对阿霉素产生多药抗药性后小鼠体内谷胱甘肽—S—转移酶的分布及瘤细胞活性的变化

<正> 肿瘤化疗失败的原因之一是多药抗药性的产生。小鼠艾氏腹水瘤对阿霉素产生多药抗药性后,可以耐受5mg·kg~(-1)的剂量。为了更清楚的了解抗药现象,我们测定了小鼠艾氏腹水瘤对阿霉素产生抗药性后肝,心及瘤细胞中谷胱甘肽—S—转移酶(GST)的活性;并比较了抗药瘤细胞及敏感瘤细胞的活性。     

Bullatacin克服肿瘤多药抗药性作用及其机理

目的：探讨bullatacin克服肿瘤多药抗药性(MDR)的作用及其机制。方法：以两对MDR细胞株及其相应的敏感株进行对比,比较两种细胞株的细胞毒、Fura-2及阿霉素细胞内积累。结果：bullatacin不仅对敏感细胞株具有很强的细胞毒活性,而且对MDR细胞株也同样具有很强的细胞毒活性,不受抗药性的影响。bullatacin能使MDR细胞内Fura-2的积累增加;也能增加MDR细胞内阿霉素的积累。结论：bullatacin具有克服MDR的作用,其作用机理与bullatacin影响MDR细胞P-gp的功能,使MDR细胞内抗癌药物积累增加有关。     

MTT方法测定培养细胞抗药水平的评价

用ＭＴＴ方法测定了多药抗药细胞ＭＣＦ－７Ａｄｒ及其敏感细胞ＭＣＦ－７ＷＴ对阿霉素、长春新碱、秋水仙碱的化学敏感性。ＭＣＦ－７Ａｄｒ与ＭＣＦ－７ＷＴ相比，对三种药物有明显的抗药性。维拉帕米可逆转ＭＣＦ－７Ａｄｒ对阿霉素的抗药性。随选择药物撤除时间的延长，ＭＣＦ－７Ａｄｒ的抗药水平逐渐下降。Ｓｏｕｔｈｅｒｎ杂交显示ＭＣＦ－７Ａｄｒ细胞有典型的ＭＤＲ１基因扩增现象。用ＭＴＴ方法测定Ｓｗｉｓｓ３Ｔ３细胞对阿霉素、长春新碱、秋水仙碱的化学敏感性，同时测定时各亚克隆株间差异较小，而分批测定时，不同批次相差很大。结果说明，ＭＴＴ方法能有效地检测出抗药细胞株的相对抗性；并提示，应在同时用完全相同的条件进行杀伤试验与ＭＴＴ测试，以避免批次间差异。     

川芎嗪和维拉帕米纠正阿霉素对小鼠艾氏腹水癌的抗药性

阿霉素(adriamycin,ADR)广泛应用于肿瘤临床,但易产生抗药性及多药抗药性(multidrug resistance,MDR),从而限制了其临床疗效的发挥。MDR与细胞表型改变有关。抗ADR的人乳腺癌细胞和小鼠SEWA癌细胞内,谷胱甘肽-S-转移酶(GST)的活性增高。ADR的MDR可被钙拮抗剂逆转,但是否与GST有关尚未见报道。     

棒烷肽抗生素G0069A的抗肿瘤作用

体外用MTT法和动物移植性肿瘤模型研究棒烷肽抗生素G0069A的抗肿瘤活性。结果表明,G0069A对多药抗药性肿瘤细胞显示高度的杀伤活性,与阿霉素、长春新碱无交叉抗药性。G0069A经iv或ip给药对小鼠肉瘤180和肝癌22有显著疗效;用小鼠可耐受剂量,对肉瘤180和肝癌22的抑制率分别达87%和74%。提示G0069A是天然来源的具有明显抗肿瘤作用的棒烷肽。     

用同系单克隆抗体对自发性小鼠乳腺癌进行免疫治疗

作者从患自发性乳腺癌的BALB/c小鼠体内,取出实体肿瘤,用等渗盐水制成细胞悬液。以200μl癌细胞悬液注入5只雌性BALB/c小鼠背部,诱导出实体癌,建立了一株称为JC的肿瘤细胞系,该细胞系保留了腺癌的特征及在BALB/c小鼠体内产生肿瘤的能力。作者报告用与JC细胞系有交叉反应的抗培养的人乳腺癌细胞的小鼠单克隆抗     

