卵巢癌多细胞球体对紫杉醇耐药及其机制的探讨

背景和目的:多细胞球体(multicellularspheroids,MCS)对传统细胞毒化疗药物的敏感性明显低于单层细胞。本实验旨在探讨卵巢癌MCS耐药的分子机制。方法:以单层细胞为对照,以三维培养方法获得的人卵巢癌A2780MSC和CAOV3MCS为模型,采用台盼蓝拒染法比较紫杉醇对单层细胞和MCS生长的抑制作用,流式细胞仪比较单层细胞和MCS细胞周期的分布和细胞凋亡率;采用流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测单层细胞及MCS的P-糖蛋白(P-glycoprotein,P-gp)表达及亚细胞分布;采用RT-PCR法检测mdr1mRNA表达水平。结果:(1)不同浓度的紫杉醇(0.2、2.0、10.0、20.0μmol/L)作用后,MCS细胞生长抑制率明显低于单层细胞(PA2780=0.003,PCAOV3=0.015);经20.0μmol/L的紫杉醇作用后,单层细胞的细胞凋亡率明显高于MCS,差异有统计学意义(PA2780=0.034,PCAOV3=0.032)。(2)流式细胞仪、蛋白质免疫印迹法、激光共聚焦显微镜检测提示P-gp在单层细胞中不表达,在MCS中表达明显升高(P均<0.05);RT-PCR证实MCS中有mdr1mRNA表达,而单层细胞中未检出其表达。(3)流式细胞仪检测提示将单层细胞培养成MCS时,G0/G1期细胞比率增加,S期和G2/M期细胞比率降低(P均<0.05)。结论:卵巢癌MCS对紫杉醇化疗耐药性增加,其高表达P-gp,并且与G0/G1期细     

MULTICELLULAR-MEDIATED EXPRESSION OF P-GP AND MRP AND RELATIONSHIP WITH CELL CYCLE PROFILES IN HUMAN OVARIAN CANCER SK-OV-3ip1 MULTICELLULAR AGGREGATES

Clinical multidrug resistance to chemotherapeutic agents is a major obstacle to treatment of patients with advanced epithelial ovarian cancer. A number of possible mechanisms have been put forward to account for drug resistance[1]. However, most of these proposed mechanisms are based largely on the study of drug-resistant variants isolated from tumor cell lines exposed to various classes of drugs in monolayer tissue culture. The impact of such mechanisms on clinical drug resistance remains spe…     

The Effect of the MDM2-p53 Loop on the Sensitivity of Ovarian Cancer Cells to Cisplatin

OBJECTIVE To explore whether MDM2 transfection can alter the MDM2-p53 autoregulatory feedback loop so as to change the sensitivity of ovarian cancer cell lines to cisplatin. METHODS The ovarian cancer cell line A2780 expressing wild-type P53 and the ovarian cancer cell line SKOV-3 with the p53 null type were stably transfected with pCMV-MDM2 or pCMV as a control. The blocked expression of P53 was determined by Western blots. Cytotoxicity was assessed using the MTT assay and the trypan blue exclusion assay. Flow cytometry was used to detect changes in the cell cycle and removal of platinum -DNA adducts was measured by atomic absorption spec-troscopy. RESULTS (1) Parental A2780 and A2780-V cells (IC50= 15.14±1.39 μmol) have similar cisplatin sensitivities, whereas sensitivity to cisplatin in A2780-M cells (IC50=7.98±1.32 μmol) was 2 to 3 fold greater (P=0.001). The trypan blue exclusion assay demonstrated that cisplatin killed a higher percentage of A2780-M cells compared to A2780-V cells. There was no significant change following MDM2 transfection in SKOV-3 cells. (2) After cisplatin treatment, A2780-M cells showed a pronounced S-phase arrest, however, A2780 cells with the intact wild-type P53, arrested primarily at the G2/M transition. (3) Platinum uptake was similar for all of the A2780 cell lines after ciaplatin treatment, but the removal of plat-inum-DNA adducts was reduced in the A2780-M cells compared with A2780-V cells. CONCLUSION MDM2 increases cisplatin cytotoxicity in ovarian cancer cells by blocking the expression of p53 through the MDM2-p53 autoregulatory feedback loop.     

