[Resistance to medicinal tumor therapy. Current status]. / Resistenzen gegenüber medikamentöser Tumortherapie. Aktueller Status.

In the past multiple mechanisms could be identified that are involved in anticancer drug resistance; however, diagnostic assays for prediction of therapy response to classical cytostatic drugs did not enter routine clinical diagnostics. Only when new targeted drugs, e.g. tyrosine kinase inhibitors or therapeutic antibodies, were introduced in oncology were diagnostics for prediction of therapy response routinely preformed. First and foremost this was the result of the development of highly standardized techniques, i.e. exact mutation analysis in functional relevant codons of genes encoding signal proteins of cancer-related signal transduction pathways targeted by the new drugs. Due to increasing costs of health systems, in the future predictive diagnostics will probably become more and more important. Therefore, it will be necessary to develop improved diagnostic assays for prediction of individual therapy response.

A snapshot of microarray-generated gene expression signatures associated with ovarian carcinoma.

It was hypothesized that analysis of global gene expression in ovarian carcinoma can identify dysregulated genes that can serve as molecular markers and provide further insight into carcinogenesis and provide the basis for development of new diagnostic tools as well as new targeted therapy protocols. By applying bioinformatics tools for screening of biomedical databases, a gene expression profile databank, specific for ovarian carcinoma, was constructed with utilizable data sets published in 28 studies that applied different array technology platforms. The data sets were divided into four compartments: (i) genes associated with carcinogenesis: in 14 studies, 1881 genes were extracted, 75 genes were identified in more than one study, and only 4 genes (PRKCBP1, SPON1, TACSTD1, and PTPRM) were identified in three studies. (ii) Genes associated with histologic subtypes: in four studies, 463 genes could be identified, but none of them was identified in more than a single study. (iii) Genes associated with therapy response: in seven studies, 606 genes were identified from which 38 were differentially regulated in at least two studies, 3 genes (TMSB4X, GRN, and TJP1) in three studies, and 1 gene (IFITM1) in four studies. (iv) Genes associated with prognosis and progression: 254 genes were found in seven studies. From these genes, merely three were identified in at least two different studies. This snapshot of available gene expression data not only provides independently described potential diagnostic and therapeutic targets for ovarian carcinoma but also emphasizes the drawbacks of the current state of global gene expression analyses in ovarian cancer.

[YB-1 as a potential target in cancer therapy]. / YB-1 als potenzielles Ziel für die Tumortherapie.

The 42-kDa multifunctional cellular protein Y-box protein 1 (YB-1) is expressed in various cancers. It is localized in the cytoplasm as well as in the nucleus. In particular, YB-1 is localized in the nuclear compartment following cellular stress, such as radiation, drug treatment, hyperthermia, or viral infection. Within the nucleus, YB-1 can act as a transcription factor, and it is involved in the regulation of important cancer-associated genes. For example, YB-1 triggers the expression of Her-2 and estrogen receptor alpha (ERalpha) in breast cancer. Thus, nuclear YB-1 appears to be a potential target for the inhibition of Her-2- and ERalpha-dependent proliferation signals, particularly with regard to resistance to Her-2-targeting drugs such as trastuzumab. In some cancers, YB-1 may be involved in regulating MDR1/P-glycoprotein, mediating classical multidrug resistance (MDR). Furthermore, YB-1 is involved in the replication of adenovirus type 5, a commonly used vector in gene therapy. Thus, YB-1 can trigger an "oncolytic" effect in YB-1 nuclear positive cancer cells treated with adenoviruses. Besides its impact as a prognostic factor, in the future the diagnostics of cellular YB-1 status may provide the basis for a virotherapy or a gene therapy incorporating adenoviruses.

Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines.

BACKGROUND: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone. METHODS: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively. RESULTS: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells. CONCLUSIONS: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines.

Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257.