MDR1基因转染的Swiss 3T3细胞经秋水仙碱孵育后多药抗药性模式改变

为观察长期药物筛选对多药抗药性模式的影响，用MDR1 cDNA转染了Swiss 3T3细胞. 秋水仙碱选择培养两个月前后，以MTT方法测定各转化子对阿霉素，长春新碱，秋水仙碱的抗药性，以确定其多药抗药性模式. 还测定了维拉帕米对各转化子的增敏效应. 在非选择培养液中生长的转化子观察到基本一致的抗药性模式，即对长春新碱高度抗性，对秋水仙碱中度抗性，对阿霉素低度抗性. 而在选择培养液中生长两个月后，各转化子的抗药性模式变得参差不齐. 结果说明，MDR1基因表达可造成一致的抗药性模式，长期秋水仙碱孵育可改变抗药性模式的均一性.     

新的肿瘤耐药相关基因ERGIC-53的研究

目的:研究ERGIC-53基因表达与肿瘤细胞耐药的关系,寻找肿瘤耐药逆转可能的新靶点。方法:采用Northern Blot检测ERGIC-53基因在K562和K562/A02中的表达差异;应用逆转录聚合酶链反应(RT-PCR)检测11种肿瘤敏感及耐药细胞系中ERGIC-53基因的表达;设计合成针对ERGIC-53基因的siRNAs,用脂质体法转染K562和K562/A02细胞,用MTT比色法和流式细胞仪检测其干扰耐药细胞中ERGIC-53基因表达后的细胞对阿霉素的敏感性。结果:ERGIC-53在K562/A02细胞中的表达明显高于亲代K562细胞(P<0.05);6种耐药细胞株中ERGIC-53基因表达的平均值约为亲代敏感细胞的2倍。siRNA转染组与对照组相比,K562/A02细胞对阿霉素的敏感性均有不同程度的提高,且在转染后72h时细胞内阿霉素积累接近其亲代敏感细胞水平。结论:ERGIC-53基因与肿瘤细胞耐药相关,明确该基因参与耐药肿瘤细胞表型的形成。     

阿霉素对裸小鼠人肝癌原位移植瘤多药耐药性的影响

目的　探讨阿霉素对裸小鼠原位移植人肝癌多药耐药性的影响 ,并研究其耐药机制。方法　人肝癌 (BEL 740 2 )裸小鼠原位移植 ,用阿霉素腹腔注射诱导耐药 ,经MTT法检测原代培养的耐药细胞对抗癌药的敏感性 ,以流式细胞仪检测癌细胞表面mdr1基因产物P170的表达及功能。以裸小鼠原位移植人肝癌模型观察阿霉素对耐药组的疗效。结果　移植瘤组织形态及生物学方面符合人肝癌特征 ,耐药细胞表面P170表达为 75 45 %± 5 6 7% ,而对照组表达仅 4 2 5 %± 1 2 8% (P <0 0 1) ,对阿霉素的耐药倍数提高了 16 7倍 ,对羟基喜树碱和表阿霉素具有交叉耐受性(13 7倍和 7 5倍 )。耐药细胞表面P170有较强的药物外排功能。诱导后的肝癌在体内对阿霉素获得了明显的抗性。结论　阿霉素较易诱导原位移植于裸小鼠的人肝癌多药耐药性的产生 ,耐药机制主要与P170的过度表达有关     

P—糖蛋白介导的多药抗性分子机制

肿瘤细胞的抗药性,被认为是临床上化疗失败的一个主要原因.逐步增加药物浓度来筛选抗药性细胞株,人们已建立了许多多抗药性株.这些抗药株不仅对筛选的药物具有抗药性,还对秋水仙素、长春新碱、阿霉素、桑红霉素、放线菌素D、氯醌等结构和功能没有联系的药物具有交叉抗药性.多药抗性机制相当复杂,除了有P-糖蛋白将药物泵出细胞外的机制外,还有拓扑异构酶Ⅱ的功能变化和各胱甘肽过氧化物酶变化引起的多药抗性等等.本文论述与P-糖蛋白有关的多药抗性机制.     

