞凋亡的影响.结果:与MCF-7相比,MCF-7/ADM细胞对ADM、DDP和TAX均具有耐药性.成功构建Med19稳定低表达的MCF-7/ADM与MCF-7细胞株,并且其对ADM、DDP、TAX的敏感性增加,药物作用IC50显著降低(均P＜0.05).MCF-7/ADM细胞Med19 mRNA和蛋白表达显著高于MCF-7细胞,Med19的敲减可降低MCF-7/ADM细胞中MDR1 mRNA与蛋白表达水平(均P＜0.05)并可增加MCF-7/ADM及MCF-7细胞中凋亡相关active Caspase-3、Bax的蛋白表达,降低Bc12的蛋白表达(均P＜0.05).此外,与NC及NC+ ADM组相比,KD组及KD+ ADM组凋亡水平显著增加(均P＜0.05).结论:Med19高表达参与乳腺癌化疗耐药,其机制可能与Med19调节MDR1的表达并影响细胞凋亡有关.     

Reversal of doxorubicin resistance in breast cancer cells by photochemical internalization

Multiple drug resistance (MDR) is a problem that seriously reduces the efficacy of many chemotherapy agents. One mechanism for MDR is increased acidification of endocytic vesicles and increased cytosol pH, so weak base chemotherapeutic agents, including doxorubicin, are trapped in endocytic vesicles and exhibit a drug resistant phenotype. Treatments that selectively reverse this accumulation may therefore reverse the MDR phenotype. Photochemical internalization (PCI) is a novel technology developed for site-specific enhancement of the therapeutic efficacy of macromolecules by selective photochemical rupture of endocytic vesicles and consequent release of endocytosed macromolecules into the cytosol. This study evaluates PCI for release of doxorubicin from endocytic vesicles in MDR cells. Two breast cancer cell lines, MCF-7 and MCF-7/ADR (the latter resistant to doxorubicin), were selected. They were found equally sensitive to photochemical treatment with the photosensitiser TPPS(2) (a) (disulfonated meso-tetraphenylporphine) and light. On exposure to doxorubicin alone, the IC(50) (drug concentration for 50% reduction in colony formation) was 0.1 microM for MCF-7 and 1 microM for MCF-7/ADR. After PCI (photochemical treatment followed by doxorubicin), the IC(50) concentration was 0.1 microM for both cell lines. Comparable changes were seen with assay of cell viability using 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). On fluorescence microscopy in MCF-7/ADR cells, doxorubicin localised in granules identified as lysosomes. After PCI, doxorubicin was released into the cytosol and entered cell nuclei, as was seen in MCF-7 cells without PCI. In conclusion, PCI reversed the MDR phenotype of doxorubicin resistant breast cancer cells by endo-lysosomal release of the drug. The technique is a promising new approach to tackling the problem of MDR.     

ＡＴＰ生物荧光法检测非小细胞肺癌胸水癌细胞对化疗的敏感性

目的　探讨ＡＴＰ生物荧光法（ＡＴＰ-ＴＣＡ）体外药敏试验预测非小细胞肺癌（ＮＳＣＬＣ）胸水治疗药物敏感性的可行性。方法　采用ＡＴＰ-ＴＣＡ检测２４例ＮＳＣＬＣ患者胸水标本,对４种含铂类二药联合方案及培美曲塞单药的敏感性。结果　２４例胸水标本的可评价率为９５.８％（２３／２４）,其中ＧＰ方案的敏感率最高,为６０.９％,其次为ＴＣ、ＴＰ及ＮＰ方案,敏感率分别为４３.５％、３９.１％和３４.８％。８例培美曲塞单药二线治疗标本的敏感率为５０.０％。在敏感检测标本中,不同方案对不同病理类型的敏感度亦不同。结论　ＮＳＣＬＣ对抗癌药物的敏感程度存在着异质性,ＡＴＰ-ＴＣＡ体外药物敏感性检测技术是一种重要的体外药物筛选方法,可以辅助选择ＮＳＣＬＣ合适的化疗药物。     

DHA在人乳腺癌中对阿霉素细胞毒活性的影响与脂质过氧化作用的关系

目的　探讨廿二碳六烯酸 (DHA)影响阿霉素 (ADM)对人乳腺癌DMA MB 435s细胞毒活性的机理是否通过DHA的脂质过氧化作用。方法　在培养的人乳腺癌DMA MB 435s细胞株中加入不同配伍的细胞毒药物、多不饱和脂肪酸 (PUFAs)、前氧化剂VitC和VitK3混合物及抗氧化剂VitE等 ,在细胞的抽提物中以TBA法每 2 4小时测定脂质过氧化产物丙二醛 (MDA)及用亚硝酸盐分光光度法测定一氧化氮 (NO)的含量 ,并作出MDA、NO含量与细胞毒性间的剂量 -效应相关直线。结果　从细胞培养的第三天开始 ,出现明显的细胞活力变化 ,同时伴有脂质过氧化物 (MDA)水平的增加及NO含量的降低。细胞抽提物中MDA及NO含量与细胞毒性之间存在直线相关关系。结论　DHA能明显增加ADM对MDA MB 435s细胞株的细胞毒活性 ,机理之一是DHA增强了瘤细胞内的脂质过氧化作用。     

三磷酸腺苷-肿瘤体外药敏检测在晚期卵巢上皮癌化疗中的成本-效果评价

目的 评价三磷酸腺苷-肿瘤体外药敏检测(adenosine triphosphate-tumor chemosensitivity assay,ATP-TCA)在晚期卵巢上皮癌化疗中的应用价值,以及对该技术应用的成本-效果进行评价.方法 回顾性分析158例在我院例行ATP-TCA检测的晚期卵巢上皮癌病例,其中原发67例...     

