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The multifunctional Y-Box protein 1 (YB-1) exerts positive and negative regulatory effects on gene expression by different mechanisms. Since transcription can be controlled by micro RNAs (miRNAs), YB-1 could also cause effects on gene expression by regulation of cellular miRNAs. To test this hypothesis, a previously established and well-characterized cell model derived from drug-sensitive (EPG85-257P/tetR/YB-1) and multidrug-resistant (EPG85-257RDB/tetR/YB-1) gastric carcinoma cells, in which the expression of YB-1 can be inhibited by tetracycline-dependent triggering of the RNA interference (RNAi) pathway, was investigated concerning their miRNA expression profiles in the presence and absence of YB-1. Microarray hybridizations demonstrated that six miRNAs (miR-96*, miR-210, miR-503, miR-623, miR-1275, miR-1290) were up-regulated more than 1.5-fold in drug-sensitive cells following YB-1 inhibition, but no differences in miRNA expression could be detected in multidrug-resistant cells. Independent validation of these findings by quantitative real-time reverse transcriptase polymerase chain reaction did not confirm these effects. Likewise, an in silico analysis of potential regulatory effects of the miRNAs on their target genes did not support the potential miRNA regulatory effects of YB-1. In conclusion, the data provide evidence that YB-1 has no direct influence on global miRNA expression pattern in different variants of gastric carcinoma cells and, therewith, does not control gene expression by regulation of miRNAs.

The use of the RNA interference (RNAi) through the expression of small hairpin RNA (shRNA) is a promising approach for efficient gene silencing for therapeutic applications. In this chapter, we describe the in vivo reversal of the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype by shRNA. For local intratumoral delivery of naked shRNA-encoding vector constructs, the nonviral jet-injection was used. This jet-injector system uses compressed air to inject small volumes (5-10 muL) of naked nucleic acid solutions into tumor tissues. Furthermore, the design of the jet-injector allows multiple injections. Under our experimental design, the delivery of plasmid DNA encoding anti-MDR shRNA by jet-injection into human MDR1/P-gp overexpressing MaTu/ADR breast cancer xenografts resulted in a decrease of MDR1 mRNA expression level to more than 90%. Accordingly, the corresponding MDR1/P-gp protein is no longer detectable in the tumors after anti-MDR1 shRNA vector injection. Furthermore, combination of two intratumoral jet-injections of anti-MDR1 shRNA vectors with two intravenous administrations of doxorubicin is sufficient for a complete reversal of the MDR phenotype in association with tumor growth inhibition.

The ATP-binding cassette (ABC)-transporter P-glycoprotein (Pgp, also known as ABCB1) is the best characterized factor involved in multidrug resistance (MDR) of cancer cells. Pgp, which is encoded by the MDR1 gene, acts as a membrane-embedded drug extrusion pump for multiple structurally unrelated cytotoxic drugs. Inhibition of the pump activity of Pgp by low-molecular weight pharmacologically active compounds as a method to reverse MDR in cancer patients has been studied extensively, but so far clinical trials have generally been disappointing. Thus, experimental strategies for overcoming MDR are under investigation. These approaches include the application of the RNA interference (RNAi) technology. RNAi is a physiological mechanism triggered by small double-stranded RNA molecules resulting in a sequence-specific gene-silencing. Besides its potential for development of novel therapeutics, RNAi also offers the possibility for specific inhibition of cellular targets in functional investigations. For specific inhibition of Pgp by triggering the RNAi pathway, transient gene-silencing by application of small interfering RNA (siRNA), and stable inhibition by transfection of MDR cancer cells with short hairpin RNA (shRNA) encoding expression cassettes encoded on plasmid DNA are described. Efficacy of RNAi on MDR1 mRNA expression level is determined by quantitative real-time RT-PCR and Northern blot. The consequences of RNAi on protein expression level are measured by Western blot and immunohistochemistry. The effects on the drug extrusion activity are measured by a drug accumulation assay based on flow cytometry, and reversal of the drug-resistant phenotype by assessment of drug-specific IC(50)-values by a cell proliferation assay based on colorimetry.

