Gene expression of metalloproteinase 11, claudin 1 and selected adhesion related genes in papillary thyroid cancer

Wstęp: Nadekspresja genów kodujących białka uczestniczące w adhezji komórkowej może być związana z cechami inwazyjności i możliwością przerzutów. Celem pracy było porównanie poziomu ekspresji wybranych genów w rakach brodawkowatych tarczycy (PTC, papillary thyroid carcinoma) w odniesieniu do odpowiadających im tkanek prawidłowych przy użyciu reakcji Q-PCR w czasie rzeczywistym. Materiał i metody: Materiał do badań stanowił całkowity RNA wyizolowany z 38 tkanek raka tarczycy i odpowiadających im tkanek zdrowych. Całkowity RNA w ilości 0,5 μg wykorzystywano w reakcji odwrotnej transkrypcji, z kolei cDNA stanowił matrycę w reakcji Q-PCR. Jako kontrolę wewnętrzną zastosowano gen GUS, którego ekspresja była stabilna we wszystkich analizowanych tkankach. Wyniki: Analizowane geny zostały wytypowane metodą mikromacierzy DNA jako charakterystyczne dla różnicowania PTC od tkanki zdrowej. Poziom ekspresji analizowanych genów, badany poprzez ilościową ocenę odpowiednich transkryptów, był podwyższony w PTC, a jej średnia wartość wynosiła: 1,08 dla CLDN1; 3,98 dla LRP4; 4,57 dla FLRT3; 26,6 dla MRC2; 2,76 dla NRCAM; 0,76 dla MMP11 i 1,35 dla EVA1 w jednostkach arbitralnych, podczas gdy w kontrolnych tkankach zdrowej tarczycy wynosiła odpowiednio: 0,145; 0,7; 0,74; 7,9; 0,85; 0,09; 0,396 jednostek. Dla potwierdzenia ich nadekspresji metodą Q-PCR zastosowano test Kołmogorowa-Smirnowa. Dla wszystkich genów różnice między tkanką prawidłową a PTC były znamienne statystycznie. Wnioski: Spośród badanych genów największe różnice w ekspresji między tkanką PTC a zdrową tkanką tarczycy wykazały geny CLDN1 (klaudyna 1) i MMP11 (metaloproteinaza 11). Gen MMP11 można zaliczyć do grupy charakterystycznej dla nowotworów złośliwych tarczycy, lecz bez wskazania na jego rodzaj, ponieważ istotny wzrost jego ekspresji występuje również w raku rdzeniastym tarczycy. Wydaje się, że gen CLDN1 mógłby być markerem raka brodawkowatego, jednak potrzebna jest weryfikacja przeprowadzonej analizy na dodatkowym materiale.Introduction: Metastasis and invasiveness of thyroid carcinoma might be correlated with over-expression of genes which encode proteins responsible for cellular adhesion. The aim of our study was to compare by means of real-time Q-PCR expression levels of chosen genes in papillary thyroid carcinoma (PTC) with respect to normal thyroid tissues. Material and methods: Total RNA was isolated from 38 paired normal and PTC samples. 0.5 μg of RNA was used in the reverse transcription reaction. cDNA was further used in Q-PCR reaction. As endogenous control we used GUS gene, as its expression was stable in all analysed samples. Results: The analyzed genes were found by microarray studies as characteristic in differentiated papillary carcinoma and normal tissue. The levels of gene expression, estimated by quantitative analysis of particular transcripts were increased in papillary thyroid carcinoma with the following average values: 0.76 for MMP11; 1.08 for CLDN1; 3.98 for LRP4; 4.57 for FLRT3; 26.6 for MRC2; 2.76 for NRCAM; and 1.35 for EVA1 (arbitrary units), whereas in normal thyroid tissues treated as control the respective values were: 0.09; 0.145; 0.7; 0.74; 7.9; 0.85 and 0.396 units. To confirm the overexpression of mentioned genes the conservative Kolmogorov- Smirnov test was used. Differences in expression between normal thyroid tissue and PTC were statistically significant. Conclusions: Among the analyzed genes two show the largest difference in expression between papillary thyroid cancer and normal tissue: MMP11 (metalloproteinase 11) and CLDN1 (claudin 1). The MMP11 gene can be characteristic for various malignant thyroid carcinomas, because its increased levels are present also in medullary thyroid carcinomas. It appears that claudin 1 may be used as a marker of PTC, however a verification on independent collection of tumors of this result is required