A newly established gastric carcinoma cell line (EPG85-257P) exhibited a high sensitivity to mitoxantrone (DHAD) as determined by a monolayer proliferation assay. The concentration to inhibit cell growth to 50% of controls (IC50) was 0.0022 micrograms/ml culture medium. The cells were continuously incubated for more than 4 months in the presence of stepwise increased concentrations of DHAD, and the IC50 was increased to 0.41 micrograms/ml, i.e., 186.4-fold. This resistant variant was named EPG85-257RNOV. The EPG85-257RNOV cells became cross-resistant to Adriamycin with enhanced IC50 by 10.5-fold and to daunomycin with enhanced IC50 by 3.9-fold. No distinct resistance was observed to vinblastine, vincristine, and colchicine. Verapamil (10(-6), 4 X 10(-6) and 10(-5) M) and cyclosporin A (10(-6), 3 X 10(-6) and 10(-5) M) did not reverse DHAD resistance. As shown by immunocytochemistry (monoclonal antibodies: C219 and JSB-1) and Northern blot analysis, DHAD resistance was not associated with the appearance of the multidrug resistance (MDR)-associated (Mr 170,000) P-glycoprotein or the overexpression of P-glycoprotein mRNA. The data indicate a chemoresistance pattern unlike typical MDR (often called "atypical" MDR). The phenotypes of parent and resistant EPG85-257 cells were compared using interference contrast microscopy, electron microscopy, and immunocytochemistry. After DHAD application the following structural characteristics were found to be associated with emergence of resistance: (a) intensive formation of surface vesicles in the resistant variant. Such vesicles were almost absent in sensitive cells; (b) the vesicles contained the selecting DHAD which was visualized by its blue color; and (c) in electron microscopy the vesicles were formed by an inner and an outer double membrane, presumably derived from the plasmalemma. These observations suggest a complex cellular mechanism responsible for DHAD resistance which includes formation of membrane vesicles, vesicular drug binding, and drug compartmentalization.

Induction of the ABC-transporters Mdr1/P-gp (Abcb1), mrpl (Abcc1), and bcrp (Abcg2) during establishment of multidrug resistance following exposure to mitoxantrone.

Resistance to mitoxantrone is often associated with enhanced drug efflux mediated by members of the superfamily of adenosinetriphosphate-binding cassette (ABC) transporters, i.e. MDR1/P-gp (ABCB1), MRP1 (ABCC1), or BCRP (ABCG2). So far it is unclear whether the same ABC-transporter is always activated from the beginning of mitoxantrone treatment to the end of drug exposure. Here, we demonstrate that the expression of all three extrusion pumps is induced by increasing levels of mitoxantrone resistance, but in the end, merely the overexpression of a dominant single drug transporter, i.e. Mdr1/P-gp, is realized. This upregulation of Mdr1/P-gp was reflected by amplification of the Mdr1/P-gp encoding gene. Short mitoxantrone exposure demonstrated that upregulation of two different transporters, Mdr1/P-gp and Bcrp, was induced. The data indicate that mitoxantrone treatment influences the expression of several ABC-transporters, but in the end, merely a single extrusion pump will be dominant.

Cloning and characterization of human cDNAs encoding a protein with high homology to rat intestinal development protein OCI-5.

We constructed a lambda complementary DNA expression library from the mitoxantrone-resistant human gastric carcinoma cell line EPG85-257RNOV. The library was screened by differential hybridization (resistant cell line against non-resistant cell variant). By this procedure we found five independent cDNA clones representing one single gene that has much higher expression in the mitoxantrone-resistant cell line EPG85-257RNOV than in the non-resistant variant EPG85-257P. One of the cDNA clones (MXR7) contains a complete open reading frame (ORF) encoding a 580-amino acid polypeptide. Amino acid and nucleotide sequence analysis revealed that this gene codes the human variant of a rat intestinal development protein OCI-5.

Cloning of a human cDNA encoding a protein with high homology to yeast methionyl-tRNA synthetase.

A composite 2779-bp cDNA that encodes human cytoplasmic methionyl-tRNA synthetase (MetRS) has been constructed from partial cDNA clones derived from the gastric carcinoma cell line EPG85-257RNOV and the nucleotide sequence has been determined. The open reading frame (ORF) encodes a 900 amino acid (aa) protein with a predicted molecular mass of 101 kDa. Northern blotting analysis of total RNA extracted from human gastric carcinoma cells demonstrated a single band with a mobility corresponding to a size of 3.0 kb.

DFNA5 (ICERE-1) contributes to acquired etoposide resistance in melanoma cells.