米非司酮对乳腺癌细胞系MCF-7/ADM裸鼠移植瘤耐药逆转作用

目的：探讨米非司酮对乳腺癌细胞系MCF-7/ADM裸鼠移植瘤耐药逆转作用。方法：以人乳腺癌细胞系MCF-7/ADM建立耐阿霉素人乳腺癌裸鼠皮下移植瘤动物模型,将成瘤裸鼠随机分为空白对照组,米非司酮组（MIF组）,阿霉素组（ADM组）,米非司酮联合阿霉素组（MIF＋ADM组）。测量裸鼠移植瘤横径与短径,计算移植瘤体积,绘制生长曲线。结果：对裸鼠干预处理4周后,MIF＋ADM组移植瘤体积[（232.5149±309.2377）mm3]最低,ADM组[（508.9648±16.2609）mm3]次之,均低于空白对照组移植瘤体积[（962.2309±261.1313）mm3],差异有统计学意义（P〈0.05）;MIF＋ADM组移植瘤体积较单独用药的MIF组及ADM组低,差异具有统计学意义（P〈0.05）。结论：米非司酮有逆转乳腺癌细胞MCF-7/ADM裸鼠移植瘤耐药性的作用。     

Acquired multidrug resistance in human K562/ADM cells is associated with enhanced autophagy

Abstract

Autophagy, as a necessary process for survival in mammalian cells deprived of nutrients or growth factors, will be activated in many tumor cells while treated with chemotherapeutic drugs, but the role of autophagy in acquired multidrug resistance of human acute myelogenous leukemia to adriamycin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental acute myelogenous leukemia cell line K562 and resistant sub cell line, K562/ADM, which were obtained by treating adriamycin with increasing concentrations. K562/ADM and K562 cells were exposed to PBS culture medium for 3 hours, then the stress-induced autophagy was measured. Real-time quantitative RT-PCR revealed the expression of LC3 mRNA was higher in K562/ADM than in K562 cells. LC3-II, as an autophagosomal marker, was more abundant in K562/ADM than in K562 cells measured by Western blotting. To determine the effect of 3-MA, a known specific inhibitor of autophagy, on overcoming acquired multidrug resistance induced by adriamycin, the MTT assay and flow cytometry were performed. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of adriamycin in acquired resistant cells (K562/ADM). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in multidrug resistance of K562/ADM cells induced by adriamycin.     

油菜素内酯对CCRF-VCR1000细胞多药抗药性的逆转作用及其机制的初步探讨

目的了解油菜素内酯（BR）对多药抗药性细胞CCRF-VCR1000耐药性的逆转及机理。方法MTT法测定耐药因子及逆转倍数；罗丹明法测定药物积累；Sulliven方法测定拓扑异构酶II催化活性；Western blotting方法检测p53蛋白表达。结果CCRF-VCR1000细胞对阿霉素、VP-16和VCR分别呈153.1，55.9和8 123.1倍耐药。BR在不具细胞毒的浓度下（0.001-10.00 μg·mL^-1）使耐药性逆转4.4-11.6倍；并使耐药细胞药物积累改善；过度表达的p53回复到敏感细胞水平。结论在无细胞毒浓度下，BR能逆转CCRF-VCR1000的耐药性；抑制P糖蛋白对药物的外排。下调异常表达的p53蛋白可能是其逆转机制之一。     

Reversal effect of Dioscin on multidrug resistance in human hepatoma HepG2/adriamycin cells

Multidrug resistance is a serious obstacle encountered in cancer treatment. Since drug resistance in human cancer is mainly associated with overexpression of the multidrug resistance gene 1 (MDR1), the promoter of the human MDR1 gene may be a target for multidrug resistance reversion drug screening. In the present study, HEK293T cells were transfected with pGL3 reporter plasmids containing the 2 kb of MDR1 promoter, and the transfected cells were used as models to screen for candidate multidrug resistance inhibitors from over 300 purified naturally occurring compounds extracted from plants and animals. Dioscin was found to have an inhibiting effect on MDR1 promoter activity. The resistant HepG2 cell line (HepG2/adriamycin) was used to validate the activity of multidrug resistance reversal by Dioscin. Results showed that Dioscin could decrease the resistance degree of HepG2/adriamycin cells, and significantly inhibit P-glycoprotein expression, as well as increase the accumulation of adriamycin in HepG2/adriamycin cells as measured by Flow Cytometric analysis. These results suggest that Dioscin is a potent multidrug resistance reversal agent and may be a potential adjunctive agent for tumor chemotherapy.     

