Regulation of MDR1 gene expression in multidrug-resistant cancer cells is independent from YB-1

The MDR1 gene encoded transmembrane ABC-transporter MDR1/P-glycoprotein can mediate the phenotype of multidrug resistance (MDR), a major obstacle in the clinical management of cancer patients. It was hypothesized that YB-1 is a fundamental regulatory factor of the MDR1 gene in tumor cells and can therewith enhance drug resistance. To analyze the potential impact of YB-1 in MDR cancer cells, two specific anti-YB-1 small interfering RNAs (siRNAs) were designed for transient triggering the gene-silencing RNA interference (RNAi) pathway in the MDR cell lines EPG85-257RDB and EPP85-181RDB as well as in their drug-sensitive counterparts EPG85-257P and EPP85-181P. Since both siRNAs showed biological activity, for stable inhibition of YB-1 corresponding tetracycline-inducible short hairpin RNA (shRNA)-encoding expression vectors were designed. By treatment of the cancer cells with these constructs, the expression of the targeted YB-1 encoding mRNA and protein was completely inhibited following tetracycline exposure. These gene-silencing effects were not accompanied by modulation of the MDR1 expression or by reversal of the drug-resistant phenotype. In conclusion, the data demonstrate the utility of the analyzed RNAs as powerful laboratory tools and indicate that YB-1 is not involved in the regulation of the MDR1 gene or the development of the drug-resistant phenotype in MDR cancer cells.     

Macrocyclic diterpenes resensitizing multidrug resistant phenotypes

Herein, collateral sensitivity effect was exploited as a strategy to select effective compounds to overcome multidrug resistance in cancer. Thus, eleven macrocyclic diterpenes, namely jolkinol D (1), isolated from Euphorbia piscatoria, and its derivatives (2–11) were evaluated for their activity on three different Human cancer entities: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) each with a variant selected for resistance to mitoxantrone (EPG85-257RN; EPP85-181RN; HT-29RN) and one to daunorubicin (EPG85-257RD; EPP85-181RD; HT-29RD). Jolkinol D (1) and most of its derivatives (2–11) exhibited significant collateral sensitivity effect towards the cell lines EPG85-257RN (associated with P-glycoprotein overexpression) and HT-29RD (altered topoisomerase II expression). The benzoyl derivative, jolkinoate L (8) demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as P-glycoprotein modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells. A regression analysis between the antiproliferative activity presented herein and the previously assessed P-glycoprotein modulatory effect showed a strong relation between the compounds that presented both high P-glycoprotein modulation and cytotoxicity.     

Chromosomal imbalances associated with drug resistance and thermoresistance in human pancreatic carcinoma cells

Resistance to therapeutic treatment is the major obstacle to advances in the successful management of pancreatic cancer. To characterize chromosomal alterations associated with different phenotypes of acquired multidrug resistance (MDR) and thermoresistance, comparative genomic hybridization (CGH) was applied to compare human pancreatic carcinoma-derived cells. This panel of cell lines consists of the parental, drug- and thermosensitive pancreatic carcinoma cell line EPP85 - 181P, its atypical MDR variant EPP85-181RNOV, the classical MDR subline EPP85-181RDB, and their thermoresistant counterparts EPP85-181P-TR, EPP85-181RNOV-TR, and EPP85 - 181RDB-TR, respectively. CGH using genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included 23 members of the family of ABC transporters, 27 members of the family of cytochrome P450 (CYP) monooxygenases, various molecular chaperones, DNA repair enzymes, and factors involved in the regulation of cell cycle and apoptosis. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.     

