Detection of drug resistance in human ovarian carcinoma

Summary Drug-resistant cancer cells with the multidrug-resistance phenotype show overexpression ofP-glycoprotein, and we therefore tested carcinoma tissue from five patients with stage III or IV ovarian cancer forP-glycoprotein using 265/F4 and C 219 monoclonal antibodies, prepared against membrane glycoproteins in colchicine-resistant CHO cells. Using immunofluorescence and immunoblotting techniques, one of the tumors showed a positive reaction. Using the pcDR 1.5 clone we found that the same cancer tissue had elevated expression of the genes responsible for multidrug resistance. The demonstration of elevatedP-glycoprotein in ovarian carcinomas indicates thatP-glycoprotein overexpression is not limited to experimental tumor models.     

Taxol-oligoarginine conjugates overcome drug resistance in-vitro in human ovarian carcinoma

Objective

Multidrug resistance is the major cause of failure of many chemotherapeutic agents. While resistance can arise from several factors, it is often dominated by drug efflux mediated by P-glycoprotein (P-gp), a membrane-bound polysubstrate export pump expressed at high levels in resistant cells. While co-administration of pump inhibitors and a drug could suppress efflux, this two-drug strategy has not yet advanced to therapy. We recently demonstrated that the reversible attachment of a guanidinium-rich molecular transporter, polyarginine, to a drug provides a conjugate that overcomes efflux-based resistance in cells and animals. This study is to determine whether this strategy for overcoming resistance is effective against human disease.

Methods

Tumor samples from ovarian cancer patients, both malignant ascites cells and dissociated solid tumor cells, were exposed to Taxol-oligoarginine conjugates designed to release free drug only after cell entry. Cell viability was determined via propidium-iodide uptake by flow cytometry. To analyze bystander effect, toxicity of the drug conjugates was also tested on peripheral blood leucocytes.

Results

Human ovarian carcinoma specimens resistant to Taxol in vitro demonstrated increased sensitivity to killing by all Taxol-transporter conjugates tested. These studies also show that the drug conjugates were not significantly more toxic to normal human peripheral blood leukocytes than Taxol.

Conclusions

These studies with human tumor indicate that oligoarginine conjugates of known drugs can be used to overcome the efflux-based resistance to the drug, providing a strategy that could improve the treatment outcomes of patients with efflux-based drug-resistance.     

CDH1基因异常甲基化在上皮性卵巢癌中的检测及临床意义

目的 探讨CDH1基因异常甲基化在上皮性卵巢癌发生发展中的作用及临床意义。方法 对中国医科大学附属第一医院妇科、辽宁省肿瘤医院妇科1999年至2006年63例上皮性卵巢癌组织原发灶、41例相应的盆腹腔转移灶、10例癌旁卵巢组织及20例正常卵巢组织,采用甲基化特异性聚合酶链式反应（MSP）法检测CDH1基因启动子区甲基化状态。结果 上皮性卵巢癌组织原发灶及相应的盆腹腔转移灶中存在CDH1基因启动子区异常甲基化,发生率分别为28.6%、39.0%,显著高于正常卵巢组织（P〈0.01）。10例癌旁卵巢组织中1例检测到CDH1基因启动子区甲基化,20例正常卵巢组织未检测到CDH1基因启动子区甲基化。CDH1基因启动子区甲基化的发生率在临床Ⅰ期和Ⅱ期显著低于Ⅲ期和Ⅳ期（P〈0.05）,在高分化癌中低于低分化癌（P〈0.05）。结论 CDH1基因启动子区异常甲基化与上皮性卵巢癌发生密切相关,并可能与上皮性卵巢癌转移、临床分期及分化程度有关。     

卵巢上皮性癌血清肿瘤标志物谱变化的临床意义

目的 探讨卵巢上皮性癌(卵巢癌)患者化疗后肿瘤标志物谱的变化及其潜在的临床意义.方法 选择1999年1月至2007年7月期间经肿瘤细胞减灭术及规范化疗的卵巢癌患者102例,对其术前、术后、每次化疗前、随访期间和复发前后的血清肿瘤标志物CA125、CA19-9和CP2的水平进行检测、分析,其中48例患者的肿瘤标志物记录完整而纳入分析,复发患者为28例,初治化疗患者20例(均为耐药病例).根据肿瘤标志物谱变化与否,分别将复发和初治化疗患者分为肿瘤标志物谱变化组与未变化组.平均随访时间为25个月.结果 (1)肿瘤标志物谱的主要变化表现为标志物的数最变化和(或)标志物的种类改变.28例复发患者中肿瘤标志物谱发生变化者占46%(13/28),20例初治化疗患者中标志物谱发生变化者占45%(9/20).(2)肿瘤标志物谱变化的复发患者中,病理类型以浆液性癌所占比例最高,为77%(10/13),而初治化疗患者中,以黏液性癌所占比例最高,为4/9.(3)复发患者肿瘤标志物谱变化组的无疾病进展期和中位总生存时间分别为22.2、60.0个月,较未变化组(分别为17.4、46.0个月)明显延长(P均<0.05);初治化疗患者肿瘤标志物谱变化组的中位总生存时间较未变化组(分别为15.9、25.0个月)明显缩短(P<0.05).结论 卵巢癌化疗期间和复发后肿瘤标志物谱可发生变化,化疗及随访期间应对肿瘤标志物进行联合检测.     

