舒林酸对卵巢癌细胞系CP70顺铂耐药性的逆转作用

目的：研究舒林酸体外逆转卵巢癌细胞系CP70的顺铂耐药性,并探讨其与β—catenin表达的关系。方法：MTT法检测单独使用舒林酸或顺铂及两药联合使用时对CP70细胞的抑制作用,免疫细胞化学和Westernblot方法检测细胞内β—catenin表达水平。结果：两药联合处理细胞后顺铂耐药逆转倍数为6．01,联合处理组的细胞中β—catenin表达明显降低。结论：舒林酸能部分逆转CP70细胞对顺铂的耐药性,且此作用可能与下调细胞的β—catenin表达有关。     

舒林酸对卵巢癌细胞系CP70顺铂耐药性的逆转作用

目的:研究舒林酸体外逆转卵巢癌细胞系CP70的顺铂耐药性,并探讨其与β-catenin表达的关系。方法:MTT法检测单独使用舒林酸或顺铂及两药联合使用时对CP70细胞的抑制作用,免疫细胞化学和Western blot方法检测细胞内β-catenin表达水平。结果:两药联合处理细胞后顺铂耐药逆转倍数为6.01,联合处理组的细胞中β-catenin表达明显降低。结论:舒林酸能部分逆转CP70细胞对顺铂的耐药性,且此作用可能与下调细胞的β-catenin表达有关。     

HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

Acquired resistance to platinum (Pt)‐based therapies is an urgent unmet need in the management of epithelial ovarian cancer (EOC) patients. Here, we characterized by an unbiased proteomics method three isogenic EOC models of acquired Pt resistance (TOV‐112D, OVSAHO, and MDAH‐2774). Using this approach, we identified several differentially expressed proteins in Pt‐resistant (Pt‐res) compared to parental cells and the chaperone HSP90 as a central hub of these protein networks. Accordingly, up‐regulation of HSP90 was observed in all Pt‐res cells and heat‐shock protein 90 alpha isoform knockout resensitizes Pt‐res cells to cisplatin (CDDP) treatment. Moreover, pharmacological HSP90 inhibition using two different inhibitors [17‐(allylamino)‐17‐demethoxygeldanamycin (17AAG) and ganetespib] synergizes with CDDP in killing Pt‐res cells in all tested models. Mechanistically, genetic or pharmacological HSP90 inhibition plus CDDP ‐induced apoptosis and increased DNA damage, particularly in Pt‐res cells. Importantly, the antitumor activities of HSP90 inhibitors (HSP90i) were confirmed both ex vivo in primary cultures derived from Pt‐res EOC patients ascites and in vivo in a xenograft model. Collectively, our data suggest an innovative antitumor strategy, based on Pt compounds plus HSP90i, to rechallenge Pt‐res EOC patients that might warrant further clinical evaluation.     

牛蒡子苷元对卵巢癌细胞耐药性的影响

目的:探讨牛蒡子苷元对人卵巢癌顺铂耐药细胞株SKOV3/DDP细胞耐药性的影响及其可能的作用机制。方法:构建卵巢癌耐药细胞株SKOV3/DDP。用不同浓度的牛蒡子苷元(1、5、10、15、20、25 mmol/L)分别处理SKOV3和SKOV3/DDP细胞,CCK-8法检测细胞生长,筛选适用浓度。随后用10 mmol/L牛蒡子苷元处理SKOV3/DDP细胞。流式细胞仪检测细胞凋亡,Western blot检测Caspase-3/9和Cleaved Caspase-3/9蛋白表达。CCK-8法检测细胞半数抑制浓度(50%inhibition concentration, IC₅₀)。RT-PCR和Western blot分别检测Survivin的mRNA和蛋白表达。在牛蒡子苷元处理的SKOV3/DDP细胞中转染Survivin过表达质粒检测IC₅₀和细胞凋亡水平。结果:不同浓度牛蒡子苷元对SKOV3和SKOV3/DDP细胞生长均有抑制作用,并且当牛蒡子苷元的浓度≥10 mmol/L时,对SKOV3/DDP细胞生长抑制作用高于SKOV3细胞(P&...     

