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The human Y-box binding protein 1 (YB-1) is known to be a promising target for cancer therapy. We have demonstrated that YB-1 plays an important role in the adenoviral life cycle by regulating the adenoviral E2-gene expression. Thus, we studied the oncolytic effect of the recombinant adenovirus Ad-Delo3-RGD, in which the transactivation domain CR3 of the E1A protein is ablated to enable viral replication only in YB-1 positive cancer cells. In vitro Southern Blot analysis and cytopathic effect assays demonstrate high anti-glioma potency, which was significantly increased in combination with temozolomide (TMZ), daunorubicin and cisplatin. Since vascular endothelial growth factor (VEGF) is thought to promote the hypervascular phenotype of primary, malignant brain tumors, we also tested Ad-Delo3-RGD in regard to the inhibition of VEGF expression. Indeed, we found that Ad-Delo3-RGD induced VEGF down regulation, which was even amplified under hypoxic conditions. Tumor-bearing nudemice treated with the YB-1 dependent oncolytic adenovirus showed significantly smaller tumors than untreated controls. Furthermore, combination therapy with TMZ led to a regression in all treated animals with complete tumor regression in 33 % of analyzed mice, which was verified by bioluminescence imaging and histological studies. In addition, histopathological evaluation revealed enhanced apoptosis and a reduction in tumor vessel formation, indicating that Ad-Delo3-RGD has an anti-angiogenic effect in addition to its oncolytic capacity in vivo. Hence, our results demonstrate that the combination therapy of YB-1 dependent virotherapy and TMZ is effective in a xenograft glioma mouse model and might be useful in a YB-1 based clinical setting., (Copyright © 2010 UICC.)

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.)

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.

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.)

MDR in human cancers is one of the major causes of failure of chemotherapy. A member of the superfamily of ABC transporters, BCRP, was demonstrated to confer an atypical MDR phenotype to tumor cells. To overcome the BCRP-mediated drug resistance, the fungal secondary metabolite TPS-A, a diketopiperazine, was analyzed with regard to its potency to reverse the BCRP-mediated drug-resistant phenotype. At concentrations of 10-50 microM, TPS-A reversed a mitoxantrone-resistant phenotype and inhibited the cellular BCRP-dependent mitoxantrone accumulation in the human gastric carcinoma cell line EPG85-257RNOV, the human breast cancer cell line MCF7/AdrVp (both exhibiting acquired BCRP-mediated MDR) and the BCRP cDNA-transfected breast cancer cell line MCF-7/BCRP clone 8. No cytotoxicity was seen at effective concentrations. These data indicate that TPS-A is a novel BCRP inhibitor., (Copyright 2003 Wiley-Liss, Inc.)

Therapy resistance is the major obstacle to advances in successful cancer treatment. To characterize chromosomal alterations associated with different types of acquired MDR and thermoresistance, we applied CGH to compare a unique panel of human gastric carcinoma cells consisting of the parental, drug-sensitive and thermosensitive cancer cell line EPG85-257P, the atypical MDR variant EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. CGH with 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 various members of the families of ABC transporters and molecular chaperones. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated., (Copyright 2002 Wiley-Liss, Inc.)

The impact of the ABC transporters breast cancer resistance protein/mitoxantrone resistance associated transporter (BCRP/MXR), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance gene-1/P-glycoprotein (MDR1/PGP) on the multidrug resistance (MDR) phenotype in chemoresistance and thermoresistance was investigated in the parental human gastric carcinoma cell line EPG85-257P, the atypical MDR subline EPG85-257RNOV, the classical MDR subline EPG85-257RDB and their thermoresistant counterparts EPG85-257P-TR, EPG85-257RNOV-TR and EPG85-257RDB-TR. Within the atypical MDR subline EPG85-257RNOV expression of BCRP/MXR and of MRP1 were clearly enhanced (vs. parental and classical MDR lines). MDR1/PGP expression was distinctly elevated in the classical MDR subline EPG85-257RDB (vs. parental and atypical MDR sublines). In all thermoresistant counterparts basal expression of BCRP/MXR, MRP1 and MDR1/PGP was increased relative to thermosensitive sublines. Although it could be shown that the overexpressed ABC transporters were functionally active, however, no decreased drug accumulations of doxorubicin, mitoxantrone and rhodamine 123 were observed. Thus, expression of BCRP/MXR, MRP1 and MDR1/PGP was found to be dependent on the appropriate type of chemoresistance; correlating with a classical or atypical MDR phenotype. Within the thermoresistant variants, however, the increase in ABC transporter expression did obviously not influence the MDR phenotype., (Copyright 2001 Wiley-Liss, Inc.)