人子宫内膜癌体外化学药物敏感性的实验研究

目的 利用人子宫内膜癌细胞株(HECCL-1)筛选对人子宫内膜癌敏感的化学药物,并探讨其作用机制。方法 采用四甲基偶氮唑蓝(MTY)法体外药敏实验检测HECCL-1细胞对18种不同浓度的化学药物的敏感性。利用流式细胞仪(FCM)进行细胞周期分析,并检测细胞凋亡率及用药前后多药耐药基因MDR1的表达。结果 MTY法结果显示,部分化学药物可显著抑制HECCL-1细胞的增殖,且呈剂量依赖效应,敏感性药物依次为阿霉素、奥铂、卡铂、顺铂、泰素、表阿霉素、放线菌素D、米托葸醌和氟脲嘧啶。FCM检测结果显示,化学药物作用后G0期、G1期细胞显著减少,S期、G2期、M期细胞显著增加。在血浆峰值浓度(PPC)下,11种化学药物作用于HECCL-1细胞后出现细胞凋亡图像。表阿霉素、氟脲嘧啶、羟基喜树碱、米托葸醌4种药物作用后诱导出MDR1阳性表达。结论 HECCL-1是一种化学药物较敏感的细胞株,多种化学药物能够显著抑制HECCL-1细胞的增殖活性,改变其细胞周期分布;诱导细胞凋亡是化学药物作用于该细胞的重要机制;获得性耐药是继续用药失败的主要原因。     

bcl—2基因家族在紫杉醇介导BJAB细胞凋亡中的作用

目的 观察抗微管新药紫杉醇对Ｂ细胞淋巴瘤细胞株ＢＪＡＢ是否具有凋亡诱导作用。并进一步研究ｂｃｌ－２基因家族在此过程中的作用。方法 将不同浓度的紫杉醇作用于ＢＪＡＢ细胞,观察其作用的时间效应及剂量效应;在光镜和电镜下观察其形态变化;用流式细胞仪分析细胞ＤＮＡ含量的改变并做ＤＮＡ片段分析;用免疫组织化学及半定量ＲＴ－ＰＣＲ法观察在紫杉醇作用过程中ｂｃｌ－２基因家族的蛋白及ｍＲＮＡ的变化。结果 紫杉醇能抑制ＢＪＡＢ细胞生长,抑制作用首先表现为Ｇ２／Ｍ期阻滞,一定时间后出现细胞凋亡,并显示剂量和时间效应。在这一过程中,ｂｃｌ－２转录及蛋白表达下降,并出现ｂｃｌ－ｘｓ的转录。结论 紫杉醇可诱导ＢＪＡＢ细胞凋亡,这为其应用于Ｂ细胞淋巴瘤的治疗提供依据。ｂｃｌ－２和ｂｃｌ－ｘｓ参与了紫杉醇介导ＢＪＡＢ细胞凋亡的基因调控。     

紫杉醇诱导胃癌细胞凋亡的实验研究

 目的　探讨紫杉醇对胃癌细胞的诱导凋亡作用及其诱导的胃癌细胞凋亡的周期时相性。方法　用Sub-G1法检测紫杉醇诱导胃癌细胞MKN-28的凋亡,API法检测紫杉醇诱导胃癌细胞凋亡的周期时相性,并分选后激光共聚焦显微镜技术（PSC）观察形态学,MTT法检测紫杉醇对临床胃癌组织细胞的敏感性。结果　Sub-G1法结果显示紫杉醇能诱导胃癌细胞的凋亡,紫杉醇（浓度10 mg／L）诱导胃癌细胞凋亡在10 h后达到高峰;API法检测结果显示紫杉醇诱导胃癌细胞发生凋亡的时相在G2／M期,PSC形态学观察到G2／M期凋亡特征;MTT法结果显示紫杉醇诱导的20例临床胃癌组织标本中,有16例抑制率大于50 %。结论　紫杉醇能诱导胃癌细胞发生凋亡,相对较敏感,其诱导的胃癌细胞凋亡具有周期时相性。     

