Návrh modelu standardizace popisu pracovních pozic - plánovaných míst v informačním systému SAP R/3 HR

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Ekonomická fakulta. Katedra (155) informatiky v ekonomic

Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

<div><p>The cysteine protease cathepsin B has been causally linked to progression and metastasis of breast cancers. We demonstrate inhibition by a dipeptidyl nitrile inhibitor (compound <b>1)</b> of cathepsin B activity and also of pericellular degradation of dye-quenched collagen IV by living breast cancer cells. To image, localize and quantify collagen IV degradation in real-time we used 3D pathomimetic breast cancer models designed to mimic the <i>in vivo</i> microenvironment of breast cancers. We further report the synthesis and characterization of a caged version of compound <b>1</b>, [Ru(bpy)₂(<b>1</b>)₂](BF₄)₂ (compound <b>2</b>), which can be photoactivated with visible light. Upon light activation, compound <b>2</b>, like compound <b>1</b>, inhibited cathepsin B activity and pericellular collagen IV degradation by the 3D pathomimetic models of living breast cancer cells, without causing toxicity. We suggest that caged inhibitor <b>2</b> is a prototype for cathepsin B inhibitors that can control both the site and timing of inhibition in cancer.</p></div

Changes to the electronic absorption of compound 2 (25 μM) in a 2% acetone aqueous solution at irradiation times, t_irr, of 0, 3, 5, 7, 10 and 15 min (λ_irr ≥ 395 nm); the * denotes the mono-aqua intermediate.

<p>Inset: t_irr = 0.0, 0.5, 1.5, and 3.0 min.</p

Uncaged inhibitor 1 reduces total and pericellular degradation, but not intracellular degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>Quantification of degraded collagen IV in entire 3D volume of MDA-MB-231 and Hs578T structures at 4 days of culture: total degraded collagen IV, black bars; pericellular degraded collagen IV, open bars; and intracellular degraded collagen IV, gray bars. DMSO (negative control), CA074/CA074Me (5 μM each; positive control) and uncaged inhibitor 1. Data shown are from 3 independent experiments (48 fields); * p < 0.05; mean ± SD.</p

Synthesis of the ruthenium-caged, nitrile-based inhibitor 2.

<p>Synthesis of the ruthenium-caged, nitrile-based inhibitor 2.</p

The ruthenium complex <i>cis</i>-[Ru(bpy)₂(MeCN)₂](PF₆)₂ (3) used for caging of inhibitor 2 does not affect degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>(A) Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Panels from left to right are DMSO control, dark-exposed ruthenium complex and light-exposed ruthenium complex. (B) Hs578T breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. See A for further details. (C) Quantification of degraded DQ-collagen IV per cell in MDA-MB-231 (left) and Hs578T (right) structures incubated with DMSO (negative control), dark-exposed ruthenium complex or light-exposed ruthenium complex. Data shown are from 3 independent experiments (48 fields); mean ± SD.</p

Uncaged inhibitor 1 reduces degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>(A) Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Panels from left to right are DMSO control and cysteine protease inhibitors (middle: 5 μM each of CA074 + CA074Me; right: uncaged inhibitor 1). (B) Hs578T breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. See A for further details. (C) Quantification of degraded DQ-collagen IV per cell in MDA-MB-231 (left) and Hs578T (right) structures exposed to DMSO (negative control), CA074/CA074Me (5 μM each; positive control) and uncaged inhibitor <b>1</b>. Data shown are from 3 independent experiments (48 fields); * ≤ 0.05; mean ± SD.</p

Light activation of caged inhibitor 2 reduces degradation of DQ-collagen IV by 3D MAME cultures of breast carcinoma cells.

<p>(A) Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Panels from left to right are DMSO control, dark-exposed caged inhibitor <b>2</b> and light-exposed caged inhibitor <b>2</b>. (B) Hs578T breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. See A for further details. (C) Quantification of degraded DQ-collagen IV per cell in MDA-MB-231 (left) and Hs578T (right) structures incubated with DMSO (negative control), dark-exposed caged inhibitor <b>2</b> or light-exposed caged inhibitor <b>2</b>. Data shown are from 3 independent experiments (48 fields); *p ≤ 0.05; **p ≤ 0.005; mean ± SD.</p

IC₅₀ values^a (μM) for compounds 1 and 2 and dark/light ratio (with and without irradiation) against human CTSB and human breast cancer cell lysates.

<p>^aInhibitory activities were determined with the fluorogenic substrate Z-Arg-Arg-AMC.</p><p>Reactions were conducted in the dark (no irradiation) and light (λ_irr = 395–750 nm) for 45 min with a 250 W tungsten halogen lamp and H₂O filter. The standard deviations were within 40% of the IC₅₀ values and the curves were plotted against log [inhibitor] with 100% activity set equal to the control reaction in the absence of inhibitor. Assay conditions: activator buffer containing 5 mM EDTA, 10 mM DTT, pH 5.2 and assay buffer containing 0.6 mM CaCl₂, 0.6 mM MgCl₂, 25 mM piperazin-N-N‘-bis[2-ethanosulfonic acid (disodium salt)], pH 7.3. DMSO was used as a negative control. Substrate concentration was 150 μM and CTSB concentration was 10 nM. Data shown are from 3 independent experiments.</p><p>IC₅₀ values<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142527#t001fn001" target="_blank">^a</a> (μM) for compounds 1 and 2 and dark/light ratio (with and without irradiation) against human CTSB and human breast cancer cell lysates.</p