[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.

Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells.

Nineteen terpenoids, including macrocyclic diterpenes, diterpenic lactones and other polycyclic diterpenes, steroids and a triterpene isolated from the methanolic extracts of Euphorbia species, were evaluated for their potential antineoplastic activity in various human cancer cell lines that were derived from three tumor entities: gastric (EPG85-257), pancreatic (EPP85-181) and colon (HT-29) carcinomas. Furthermore, different multidrug-resistant variants of these cancer cell lines with over-expression of MDR1/P-gp or no MDR1/P-gp expression were also investigated. In parental drug-sensitive cell lines, the tested compounds showed a moderate/weak antiproliferative effect or were inactive. Most of them were found more effective in drug-resistant cells than in the parental, drug-sensitive ones, and some of them showed high antineoplastic efficacy in classical or atypical drug-resistant cells. The most active compounds were the lathyrane diterpenes latilagascenes C and D, and the diterpenic lactones 3beta-acetoxy-helioscopinolide B and helioscopinolide E which exhibited high antineoplastic activities against the drug-resistant subline EPG85-257RDB derived from gastric carcinoma. In addition, the macrocyclic lathyrane diterpene jolkinol B was found to be highly effective in the multidrug-resistant variant HT-29RNOV.

Adenovirus-based virotherapy enabled by cellular YB-1 expression in vitro and in vivo.

We have earlier described the oncolytic adenovirus vector dl520 that was rendered cancer-specific by deletion of the transactivation domain CR3 of the adenoviral E1A13S protein; this deletion causes antitumor activity in drug-resistant cells displaying nuclear YB-1 expression. We hypothesized that the anticancer activity of dl520 could be further improved by introducing the RGD motif in the fiber knob and by deletion of the adenoviral E1B19K protein (Ad-Delo3-RGD). In this study, the in vitro and in vivo antitumor activity of Ad-Delo3-RGD was investigated focussing on two pancreatic cancer cell lines MiaPaCa-2 and BxPC3 alone and in combination with cytotoxic drugs. Furthermore, luciferin-based bioluminescence imaging was established to study the therapeutic response in vivo. In addition, to confirm the specificity of Ad-Delo3-RGD for YB-1 a tetracycline-inducible anti-YB-1 shRNA-expressing cell variant EPG85-257RDB/tetR/YB-1 was used. This TetON regulatable expression system allows us to measure adenoviral replication by real-time PCR in the absence of YB-1 expression. The results confirmed the YB-1 dependency of Ad-Delo3-RGD and showed that Ad-Delo3-RGD has potent activity against human pancreatic cancer cells in vitro and in vivo, which was augmented by the addition of paclitaxel. However, although high replication capacity was measured in vitro and in vivo, complete tumor regression was not achieved, indicating the need for further improvements to treat pancreatic cancer effectively.

[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.

An overview of cancer multidrug resistance: a still unsolved problem.

Although various mechanisms involved in anticancer multidrug resistance (MDR) can be identified, it remains a major problem in oncology. Beyond that, the introduction of new "targeted" drugs have not solved the problem. On the contrary, it has been demonstrated that the "classical" MDR-associated mechanisms are similar or identical to those causing resistance to these novel agents. These mechanisms include the enhanced activity of drug pumps, i.e. ABC or alternative transporters; modulation of cellular death pathways; alteration and repair of target molecules; and various less common mechanisms. Together they build a complex network of cellular pathways and molecular mechanisms mediating an individual MDR phenotype. Although the application of new high throughput "-omics" technologies have identified multiple new gene-/protein expression signatures or factors associated with drug resistance, so far none of these findings has been useful for creating improved diagnostic assays, for prediction of individual therapy response, or for development of updated chemosensitizers.

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.

Expression of aminopeptidase N/CD13 in human ovarian cancers.

Aminopeptidase N/CD13 (EC 3.4.11.2) is suggested to play a role in cancer cells invasion, and its activity can be inhibited using specific inhibitors. CD13 inhibitors evoke apoptosis of CD13-positive cancer cells. However, expression of CD13 has not been described in specimens obtained from ovarian carcinomas. Thus, in the present study, the expression of CD13 and its significance was examined in samples of ovarian cancers. The analyses were performed on sections originating from 73 tumor samples (43 from primary laparotomies [PL] and 30 from secondary cytoreductions [SCRs]). Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD13. The analysis demonstrated no relationships between the expression of CD13 on one hand and clinical variables and pathologic variables of the patients on the other hand. Expression of CD13 was demonstrated to be significantly more pronounced in samples obtained in PLs as compared to samples from SCRs (P < 0.001). Thus, the data indicate that a potential treatment of ovarian carcinoma with CD13 inhibitors should be performed before chemotherapy or in parallel to first-lapse chemotherapy.