One of the leading causes of chemotherapy failure in non-Hodgkin's lymphomas (NHLs) is multidrug resistance (MDR). MDR can be associated with expression of members of the family of ABC-transporters. Since a correlation between expression of cyclooxygenase-2 (COX-2) and MDR in various cancer cells was described, the expression of COX-2 and the ABC-transporters MDR1/P-glycoprotein (P-gp), MRP1, MRP2 and BCRP was examined in 56 previously non-treated patients by immunohistochemistry. The data show that: i) P-gp is not expressed in non-treated NHLs; ii) MRP2 can be localized in the nuclear membranes of NHL cells; iii) expression of MRP2 in the cytoplasm membrane correlates with clinical response; iv) elevated expression of BCRP is typical for the patients, who did not respond to primary chemotherapy and for cases with shorter progression-free survival time in a 30 months follow-up; and v) there is a strong correlation between COX-2 and MRP1, MRP2 and BCRP. It can be concluded that: i) BCRP may be a crucial factor involved in primary resistance of NHLs, thus it may be useful for prediction of chemotherapeutic treatment and risk of relapse; and ii) since there is strong correlation between COX-2 expression and MDR in NHLs, the application of COX-2 inhibitors may be considered for chemosensitization.

Objective: In this study, we aimed to evaluate the influence of proinflammatory cytokines on ABCG2 expression and function in human MCF-7 breast cancer cell line and its mitoxantrone-resistant derivative MCF-7/MX., Methods: The effects of proinflammatory cytokines on ABCG2 mRNA expression were studied using real-time PCR method. Cytokine-mediated modification of ABCG2 protein expression and function was investigated by means of flow cytometry., Results: Significant inductions in the ABCG2 mRNA levels, protein expression, and activity were observed in IL-1 beta and TNF-alpha-treated MCF-7 cells. IL-6 increased ABCG2 protein, but had no effects on ABCG2 mRNA and function in MCF-7 cells. Although IL-1 beta did not alter mRNA and protein levels of the transporter in MCF-7/MX cells, ABCG2-mediated efflux was significantly increased in IL-1 beta-treated MCF-7/MX cells. TNF-alpha-treated MCF-7/MX cells also demonstrated greater ABCG2 protein expression and function without any changes in mRNA levels of the transporter. Neither ABCG2 mRNA nor its protein expression and function were affected by IL-6 in MCF-7/MX cells., Conclusion: IL-1 beta and TNF-alpha induce ABCG2 mRNA and protein expression and increase its activity in breast cancer cell line MCF-7. In MCF-7/MX cells these cytokines modulate ABCG2 protein expression and/or function, but they have no influence on the transporter mRNA levels.

Resistance to anticancer therapy is a major obstacle for successful management of patients in oncology. Although in the past, various biological mechanisms involved in therapy resistance, in particular multidrug resistance, have been identified, cancer patients did not really benefit. The mechanisms include the enhanced activity of drug extrusion pumps, modulation of cellular death pathways, alteration and repair of target molecules and various other mechanisms. Together they build a complex network mediating an individual therapy-resistant phenotype. The improved description of this multifactorial network should be useful for prediction of treatment response and would allow to design an individual-tailored therapy regiment. Proteome analyzing technologies appear as powerful tools for identifying new factors and protein expression profiles associated with anticancer therapy resistance. In the last years, the application of proteomic techniques identified multiple new factors or protein expression signatures in drug-resistant cell models and cancerous tissues. However, the functional role and the clinical impact of these findings are not yet clarified. So far, none of the proteomic data were useful for the development of improved diagnostic tests, for prediction of individual therapy response or for development of updated chemosensitizers. Here, the previous therapy resistance-related proteome data and future perspectives will be discussed., (Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Betulinic acid is a triterpene isolated from the bark of many plants that exhibits cytotoxicity in several cancer cell lines and is capable of inducing apoptosis. In this study, we examined the cytotoxic activity and apoptotic ability of betulinic acid in the drug-sensitive (MeWo) and drug-resistant melanoma MeWo CIS (cisplatin), MeWo ETO (etoposide), MeWo VIN (vinblastin) and MeWo FOTE (fotemusine) cell lines, as well as in the normal melanocyte NHEM-neo cell line. The results show that betulinic acid exhibited significant cytotoxicity on all the cell lines. However, a sulphorhodamine B cell proliferation assay and immunocytochemical analysis of Ki67 expression revealed the strongest cytotoxicity on the normal melanocyte cell line, NHEM-neo. Flow cytometry and immunocytochemical analysis of caspase 3 expression was used to confirm cell death by apoptosis. In conclusion, betulinic acid is a potential candidate for anticancer research, and may also have an application in the cosmetics industry.