小儿造血干细胞移植后出血性膀胱炎的临床特征与危险因素分析

目的 分析小儿造血干细胞移植(HSCT)后出血性膀胱炎(HC)的临床特点，探讨其发病危险因素。 方法 对1998年10月至2004年6月中山大学附属二院儿科完成的52例小儿HSCT后11例HC的临床资料进行回顾分析。 结果 11例HC中轻度(Ⅰ~Ⅱ度)6例，重度(Ⅲ~Ⅳ度)5例；早发性4例，迟发性7例；发病时间为术后+2d至+25d(中位数为+15d)，病程3~60d(中位数为17d)。临床表现均有血尿，其中典型尿频、尿急、尿痛及肉眼血尿7例。HC患儿组中性粒细胞植入时间和血小板植入时间与非HC患儿组比较差异无显著性(P>0.05)。受者移植年龄≥6岁、aGVHD阳性、CMV感染组的HC发生率分别高于年龄<6岁(321%和83%，P<0.05)、GVHD阴性(34.6%和7.7%，P<0.05)、CMV未感染组(62.5%和13.6%，P<0.05)。 结论 小儿HSCT后HC有其自身的临床特征；受者移植年龄≥6岁、aGVHD阳性、CMV感染为其发生的危险因素。     

卵巢上皮性癌ERCC1表达与顺铂化疗耐药关系的研究

目的:探讨卵巢上皮性癌组织中ERCC1表达水平与顺铂化疗耐药的相关性。方法:应用免疫组化SP法检测42例卵巢癌组织中ERCC1的表达并分析其与顺铂化疗耐药的关系。结果:卵巢癌组织ERCC1表达水平与卵巢上皮性癌患者年龄、手术分期及组织学类型无关,而与卵巢上皮性癌患者以顺铂为基础联合化疗的耐药性有关。卵巢癌化疗耐药组ERCC1的阳性表达率为77.27%,明显高于敏感组35.00%,组间差异有统计学意义(P<0.05)。结论:ERCC1在卵巢上皮性癌组织中的表达水平与顺铂化疗耐药有关。     

In vitro chemosensitivity testing in the treatment of ovarian carcinoma

Summary In vitro chemosensitivity to cisplatinum, adriamycin, cyclophosphamide, and 5-fluorouracil was investigated in 58 cases of ovarian carcinoma using Volm's short-term test. These in vitro results were retrospectively correlated with the relapse-free interval. Operative treatment in all patients (FIGO stage I (5), III (43), IV (10)) comprised maximum debulking procedure including hysterectomy, adnexectomy, omentectomy, pelvic and in most cases additionally paraaortic lymphadenectomy. Subsequently, all patients were treated with the cisplatinum-epirubicin-cyclophosphamide regimen. 33/58 tumors (66%) were sensitive in vitro (inhibition of nucleic acid precursor incorporation of more than 45% as compared to untreated controls). The median relapse-free interval of patients with sensitive tumors was significantly longer than that of those with resistant carcinomas (30.3 versus 22.6 months, respectively;P<0.05). Histopathological evaluation showed the majority of serous cystadenocarcinomas to be sensitive (26/33=79%,P<0.05).     

