The xc- cystine/glutamate antiporter: a mediator of pancreatic cancer growth with a role in drug resistance

The x(c)(-) cystine transporter enhances biosynthesis of glutathione, a tripeptide thiol important in drug resistance and cellular defense against oxidative stress, by enabling cellular uptake of cystine, a rate-limiting precursor. Because it is known to regulate glutathione levels and growth of various cancer cell types, and is expressed in the pancreas, we postulate that it is involved in growth and drug resistance of pancreatic cancer. To examine this, we characterised expression of the x(c)(-) transporter in pancreatic cancer cell lines, MIA PaCa-2, PANC-1 and BxPC-3, as subjected to cystine-depletion and oxidative stress. The results indicate that these cell lines depend on x(c)(-)-mediated cystine uptake for growth, as well as survival in oxidative stress conditions, and can modulate x(c)(-) expression to accommodate growth needs. Immunohistochemical analysis showed that the transporter was differentially expressed in normal pancreatic tissues and overexpressed in pancreatic cancer tissues from two patients. Furthermore, gemcitabine resistance of cells was associated with elevated x(c)(-) expression and specific x(c)(-) inhibition by monosodium glutamate led to growth arrest. The results suggest that the x(c)(-) transporter by enhancing glutathione biosynthesis plays a major role in pancreatic cancer growth, therapy resistance and represents a potential therapeutic target for the disease.     

Protection of platinum-DNA adduct formation and reversal of cisplatin resistance by anti-MRP2 hammerhead ribozymes in human cancer cells

Resistance to platinum-containing antineoplastic drugs is the major limitation in their clinical use. To elucidate the role of the ABC transporter MRP2 in platinum drug resistance, its expression was analyzed in human cisplatin-resistant cell lines: the ovarian carcinoma line A2780RCIS, the adrenocortical carcinoma line D43/86RCIS and the melanoma line MeWoCIS1. All these cells showed overexpression of MRP2. For reversal of platinum resistance, 2 anti-MRP2 hammerhead ribozymes were introduced into A2780RCIS cells. Both ribozymes showed gene-silencing activities and reversed the drug-resistant phenotype. Moreover, formation of platinum-induced intrastrand cross-links was measured in DNA. The level of DNA platination corresponded inversely to the level of MRP2 expression and was accompanied by increased caspase-3-dependent apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity was not altered; the decrease in platinum-DNA adduct formation was rather a reflection of the protecting activity of MRP2. In conclusion, functional inhibition of MRP2 might be a promising strategy in the reversal of resistance to platinum-based anticancer drugs. This was reflected by the specific inhibition of MRP2 by ribozyme technology, indicating that this gene therapeutic approach may be applicable as a specific means to overcome platinum resistance in human neoplasms.     

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。     

雷帕霉素增强卵巢癌细胞系对顺铂敏感性的机制研究

目的：研究雷帕霉素对卵巢癌细胞系顺铂敏感性的影响,以及PI3K/AKT/mTOR信号通路（简称mTOR信号通路）与卵巢癌细胞系顺铂耐药的相关性;初探雷帕霉素增强卵巢癌细胞系顺铂敏感性的分子机制。方法采用CCK-8法检测卵巢癌顺铂耐药细胞系SKOV3/DDP的耐药指数（resistance index,RI）、细胞增殖抑制率;采用克隆平板实验观察不同用药方案对卵巢癌顺铂非耐药细胞系及SKOV3细胞系两种细胞系克隆形成的影响;采用蛋白质印迹法（Western blot）检测两种细胞系的蛋白表达差异。结果①SKOV3/DDP细胞系的RI为6.10,属中度耐药。②在SKOV3细胞系中,顺铂联合雷帕霉素作用24 h、48 h后的细胞增殖抑制率明显高于单用顺铂组,差异有统计学意义（P＜0.01）,两组间的72 h细胞增殖抑制率无明显的统计学意义（P＞0.05）;顺铂联合雷帕霉素作用于SKOV3/DDP细胞系24 h、48 h、72 h后的细胞增殖抑制率均明显高于单用顺铂组,差异有统计学意义（P＜0.01）。③雷帕霉素联合顺铂作用于SKOV3细胞系4 h后,其克隆形成抑制率明显高于单用顺铂组,差异有统计学意义（P＜0.01）。④SKOV3/DDP细胞系比SKOV3细胞系p-mTOR、p-AKT的表达升高,而mTOR、AKT的表达则相似。⑤联合用药组比单用顺铂组的PARP断裂增加。⑥雷帕霉素作用SKOV3细胞系24 h ,BCL2表达下调,LC3B由LC3BⅠ向LC3BⅡ转化增加;在SKOV3/DDP细胞系中未发现这两种作用。结论①在体外培养条件下,雷帕霉素能增强SKOV3及SKOV3/DDP细胞系对顺铂的敏感性。②mTOR信号通路激活可能在卵巢癌细胞系顺铂耐药机制中起重要作用。③雷帕霉素增强SKOV3细胞系对顺铂敏感性的分子机制包括：增强顺铂所致的DNA断裂、下调抗凋亡蛋白BCL2及引起细胞自噬;雷帕霉素增强SKOV3/DDP对顺铂敏感性分子机制可能与增强顺铂所致的DNA断裂有关。     