Ovarian carcinoma has the highest mortality rate among gynaecological malignancies. In this project, we investigated the hypothesis that molecular markers are able to predict outcome of ovarian cancer independently of classical clinical predictors, and that these molecular markers can be validated using independent data sets. We applied a semi-supervised method for prediction of patient survival. Microarrays from a cohort of 80 ovarian carcinomas (TOC cohort) were used for the development of a predictive model, which was then evaluated in an entirely independent cohort of 118 carcinomas (Duke cohort). A 300-gene ovarian prognostic index (OPI) was generated and validated in a leave-one-out approach in the TOC cohort (Kaplan-Meier analysis, p = 0.0087). In a second validation step, the prognostic power of the OPI was confirmed in an independent data set (Duke cohort, p = 0.0063). In multivariate analysis, the OPI was independent of the post-operative residual tumour, the main clinico-pathological prognostic parameter with an adjusted hazard ratio of 6.4 (TOC cohort, CI 1.8-23.5, p = 0.0049) and 1.9 (Duke cohort, CI 1.2-3.0, p = 0.0068). We constructed a combined score of molecular data (OPI) and clinical parameters (residual tumour), which was able to define patient groups with highly significant differences in survival. The integrated analysis of gene expression data as well as residual tumour can be used for optimized assessment of the prognosis of platinum-taxol-treated ovarian cancer. As traditional treatment options are limited, this analysis may be able to optimize clinical management and to identify those patients who would be candidates for new therapeutic strategies.

Topoisomerase IIalpha (Top IIalpha) is a nuclear enzyme that plays a central role in DNA metabolism, and is a molecular target for a variety of chemotherapeutic agents. Top IIalpha has recently gained attention as a biomarker for therapy response and patient survival. In this study, we attempted to assess the feasibility of measuring Top IIalpha gene expression in RNA, isolated from archival formalin-fixed paraffin-embedded tissue specimens, which are used routinely in pathology laboratories. We have employed a new technique on the basis of magnetic particles' separation and purification of nucleic acids, and evaluated both protein and mRNA expressions from the same routinely processed tissue blocks. We investigated the expression of Top IIalpha mRNA and protein by real-time reverse transcription polymerase chain reaction and immunohistochemistry, in a cohort of 133 primary ovarian carcinomas, and evaluated the association between Top IIalpha expression and clincopathological variables as well as patient outcome. Elevated Top IIalpha mRNA expression was observed in high-grade tumors (P=0.003) and advanced stage disease (P=0.011). In univariate Kaplan-Meier analysis, patients with higher expression of Top IIalpha nuclear protein had a significantly decreased overall survival (P=0.045). Interestingly, we detected cytoplasmic protein expression of Top IIalpha in a subset of samples. Cytoplasmic expression of Top IIalpha was associated with the expression of chromosomal region maintenance/exportin 1 (CRM1)-a nuclear export protein (P=0.008). Our study suggests that Top IIalpha overexpression is involved in the progression of ovarian cancer in a subset of the patients. Our results encourage the further evaluation of the prognostic and predictive values of Top IIalpha expression in ovarian carcinoma, which might help to assess the patients' risk profile, and the planning of an individualized therapy.

Purpose: Novel multidrug resistance (mdr) modulators have been proved as inhibitors of P-glycoprotein (P-gp). We first investigated the in vitro effects of selected compounds in human cancer cells on multidrug resistance reversal effects compared to drug standards and on P-gp induction to characterize the potential of the compounds as clinical candidates., Methods: The uptake of daunorubicin into a parental cancer cell line and P-gp expressing subcell line in presence of the modulators was characterized by flow cytometry. Induction of P-gp was investigated in P-gp expressing and non-expressing cancer cell lines on the RNA level by real-time quantitative polymerase chain reaction (RTQ-PCR) and protein quantification. Results were additionally confirmed by northern blot techniques and functionality assays in selected cell lines., Results: The novel modulators showed activities as mdr reversers in a P-gp specific human cancer cell model with mainly increased uptake rates of daunorubicin into the drug-resistant cell line. H17 proved to be more active than cyclosporine A as a known strong mdr modulator. The induction studies revealed practically no induction potential of the compounds in usual short-time drug application regimes in all cell lines. Furthermore, the novel modulators did not increase the efflux of a P-gp model substrate in the functionality model assay. This confirmed the results of non-P-gp induction which was observed on both the RNA and the protein levels., Conclusions: The novel mdr modulators proved as perspective candidates for further clinical studies because they turned out to be highly active in human cancer cell models. Furthermore, they showed no potential to induce the transmembrane efflux pump P-gp. This is a significant advantage compared to modulators which failed in clinical trials because of induction-effects that increase cellular resistances and, moreover, side effects in normal cells.