Predictability of in vivo chemosensitivity by in vitro MTT assay with reference to the clonogenic assay

The MTT assay reported by Mosmann is a rapid and convenient colorimetric assay for cellular growth and survival in vitro. In this paper, the MTT assay was modified as a chemosensitivity test, and its potential was investigated. Using 10 human tumor xenografts in athymic nude mice, the predictability of in vitro antitumor effects of drugs using the MTT assay was compared with that using the clonogenic assay. The MTT assay showed excellent reproducibility, and the predictable rate in this assay was 86.7%, with 100% true-positive and 77.8% true-negative rates, almost equivalent to the 90.0% predictable rate of the clonogenic assay. This method also has several advantages with respect to rapidity, quantitation, management of many samples, and cell number required for the assay. Application of this assay to chemosensitivity testing seems to be valuable and useful.     

ATP生物荧光肿瘤药敏在复发卵巢癌并腹水化疗中的应用价值

Objective:To investigate the clinical value and application of ATP based bioluminescence tumor chemo-sensitivity assay (ATP-TCA) in the chemotherapy for ascites caused by recurrent ovarian cancer.Methods:More than 10 kinds of chemotherapeutic drugs or combinations were applied and 35 ascites specimens from recurrent ovarian cancer were analyzed by ATP-TCA.Sensitivity of chemotherapeutic drugs was assessed.After 2-4 chemotherapeutic cycles,clinical outcomes were analyzed,which were compared with those of 40 cases by empirical regimens.Results:32 of 35 specimens were evaluated with an overall evaluation rate of 91%.The assay results suggested that chemo-naive patients responded to chemotherapeutic drugs with individualized profiles.The sensitivity rates of GEM,EPI,OXA,DDP,CBP,ADM,VP-16,CTX,NVB,5-FU,PTX and TXT were 40%,30%,33%,29%,33%,38%,25%,33%,38%,33%,25% and 20%,respectively.While the sensitivity rates of combinations GEM EPI,GEM CBP,GEM DDP,NVB DDP,CTX ADM DDP,CTX ADM,DDP VP-18,OXA 5-FU,VP-16 IFO,PTX DDP,TXT CBP,VCR CTX MTX,DDP CPT-11,OXA CPT-11,and DTIC CTX were 47%,50%,36%,44%,30%,33%,27%,33%,40%,27%,23%,14%,28%,30% and 17%,respectively.In vitro results correlated well with clinical outcomes.Objective response rate (RR) in chemo-sensitivity-guided group was of significance compared with that in empirical-regimen-guided group.Conclusion:ATP-TCA is a choice for the screening of chemotherapeutic drugs against ascites caused by recurrent ovarian cancer with excellent sensitivity and reliability.ATP-TCA assay results correlate well with clinical outcomes,suggesting its clinical value in the management of difficult-to-manage therapeutic situations such as ascites in recurrent ovarian cancer.     

In vitro potentiation by lonidamine of the cytotoxic effect of adriamycin on primary and established breast cancer cell lines

Summary Lonidamine is a new potential chemotherapeutic agent, relatively non-toxic, that can positively modulate the efficacy of several antineoplastic drugs. We evaluated the response of two established human breast cancer cell lines (MCF-7 and BRC-230) and of 20 primary breast cancer cell lines to lonidamine, either alone or in combination with adriamycin, the drug most widely used in the management of breast cancer. Different schedules were tested by varying either concentration of the drugs (LND: 10–150µg/ml; ADM: 0.10–0.15µg/ml), or time of exposure (1–96 hours), or sequence of administration (ADM LND; LND ADM; ADM + LND). Our results indicate slight sensitivity of the cell lines to lonidamine when used alone, whereas an increase of efficacy was noted when lonidamine was added for at least 24 hours after a 4 hour exposure to adriamycin. Such efficacy was significantly greater than that expected from an additive effect between the two drugs. We conclude that lonidamine, when given according to an appropriate schedule, enhances,in vitro, the efficacy of adriamycin. A correct employment of lonidamine in the management of breast cancer might therefore potentiate the therapeutic effect of adriamycin.     

Guggulsterone and bexarotene induce secretion of exosome‐associated breast cancer resistance protein and reduce doxorubicin resistance in MDA‐MB‐231 cells

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA‐MB‐231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP‐associated exosomes, while siRNA‐mediated knockdown or GW4869‐mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor‐labeled breast cancer stem‐like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.