Multivariate analysis of oestrogen receptor alpha, pS2, metallothionein and CD24 expression in invasive breast cancers.

Determination of oestrogen receptor alpha (ER) represents at present the most important predictive factor in breast cancers. Data of ours and of other authors suggest that promising predictive/prognostic factors may also include pS2, metallothionein (MT) and CD24. Present study aimed at determining prognostic and predictive value of immunohistochemical determination of ER, pS2, MT, and CD24 expression in sections originating from 104 patients with breast cancer. An univariate and multivariate analysis was performed. Both univariate and multivariate analyses demonstrated that cytoplasmic-membranous expression of CD24 (CD24c-m) represents a strong unfavourable prognostic factor in the entire group and in most of the subgroups of patients. In several subgroups of the patients also a prognostic value was demonstrated of elevated expression of pS2 and of membranous expression of CD24. Our studies demonstrated that all patients with good prognostic factors (higher ER and pS2 expressions, lower MT expression, CD24c-m negativity) survived total period of observation (103 months). The study documented that cytoplasmic-membranous expression of CD24 represented an extremely strong unfavourable prognostic factor in breast cancer. Examination of the entire panel of the studied proteins permitted to select a group of patients of an exceptionally good prognosis.

Unfavorable prognostic value of CD24 expression in sections from primary and relapsed ovarian cancer tissue.

Expression of CD24 represents a poorly recognized, unfavorable prognostic factor. Expression of the protein is supposed to facilitate extravasation of tumor cells. Our study aimed at examination of prognostic significance of CD24 estimation in samples obtained from primary surgeries (PS) and secondary cytoreductions (SCR) (after chemotherapy) in ovarian cancer patients. The analyses were performed on sections originating from 73 tumor samples. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies against CD24. Kaplan-Meier's analysis showed that a significantly shorter overall survival time and progression-free time was demonstrated to characterize cases with cytoplasmic membranous expression of CD24 (CD24c-m) (P < 0.001). The calculations performed demonstrated also a significantly higher proportion of CD24c-m positive cases in sections from SCR as compared to that from PS (P= 0.04) and in cases of progressive disease as compared to complete response at PS and SCR (P= 0.002 and P= 0.05, respectively). Summing up, in this study, we have demonstrated a negative prognostic significance of a cytoplasmic membranous expression of CD24 in cases of ovarian cancer.

Topoisomerase 1A, HER/2neu and Ki67 expression in paired primary and relapse ovarian cancer tissue samples.

In the present study we examined prognostic value of immunohistochemical estimation of topoisomerase 1A (TOP 1A) and HER-2/neu expression in ovarian cancers treated with platinum-based drugs but not with topotecan and the relation between expression of these proteins on the one hand and intensity of proliferation (Ki67) on the other. The analyses were performed on 73 samples of ovarian carcinoma originating from 43 first-look laparotomies (FLL) and, in 30 cases, from secondary cytoreductions (SCR)(after chemotherapy) from the same patients. In paraffin sections immunohistochemical reactions were performed using antibodies directed to HER-2/neu, TOP 1A and Ki67. Kaplan-Meier's analysis disclosed a shorter overall survival time in cases with augmented expression of TOP 1A at FLL and with higher expression of Ki67 at SCR. A shorter progression-free time was detected in cases with higher proportion of Ki67 positive cells at FLL. No relationship could be disclosed between HER-2/neu expression and the studied clinicopathological parameters. The studies confirmed high value of Ki67 estimation. The augmented expression of TOP 1A was demonstrated to represent an unfavourable prognostic factor. Thus, in cases with elevated expression of TOP 1A application of topotecan-based therapeutic schemes should be considered.

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.

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.

Characterization of the differential protein expression associated with thermoresistance in human gastric carcinoma cell lines.

Resistance to chemotherapeutic agents is one of the major problems faced during palliative therapy of tumor cells. Thus, chemotherapy is frequently combined with other modes of therapy such as radiation therapy and/or hyperthermia. Tumor cells respond to heat stress with development of thermotolerance and the interactions between chemo- and thermoresistance phenomena are not clearly understood. In this paper, we analyze the differential protein expression in vitro in human stomach cancer cells, their chemoresistant and thermoresistant counterparts using proteomics. The immediate aim was to identify sets of proteins that may lead to the development of thermoresistance. Based on these results, we aim to develop functional tests and methods for the modulation of thermoresistance and chemoresistance phenomena that may assist the therapy of inoperable cancers.

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.

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.

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.

Acquired resistance of melanoma cells to the antineoplastic agent fotemustine is caused by reactivation of the DNA repair gene MGMT.

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.