Head and neck cancers are treated by a combination of surgery, radiotherapy and/or chemotherapy. The clinical success of cisplatin-based chemotherapy, mostly in combination with 5-FU or a taxane, is however limited by multifactorial intrinsic or acquired resistance. So far, known genes involved in cisplatin resistance do not sufficiently allow the prediction of cancer chemosensitivity. Thus, the purpose of this study was to search for further genes involved in cisplatin resistance by differential gene expression analysis of the parental tongue cancer cell line Cal27 and its 10-fold more resistant sub-cell line Cal27cis, which was obtained by treating Cal27 with increasing concentrations of cisplatin. As found by the suppression subtractive hybridization, expression of DKK1, an inhibitor of canonical WNT signaling, was decreased in Cal27cis. Microarray analysis, qPCR and ELISA confirmed the approximately 2-fold difference in expression. Cisplatin treatment and serum starvation increased by 2-fold the secretion of DKK1 in Cal27 and Cal27cis, thus rendering DKK1-levels significantly different in both cell lines under basal and stress conditions. Recombinant overexpression of DKK1 in Cal27 and Cal27cis resulted in clonal cell lines, which were both 2.2- to 3-fold more sensitive toward cisplatin in cell viability (MTT) and in proliferation (BrdU) assays. In conclusion, acquired (10-fold) resistance of Cal27 against cisplatin is associated with decreased DKK1 expression and could partially be reversed by DKK1 overexpression, thus suggesting DKK1 and the WNT signaling pathway as a marker and target for cisplatin chemosensitivity., ((c) 2008 Wiley-Liss, Inc.)

Novel 3,9-diazatetraasteranes have been synthesized with varied aromatic substitution patterns and evaluated as P-glycoprotein (P-gp) inhibitors. Structure-activity relationships (SAR) are discussed in relation to determined physicochemical properties. The potential to induce P-gp expression has been evaluated in cancer cell lines. The bioanalytical results indicate favorable noninducing properties compared to P-gp inducing drug standard.

Background: The primary method of treatment of non-small cell lung cancer (NSCLC) in stage IIIB and IV is chemotherapy. Previous data suggested a correlation between cyclooxygenase-2 (COX-2) expression and the multidrug-resistant phenotype of cancer cells., Materials and Methods: In this prospective study, 32 patients with NSCLC in stage IIIB and IV from 1,078 patients were included. The expression of COX-2 as well as the expression of the ABC transporters MDR1/P-glycoprotein (MDR1/P-gp), BCRP and MRP1 were detected immunohistochemically., Results: Univariate and multivariate analyses demonstrated no prognostic or predictive significance of these proteins. It was merely demonstrated that complete or partial response are favourable factors for prediction of longer progression-free survival time. However, a strong positive correlation between the expression of COX-2, MDR1/P-gp and BCRP was found in NSCLC., Conclusion: These data suggest no clinical impact for the expression of MDR1/P-gp, MRP1, BCRP or COX-2 in NSCLC, but a putative coregulation of COX-2 and MDRI/P-gp and BCRP in NSCLC.

Decreased expression of p16 may result from hypermethylation of the promoter or from deletion of the gene. It can lead to intensified proliferation of neoplastic cells and to cytostatic drug resistance. The study was aimed at the examination of prognostic value of p16 expression in relation to Ki67 and caspase-3 in ovarian cancers using immunohistochemistry. The immunohistochemical studies were performed on 73 paraffin-embedded samples of ovarian cancers from 43 patients and samples from 6 healthy ovaries. We have used monoclonal antibodies against p16. ABC method and DAB were used for antigens visualisation. The intensity of the immunohistochemical reactions was appraised using the semi-quantitative IRS scale. In healthy ovaries we have shown strong reaction in the nuclei of surface epithelium. In the case of studied ovarian cancers, the reaction of a nuclear and cytoplasmic localization was obtained. The mean overall immunoreactivity score of nuclear p16 expression amounted to 5.30+/-3.44 SD in primary laparotomy material and 6.61+/-4.34 SD in secondary cytoreduction material. Statistical analysis demonstrated that lower p16 expression was typical of the younger patients and the patients who died. Kaplan-Meier's analysis proved that lower expression of p16 was characteristic of cases with shorter overall survival. In the present study we have demonstrated that lowered p16 expression represented an unfavourable prognostic index in ovarian cancer. Lowered p16 expression was also typical for chemotherapy-resistant ceases (cases of lower caspase-3 and higher Ki67 at secondary cytoreduction expression).