同步放化疗对宫颈鳞癌细胞增殖、凋亡及细胞周期的影响

目的:探讨局部晚期宫颈鳞癌细胞对同步放化疗应答的分子机制。方法:49例患者被分为2组:单纯放疗组(RT)和同步放化疗组(CCRT)。在治疗前和治疗中[RT组:放疗10 Gy后;CCRT组:放疗10 Gy+(DDP+5-FU)×1个周期]分别活检留取标本,用FCM、TUNEL及免疫组化检测细胞周期、凋亡以及PCNA的表达。结果:RT组和CCRT组治疗中较治疗前AI及凋亡阳性率均明显升高(P<0.05,P<0.001),治疗中CCRT组凋亡率显著高于RT组(P=0.03);PCNA的表达CCRT组较RT组降低更明显(P=0.005)。治疗中RT组细胞周期大部分被阻滞在G2/M期,而CCRT组大部分被同时阻滞在S和G2/M期。结论:CCRT治疗局部晚期宫颈鳞癌中化疗和放疗有协同作用,其机制可能为通过抑制细胞增殖以及阻滞细胞周期于S和G2/M期,使细胞周期同步化,继而诱导肿瘤细胞的凋亡实现的。     

Taxol-induced bcl-2 phosphorylation in ovarian cancer cell monolayer and spheroids

Expression of Bcl-2, Bax, p53 and induction of apoptosis were studied in cisplatin or Taxol treated monolayer and spheroid cultures of ovarian cancer cell lines (SKOV-3, UL-1, UL-3C). While cisplatin (15-75 microg/ml) induced apoptosis in monolayer and spheroid cultures, Taxol (100-800 nM) induced fragmentation in monolayers only. Cisplatin induced up to 5-fold DNA fragmentation in monolayers, while 3-fold (UL-3C, SKOV-3), and 1.5-fold (UL-1) in spheroids. Taxol treatment of monolayers resulted in the characteristic phosphorylation of Bcl-2, which was not demonstrated in spheroid cultures. Bax expression was reduced in spheroids following cisplatin or Taxol treatment, while p53 levels remained unchanged.     

多烯紫杉醇诱导的卵巢癌SK-OV-3多倍体肿瘤巨细胞增殖、迁移和凋亡的特性研究

目的:探讨多烯紫杉醇（Docetaxel,Doc）诱导的卵巢癌SK-OV-3多倍体肿瘤巨细胞增殖、迁移和凋亡的特性。方法:0.8 μmol/L Doc处理SK-OV-3细胞16 h,更换为完全培养液继续培养细胞至第3天和第5天。免疫荧光染色法观察细胞形态和增殖,流式细胞术检测细胞倍性、周期和凋亡,划痕实验检测细胞迁移能力,Western blot检测凋亡相关蛋白表达。结果:Doc处理后SK-OV-3细胞体积变大,细胞核增多,细胞增殖能力降低。Doc（3 day）组和Doc（5 day）组多倍体细胞（>4 N）百分比高于对照组（P＜0.01）。Doc诱导Doc（16 h）组发生细胞周期阻滞。Doc（5 day）组细胞迁移能力低于对照组（P＜0.01）。Doc（3 day）组和Doc（5 day）组早期凋亡细胞百分比高于对照组（P＜0.05）。Doc（16 h）组、Doc（3 day）组和Doc（5 day）组促凋亡蛋白表达逐渐上调,抗凋亡蛋白表达下调。结论:Doc诱导卵巢癌SK-OV-3细胞形成多倍体肿瘤巨细胞,SK-OV-3多倍体肿瘤巨细胞增殖和迁移能力降低,早期凋亡增加,这为探索多倍体肿瘤巨细胞特性提供了研究基础。     

Taxol cytotoxicity on human leukemia cell lines is a function of their susceptibility to programmed cell death