Molecular profiling of platinum resistant ovarian cancer

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p<0.01). The findings of this study are the discovery of a gene signature that classifies the tumours, according to their response, and a 9-gene set that determines resistance in an independent validation set that outperforms patient and tumour characteristics. A larger independent multicentre study should further confirm whether this 9-gene set can identify the patients who will not respond to platinum-based chemotherapy and could benefit from other therapies.     

Identification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer

Background:

Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer.

Methods:

For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated.

Results:

The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment.

Conclusion:

Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.     

Mechanism of drug resistance and reversal with ligustra-zine and cyclosporin A in cisplatin-induced human epithelial ovarian cancer resistant cell line 3Ao/cDDP

Epithelial ovarian cancer is the leading cause of death among gynecologic malignancies. Chemotherapy plays an irreplaceable role in the treatment of patients. Protocols based on platinum compounds have significantly improved the overall response and clinical complete response but only less than a quarter of patients with advanced disease will be alive 5 years later.[1] Diagnosis at an advanced stage and development of resistance to chemotherapy, despite remarkable initial chemosensitivity, acc…     

Downregulation of miR‐29 contributes to cisplatin resistance of ovarian cancer cells

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin‐resistant cells expressed a lower level of miR‐29a/b/c. Manipulation of microRNA‐29 (miR‐29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR‐29a/b/c increased the ability of cells to escape cisplatin‐induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal‐regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR‐29 decreased the amount of the active form of caspase‐9 and caspase‐3. Ectopic expression of miR‐29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR‐29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR‐29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.     

Dacomitinib improves chemosensitivity of cisplatin-resistant human ovarian cancer cells

Drug resistance hinders effectiveness of human ovarian cancer (OC) therapies, such as cisplatin or paclitaxel therapy. Although dacomitinib, a novel anticancer agent is used against multiple types of cancers, such as non-small cell lung cancer, head and neck cancer, few studies report its effectiveness in drug-resistant human OC cells. In the present study, would healing, microplate spectrophotometer analysis, flow cytometry analysis, western blotting and Gene Expression Omnibus (GEO) analysis were used to detect the synergistic effect of dacomitinib and cisplatin in human OC SKOV-3 or OV-4 cells. Co-administration of dacomitinib and cisplatin significantly reduced viability and promoted cell apoptosis of drug resistant OC cells. In addition, dacomitinib increased Cadherin 1 (CDH1) levels and decreased P-glycoprotein (P-GP) levels in cisplatin-resistant OC cells. In addition, GEO analysis demonstrated that dacomitinib inhibited the epidermal growth factor receptor (EGFR) signaling pathway. In summary, dacomitinib improves chemosensitivity of cisplatin in human OC by regulating CDH1 and P-GP protein levels and inhibiting the EGFR signaling pathway.     

hMS H2重组质粒对人卵巢癌细胞顺铂敏感性的影响

目的：研究hMSH2重组质粒对卵巢癌顺铂敏感性方面的影响。方法：构建重组真核表达质粒pCAN－hMSH2,通过酶切及测序法对重组质粒进行鉴定。采用脂质体转染技术对卵巢癌耐药细胞SKOV3／DDP进行瞬时转染,对照组为未转染细胞和SKOV3敏感细胞;hMSH2在细胞内的表达变化由Western blotting和RT－PCR进行检测;四甲基偶氮唑蓝（MTT）法对细胞的顺铂敏感性进行检测;耐药细胞的凋亡情况通过Hoechst染色法检测。结果：重组质粒pCAN－hMSH2转染进SKOV3／DDP后,经RT－PCR和Western blotting检测证实转染成功,hMSH2的表达得到增强;MTT结果提示转染后的卵巢癌耐药细胞对顺铂的敏感性明显增强;Ho-echst染色发现,转染后耐药细胞的凋亡明显增强。结论：hMSH2基因转染后能提高其在SKOV3／DDP细胞中的表达,增强SKOV3／DDP细胞对顺铂的敏感性,促进在顺铂作用下的凋亡。     