Background: Numerous experimental studies have described the capacity of myofibroblasts to stimulate mammary cancer cells in a paracrine manner. Until now, the prognostic significance of myofibroblasts present in breast cancer has not been examined., Patients and Methods: In paraffin sections, originating from 45 patients with primary invasive breast cancer, immunohistochemical reactions were performed using antibodies directed against smooth muscle actin, Ki-67, VEGF, bFGF and UPA., Results: The cases with higher content of myofibroblasts in the tumour tissue manifested higher grade, more pronounced expression of Ki-67, VEGF and bFGF and shorter overall survival and relapse-free survival., Conclusion: The present study for the first time documents the unfavourable prognostic significance of myofibroblasts in tissues of invasive ductal mammary carcinomas.

Elevated metallothionein (MT) expression in ovarian cancers treated with cisplatin-based schemes represents an unfavorable prognostic index. MT expression is significantly higher in tumor samples obtained after chemotherapy. The present study aimed at examining MT expression in ovarian carcinoma cells sensitive (A2780) or resistant (A2780RCIS) against platinum drug treatment as well as examining effects of exposure to cisplatin on MT expression. Subcellular expression of MT was evaluated also in samples originating from 73 ovarian tumors. Cisplatin-resistant A2780RCIS cells were exposed to increasing cisplatin concentrations, and the subcellular expression of MT was determined by immunocytochemistry. The studies demonstrated that cisplatin-resistant A2780RCIS cells exposed to cisplatin typically manifested a nuclear MT expression. The study demonstrated also that exposure to cisplatin was paralleled by growing MT expression in cell nuclei. The nuclear expression of MT was also found to be specific for ovarian cancers of poor clinical outcome. No relationship could be demonstrated between cytoplasmic expression of MT and clinical variables. Nuclear MT expression is induced by cisplatin and seems to protect DNA in the cells from toxic effects of the drug.

An obstacle in chemotherapy of ovarian cancer is the development of drug resistance. Taxol (paclitaxel)-resistance-associated gene-3 (TRAG-3/CSAG2) was found to be overexpressed in a paclitaxel-resistant ovarian carcinoma cell line. However, clinical impact of TRAG-3 in ovarian carcinoma has not been demonstrated previously. For demonstration of potential clinical impact of TRAG-3, immunohistochemistry was applied to determine TRAG-3 protein expression in specimens obtained from ovarian carcinoma patients (n=37) who received a paclitaxel-based chemotherapy at two different time points, initial laparotomy before chemotherapy, and secondary cytoreduction after chemotherapy. The TRAG-3-specific immunohistochemical staining was correlated with clinical outcome. In ovarian carcinoma specimens obtained at the initial laparotomy, an advantage in overall (P < 0.001) and progression-free (P = 0.003) survival for patients with weak TRAG-3 expression could be demonstrated. Tumor specimens excised at secondary cytoreduction procedure were not predictive for clinical outcome. In summary, TRAG-3 was found to be a prognostic factor for the prediction of clinical outcome after the application of paclitaxel-based chemotherapy.

Resistance to cytotoxic chemotherapy is the main cause of therapeutic failure and death in women suffering from ovarian carcinoma. The standard first-line chemotherapy of ovarian cancer consists of a combination of a taxane and a platinum-containing drug. Thus, the cellular and molecular mechanisms involved in resistance against these compounds are of vital importance in the context of chemotherapy of ovarian cancer. This review will discuss the current state of knowledge of drug resistance-associated factors and their impact on clinical chemotherapy response in ovarian carcinoma as well as different strategies for reversal of drug resistance.

Background: The membrane cofactor protein CD46 represents a complement inhibitor, which protects autologous cells from complement - mediated cytotoxicity. CD46 may exhibit the potential to protect tumor cells from the immune responses of the host. The present study aimed to evaluate the prognostic significance of CD46 expression in ovarian cancers., Materials and Methods: The analyses were performed on 73 ovarian cancer samples. Immunohistochemical reactions were performed on paraffin sections of tumors using monoclonal antibodies directed against CD46. The immunohistochemical reactions and the clinical observations results were subjected to statistical analysis., Results: Expression of CD46 was demonstrated in 60% of primary laparotomy cases and in 70% secondary cytoreduction cases. Kaplan-Meier analysis showed that a significantly shorter revival-free time was linked to cases with CD46 expression at PL (p= 0.01)., Conclusion: Ovarian cancers manifest CD46 expression that is linked to a less favourable prognosis.