卵巢上皮性癌DNA异常甲基化模式的建立及其应用

目的 建立卵巢上皮性癌(卵巢癌)的DNA异常甲基化模式,探讨其在寻找新的卵巢癌特异性标志物中的应用价值.方法 用激光显微切割技术从20例卵巢癌组织冰冻切片中获取的肿瘤细胞作为实验对象,用原代培养的5例正常卵巢上皮细胞作为对照,用基于芯片技术的差异甲基化杂交(DMH)方法检测卵巢癌的DNA异常甲基化模式.选择7个DMH结果显示在卵巢癌中低甲基化的基因启动子区胞嘧啶-磷酸-鸟嘌呤二核苷酸岛(CGI),用甲基化实时荧光定量PCR技术检测其在87例卵巢癌和42例卵巢良性病变患者病变组织中的甲基化状态.结果 182个过甲基化位点和64个低甲基化位点(阳性率为25%以上的位点分别有18个和31个)组成了卵巢癌的DNA异常甲基化模式.87例卵巢癌和42例卵巢良性病变患者组织DNA中,基因LSM2、EGFLAM和CDKN2A的甲基化率依次为11%(10/87)和33%(14/42)、8%(7/87)和21%(9/42)、9%(8/87)和31%(13/42),与卵巢良性病变相比,卵巢癌中3个基因甲基化率均有显著下降,分别比较,差异均有统计学意义(P＜0.05).结论 建立卵巢癌的DNA异常甲基化模式是卵巢癌研究中非常重要的基础环节.基因EGFLAM、CDKN2A和LSM2启动子区CGI有可能成为新的卵巢癌特异性的低甲基化肿瘤标志物.     

上皮性卵巢癌组织中金属硫蛋白与肺耐药蛋白的表达及意义

目的　探讨上皮性卵巢癌中金属硫蛋白 (MT)及肺耐药蛋白 (LRP)的表达及其临床意义。方法　采用免疫组化SP法及半定量分析方法 ,检测 1995～ 2 0 0 1年上皮性卵巢癌 6 4例 ,复发后二次手术癌组织 17例 ,卵巢良性上皮性肿瘤 16例及正常卵巢组织 14例中MT及LRP的表达 ,并进行相关临床病理因素分析。结果　MT在上皮性卵巢癌中的表达显著高于卵巢良性肿瘤及正常卵巢组织 (P <0 0 1) ,MT表达与细胞分级和腹水相关 ;在上皮性卵巢癌中LRP的阳性率为 78 1% ,与卵巢良性肿瘤及正常卵巢组织相比差异有显著性意义(P <0 0 1) ;化疗后癌组织中MT的阳性表达率和LRP的强阳性表达率与初治卵巢癌组织中相应的表达率差异有显著性意义 ;MT与LRP表达存在相关性 ,两者同时表达者术后生存时间短 ,易于复发。结论　MT、LRP在上皮性卵巢癌中较恒定表达 ,化疗可以增加癌组织中MT、LRP的表达。联合检测MT、LRP可预测卵巢癌的铂类联合化疗疗效 ,是判断其预后的可靠指标     

4种肿瘤标志物对上皮性卵巢癌定性诊断价值的初步研究

目的：探讨多胺（ＰＡ）、ＣＡ１２５、ＣＡ１５．３和ＣＡ１９．９在定性诊断上皮性卵巢癌中的价值。方法：应用ＨＰ％Ｍ高效液相色谱仪和ＨＰ１０４０Ａ荧光检测器或酶联免疫吸附法测定上皮性卵巢癌４０例和卵巢良性肿瘤１８例血清中ＰＡ、ＣＡ１２５、ＣＡ１５．３和ＣＡ１９．９水平。结果：４种标志物中，ＰＡ诊断卵巢癌的敏感性、阳性预测率、阴性预测率和预测准确率最高，其次是ＣＡ１２５。结论：ＰＡ对人类恶性肿瘤缺乏特异性，但可作为鉴别卵巢良、恶性病变的有价值标志物。联合测定ＰＡ和ＣＡ１２５时，其敏感率为９４．９％。因此，联合测定ＰＡ和ＣＡ１２５可作为筛查卵巢良、恶性肿瘤的方法。     

BAG3 upregulates Mcl-1 through downregulation of miR-29b to induce anticancer drug resistance in ovarian cancer

Objective

Ovarian cancer is the leading cause of death from gynecologic cancer, reflecting its often late diagnosis and its chemoresistance. We identified a set of microRNAs whose expression is altered upon BAG3 knockdown. Our primary objective was to examine the relationships between BAG3, miR-29b and Mcl-1, an antiapoptotic Bcl-2 family protein, in ovarian cancer cells.

Methods

Ovarian cancer cells were cultured and their responsiveness to paclitaxel was tested. Microarray analysis was performed to identify microRNAs differentially expressed in ES2 BAG3 knockdown ovarian cancer cells and their control cells. Primary ovarian cancer tissues were obtained from 56 patients operated on for ovarian cancer. The patients' clinical and pathological data were obtained from their medical records.