Resistance to drug treatment is a common observation in malignant melanoma. In order to analyze alterations in mRNA expression profiles associated with drug resistance in melanoma cells we previously established a panel of various drug-resistant cell variants derived from the human melanoma line MeWo and compared the mRNA expression profiles by a differential display technique. By that approach it could be demonstrated that the expression level of a mRNA encoded by a gene found to be mutated in non-syndromic hearing impairment, DFNA5 (ICERE-1), was distinctly decreased in the 33-fold etoposide-resistant melanoma cell line MeWo ETO 1. To evaluate the hypothesis that a decrease in DFNA5 mRNA expression level contributes to the acquired etoposide resistance phenotype exhibited by MeWo ETO 1 cells, this drug-resistant line was stably transfected with the DFNA5-encoding cDNA. Transfected clones showed a 30-35% reduced etoposide susceptibility by comparing the IC(25), IC(50) and IC(75) values of these clones with those displayed by the non-transfected, etoposide-resistant melanoma cell line MeWo ETO 1 and controls. Furthermore, etoposide exposure of stable DFNA5 transfectants resulted in an increase of caspase-3-mediated apoptotic events in DFNA5-transfected clones in comparison to MeWo ETO 1 cells and controls. The data therefore demonstrate that a decrease in DNFA5 mRNA expression level is associated with an increased etoposide resistance in melanoma cells due to an elevated cellular susceptibility to trigger a caspase-3-depending signal pathway leading to programmed cell death.

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.

Kinetic characterization of ribozymes directed against the cisplatin resistance-associated ABC transporter cMOAT/MRP2/ABCC2.

The enhanced expression of the human ABC transporter, cMOAT (MRP2/ABCC2), is associated with resistance of tumor cells against platinum-containing compounds, such as cisplatin. Therefore, cMOAT represents an interesting candidate factor for modulation of antineoplastic drug resistance. Two different hammerhead ribozymes, which exhibit high catalytic cleavage activities towards specific RNA sequences encoding cMOAT, were designed. Cleavage sites of these ribozymes are the GUC sites in codons 704 and 708 of the open reading frame in the cMOAT-specific mRNA molecule. Hammerhead ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. cMOAT-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using RNA prepared from the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS overexpressing the cMOAT-encoding transcript. In a cell-free system, both anti-cMOAT ribozymes cleaved their substrate in a highly efficient manner at a physiologic pH and temperature. The cleavage reaction was dependent on time and ribozyme:substrate ratio for determining specific kinetic parameters.

Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance-associated ABC transporter BCRP/MXR/ABCG2.

Breast cancer resistance protein (BCRP) is a recently identified new member of the superfamily of ATP-binding cassette transporters. BCRP is a "half transporter" that may homo- or heterodimerize to form an active transport complex. A considerable overexpression of BCRP was reported from various atypical multidrug-resistant tumor cell lines, in particular from those which were established by treatment with mitoxantrone. Thus, BCRP represents a very interesting candidate molecule for reversal of a drug-resistant phenotype. Six hammerhead ribozymes directed against the BCRP-encoding mRNA were designed and tested for their ability to cleave their target molecule. The anti-BCRP ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. BCRP-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using total RNA prepared from the atypical multidrug-resistant gastric carcinoma cell line EPG85-257RNOV exhibiting a high BCRP mRNA expression level. One anti-BCRP ribozyme was found to show a very high endoribonucleolytic cleavage activity at physiologic pH and temperature. This ribozyme was characterized in a cell-free system with regard to its specific kinetic parameters using large target molecules.

Selection of a high activity ribozyme against cytostatic drug resistance-associated glypican-3 using an in vitro assay containing total tumor RNA.

We tested 11 hammerhead ribozymes for their ability to bind and cleave RNA transcripts of the cytostatic drug resistance-associated glypican-3-encoding gene (GPC3, MXR7, OCI-5). To select the optimum target sequence, the activity of each hammerhead ribozyme was tested in a short in vitro assay using truncated RNA substrates without time-consuming cloning procedures. Glypican-3-derived RNA was cleaved effectively by 3 of 11 hammerhead ribozymes. One of these, the hammerhead ribozyme Rz967, recognized the GUC sequence at nucleotides +965 to +967 of the open reading frame of the glypican-3-encoding mRNA. Rz967 cleaved in vitro-transcribed fragments derived from glypican-3 mRNA (nucleotides +803 to +1036) most efficiently. Cleavage efficiency was confirmed by a rapid in vitro assay using full-length total tumor cell RNA. We were able to demonstrate that this new in vitro assay is suitable for the selection of hammerhead ribozymes that have the capability to cleave glypican-3-encoding mRNA in human tumors.