Twenty previously synthesized fused heterocyclic DNA-topoisomerase II (Topo II)-inhibiting compounds were investigated for their potential efficacy in various human cancer cell lines that were derived from different tumor entities. Moreover, different multidrug-resistant variants of these cancer cell lines with decreased Topo II expression were investigated. In parental, drug-sensitive cells merely the compounds BD3 and G35 showed efficacies, in terms of microM, which were similar to that of the classical Topo II inhibitor etoposide. On the other hand, most of the tested heterocyclic compounds were found more effective in drug-resistant cells than in the parental, drug-sensitive ones, and some of the compounds showed high antineoplastic efficacy in several drug-resistant cell models. Compounds BD13, BD14 and BD16 exhibited high antineoplastic activities against the drug-resistant sublines EPG85-257RNOV and EPG85-257RDB derived from gastric carcinoma, EPP85-181RNOV and EPP85-181RDB derived from pancreatic carcinoma, MCF-7/Adr derived from breast cancer, D79/86RNOV derived from fibrosarcoma, and MeWoETO1 derived from melanoma. Furthermore, compound D23 was found highly efficient in the multidrug-resistant variants HT-29RNOV and HT-29RDB derived from colon carcinoma, and compound D24 exhibited the highest antineoplastic activity among the tested compounds in the drug-resistant subline MDA-MB-231ROV derived from breast cancer. In conclusion, compounds BD 13, BD 14, BD 16, D 23 and D 24 may be useful for the treatment of different multidrug-resistant cancer cells with cross resistance against "classical" Topo II-targeting drugs.

Cancer patients with tumors of similar grading, staging and histogenesis can have markedly different treatment responses to different chemotherapy agents. So far, individual markers have failed to correctly predict resistance against anticancer agents. We tested 30 cancer cell lines for sensitivity to 5-fluorouracil, cisplatin, cyclophosphamide, doxorubicin, etoposide, methotrexate, mitomycin C, mitoxantrone, paclitaxel, topotecan and vinblastine at drug concentrations that can be systemically achieved in patients. The resistance index was determined to designate the cell lines as sensitive or resistant, and then, the subset of resistant vs. sensitive cell lines for each drug was compared. Gene expression signatures for all cell lines were obtained by interrogating Affymetrix U133A arrays. Prediction Analysis of Microarrays was applied for feature selection. An individual prediction profile for the resistance against each chemotherapy agent was constructed, containing 42-297 genes. The overall accuracy of the predictions in a leave-one-out cross validation was 86%. A list of the top 67 multidrug resistance candidate genes that were associated with the resistance against at least 4 anticancer agents was identified. Moreover, the differential expressions of 46 selected genes were also measured by quantitative RT-PCR using a TaqMan micro fluidic card system. As a single gene can be correlated with resistance against several agents, associations with resistance were detected all together for 76 genes and resistance phenotypes, respectively. This study focuses on the resistance at the in vivo concentrations, making future clinical cancer response prediction feasible. The TaqMan-validated gene expression patterns provide new gene candidates for multidrug resistance.

Background: In breast cancer, the expression of CD24 represents a poorly recognised unfavourable prognostic factor. CD24 has been described to be potentially down-regulated by estrogen receptor alpha (ER). The present study was aimed at examining the predictive value of CD24 expression in tamoxifen-treated breast cancer cases., Materials and Methods: Sixty patients with primary invasive ductal breast cancers with post-operative tamoxifen treatment were enrolled in the study. Immmunohistochemical reactions were performed using monoclonal antibodies directed against CD24 and ER., Results: Cases demonstrating cytoplasmic-membranous expression of CD24 (CD24c-m) proved to be characterised by a significantly lower expression of ER as compared to CD24c-m-negative cases. A multivariate progression analysis based on the Cox proportional hazard model demonstrated that CD24c-m expression is an independent prognostic factor for poor overall survival., Conclusion: The data from the present study suggested that CD24c-m expression is specific for tamoxifen-resistant breast cancer cases. CD24 should be subjected to comprehensive studies as a marker of resistance to tamoxifen treatment.