Taxol is the prototype of a class of antineoplastic drugs that target microtubules. It enhances tubulin-monomer polymerization and stabilizes tubulin polymers, increasing the fraction of cells in the G2 or M phase of the cell cycle. We report that treatment of HL-60 and U937 myeloid cell lines with 1–10 M taxol induces DNA fragmentation and the appearance of morphological features consistent with the process of apoptosis. Taxol-induced apoptosis is inhibited neither by cycloheximide nor by actinomycin D and therefore appears to be independent of new protein synthesis. Taxol causes arrest in the G2 phase of the cell cycle and affects cell viability but does not induce DNA fragmentation in the K562 erythromyeloid cell line. Protein-synthesis inhibitors, colcemid, ionomycin, and starvation, known to trigger apoptosis, proved ineffective as well. These results suggest that the antineoplastic effect of taxol is mediated in susceptible cell lines by induction of the apoptotic machinery and that K562 partial resistance may depend upon the intrinsic inability of these tumor cells to undergo apoptosis.Abbreviations CHX cycloheximide - Act D actinomycin D - PI propidium iodide     

腺病毒介导p16基因的表达及其对肺癌细胞的周期阻滞作用

目的:以增殖缺陷型腺病毒载体介导p16基因感染人肺腺癌细胞系,观察并鉴定该基因在细胞中的表达及其对细胞周期和细胞凋亡的影响.方法:采用293细胞内同源重组的方法,制备重组腺病毒Ad5-p16,感染肺腺癌细胞SPC-A1;免疫组化方法鉴定P16蛋白的表达;台盼蓝染色计数活细胞数,绘制细胞生长曲线;流式细胞术(FCM)分析细胞周期及细胞凋亡的变化.结果:以M0I=10的重组增殖缺陷型腺病毒Ad5-p16感染SPC-A1细胞48 h后,P16蛋白表达的阳性细胞比率为71%;以MOI=100感染SPC-A1细胞后第2天,细胞开始出现生长抑制及细胞病变;FCM分析发现细胞在感染腺病毒后出现细胞周期阻滞和细胞凋亡.结论:以腺病毒为载体介导p16基因在SPC-A1细胞内表达,可发挥抑制细胞生长、诱发细胞凋亡的作用.本项基因治疗策略以直接调控细胞周期的抑癌基因为靶向治疗基因,为肺癌基因治疗提供了可靠的理论依据.     

Bortezomib induces G2-M arrest in human colon cancer cells through ROS-inducible phosphorylation of ATM-CHK1

Colorectal cancer (CRC) is one of the most common cancers; however, the development of drugs to treat the condition has reached a plateau. Bortezomib (PS-341, Velcade?) is a proteasome inhibitor approved for the treatment of hematological malignancies, including multiple myeloma. A few trials of bortezomib, alone or in combination chemotherapy, for CRC patients have been reported; however, the results were largely inconclusive. This may be related to a lack of understanding of the drug's mechanism of action. Although bortezomib is reported to induce apoptosis and cell cycle arrest in various human cancer cells, the inhibitory mechanism involved is not clear. In this study, the effect of bortezomib as a treatment for human CRC was examined in vitro using three CRC cell lines. Bortezomib induced G2-M arrest in CRC cells. Investigation of G2-M phase-related cell cycle proteins involved in the response to bortezomib revealed that the ataxia telangiectasia mutated (ATM)-cell cycle checkpoint kinase 1 (CHK1) pathway, but not ATM and Rad3-related (ATR), was activated, resulting in the inactivation of cdc2. Bortezomib caused an increase in intracellular reactive oxygen species (ROS) and treatment with the ROS scavenger NAC inhibited phosphorylation of ATM leading to a decrease in the number of cells in G2-M phase. Thus, increased ROS levels after exposure to bortezomib resulted in ATM phosphorylation. In addition, knockdown of endogenous ATM by RNA interference resulted in decreased sensitivity to bortezomib. These results suggest that bortezomib induces G2-M arrest through ROS-inducible ATM phosphorylation and demonstrate that bortezomib is a potential candidate for further investigations in the treatment for CRC patients.     