Antitumor effect of RGD-4C-GG-D(KLAKLAK)2 peptide in mouse B16(F10) melanoma model

Vasculature targeting agents have been tested as cancer therapeutics for the past few years. Such therapy could be accomplished using, for example, bifunctional (two-domain) peptides. RGD-4C-GG-D(KLAKLAK)2, a peptide designed by Ellerby and coworkers (1999) (full sequence: ACDCRGDCFCGGKLAKLAKKLAKLAK), binds selectively to αVβ3 integrin receptors expressed in tumor neovasculature and, after internalization, effectively induces apoptosis of endothelial cells. The aim of this study was to examine if RGD-4C-GG-D(KLAKLAK)2 would efficiently target cells, among them B16(F10), that overexpress αVβ3 receptors, and whether it would be suitable for therapeutic treatment of primary B16(F10) murine melanoma tumors. Thus, the peptide would target two distinct tumor compartments: that formed by endothelium of blood vessels and that made up of neoplastic cells. The therapeutic peptide was recognized and did induce apoptosis in B16(F10) cell line. Tumor growth inhibition was observed following direct intratumoral administration. However, cessation of peptide administration led to rapid tumor growth and death of the animals

Preclinical characterization of CPL302-253, a selective inhibitor of PI3Kδ, as the candidate for the inhalatory treatment and prevention of Asthma.

Asthma is a common chronic inflammatory disease. Although effective asthma therapies are available, part of asthmatic population do not respond to these treatment options. In this work we present the result of development of CPL302-253 molecule, a selective PI3Kδ inhibitor. This molecule is intended to be a preclinical candidate for dry powder inhalation in asthma treatment. Studies we performed showed that this molecule is safe and effective PI3Kδ inhibitor that can impact many immune functions. We developed a short, 15-day HDM induced asthma mouse model, in which we showed that CPL302-253 is able to block inflammatory processes leading to asthma development in vivo

A PDE10A inhibitor CPL500036 is a novel agent modulating striatal function devoid of most neuroleptic side-effects

Background: Phosphodiesterase 10A (PDE10A) is expressed almost exclusively in the striatum and its inhibition is suggested to offer potential treatment in disorders associated with basal ganglia. We evaluated the selectivity, cytotoxicity, genotoxicity, pharmacokinetics and potential adverse effects of a novel PDE10A inhibitor, CPL500036, in vivo. Methods: The potency of CPL500036 was demonstrated by microfluidic technology, and selectivity was investigated in a radioligand binding assay against 44 targets. Cardiotoxicity in vitro was evaluated in human ether-a-go-go related gene (hERG)-potassium channel-overexpressing cells by the patch-clamp method and by assessing key parameters in 3D cardiac spheroids. Cytotoxicity was determined in H1299, HepG2 and SH-SY5Y cell lines. The Ames test was used for genotoxicity analyses. During in vivo studies, CPL500036 was administered by oral gavage. CPL500036 exposure were determined by liquid chromatography–tandem mass spectrometry and plasma protein binding was assessed. The bar test was employed to assess catalepsy. Prolactin and glucose levels in rat blood were measured by ELISAs and glucometers, respectively. Cardiovascular safety in vivo was investigated in dogs using a telemetry method. Results: CPL500036 inhibited PDE10A at an IC(50) of 1 nM, and interacted only with the muscarinic M2 receptor as a negative allosteric modulator with an IC(50) of 9.2 µM. Despite inhibiting hERG tail current at an IC(25) of 3.2 μM, cardiovascular adverse effects were not observed in human cardiac 3D spheroids or in vivo. Cytotoxicity in vitro was observed only at > 60 μM and genotoxicity was not recorded during the Ames test. CPL500036 presented good bioavailability and penetration into the brain. CPL500036 elicited catalepsy at 0.6 mg/kg, but hyperprolactinemia or hyperglycemic effects were not observed in doses up to 3 mg/kg. Conclusion: CPL500036 is a potent, selective and orally bioavailable PDE10A inhibitor with a good safety profile distinct from marketed antipsychotics. CPL500036 may be a compelling drug candidate