Results

BAG3 knockdown increased the chemosensitivity to paclitaxel of ES2 ovarian clear cell carcinoma cells to a greater degree than AMOC2 serous adenocarcinoma cells. qRT-PCR analysis showed that miR-29b expression was significantly upregulated in primary cancer tissue expressing low levels of BAG3, as compared to tissue expressing high levels. Moreover, levels of miR-29b correlated significantly with progression-free survival. Upregulation of miR-29b also reduced levels of Mcl-1 and sensitized ES2 cells to low-dose paclitaxel.

Conclusions

BAG3 knockdown appears to downregulate expression of Mcl-1 through upregulation of miR-29b, thereby increasing the chemosensitivity of ovarian clear cell carcinoma cells. This suggests that BAG3 is a key determinant of the responsiveness of ovarian cancer cells, especially clear cell carcinoma, to paclitaxel and that BAG3 may be a useful therapeutic target for the treatment of ovarian cancer.     

Survival outcomes in patients with recurrent ovarian cancer who were treated with chemoresistance assay-guided chemotherapy

OBJECTIVE: The purpose of this study was to determine the outcome of patients with recurrent ovarian carcinoma after extreme drug resistance assay-directed therapy. STUDY DESIGN: Fifty women who were treated with chemotherapy based on extreme drug resistance assay guidance were compared with 50 well-balanced control subjects who were treated empirically. RESULTS: In the platinum-sensitive group, patients with extreme drug resistance-directed therapy had an overall response rate of 65% compared with 35% in the patients who were treated empirically (P=.02). The overall and progression-free median survival were 38 and 15 months in the extreme drug resistance assay group compared with 21 and 7 months in the control group, respectively (P=.005, overall; P=.0002, progression free). In the platinum-resistant group, there was no improved outcome in the patients who underwent assay-guided therapy. In multivariate analysis, platinum-sensitive disease, extreme drug resistance-guided therapy and early stage of disease were independent predictors for improved survival. CONCLUSION: In this retrospective analysis, our results indicate an improved outcome in patients with recurrent ovarian carcinoma who have platinum sensitive disease and who underwent extreme drug resistance-directed chemotherapy. Randomized, prospective, controlled trials are needed.     

TWEAK调控凋亡增加上皮性卵巢癌细胞对铂类药物敏感性的研究

目的：探讨肿瘤坏死因子样凋亡微弱诱导剂（TWEAK）在调控上皮性卵巢癌（EOC）细胞对顺铂（DDP）敏感性中发挥的作用。方法：不同浓度TWEAK刺激A2780/cDDP细胞后,采用CCK-8法检测细胞的增殖能力及DDP半数抑制浓度（IC50）的变化;采用流式细胞术检测细胞凋亡率的变化;采用透射电镜检测细胞核染色质形态和凋亡小体的变化;Western blot法检测细胞凋亡相关蛋白caspase 3的活化情况。结果：TWEAK对A2780/cDDP细胞的增殖能力无影响,但能显著增加A2780/cDDP细胞对DDP的敏感性。流式结果显示,随着TWEAK剂量增加,A2780/cDDP细胞的凋亡显著增加。TWEAK刺激后A2780/cDDP细胞中caspase 3活化体表达增加,细胞核染色质发生形态变化,诱导了细胞的凋亡及凋亡小体形成。结论：TWEAK通过活化凋亡相关通路,促进EOC细胞凋亡,提高了DDP耐药EOC细胞对DDP的敏感性。这可能为临床开创逆转EOC铂类耐药的治疗新途径提供理论依据。     

Use of in vitro method to predict response of human ovarian carcinoma cells to chemotherapeutic agents

In vitro tissue culture and sensitivity tests were carried out on 16 cases of ovarian cancer to select the right anticancer drug(s) on an individual basis. The predictive accuracy of the in vitro drug sensitivity tests were 81.8%. The drug(s) suggested by in vitro tests did not reveal any correlation to the type of ovarian cancer. The drug sensitivity of cells from primary growth was different from that of secondary growth. The in vitro screening test also provided the choice of the next best drug(s), once the tumor had become resistant to a particular drug(s). Thus, the overall prognosis of patients at an advanced stage of malignancy could be improved with the help of in vitro predictive tests.     