Effects of adriamycin and etoposide on the replication of adenovirus 5 in sensitive and resistant human tumour cells

Adenovirus is a potential probe for identifying and understanding drug sensitivity in primary, nonproliferating cultures of human normal and tumour cells but the scope and limitations of such an approach first need to be evaluated in established cell lines. For this purpose we have identified an ovarian tumour cell line (CI-80-13S) with natural resistance to adriamycin, etoposide and crosslinking agents compared with other human tumour lines. Resistance to adriamycin correlated poorly with resistance to etoposide in these cell lines (r = 0.05). Adenovirus replication in drug-treated cells (viral capacity) was found to be differentially inhibited in sensitive cells when the drug was administered to cells simultaneously with infection (adriamycin) or 20 hr after infection (etoposide). Viral capacity could not be inhibited by more than 90% in sensitive cells. In contrast, no such plateau was exhibited in the dose-responses of cell survival or inhibition of cellular DNA synthesis, both of which distinguished sensitive from resistant cells. Adenovirus was not inactivated by preincubation with high doses of adriamycin or etoposide, thus confirming that no functionally-relevant damage is directly induced by these agents in DNA. Uptake of adriamycin and etoposide was similar in sensitive and resistant cells and both agents blocked cells in the G2 phase of the cell cycle. Protein-linked DNA was induced in sensitive cells. The results indicate that (a) these drugs have two dose-dependent effects in cells, one of which does not inhibit replication of adenovirus; and (b) inhibition of adenovirus replication could in principle be used to predict sensitivity to adriamycin and etoposide.     

耐阿霉素人骨肉瘤细胞株的建立及其耐药机制探讨

目的诱导并建立耐阿霉素(ADM)的人骨肉瘤细胞株并探讨其耐药机制。方法采用逐步增加药物剂量冲击诱导方法诱导人成骨肉瘤原代Saos-2细胞株;MTT法检测原代与耐药细胞株对ADM、顺铂(DDP)、甲氨蝶呤(MTX)、异环磷酰胺(IFO)、表柔比星(EPI)、比柔比星(THP)、紫杉醇(Paclitaxel,PTX)药物敏感性;利用光学显微镜、透射电镜观察细胞形态及超微结构变化;RT-PCR和IHC法分别检测多药耐药基因1(MDR1)、多药耐药相关蛋白(MRP)基因及其相应蛋白(P-gp)、MRP的表达。结果经167d的诱导,建立Saos-2/ADM1、Saos-2/ADM4细胞株,其对ADM的耐药指数分别为原代细胞株的49.8和74.6倍;耐药细胞株对MTX、EPI、THP、PTX亦产生不同程度的交叉耐药(P<0.05),对DDP仍然敏感(P>0.05);光镜观察Saos-2/ADM1、Saos-2/ADM4细胞株细胞体积增大,多核现象较原代Saos-2细胞株明显增加;透射电镜显示Saos-2/ADM1、Saos-2/ADM4细胞株表面突起较原代Saos-2细胞株减少、且核仁增大增多;细胞生长曲线显示耐药细胞株增殖能力下降。MDR1mRNA、MRPmRNA和P-gp、MRP在各耐药细胞株表达阳性。结论MDR1 mRNA、MRP mRNA及其相应蛋白参与了耐药细胞株耐药的形成,这些骨肉瘤耐药细胞株为进一步研究骨肉瘤耐药特征及逆转方法打下了基础。     

Antitumour effect of abrin on transplanted tumours in mice

Abrin, a galactose specific lectin was purified using sepharose 4B affinity column from seeds of Abrus precatorius. It exhibited antitumour activity in mice when used at a sublethal dose of 7.5 micrograms/kg every alternate day for 10 days. Both intralesional and intraperiloneal (i.p.) administration of abrin was effective in reducing solid tumour mass development induced by Dalton's Lymphoma Ascites (DLA) and Ehrlich's Ascites Carcinoma (EAC) cells. DLA cell line was more sensitive to abrin than EAC. Abrin when injected i.p. increased the life span of ascites tumour bearing mice. Abrin when used simultaneously with tumour cells brought about maximum antitumour effect. On developed tumour masses, abrin administration brought about significant reduction in tumour volume, especially in DLA induced tumours. Prophylactic administration of abrin was found ineffective.     

Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines

One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1–1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH–adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1–1 at pH 7.2. Indeed, their synthetic C(7)-aglycon–GSH conjugates exerted a strong inhibitory effect on GST P1–1, with K_i at 25° in the 1–2 μM range, scarcely dependent on their stereochemistry at C(7).