Synthesis,in silico and in vitro studies of new 1,4-dihydropiridine derivatives for antitumor and P-glycoprotein inhibitory activity

P-glycoprotein (P-gp) is one of the cell membrane pumps which mediate the efflux of molecules such as anticancer drugs to the extracellular matrix of tumor cells. P_gp is a member of the ATP-binding cassette (ABC) transporter family that is implicated in cancer multidrug resistance (MDR). Since MDR is a contributor to cancer chemotherapy failure, modulation of efflux pumps is a viable therapeutic strategy. In this study, new synthetic 1,4 dihydropiridine (DHP) derivatives containing thiophenyl substitution were tested as inhibitors of P-gp. Efflux assay was conducted to evaluate the intracellular accumulation of Rhodamine123 (Rh123) as a pump substrate. MTT assay, cell cycle analysis and in silico methods were also examined. Flow cytometric analysis revealed that synthetic DHP derivatives (15 µM) increased intracellular concentration of the substrate by 2–3 folds compared with verapamil as a standard P-gp inhibitor. MTT assay on EPG85-257P and its drug-resistant EPG85-257RDB cell line revealed antitumor effects (30–45%) for new DHP derivatives at 15 µM following 72 h incubation. However, MTT test on normal cell line showed negligible toxic effects. Finally combination of synthetic derivatives with doxorubicin showed that these compounds decrease IC50 of doxorubicin in resistant cell lines from 9 to 1.5 µM. Sub-G1 peak-related apoptotic cells showed a stronger effect of synthetic compounds at 5 µM compared with verapamil. Molecular dynamic results showed a high binding affinity between DHP derivative and protein at drug binding site. Findings of these biological tests indicated the antitumor activity and P-gp inhibitory effects of new 1,4-DHP derivatives.     

Complete reversal of ABCG2-depending atypical multidrug resistance by RNA interference in human carcinoma cells

In the chemotherapeutic treatment of patients with disseminated neoplasms, multidrug resistance (MDR) is a major obstacle. ABCG2 (BCRP/MXR), a member of the superfamily of adenosine triphosphate-binding cassette (ABC) transporters, was demonstrated to be associated with "atypical" forms of multidrug-resistant phenotypes of cancer cells. To overcome the ABCG2-depending MDR, two specific anti-ABCG2 small interfering RNAs (siRNAs) were designed for transient triggering of the gene-silencing RNA interference (RNAi) pathway in the human gastric carcinoma cell line EPG85-257RNOV, exhibiting an atypical MDR phenotype. Because both siRNAs showed biological activity, for stable inhibition of ABCG2 corresponding short hairpin RNA (shRNA) expression vectors were constructed. By treatment of EPG85-257RNOV cells with these constructs, expression of the targeted ABCG2-encoding mRNA and transport protein was inhibited completely. Furthermore, anti-ABCG2 shRNA-treated cells increased cellular drug accumulation to the same level measured in drug-sensitive parental cells. These effects were accompanied by complete reversal of the drug-resistant phenotype. Thus, the data indicate that siRNA- and shRNA-mediated RNAi-based gene therapy may be applicable in preventing and reversing ABCG2-depending atypical MDR.     

Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone

Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters. Here we demonstrate that a member of the ABC-transporter family, the heterodimer 'transporter associated with antigen processing' (TAP), physiologically involved in major histocompatibility complex class I-restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85-257RNOV exhibiting a mitoxantrone-resistant phenotype. This tumor cell line shows an atypical MDR phenotype in the absence of 'P-glycoprotein' or 'MDR-associated protein' overexpression but with an enforced 'breast cancer resistance protein' expression level. Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug-sensitive parental gastric carcinoma cell line EPG85-257P conferred a 3.3-fold resistance to mitoxantrone but not to alternative anti-neoplastic agents. Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation. Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.     

Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells

Delivery of RNA interference (RNAi)-mediating agents to target cells is one of the major obstacles for the development of RNAi-based therapies. One strategy to overcome this barrier is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. In this study, the tkRNAi approach was used for modulation of the “classical” ABCB1-mediated multidrug resistance (MDR) in human cancer cells. Subsequent to treatment with anti-ABCB1 shRNA expression vector bearing E. coli, MDR cancer cells (EPG85 257RDB) showed 45% less ABCB1 mRNA expression. ABCB1 protein expression levels were reduced to a point at which merely a weak band could be detected. Drug accumulation was enhanced 11-fold, to an extent that it reached 45% of the levels in non-resistant cells and resistance to daunorubicin was decreased by 40%. The data provide the proof-of-concept that tkRNAi is suitable for modulation of “classical” MDR in human cancer cells. Overall, the prototype tkRNAi system tested here did not yet attain the levels of gene silencing seen with conventional siRNAs nor virally delivered shRNAs; but the tkRNAi system for gene-silencing of ABCB1 is still being optimized, and may become a powerful tool for delivery of RNAi effectors for the reversal of cancer MDR in future.     