微小RNA-16逆转上皮性卵巢癌顺铂耐药的研究

目的:研究微小RNA-16(miR-16)在人上皮性卵巢癌顺铂耐药细胞中的表达及其与顺铂耐药的关系。方法:脂质体lipofectamine2000介导miR-16成熟体模拟物(mimics)及阴性对照片段(NC)分别转染人卵巢癌顺铂耐药细胞株SKOV3/DDP。实时荧光定量PCR检测各组细胞中miR-16的表达;蛋白印迹法检测细胞中Bcl-2蛋白、c-myc蛋白的表达;四甲基偶氮唑蓝(MTT)比色法检测细胞的顺铂半数抑制浓度(IC50);caspase-3活性检测试剂盒检测细胞内caspase-3酶的活性;流式细胞仪检测细胞的凋亡率。结果:(1)SKOV3/DDP细胞顺铂耐药指数(RI)为5.33;(2)SKOV3/DDP细胞中miR-16表达水平明显低于SKOV3细胞,约为后者的1/10(P=0.000),转染miR-16 mimics后SKOV3/DDP细胞中miR-16水平升高约660倍(P=0.001);(3)SKOV3/DDP细胞Bcl-2、c-myc蛋白的相对表达量较SKOV3细胞明显升高,转染miR-16 mimics后SKOV3/DDP细胞Bcl-2、c-myc蛋白的相对表达量明显降低(P<0.01);(4)SKOV3/DDP细胞转染miR-16 mim-ics后,顺铂IC50(14.19±0.06)较转染NC组(22.52±0.60)明显降低(P=0.002),且顺铂作用后caspase-3的酶活力单位也明显升高(P=0.000);(5)顺铂(20μg/ml)作用24h、48h后,SKOV3组凋亡率明显高于SKOV3/DDP组,转染miR-16 mimics的SKOV3/DDP细胞凋亡率约为转染NC细胞的1.5倍(P<0.01)。结论:miR-16可能通过靶向下调上皮性卵巢癌细胞Bcl-2蛋白,并协同调节c-myc蛋白的表达,增强卵巢癌顺铂耐药细胞对顺铂诱导凋亡的敏感性,发挥逆转耐药的作用。     

Fn14在上皮性卵巢癌中的表达与化疗耐药及预后相关性研究

目的:探讨成纤维细胞生长因子诱导14(Fn14)在上皮性卵巢癌(EOC)中的表达水平及其与化疗疗效及预后的关系。方法:采用免疫组化SP法检测84例EOC组织中Fn14表达,并结合临床资料分析Fn14表达水平在EOC患者中的意义。结果:Fn14在EOC中具有不同程度的阳性表达,其表达水平与化疗敏感性和淋巴结转移有关(P0.050),而与患者年龄、肿瘤组织类型、病理分级、FIGO手术分期无明显相关性(P0.05)。生存分析发现,Fn14高表达患者的生存预后比低表达者好。结论:Fn14表达水平与EOC化疗耐药和预后相关,Fn14可能成为一个新的判断化疗疗效和评估预后的指标。     

The epigenetics of ovarian cancer drug resistance and resensitization

Ovarian cancer is the most lethal of all gynecologic neoplasms. Early-stage malignancy is frequently asymptomatic and difficult to detect and thus, by the time of diagnosis, most women have advanced disease. Most of these patients, although initially responsive, eventually develop and succumb to drug-resistant metastases. The success of typical postsurgical regimens, usually a platinum/taxane combination, is limited by primary tumors being intrinsically refractory to treatment and initially responsive tumors becoming refractory to treatment, due to the emergence of drug-resistant tumor cells. This review highlights a prominent role for epigenetics, particularly aberrant DNA methylation and histone acetylation, in both intrinsic and acquired drug-resistance genetic pathways in ovarian cancer. Administration of therapies that reverse epigenetic "silencing" of tumor suppressors and other genes involved in drug response cascades could prove useful in the management of drug-resistant ovarian cancer patients. In this review, we summarize recent advances in the use of methyltransferase and histone deacetylase inhibitors and possible synergistic combinations of these to achieve maximal tumor suppressor gene re-expression. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize ovarian tumors to the proven cytotoxic activities of conventional chemotherapeutics.     

树突状细胞体外诱导抗卵巢癌免疫的实验研究

目的 观察人外周血树突状细胞(Dendritic cells,DC),体外能否诱导抗卵巢癌免疫应答。方法 用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤坏死因子α(TNF-α)从健康女性外周血分化诱导DC,以源于人卵巢癌细胞系HO-8910的肿瘤抗原粗提物冲击致敏DC,将致敏DC、同源淋巴细胞和卵巢癌细胞共育,观察负载抗原DC体外诱导淋巴细胞对HO-8910细胞的杀伤作用,同时设不同类型肿瘤细胞(Eca-109和PC-12)作为对照。MTT法测定细胞杀伤活性。结果 经卵巢癌细胞HO-8910肿瘤抗原脉冲致敏的DC能诱导淋巴细胞特异性地杀伤卵巢癌细胞。结论 用GM-CSF、IL-4和TNF-α从人外周血诱生的DC能从卵巢癌细胞HO-8910冻融物有效递呈抗原并诱导出高效而特异的抗卵巢癌免疫反应。