Acquired resistance to antineoplastic agents is a frequent obstacle in tumor therapy. Malignant melanoma cells are particularly well known for their unresponsiveness to chemotherapy; only about 30% of tumors exhibit a transient clinical response to treatment. In our study, we investigated the molecular mechanism of acquired resistance of melanoma cells (MeWo) to the chloroethylating drug fotemustine. Determination of O(6)-methylguanine-DNA methyltransferase (MGMT) activity showed that MeWo cells that acquired resistance to fotemustine upon repeated treatment with the drug display high MGMT activity, whereas the parental cell line had no detectable MGMT. The resistant cell lines exhibit cross-resistance to other O(6)-alkylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine. Acquired resistance to fotemustine was alleviated by treatment with the MGMT inhibitor O(6)-benzylguanine demonstrating that reactivation of MGMT is the main underlying cause of acquired alkylating drug resistance. As compared with control cells, both MGMT mRNA and MGMT protein were expressed at a high level in fotemustine resistant cells. Southern blot analysis proved that the MGMT gene was not amplified. There was also only an insignificant difference in the CpG methylation pattern of the MGMT promoter whereas a clear hypermethylation in the body of the gene was observed in fotemustine resistant cells. The conclusion that hypermethylation is responsible for reactivation of the MGMT gene gained support by the finding that MGMT activity significantly declined and cells reverted (partially) to the parental sensitive phenotype upon treatment with 5-azacytidine. This is the first report of acquired resistance to a chloroethylating antineoplastic drug of melanoma cells due to gene hypermethylation., (Copyright 2001 Wiley-Liss, Inc.)

Malignant melanoma displays strong resistance against various antineoplastic drugs. The mechanisms conferring this intrinsic resistance are unclear. To better understand the molecular events associated with drug resistance in melanoma, a panel of human melanoma cell variants exhibiting low and high levels of resistance to 4 commonly used drugs in melanoma treatment, i.e., vindesine, etoposide, fotemustine and cisplatin, was characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Of 269 mRNA fragments found to be altered in expression level by DDRT-PCR, a total of 11 cDNA clones was characterized after confirmation of a differential expression pattern by Northern blot analyses. These clones include 3 genes (DSM-1, DSM-3 and DSM-5) of known function, 4 previously sequenced genes (DSM-2, DSM-4, DSM-6 and DSM-7) of uncharacterized function and 4 novel genes (DSM-8-DSM-11) without match in GenBank. All of these genes exhibited altered mRNA expression in high level etoposide-resistant cells, whereby 7 genes (DSM-1-DSM-6 and DSM-8) were found to be decreased in the transcription rate in these etoposide-resistant cells. The mRNA synthesis of the remaining genes (DSM-7 and DSM-9-DSM11) was enhanced in high level etoposide-resistant melanoma cells. The expression of 5 (DSM-5 and DSM-7-DSM-10) of the cloned cDNA encoding mRNAs was modulated in various independently established drug-resistant melanoma cells, indicating to be associated with drug resistance. Further characterization of these genes may yield inside into the biology and development of drug resistance in malignant melanoma., (Copyright 2000 Wiley-Liss, Inc.)

Malignant melanoma is well known for its primary unresponsiveness to chemotherapy. The mechanisms conferring this intrinsic resistance are unclear. In this study, we investigated the role of genes involved in DNA repair in a panel of human melanoma cell variants exhibiting low and high levels of resistance to 4 commonly used drugs in melanoma treatment, i.e., vindesine, etoposide, fotemustine and cisplatin. We show that in melanoma cells exhibiting resistance to cisplatin, etoposide and vindesine, the nuclear content of each of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2 and hMSH6 was reduced by 30-70%. A decreased expression level of up to 80% of mRNAs encoding hMLH1 and hMSH2 was observed in drug-resistant melanoma cells selected for cisplatin, etoposide and fotemustine, while vindesine-selected cells showed only moderate reduction. In melanoma cells that acquired resistance to fotemustine, the amount of nuclear MMR proteins was nearly unaltered, whereas the activity of O6-methylguanine-DNA methyltransferase (MGMT) was considerably enhanced. Activity of N-methylpurine-DNA glycosylase (MPG) was not significantly altered in any of the drug-resistant melanoma cells. Our data indicate that modulation of both MMR components and MGMT expression level may contribute to the drug-resistant phenotype of melanoma cells.

An established gastric-carcinoma cell line, EPG85-257P, is extremely sensitive to mitoxantrone (IC50, 0.12 ng/ml). Stepwise selection with mitoxantrone for 3 years resulted in a cell line (EPG85-257RN) that is 7,056-fold resistant to mitoxantrone (IC50, 846 ng/ml) and displays cross-resistance to the topoisomerase(topo)-II poisons ametantrone (411x), etoposide (112x) and teniposide (60x) as well as the topo-I poisons 7-ethyl-10-hydroxycamptothecin (331x) and topotecan (58x). We now show that this resistance is multifactorial. Western blotting revealed a 5-fold decrease in topo-IIalpha polypeptide in the mitoxantrone-resistant cells. Immunohistochemistry and Western blotting failed to demonstrate P-glycoprotein overexpression. Formation of trapped topo-II-DNA complexes in the resistant cells required higher mitoxantrone concentrations than in parental cells, even though nuclei isolated from the EPG85-257RN cells formed cleavage complexes normally. In agreement with these observations, which suggest the possibility of a defect in mitoxantrone accumulation, examination of mitoxantrone accumulation in both cell lines by confocal laser microscopy revealed that the EPG85-257RN cells accumulate less mitoxantrone at steady state. From these results, we propose that mitoxantrone accumulation, along with alterations in topo-IIalpha expression, contribute to the resistance to mitoxantrone observed in these cells.