多药耐药基因在肝细胞癌中的表达及与p53、PCNA表达的相关性研究

目的　研究肝细胞癌 (HCC)组织中多药耐药基因MDR 1与p5 3蛋白及增殖细胞核抗原 (PCNA)表达的关系 ,旨在从基因水平进一步探讨预测化学治疗效果的可行性。方法　利用免疫组织化学方法 (S P法 )研究 30例肝穿活检的肝癌组织中MDR 1、p5 3和PCNA的表达。结果　 30例肝细胞癌中MDR 1的阳性表达率为 5 6 6 7% ,MDR 1阳性表达与组织学分级无关 (P >0 0 5 )。MDR 1表达与 p5 3、PCNA表达间无相关性 (P >0 0 5 )。结论　通过检测MDR 1基因可以对肝细胞癌病人进行化学治疗敏感性预测 ;肝细胞癌中MDR 1基因表达不依赖于 p5 3和细胞增殖这些因素。     

Extra-pair paternity does not result in differential sexual selection in the mutually ornamented black swan (Cygnus atratus)

We studied patterns of parentage in 85 broods (332 cygnets) of black swans during three breeding seasons, using a set of eight polymorphic microsatellite markers. We detected both intraspecific brood parasitism (IBP; < 5% of cygnets per year) and extra-pair paternity (EPP). In these years, 10-17% (mean = 15.1%) of cygnets resulted from EPP, and 27-40% (mean 37.6%) of broods contained at least one extra-pair cygnet. Compared with levels of EPP in closely related species with similar life histories, these values are unexpectedly high. EPP in black swans appears unrelated to ecological factors (breeding density and synchrony) or genetic factors (genetic similarity between pair members or genetic quality of the offspring). We found no evidence that a mutual sexual feather ornament known to play a role in social mate choice in black swans (curled wing feathers) is involved in extra-pair mate choice. EPP does not lead to greater variance in reproductive success in males, relative to females in this species. We therefore suggest that EPP does not result in differential sexual selection on males and females, explaining why they are ornamented to the same degree.     

The ruthenium complexes cis-(dichloro)tetramineruthenium(III) chloride and cis-tetraammine(oxalato)ruthenium(III) dithionate overcome resistance inducing apoptosis on human lung carcinoma cells (A549)

Lung cancer is one of the leading causes of death in the world, and non-small cell lung carcinoma accounts for approximately 75–85 % of all lung cancers. In the present work, we studied the antitumor activity of the compound cis-(dichloro)tetramineruthenium(III) chloride {cis-[RuCl₂(NH₃)₄]Cl} against human lung carcinoma tumor cell line A549. The present study aimed to investigate the relationship between the expression of MDR1 and CYP450 genes in human lung carcinoma cell lines A549 treated with cisCarboPt, cisCRu(III) and cisDRu(III). The ruthenium-based coordinated complexes presented low cytotoxic and antiproliferative activities, with high IC₅₀ values, 196 (±15.49), 472 (±20.29) and 175 (±1.41) for cisCarboPt, cisCRu(III) and cisDRu(III), respectively. The tested compounds induced apoptosis in A549 tumor cells as evidenced by caspase 3 activation, but only at high concentrations. Results also revealed that the amplification of P-gp gene is greater in A549 cells exposed to cisCarboPt and cisCRu(III) than cisDRu(III). Taken together all these results strongly demonstrate that MDR-1 over-expression in A549 cells could be associated to a MDR phenotype of these cells and moreover, it is also contributing to the platinum, and structurally-related compound, resistance in these cells. The identification and characterization of novel mechanisms of drug resistance will enable the development of a new generation of anti-cancer drugs that increase cancer sensitivity and/or represent more effective chemotherapeutic agents.     