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.     

RON and cisplatin resistance in ovarian cancer cell lines

RON (recepteur d'origine nantais) tyrosine kinase receptor has revealed its tumorigenic potential in recent studies. RON was reported to be overexpressed in 55% of primary ovarian carcinoma samples and furthermore its activation increases cell motility and invasiveness. In this study, we investigated the correlation between RON expression and chemoresistance in ovarian cancer cells. In A2780 cells, a model featured by high chemosensitivity to cisplatin, stable overexpression of RON was able to reduce sensitivity to this agent, while incubation with a blocking anti-RON antibody (ID1) increased the cisplatin-induced growth inhibition effect. Moreover, we observed an increased RON expression both at the mRNA and protein level in A2780 cells made resistant to doxorubicin and paclitaxel (A2780ADR and TC 1, respectively), two cell lines exhibiting a collateral resistance to cisplatin. OVCAR-3 cells, showing high levels of RON expression, also displayed inherent cisplatin resistance. The morphology observed in these resistant cells is consistent with a scattering phenotype and a RON-activated state. RON expression levels were monitored upon hypoxia. A 2.5-fold increase of RON expression was noticed in response to hypoxia in OVCAR-3 cells, in parallel with a decrease of E-cadherin mRNA. Altogether these results suggest an involvement of RON in the acquisition of cisplatin resistance and highlight the importance of this factor as a promising target for combination with cisplatin-based chemotherapy in ovarian cancer.     

干扰素介导的跨膜蛋白1与卵巢上皮性癌耐药相关性研究

目的:探讨干扰素介导的跨膜蛋白1（IFITM1）与卵巢上皮性癌化疗耐药性的相关性。方法:应用免疫组化测定68例初治卵巢上皮性癌组织和卵巢癌顺铂敏感细胞株A2780、顺铂耐药细胞株CP70中IFITM1的表达,Western blotting检测两种卵巢癌细胞株中IFITM1蛋白的表达。结果:免疫组织化学显示铂类化疗敏感性卵巢癌组和耐药性卵巢癌组的表达强度之间的差异有统计学意义（P=0.043）,Western blot实验显示卵巢癌CP70细胞株中IFITM1表达水平明显高于A2780细胞株。结论:IFITM1表达强度与卵巢癌铂类化疗的敏感性相关,检测IFITM1对判断卵巢癌化疗疗效具有临床意义。     

Identification of genes associated with platinum drug sensitivity and resistance in human ovarian cancer cells

Platinum-based chemotherapeutic regimens are ultimately unsuccessful due to intrinsic or acquired drug resistance. Understanding the molecular basis for platinum drug sensitivity/resistance is necessary for the development of new drugs and therapeutic regimens. In an effort to identify such determinants, we evaluated the expression of approximately 4000 genes using cDNA microarray screening in a panel of 14 unrelated human ovarian cancer cell lines derived from patients who were either untreated or treated with platinum-based chemotherapy. These data were analysed relative to the sensitivities of the cells to four platinum drugs (cis-diamminedichloroplatinum (cisplatin), carboplatin, DACH-(oxalato)platinum (II) (oxaliplatin) and cis-diamminedichloro (2-methylpyridine) platinum (II) (AMD473)) as well as the proliferation rate of the cells. Correlation analysis of the microarray data with respect to drug sensitivity and resistance revealed a significant association of Stat1 expression with decreased sensitivity to cisplatin (r=0.65) and AMD473 (r=0.76). These results were confirmed by quantitative RT-PCR and Western blot analyses. To study the functional significance of these findings, the full-length Stat1 cDNA was transfected into drug-sensitive A2780 human ovarian cancer cells. The resulting clones that exhibited increased Stat1 expression were three- to five-fold resistant to cisplatin and AMD473 as compared to the parental cells. The effect of inhibiting Jak/Stat signalling on platinum drug sensitivity was investigated using the Janus kinase inhibitor, AG490. Pretreatment of platinum-resistant cells with AG490 resulted in significant increased sensitivity to AMD473, but not to cisplatin or oxaliplatin. Overall, the results indicate that cDNA microarray analysis may be used successfully to identify determinants of drug sensitivity/resistance and future functional studies of other candidate genes from this database may lead to an increased understanding of the drug resistance phenotype.     