Single genes, which can be used to predict response to therapy in breast cancer, including estrogen receptor (ER), HER-2, metallothionein and the ABC transporters are discussed. With the exception of the ER status, no single tumor marker has been shown to possess a sufficient predictive value to render it clinically useful. To achieve greater predictive power, multiple markers need to be examined and correlated with response to chemotherapy. With the advent of high-throughput quantification of gene expression, simultaneous assessment of thousands of genes is now possible, which allows identification of expression patterns in different breast cancers that might correlate with and, thereby, predict survival or response to treatment. Recent studies using microarrays to investigate survival prediction, chemotherapy resistance and therapy response are discussed. In vivo and in vitro experiments are discussed. Particular interest is given to anthracycline treatment, where in vitro drug resistance data may be useful for patient prognosis prediction. However, different microarray platforms can provide different results for the same experiment. A recommended statistical pathway is still not yet accepted. These problems have to be solved before future diagnostic applications using cDNA microarrays can be developed. In the near future, it can be expected that various microarray studies will be available to analyze hundreds to thousands of patients, selecting and validating predictive gene expression signatures., (Copyright© 2005 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

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., ((c) 2005 Wiley-Liss, Inc.)

Multidrug resistance (MDR) of human cancers is the major cause of failure of chemotherapy. To better understand the molecular events associated with the development of different types of MDR, two different multidrug-resistant gastric carcinoma cell lines, the MDR1/P-glycoprotein-expressing cell line EPG85-257RDB and the MDR1/P-glycoprotein-negative cell variant EPG85-257RNOV, as well as the corresponding drug-sensitive parental cell line EPG85-257P, were used for analyses of the mRNA expression profiles by cDNA array hybridization. Of more than 12,000 genes spotted on the arrays, 156 genes were detected as being significantly regulated in the cell line EPG85-257RDB in comparison to the non-resistant cell variant, and 61 genes were found to be differentially expressed in the cell line EPG85-257RNOV Seventeen genes showed a differential expression level in both multidrug-resistant gastric carcinoma variants. The impact of these alterations in gene expression levels in different multidrug-resistant gastric carcinoma cell variants is discussed.

CD24 is a small membranous protein which may participate in invasion of tumor cells. Present study aimed at evaluation of prognostic significance linked to immunohistochemical demonstration of CD24 expression and the proliferation index, Ki67 expression in ovarian cancers. The immunohistochemical reactions with monoclonal CD24- and Ki67-specific antibodies were performed in paraffin sections originating from 30 patients with ovarian cancer treated using cisplatin and paclitaxel. Results of the reactions and analysis of the clinical course of the patients were subjected to statistical analysis. Cases with cytoplasmic-membranous expression of CD24 (CD24c-m) were found to exhibit significantly shorter overall survival time (P = 0.0002) and progression-free period (P = 0.0005). Cases with membranous expression of CD24 (CD24m) manifested a longer overall survival time (P = 0.022). No relationship was disclosed between expression of Ki67 on the one hand and survival time and CD24 expression on the other. As documented using chi square test, expression of CD24c-m predisposed to relapses (P = 0.012), progression (P = 0.0362) and to death (P = 0.0034). Deaths were encountered significantly less frequently in cases with CD24m expression (P = 0.0465). The studies demonstrated that CD24c-m represented a strongly unfavorable prognostic indicator. The antigen represents an interesting target in the search for novel therapeutic methods. The more aggressive course of cases with CD24c-m expression was not linked to more intense proliferation of the tumor cells.

Background: Ribozymes are an effective tool to reduce the mRNA levels of specific target genes. Overexpression of the drug transport protein, ABCG2, has been associated with multidrug resistance in cancer cells., Materials and Methods: An expression plasmid encoding a hammerhead ribozyme against the ABCG2 gene was stably transfected into multidrug-resistant MCF7/MX cells that express very high levels of the ABCG2 protein. The effect of the ribozyme was determined by quantitative real-time RT-PCR, Western blot and cytotoxicity assays., Results: The ribozyme reduced ABCG2 mRNA levels to less than 10% of control values, which resulted in the concomitant reduction of ABCG2 protein levels and sensitization of the cells to mitoxantrone and methotrexate., Conclusion: The ribozyme used was highly effective in reducing the expression of its target gene, ABCG2, and was able to modulate the associated multidrug-resistant phenotype.

Proteomics, the global analysis of expressed cellular proteins, provides powerful tools for the detailed comparison of proteins from normal and neoplastic tissue. In particular, cancer cell culture models are suited for applying proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry, to identify specific protein expression profiles and/or proteins that may be associated with a defined phenotype of the cancer cells. As an instance of such an application of proteomics techniques, the detailed proteome analyses of different drug-resistant and thermoresistant cancer cell lines will be discussed. Finally, the potential roles of newly identified factors in a distinct biological mechanism have to be proven by functional studies. This experimental validation strategy will be discussed for two different factors identified by 2D-PAGE analyses of drug-resistant carcinoma cell lines, the "transporter associated with antigen presentation 1" (TAP1) and 14-3-3sigma.