MDR1反义RNA降低细胞KB_(v200)的耐药性

由ＭＤＲ１基因过度表达所引起的肿瘤细胞对化疗药物的耐药性,是导致化疗失败的主要原因之一．针对ＭＤＲ１中一段包含转录启始位点、翻译启始位点和转录正调控区的序列,设计了反义ＲＮＡ并将其克隆到逆转录病毒载体ｐＬＸＳＮ上．用脂质体包裹载体导入ＭＤＲ１高表达的耐药细胞ＫＢｖ２００中,在反义ＲＮＡ转染的细胞中,ＭＤＲ１在ｍＲＮＡ和蛋白水平的表达都有下降,细胞内药物的浓度有所提高,对长春新碱、阿霉素的耐药性分别下降了６５％和４７％．实验结果表明,反义ＲＮＡ对ＭＤＲ１的表达有抑制作用,从而使肿瘤细胞内的药物浓度升高,其耐药程度下降．     

沉默MDR1基因增强急性早幼粒白血病耐药细胞HT9药物敏感性

该研究利用短发卡RNA（small hairpinin RNA,shRNA）表达载体沉默HT9急性早幼粒白血病耐药细胞MDRl基因表达,以提高细胞对三尖杉酯碱、阿霉素的敏感性。通过设计合成编码shRNA的DNA模板序列,定向克隆到pSilencer2．1－u6neo质粒,成功构建1个P－gP蛋白基因特异的shRNA表达载体,稳定电转染HT9细胞,实时荧光定量PCR分析MDRlmRYAg表达,Westem blot检测细胞P—gp蛋白表达,流式细胞术检测P—gP蛋白外排泵功能,MTT法检测细胞对药物敏感性。结果显示,成功构建了shRNA表达载体pSilencer2-1-U6neo—MDRl,转染HT9细胞后,PCR检测重组质粒整合到HT9／sh－2－1－1细胞基因组DNA,获得稳定遗传;HT9／sh－2－1—1细胞MDRlmRNA表达降低了78．84％（P〈0．01）,P—gP蛋白表达降低了48．27％（P〈0．05）,细胞内Rh0123相对荧光强度由（10．8±0．58）％升高至（73．56±1．37m;转染细胞对三尖杉酯碱、阿霉素敏感性明显增强,IC50分别由（2．06±0．15）gmol／L降至（0．57±0．01）gmol／L、（4．04±017）gmol／L降至（1．56±0．05）μtmol／L。提示shRNA干扰表达载体pSilencer2．1－U6neo—MDRl能够稳定、持久地抑制MDRI基因表达,并能有效增强HT9细胞对三尖杉酯碱、阿霉素的敏感性。     

Baylisascaris procyonis (Nematoda: Ascaridoidea) eggs in raccoon (Procyon lotol) latrine scats in Orange County,California

Baylisascaris procyonis is the common intestinal nematode of the raccoon and is well-recognized as a cause of visceral, ocular, and neural larva migrans in many species of wild and domestic birds and mammals, including humans. To develop data on the prevalence of B. procyonis in Orange County, California, 800 distinct raccoon latrine sites were sampled in 4 spatial zones from 15 January to 31 December 2000. Counts of fecal eggs per gram (EPG) were determined and evaluated with reference to spatial zone and season of collection. No significant differences in EPG were noted among the spatial zones. However, EPG exhibited a significant rise (37,730 +/- 1,865) in the fall and a significant decline (26,204 +/- 1,446) in the winter (ANOVA, P = 0.045). The overall egg prevalence was 100%, and the overall mean EPG was 30,265 +/- 867.     

Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line.

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiate in vitro by replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60-90%) of CEA release.