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.     

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…     

Modulation of paclitaxel resistance by annexin IV in human cancer cell lines

A recurring problem with cancer therapies is the development of drug resistance. While investigating the protein profile of cells resistant to a novel antimitotic compound (A204197), we discovered an increase in annexin IV expression. When we examined the annexin IV protein expression level in a paclitaxel-resistant cell line (H460/T800), we found that annexin IV was also overexpressed. Interestingly a closely related protein, annexin II, was not overexpressed in H460/T800 cells. Immunostaining with either annexin II or IV antibody revealed that annexin IV was primarily located in the nucleus of paclitaxel-resistant H460/T800 cells. Short-term treatment of H460 cells with 10 nM paclitaxel for up to 4 days resulted in induction of annexin IV, but not annexin II expression. In addition, there was an increase in annexin IV staining in the nucleus starting at day 1. Furthermore, cells pretreated with 10 nM paclitaxel for 4 days resulted in cells becoming approximately fivefold more resistant to paclitaxel. Transfection of annexin IV cDNA into 293T cells revealed that there was a threefold increase in paclitaxel resistance. Thus our results indicate that annexin IV plays a role in paclitaxel resistance in this cell line and it is among one of the earliest proteins that is induced in cells in response to cytotoxic stress such as antimitotic drug treatment.     

DHMEQ enhances the cytotoxic effect of cisplatin and carboplatin in ovarian cancer cell lines

Ovarian cancer (OvCa) is one of the most lethal gynaecological malignancies. It is diagnosed mostly in advanced stages. Due to a lack of appropriate early detection markers and non-ambiguous symptoms, the five-year survival rate is significantly reduced. Despite a primary good response to platinum-based therapy, approximately 70% of patients will develop a chemoresistance phenotype. The activation of the NF-κB signalling pathway plays a crucial role in this process. It is responsible for increasing cell viability, cell cycle progression and induces growth and migration of neoplastic cells. A few independent studies have yet suggested a high correlation between activation of NF-κB and poor outcome in OvCa patients. Thus, developing inhibitors of the NF-κB pathway has become a new target of cancer therapies. One of the promising compounds is DHMEQ (dehydroxymethylepoxyquinomicin). Our preliminary studies indicated that DHMEQ combined with cisplatin (CDDP) or carboplatin (CBP) enhanced apoptosis in the A2780 cell line and caused cell cycle arrest in the G2/M phase in the SKOV3 cell line, but not in the normal cell line MRC-5 pd19. Moreover, the combination of those agents caused decreased motility of cells, especially with the CBP. However, the invasion of cells was not changed significantly. The analysis of drug interactions using CompuSyn software has revealed that observed effect of the doses used in the study was antagonistic, but the DRI guidelines and in vitro observation of biological response indicate that a combination of DHMEQ with CDDP or CBP could be a novel proposal in ovarian cancer treatment.     

HS3ST1对肺癌细胞增殖、迁移、侵袭及顺铂耐药的作用机制研究

目的:研究HS3ST1对肺癌细胞A549增殖、迁移、侵袭及顺铂耐药的影响.方法:利用慢病毒感染并构建HS3ST1过表达细胞株;利用Western blot、RT-PCT检测HS3ST1的表达;利用CCK8实验、Transwell迁移实验和细胞毒性实验检测HS3ST1对A549细胞增殖、迁移、侵袭和顺铂耐药的影响;利用W...     

Mechanisms of synergism between cisplatin and gemcitabine in ovarian and non-small-cell lung cancer cell lines.