Low drug resistance to both platinum and taxane chemotherapy on an in vitro drug resistance assay predicts improved survival in patients with advanced epithelial ovarian,fallopian and peritoneal cancer

The objective of this study was to evaluate the role of an in vitro drug resistance assay to platinum and taxane in the management of advanced epithelial ovarian, fallopian and primary peritoneal cancer. All patients with FIGO Stage IIIc and IV who received postoperative chemotherapy with platinum and taxane for more than 4 courses after the initial cytoreductive surgery between 1995 and 2008 were evaluated. Patients who received neoadjuvant chemotherapy were not included. An in vitro drug resistance assay (EDR Assay, Oncotech, Tustin, CA) was used to determine drug resistance for each patient's tumor tissue. Level of drug resistance was described as extreme (EDR), intermediate (IDR), or low (LDR). Response to chemotherapy and survival were correlated to the EDR Assay. Of the 335 patients who underwent primary cytoreductive surgery, 173 cases met the criteria for statistical evaluation. The 58 patients (33.5%) whose tumors had LDR to both platinum and taxane had statistically improved progression‐free survival and overall survival (OS) compared with the 115 patients (66.5%) who demonstrated IDR or EDR to platinum and/or taxane (5‐year OS rates, 41.1% vs. 30.9%, p = 0.014). The 5‐year OS rates for the 28 (16.2%) cases that had optimal cytoreduction with LDR to both platinum and taxane was significantly improved over the 62 (35.8%) cases that were suboptimally cytoreduced with IDR or EDR to platinum and/or taxane (54.1% vs. 20.4%, respectively, p < 0.001). In conclusion, LDR to both platinum and taxane chemotherapy, as determined by an in vitro drug resistance assay, independently predicts improved survival in patients with advanced epithelial ovarian, fallopian and peritoneal cancer, especially in those patients who undergo optimal primary cytoreduction. © 2009 UICC     

Increased sensitivity to platinum drugs of cancer cells with acquired resistance to trabectedin

Background:

In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo.

Methods:

Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts.

Results:

402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G₂/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks.

Conclusions:

Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.     

卵巢癌肿瘤干细胞耐药性及其治疗的研究进展

肿瘤干细胞是肿瘤组织内具有自我更新能力及无限增殖潜能的一群细胞,对外源细胞毒性药物耐受。卵巢癌肿瘤干细胞在卵巢癌发生、化疗耐药和复发过程中起重要作用。传统治疗对减小肿瘤体积有效,但是治疗后残留的卵巢癌干细胞成为卵巢癌复发和难治的根源,针对卵巢癌肿瘤干细胞的靶向治疗可能在抗卵巢癌治疗中具有重要作用,为临床彻底治愈卵巢癌带来希望。     

Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function

Resistance to cisplatin-based chemotherapy is a major cause of treatment failure in human ovarian cancer. Wild-type TP53 status is often, but not always, associated with cisplatin sensitivity, suggesting that additional factors may be involved. Overexpression/activation of the phosphatidylinositol-3-kinase/Akt pathway is commonly observed in ovarian cancer, and Akt activation is a determinant of chemoresistance in ovarian cancer cells, an effect that may be due, in part, to its inhibitory actions on p53-dependent apoptosis. To that end, we examined the role and regulation of p53 in chemosensitive ovarian cancer cells, as well as in their chemoresistant counterparts, and investigated if and how Akt influences this pathway. Cisplatin induced apoptosis in chemosensitive, but not chemoresistant cells, and this was inhibited by downregulation of p53. Cisplatin upregulated PUMA in a p53-dependent manner, and the presence of PUMA was necessary, but not sufficient for cisplatin-induced apoptosis. p53 was phosphorylated on numerous N-terminal residues, including Ser15, Ser20, in response to cisplatin in chemosensitive, but not chemoresistant cells. Furthermore, activation of Akt inhibited the cisplatin-induced upregulation of PUMA, and suppressed cisplatin-induced p53 phosphorylation, while inhibition of Akt increased total and phospho-p53 contents and sensitized p53 wild-type, chemoresistant cells to cisplatin-induced apoptosis. Finally, mutation of Ser15 and/or Ser20, but not of Ser37, to alanine significantly attenuated the ability of p53 to facilitate CDDP-induced apoptosis, and this was independent of PUMA expression. These results support the hypothesis that p53 is a determinant of CDDP sensitivity, and suggest that Akt contributes to chemoresistance, in part, by attenuating p53-mediated PUMA upregulation and phosphorylation of p53, which are essential, but independent determinants of sensitivity to CDDP-induced apoptosis.     