Purpose: We investigated p53 and its downstream effectors p21WAF1/CIP1, BAX, and hMSH2 as well as the proliferation marker Ki-67 (mki-67/MIB-1) in patients undergoing preoperative radiochemotherapy for rectal carcinoma to identify prognostic and predictive factors. The focus of this study was on the dynamics of these genetic markers in a longitudinal study-that is, before and after radiochemotherapy., Patients and Methods: Expression of p53, BAX, p21WAF1/CIP1, Ki-67, and hMSH2 was investigated by immunohistochemistry in pre- and posttherapeutic tumor samples in 66 patients. Tumor DNA was screened for p53 mutations by single-strand conformation polymorphism-polymerase chain reaction (SSCP-PCR). Paired tumor samples (pretherapy and posttherapy) were collected prospectively., Results: Patients with a decrease in p21 expression following radiochemotherapy had better disease-free survival (P =.03). Similarly, patients with an increase in proliferative activity as measured by increased Ki-67 expression posttherapy had better disease-free survival (P <.005). In addition, we observed a significantly better prognosis for patients with high hMSH2 expression. In contrast, pretherapeutic levels of p53, BAX, or p21 expression and p53 mutation had no prognostic value, indicating that the combination of radiotherapy and chemotherapy might override defects in these genes., Conclusion: These findings are novel and support the clinical relevance of p21 in the suppression of both proliferation and apoptosis. Thus, the dynamic induction of p21WAF1/CIP1 was associated with a lower proliferative activity but an ultimately worse treatment outcome following neoadjuvant radiochemotherapy and tumor resection. Induction of p21, therefore, represents a novel resistance mechanism in rectal cancer undergoing preoperative radiochemotherapy.

Resistance to various anti-neoplastic agents is a common observation in clinical management of melanoma. The biologic mechanisms conferring these different drug-resistant phenotypes, including resistance against the commonly used anti-cancer drug cisplatin, are unclear. In order to elucidate the role of the membrane adenosine triphosphate binding cassette-transporter cMOAT (canalicular multispecific anion transporter) (MRP2/ABCC2) in cisplatin resistance of melanoma, the expression of this protein was analyzed in the platinum drug-resistant cell line MeWo CIS 1. Cisplatin-resistant melanoma cells showed a distinct overexpression of cMOAT on mRNA and protein level. This observation was accompanied by a reduced formation of platinum-induced intrastrand cross-links in the nuclear DNA measured by an immunocytologic assay. This decrease in DNA platination was accompanied by an accelerated re-entry into the cell cycle after the typical cisplatin-induced G2 arrest, and a resistance to undergo apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity for Pt-d(GpG) adducts was not elevated in platinum drug-resistant melanoma cells. The decrease in platinum-DNA adduct formation in cisplatin-resistant melanoma cells was rather a reflection of the protecting activity of the transporter cMOAT. In conclusion, the functional inhibition of cMOAT might be a promising strategy in the reversal of resistance to platinum-based anti-cancer drugs in human melanoma.

The phenomenon of multidrug resistance (MDR) in human cancers is the major cause of failure of chemotherapy. To better understand the molecular events associated with the development of different types of MDR, a classical MDR P-glycoprotein expressing gastric carcinoma cell line and an atypical MDR P-glycoprotein-negative variant were analyzed by cDNA array hybridization. Of 588 cDNAs spotted on the array, a total of 9 genes showed differences in mRNA expressions. An enhanced expression level of 3 genes could be detected in both different MDR models (HLH, IR21, CCT5, Tx P-1), 4 genes were overexpressed solely in classical MDR cells (Hsp27, Rcl, aldehyde dehydrogenase 1, vimentin), whereas the expression of one gene was increased in atypical MDR cells (ProTa). Furthermore, the mRNA expressions of 3 genes were down-regulated in atypical MDR cells (Hsp27, vimentin, JNK2), whereas a decrease in mRNA expression in classical MDR cells could be determined for none of them. These differences were confirmed by a semiquantitative RT-PCR approach. Further characterization of these factors may provide more insight into the biology and development of different types of MDR in human gastric carcinomas. Moreover, these genes may be potential candidate factors for the diagnostics and/or prognosis of clinical drug resistance.