Functional evaluation of multidrug resistance transporter activity in surgical samples of solid tumors.

Determination of multidrug resistance (MDR) activity of tumor cells could provide important information for the personalized therapy of cancer patients. The functional calcein assay (MultiDrug Quant Assay, Solvo Biotechnology, Budaörs, Hungary) has been proven to be clinically valuable in hematological malignancies by determining the transporter activity of MDR protein 1 (MDR1, ATP-binding cassette protein [ABC] B1, P-glycoprotein-170) and MDR-related protein 1 (MRP1, ABCC1). In this study, we evaluated if the same functional test was adaptable for the analysis of MDR activity in solid tumors. For this purpose, tissue specimens of human colorectal cancer samples were subjected to limited enzymatic digestion by collagenase to provide a single-cell suspension; dead cells were excluded by 7-aminoactinomycin D staining, and epithelial cancer cells were detected by Cy5-conjugated anti-BerEP4 monoclonal antibody. The transporter functions of MDR1 and MRP1 in viable epithelial cells were assessed by flow cytometry detecting the intracellular accumulation of calcein dye after exposing cells to various MDR inhibitors. Collagenase disintegration preserved the MDR activity and the antigenicity of tumor cells. Thus using the extended calcein assay provided sufficient viable and functionally active tumor cells from surgical biopsies to determine the functional MDR activity. In conclusion, the newly described modified calcein assay may be applicable for evaluating the MDR phenotype in solid tissue specimens from colorectal forceps biopsy to surgical samples.

[Epidermal growth factor receptor (EGFR): therapeutic target in the treatment of lung adenocarcinoma]. / Epidermális növekedési faktor receptor (EGFR): célpont a tüdo adenocarcinomájának kezelésében.

Revolution in biotechnology made possible to identify those gene errors, which via their encoded proteins (mostly kinase enzymes) are key players in tumor development, growth and progression, and could be considered as molecular targets in tumor diagnosis and therapy. Activity of EGFR (epidermal growth factor receptor), an outstanding representative of the regulatory cell surface receptors, can be inhibited by drugs proved for clinical use. In the past year many groups observed that those lung adenocarcinoma cells, which contain activating mutation in the tyrosine kinase domain of EGFR show remarkable sensitivity to anti-EGFR compounds. The basis of the effective therapy is the identification of the mutations. The clinical advantage of EGFR is an example from the coming age of tumor chemotherapy, when the presence of molecular targets will guide the therapeutic choice.

Comparison of ELISA-based tyrosine kinase assays for screening EGFR inhibitors.

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu,Tyr)l:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431 cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.

TT232, a novel signal transduction inhibitory compound in the therapy of cancer and inflammatory diseases.

TT-232 is a structural analogue of somatostatin exhibiting strong and selective growth-inhibitory effects, inhibition of neurogenic inflammation, as well as general anti-inflammatory and analgesic potential without the wide-ranging endocrine side effects of the parent hormone and its "traditional" analogues. The anti-inflammatory action of TT-232 is mediated through the SSTR4 receptor, and its antitumor activity is mediated through the SSTR1 receptor and by the tumor-specific isoform of pyruvate kinase. Its mechanism of action is in line with a new era of molecular medicine called signal transduction therapy, where "false" intracellular or intercellular communication is inhibited or corrected without interfering with basic cell functions and machinery. TT232 has passed phase I clinical trials without toxicity and significant side effects, and phase II studies are running for oncological and anti-inflammatory indications, respectively. This compound has the perspective to become the first drug in molecularly targeted therapy of inflammation where a combined effect of anti-inflammatory, analgesic, and neurogenic inflammation-inhibiting activity can be achieved.