Involvement of the DNA mismatch repair system in antineoplastic drug resistance.

Different types of antineoplastic drugs, such as the alkylating agents busulfan, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, procarbazine and temozolomide, the antimetabolites, mercaptopurine and 6-thioguanine, the platinum compounds carboplatin and cisplatin, the anthracycline doxorubicin and the epipodophyllotoxine etoposide act by damaging DNA directly or indirectly. Increasing evidence has shown that tumours could acquire resistance to these drugs by loss of DNA-mismatch repair (MMR) activity. This phenomenon is caused by a decreased MMR-dependent stimulation of signal-transduction pathways causing programmed cell death. Simultaneously, the mutation rate in MMR-deficient tumours is increasing, which could lead to additional secondary drug/resistance phenotypes to other antineoplastic agents. In addition to this, an enhanced mutation rate may contribute to increased phenotypic variation and therefore the clinical aggressiveness of primary tumours and their metastases.

Expression of a glypican-related 62-kDa antigen is decreased in hepatocellular carcinoma in correspondence to the grade of tumor differentiation.

A monoclonal mouse antibody, Be-F4, was generated by means of immunization with a synthetic oligopeptide. In Western blots, this antibody recognizes an antigen with an apparent molecular mass of 62 kDa, termed p62. Immunohistochemical analysis of p62 in hepatocellular carcinoma (HCC) specimens (n=33) and in corresponding non-cancerous liver tissue was performed using monoclonal antibody Be-F4. All non-neoplastic hepatic cells showed, without exception, a moderate or strong staining intensity of the 62-kDa antigen, recognized by Be-F4. In contrast to the non-neoplastic hepatocytes, the cellular p62 content was unambiguously reduced in all malignant cells. The extent of decrease of p62 corresponded to the grade of histological differentiation of HCC cells (P<0.001). Using a semiquantitative scoring system, the median of p62 expression, which was 2.1 for normal hepatocytes, was significantly reduced to 1.2 for G1, 1.0 for G2, and 0.2 for G3 HCCs. These data suggest that neoplastic transformation is associated with a reduced p62 content.

New silicon compounds as resistance modifiers against multidrug-resistant cancer cells.

The efficiency of chemotherapy is often decreased by the development of resistance of cancer cells to cytostatic drugs. This phenomenon is in most cases caused by the activity of the various ABC transporters, multidrug-resistance (MDR) gene-encoded p-glycoproteins, that pump anticancer drugs out of the cells. The inhibition of the activities of the MDR proteins MDR1 and MRP was investigated via the administration of two new organosilicon compounds, alis-409 and alis-421. The study was focused on the inhibition of MDR by blocking the ADR1 gene expression and through the inhibition of the pump-function of mdr-p-glycoprotein, in human breast cancer cell lines expressing mrp and prostate cancer cell line (PC-3). Apoptosis induction and the interaction between epirubicin and the silicon-substituted compounds were studied in human MDR-1 gene-transfected mouse lymphoma and its parent cell line, Colo320/MDR-LRP and sensitive subline Colo205, by means of rhodamine 123 accumulation. The activity of MRP1 p-glycoprotein was studied in human breast cancer cell lines such as HTB-26/MRP1 and two MRP-negative breast cancer cell lines, T47D and MCF7, by carboxyfluorescein accumulation, and on a stomach cancer cell line. The activity of MRP in 257P/MDR and its drug-sensitive derivative were studied in human stomach cancer cells by daunorubicin accumulation in a flow cytometer. The two representative organosilicon derivatives, alis-409 and alis-421, showed antiproliferative effects without apoptosis induction. The drug accumulation in the human MDR1 gene-transfected mouse lymphoma cells was increased without down-regulation of the MDR1 gene expression tested by RT-PCR assay. The rhodamine uptake was increased in L5178/MDR1 and Colo320/MDR1-LRP, but not drug-sensitive human breast cancer MCF-7 and T47D, and L5178 mouse lymphoma parent cells in the presence of alis-409 and alis-421. The MRP-mediated carboxyfluorescein accumulation in HTB-26/MRP human breast cancer cells and daunorubicin accumulation in human stomach cancer cells 257P/MDR were not modified by these alis compounds. A synergistic interaction between epirubicin and the silicon-substituted resistance modifiers was found only in MDR1-mediated MDR in the case of colo-320/MDR1-LRP cells and mouse lymphoma cells transfected with the human MDR1 gene. The results indicate that the organosilyl derivatives specifically act on MDR1 p-glycoprotein 170. The alis compounds act on pgp170 in a way which is similar to verapamil isomers.

Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells.

The therapy of advanced cancer using chemotherapy alone or in combination with radiation or hyperthermia yields an overall response rate of about 20-50%. This success is often marred by the development of resistance to cytostatic drugs. Our aim was to study the global analysis of protein expression in the development of chemoresistance in vitro. We therefore used a cell culture model derived from the gastric carcinoma cell line EPG 85-257P. A classical multidrug-resistant subline EPG85-257RDB selected to daunorubicin and an atypical multidrug-resistant cell variant EPG85-257RNOV selected to mitoxantrone, were analysed using two-dimensional electrophoresis in immobilized pH-gradients (pH 4.0-8.0) in the first dimension and linear polyacrylamide gels (12%) in the second dimension. After staining with coomassie brilliant blue, image analysis was performed using the PDQuest system. Spots of interest were isolated using preparative two-dimensional electrophoresis and subjected to microsequencing. A total of 241 spots from the EPG85-257RDB-standard and 289 spots from the EPG85-257RNOV-standard could be matched to the EPG85-257P-standard. Microsequencing after enzymatic hydrolysis in gel, mass spectrometric data and sequencing of the peptides after their fractionation using microbore HPLC identified that two proteins annexin I and thioredoxin were overexpressed in chemoresistant cell lines. Annexin I was present in both the classical and the atypical multidrug-resistant cells. Thioredoxin was found to be overexpressed only in the atypical multidrug-resistant cell line.

Expression of the novel mitoxantrone resistance associated gene MXR7 in colorectal malignancies.

It was shown previously that a glypican encoding gene (MXR7/GPC3/OCl-5) was associated with mitoxantrone resistance in vitro. This study describes and investigation of an association between multidrug resistance and MXR7 in surgical cryo-specimens of 51 gastrointestinal tumors. The mRNA expression levels differ widely according to tumor species. In primary colorectal cancers, the level of MXR7 mRNA expression correlated loosely with the mRNA expression level of the multidrug-resistance-associated protein (MRP). In colorectal metastases an elevated MXR7 mRNkA expression level was observed when compared to primary colorectal carcinomas.

Surgical procedures for primary, metastatic or adjacent parotid tumours.

OBJECTIVE: The retrospective analysis of the surgical procedures in primary parotid and metastatic or adjacent parotid tumors. PATIENTS AND METHODS: Retrospective review of the records of 145 patients operated on for primary, metastatic or adjacent parotid tumors revealed 85 patients with benign tumors, 24 with primary malignant tumors, 19 with squamous skin carcinomas, 12 with skin melanomas, 3 with basocellular carcinomas and 2 with sarcomas of the parotid region. The analysis included the type of parotidectomy, the need for facial nerve sacrifice (FNS), type of neck dissection and soft part reconstruction. RESULTS: Superficial parotidectomy was performed in 81% of the benign parotid tumors and 100% of skin melanomas. Total parotidectomy was frequent in malignant parotid tumors (62%), epidermoid skin tumors (64%) and in basocellular/sarcomas of the parotid region (80%). Skin graft or flaps was infrequent in primary malignant tumors (12.5%), and frequent in epidermoid skin tumors (74%), melanomas (58%) and basocellular/sarcomas (100%). FNS was necessary in primary malignant (25%), adjacent epidermoid (37%), melanomas (17%) and basocellular/sarcomas (80%). Details on neck dissections are provided. CONCLUSIONS: Superficial parotidectomy was an adequate procedure for most benign parotid tumors and for melanoma patients. In primary malignant and adjacent or metastatic skin tumors, total parotidectomy, neck dissection and soft part reconstruction were frequent procedures. FNS and soft part reconstruction should be anticipated more frequently in squamous/basocellular skin tumors or sarcomas adjacent to the parotid gland.