低氧对紫杉醇诱导的人卵巢癌细胞A2780凋亡的影响及其机制

背景与目的低氧作为实体瘤的特征和重要的生存微环境,可能与化疗耐药相关.我们认为低氧与卵巢癌化疗耐药可能相关.本研究建立低氧模型,探讨低氧、低氧诱导因子1 α(HIF-1α)对紫杉醇诱导的人卵巢癌细胞A2780凋亡的影响.方法体外培养的A2780细胞中加入化学性低氧诱导剂氯化钴(CoCl2),诱导并制作低氧模型.将细胞分为4组A组(对照)、B组(常氧培养加紫杉醇)、C组(低氧培养加紫杉醇)、D组(低氧培养加Decoy加紫杉醇).用诱骗法(Decoy)阻断HIF-1α功能,Western blot、RT-PCR、TUNEL和流式细胞术分别检测各组细胞HIF-1α蛋白、mRNA的表达水平及细胞凋亡情况.结果CoCl2能明显增加A2780细胞HIF-1α蛋白的表达,而对其mRNA的表达无明显影响,Decoy法阻断HIF-1α的功能,对其蛋白和mRNA的表达无明显影响.TUNEL检测发现,B组凋亡指数[AI(41.12±25.65)%]显著高于C组[AI(24.12±15.19)%](P＜0.05);低氧阻断了HIF-1α功能后,D组凋亡指数[AI(35.19±21.73)%]较C组明显增加(P＜0.05).流式细胞仪检测细胞凋亡率结果与TUNEL检测结果类似.结论低氧能保护A2780细胞抵抗化学治疗,HIF-1α可能在紫杉醇诱导的细胞凋亡中发挥重要的抗凋亡作用.     

Treatment with paclitaxel alone rather than combination with paclitaxel and cisplatin may be selective for cisplatin-resistant ovarian carcinoma

BACKGROUND: We have previously reported that paclitaxel (taxol) results in cisplatin sensitization to human ovarian cancer cells with cisplatin resistance in vitro. This study was designed to determine effects of taxol and its combination with cisplatin on growth of cisplatin-sensitive cell line (KF28) and the cisplatin-resistant counterpart (KFr13) in nude mice. METHODS: From 14 days after tumor inoculation treatment was initiated. Taxol (3 mg/kg) and cisplatin (2 mg/kg) were administered i.p. once a week for 5 weeks. RESULTS: In nude mice bearing cisplatin-sensitive cells (KF28), taxol followed by cisplatin and cisplatin plus taxol inhibited significantly (P < 0.05) the tumor growth rate compared with that in nude mice treated with cisplatin alone or taxol alone and cisplatin followed by taxol. On the other hand, in nude mice bearing cisplatin-resistant KFr13 cells, treatment with taxol alone inhibited completely the tumor growth rate, whereas no schedule-dependent interaction of taxol with cisplatin was observed. CONCLUSION: These results suggest that treatment with taxol alone may be superior to combination of taxol with cisplatin in patients with cisplatin-resistant ovarian carcinoma.     

Effects of Taxol on three different types of lymphoma cell lines

Lymphomaisacommonmalilgnanttumor.Relapseandrefractorycasesareoftenseenduringroutinetherapy.Itisnecessarytosearchforpotentdrugsandtostudytheirmechanisms.Taxol(Paclitaxel)isanewanticanceragent,[']whichiseffectiveinanumberofcasesintumortherapy,suchasadvancedovarianandbreastcancer,prostatecancerandnon-smallcelllungcancer.However,littleisknowninlymphomas.Ithasbeenreportedthattaxolismoreeffectiveinrelapseandmiddlegradelymphomasthaninothertypesofthetumor.[2]Itcanobviouslyinhibitthesyngeniclymphomat…     

常规化疗药物诱导卵巢癌OVCAR-3细胞凋亡特点的分析

目的：观察抗卵巢癌药物顺铂、紫杉醇和阿霉素对卵巢癌细胞系ＯＶＣＡＲ－３体外生长和生存的影响并分析由它们所致的细胞死亡性质。方法：采用细胞形态学观察、细胞动力学检测及ＤＮＡ片段化分析等细胞和分子生物学方法,研究化疗药物对卵巢癌细胞的凋亡诱导作用。结果：上述药物在抑制ＯＶＣＡＲ－３细胞生长的同时可不同程度地诱导细胞凋亡。其中,紫杉醇诱导细胞凋亡的能力最强;较低剂量紫杉醇（１０＾－８Ｍ）和顺铂（２μｇ／     

Epothilone B analogue (BMS-247550)-mediated cytotoxicity through induction of Bax conformational change in human breast cancer cells.