miRNAs and ovarian cancer: a miRiad of mechanisms to induce cisplatin drug resistance

Ovarian cancer is the most aggressive gynecological cancer. One reason for the low 5-year survival rate of under 40% is that ovarian tumors usually acquire resistance to the platinum-based compounds used to treat them. Resistance to one such compound, cisplatin, can arise via numerous mechanisms that can be categorized as pre-, post-, on- or off-target. Pre-target mechanisms prevent accumulation of cisplatin in the cell, on-target mechanisms allow DNA damage to be repaired more efficiently, post-target mechanisms prevent the damage from inducing apoptosis and off-target mechanisms increase resistance via unrelated compensatory mechanisms. miRNAs are short non-coding RNAs that influence cellular function by repressing gene expression. Here we describe how miRNAs can induce cisplatin resistance in ovarian cancer cells via pre-, post-, on- and off-target mechanisms. A better understanding of how miRNAs feed into the mechanisms of drug resistance will inform the rational design of combination therapies for ovarian cancer.     

Identification of genes whose expression is associated with cisplatin resistance in human ovarian carcinoma cells

The goal of this study was to identify genes consistently differentially expressed in multiple pairs of isogenic cisplatin (DDP)-sensitive and resistant human ovarian carcinoma cell lines using microarray-based expression profiling. Expression profiling was carried out on six pairs of ovarian carcinoma cells lines growing under identical conditions; each cell expression profile was independently replicated six times. No genes were differentially expressed in all six pairs of cells or even in even in any five of the six pairs. Eighteen genes and 1 EST were upregulated, and four genes and 1 EST were downregulated, in at least four cell pairs. Of these, only metallothionein 2A has previously been implicated in DDP resistance. Among the genes identified on the basis of six replicates, an average of 24.8% would have been missed if only five replicates had been performed, and 38.3% would have been missed with only four replicates. The genes did not identify a dominant biochemical pathway or ontology category as being linked to DDP resistance; however, hierarchical clustering provided evidence for two classes DDP-resistant phenotypes within which there are additional cell pair-specific alterations. Many of the genes identified in this study play important roles in cell surface interactions and trafficking pathways not previously linked to DDP resistance. The genes discovered by this extensively replicated analysis are candidates for prediction of DDP responsiveness in ovarian cancer patients.     

MicroRNA-mRNA functional pairs for cisplatin resistance in ovarian cancer cells

Ovarian cancer is the leading cause of death in women worldwide. Cisplatin is the core of first-line chemotherapy for patients with advanced ovarian cancer. Many patients eventually become resistant to cisplatin, diminishing its therapeutic effect. MicroRNAs （miRNAs） have critical functions in diverse biological processes. Using miRNA profiling and polymerase chain reaction validation, we identified a panel of differentially expressed miRNAs and their potential targets in cisplatin-resistant SKOV3/DDP ovarian cancer cells relative to cisplatin-sensitive SKOV3 parental cells. More specifically, our results revealed significant changes in the expression of 13 of 663 miRNAs analyzed, including 11 that were up-regulated and 2 that were down-regulated in SKOV3/DDP cells with or without cisplatin treatment compared with SKOV3 cells with or without cisplatin treatment. miRNA array and mRNA array data were further analyzed using Ingenuity Pathway Analysis software. Bioinformatics analysis suggests that the genes ANKRD17, SMC1A, SUMO1, GTF2H1, and TP73, which are involved in DNA damage signaling pathways, are potential targets of miRNAs in promoting cisplatin resistance. This study highlights candidate miRNA-mRNA interactions that may contribute to cisplatin resistance in ovarian cancer.     