2',2'-Difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum (cisplatin, CDDP) are active agents against ovarian cancer and non-small-cell lung cancer (NSCLC). CDDP acts by formation of platinum (Pt)-DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index (CI; CI < 1 indicates synergism). With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the CIs after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all CIs were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt-DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines (1.6-, 1.4-, 2.9- and 1.6-fold respectively). This increase in Pt-DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt-DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt-DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt-DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA.     

Low expression of MYCN promotes cisplatin resistance by suppressing cisplatin-induced apoptosis in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer-associated mortality. Cisplatin is one of the most effective chemotherapeutic drugs used in EOC; however, its use can lead to relapse due to cisplatin resistance. MYCN sensitizes neuroblastoma to undergo cisplatin-induced apoptosis. However, to the best of our knowledge, there have been no studies to date on the association between MYCN and cisplatin resistance in EOC. Therefore, the present study assessed this association. Datasets from The Cancer Genome Atlas database were used. The overall survival (OS) of patients receiving platin-based therapy was analyzed using Kaplan-Meier Plotter software. RNA sequencing data of 300 patients with EOC were downloaded from cBioportal. The co-expressed genes were subjected to ‘Kyoto Encyclopedia of Genes and Genomes’ analysis using DAVID software. For gene set enrichment analysis, the expression matrix was separated according to the median expression of MYCN, which was selected for hallmark gene set enrichment. Immunohistochemistry was used to assess MYCN expression in EOC tissue. Western blotting was used to evaluate MYCN, p53, Bax and Bcl-2 protein expression levels in EOC cells. Cell viability and apoptosis were assessed using Cell Counting Kit-8 and flow cytometry, respectively. The results demonstrated that MYCN upregulation was associated with increased cisplatin sensitivity and prolonged OS of patients with EOC and patients receiving platin-based therapy. Cisplatin downregulated MYCN expression in cisplatin-sensitive, but not resistant, EOC cells. The genes co-expressed with MYCN were primarily involved in pathways involved in ‘chemotherapeutic resistance’ and ‘apoptosis’. MYCN enriched the apoptosis and p53 signaling pathways in hallmark gene sets. Cells in which MYCN was knocked down demonstrated significantly increased cisplatin resistance; however, MYCN overexpression in cisplatin-resistant cells restored cisplatin sensitivity. Collectively, the present study demonstrated that MYCN downregulation promoted cisplatin resistance by suppressing cisplatin-induced apoptosis in EOC.     

Induction of p53 and drug resistance following treatment with cisplatin or paclitaxel in ovarian cancer cell lines

Treatment failures result from resistance to chemotherapy in ovarian cancer. The effect of cisplatin and paclitaxel treatments on chemosensitivity was studied in ovarian cancer cells developed from a patient with stage IIIC disease. Cells (UL-3A, UL-3B) that recovered from cisplatin (Cis) and paclitaxel (Tax) treatments showed higher levels of p53, mdr-1 and chemoresistance than untreated controls. EC50 values of Cis and Tax for UL-3A clones were 7.2-34.6, average 20.9 microg/ml, while UL-3B clones ranged from 11.8-252.0 microg/ml, with a mean value of 73.2 microg/ml for Cis, and 260.0-4400.0 nM (mean 2555.0 nM) for Tax. Selection pressures during treatment may contribute to drug resistance.     

Accumulation of intracellular platinum is correlated with intrinsic cisplatin resistance in human bladder cancer cell lines

We investigated the mechanism of intrinsic resistance to cisplatin in human transitional cell cancer (TCC) using 7 human bladder cancer cell lines, which were derived from untreated TCC of the urinary bladder. The sensitivity to cisplatin was different from cell line to cell line, and a 15-fold difference was observed between the most sensitive line and the most resistant line. No significant correlation was observed between the content of intracellular glutathione and the resistance to cisplatin. In contrast, a positive correlation was seen between intracellular cisplatin accumulation and cisplatin resistance. The expression of drug resistance-related genes including glutathione S-transferase pi, gamma-glutamyl-cysteine synthetase, multidrug resistance-1, multidrug resistance-associated protein, DNA topoisomerase I, topoisomerase II, human canalicular multispecific organic anion transporter, and thioredoxin was not significantly related to cisplatin resistance. These data suggest that intracellular cisplatin may contribute to intrinsic cisplatin resistance and may therefore be a useful biomarker to predict cisplatin sensitivity in human untreated TCC.