Epothilone B is a novel nontaxane antimicrotubule agent that is active even against paclitaxel (Taxol)-resistant cancer cells. The present study further explores the mechanisms underlying epothilone B-mediated cytotoxicity in human breast cancer cells. We show that BMS-247550 (EpoB), a novel epothilone B analogue, induces cell cycle arrest at the G(2)-M phase transition and subsequent apoptotic cell death of MDA-MB-468 (468) cells. Treating cells with EpoB triggers a conformational change in the Bax protein and its translocation from the cytosol to the mitochondria, which is accompanied by cytochrome c release from the inter-membrane space of mitochondria into the cytosol. Overexpression of Bcl-2 delays Bax conformational change, cytochrome c release, and apoptosis induced by EpoB. Conversely, the Bcl-2 antagonist Bak-BH3 peptide or HA14-1 compound abrogates the antiapoptotic effects of Bcl-2 and enhances apoptosis of 468 cells pretreated with EpoB (to induce mitotic arrest). In synchronized 468 cells, EpoB is more potent in inducing Bax conformational change and apoptosis at G(2)-M phase compared with G(1)-S phase of the cell cycle. Taken together, these findings demonstrate that EpoB induces apoptosis through a Bcl-2-suppressible pathway that controls a conformational change of the proapoptotic Bax protein. The enhanced cytotoxicity of EpoB by blocking Bcl-2 at mitochondria implies a potential application of the combination of EpoB and Bcl-2 antagonists in the treatment of human breast cancer.     

Taxol sensitizes human astrocytoma cells to radiation.

Taxol is a chemotherapeutic drug which acts by stabilizing microtubules, preventing normal mitosis and resulting in a block of the cell cycle at G2 and M. The drug is isolated from the yew, Taxus sp. L., and is currently being evaluated in a series of Phase II and Phase III clinical trials. Taxol blocks cells in the most radiosensitive phases of the cell cycle and thus could act as a cell cycle-specific radiosensitizer. We report the results of combined taxol-radiation exposures in the human Grade III astrocytoma cell line, G18. Taxol is a potent inhibitor of G18 cell division; a concentration of 10 nM is cytostatic for a cell population observed for at least two doubling times. Cell survival curves for G18 cells showed a significant concentration-dependent interaction between taxol and radiation. Treatment of G18 cells with a fixed taxol concentration and radiation dose showed the interaction to be dependent on the duration of taxol exposure and consequently the fraction of cells in the G2 or M phase of the cell cycle. The sensitizer enhancement ratio for 10 nM taxol at 10% survival is 1.8 and, for 1 nM taxol, it is 1.2. These results suggest that appropriate combinations of taxol have a more than additive interaction in human tissue culture and may have a role in clinical protocols.     

Modifications of cell-cycle induced by dpr, a new platinum-triamine complex containing procaine

cis-diamminechloro-[2-(diethylamino)ethyl 4-amino-benzoate, N4]-chlorideplatinum(II) monohydrochloride monohydrate (DPR) is a new platinum-triamine complex containing as ligand the local anesthetic procaine. In this study DPR was compared to the parent compound cisplatin (cis-DDP) in order to study the influence of both molecules on the cell cycle phases, and particularly on the induction of apoptosis. P388 murine leukemic cells were used as cellular model, and were exposed in vitro to either compound, continuously for 24 hours. At the end of the incubation, the thymidine uptake, the trypan blue dye exclusion assay, and the flow cytometry were assessed. Both the cytotoxic activity and the inhibition of DNA synthesis evaluated after 24 h incubation with DPR or cis-DDP were comparable. Moreover, cell cycle was modified in a comparable manner by both molecules. In particular the induction of the apoptotic effect was similarly induced by the same concentrations of the compounds and time exposure. In conclusion, DPR and cis-DDP seem to have a similar effect on the cell cycle of P388 leukemic cells and particularly on the induction of the programmed cell death.