RIP1蛋白在卵巢癌中的表达及其对卵巢癌细胞增殖和化疗敏感性的影响

目的:探讨RIP1对卵巢癌细胞增殖和化疗敏感性的影响。方法:通过实时定量PCR法检测RIP1在卵巢癌组织和癌旁组织中的表达;免疫组化方法分析卵巢癌组织和癌旁组织中RIP1蛋白的表达;实时定量PCR法以及Western blot法检测卵巢上皮细胞及卵巢癌细胞中RIP1的表达;通过在SKOV3卵巢癌细胞中敲除RIP1蛋白及在A2780卵巢癌细胞中过表达RIP1蛋白,采用MTT法检测其增殖能力的改变,并联合克隆形成法考察细胞对顺铂敏感性的变化。结果:相比于癌旁组织以及卵巢上皮细胞,RIP1在人卵巢癌组织和卵巢癌细胞中表达有不同程度升高,其中在SKOV3细胞中升高最为显著（P＜0.01）,在A2780细胞中不显著（P＞0.05）;在SKOV3细胞中敲除RIP1,96 h后细胞增殖能力明显降低（P＜0.01）;同时,在A2780细胞株中过表达RIP1,72 h后细胞增殖能力明显升高（P＜0.05）。RIP1敲除后,顺铂对SKOV3细胞的杀伤作用明显升高（P＜0.05）,同时顺铂的IC50降低;RIP1过表达后,顺铂对A2780细胞的杀伤作用明显降低（P＜0.05）,同时顺铂的IC50升高。结论:RIP1作为一种癌基因,能够促进肿瘤细胞的增殖,并降低肿瘤细胞对化疗药物的敏感性。     

MiR-497 decreases cisplatin resistance in ovarian cancer cells by targeting mTOR/P70S6K1

The mechanism of cisplatin resistance in ovarian cancer is not clearly understood. In the present investigation, we found that the expression levels of miR-497 were reduced in chemotherapy-resistant ovarian cancer cells and tumor tissues due to hypermethylation of miR-497 promoter. Low miR-497 expression levels were associated with chemo-resistant phonotype of ovarian cancer. By analyzing the expression levels of miR-497, mTOR and p70S6K1 in a clinical gene-expression array dataset, we found that mTOR and p70S6K1, two proteins correlated to chemotherapy-resistance in multiple types of human cancers, were inversely correlated with miR-497 levels in ovarian cancer tissues. By using an orthotopic ovarian tumor model and a Tet-On inducible miR-497 expression system, our results demonstrated that overexpression of miR-497 sensitizes the resistant ovarian tumor to cisplatin treatment. Therefore, we suggest that miR-497 might be used as a therapeutic supplement to increase ovarian cancer treatment response to cisplatin.     

基于microRNA调控网络预测卵巢癌多药耐药相关基因

目的:基于microRNA(miRNA)调控网络预测卵巢癌多药耐药相关基因。方法:综合运用文本挖掘、网络构建和预测等生物信息学分析方法,挖掘卵巢癌化疗耐药相关miRNA和miRNA-靶基因数据,并构建miRNA调控网络,利用已知miRNA对卵巢癌多药耐药相关基因进行预测。结果:文本挖掘出11个与卵巢癌化疗耐药相关的miRNA,包括miR-130a、miR-214、let-7i、miR-125b、miR-376c、miR-199a、miR-93、miR-141、miR-130b、miR-193b*和miR-200c。在miRNA靶基因预测数据软件Target Scan中挖掘出47 077个miRNA-靶基因数据,而Pic Tar挖掘出1 675个miRNA-靶基因数据。在miRNA调控网络中,神经素1基因(neuropilins,NRP1)是最重要的Hub-基因。结论:利用已知miRNA构建miRNA调控网络进而预测卵巢癌多药耐药相关基因是一种行之有效的方法。NRP1极有可能在卵巢癌化疗耐药形成中扮演着重要的角色,是卵巢癌潜在的药物治疗靶点。