Elementy prawnonaturalne w stosowaniu Konstytucji RP [Natural-Law Elements in Application of the Constitution of the Republic of Poland]

Recognizing inherent and inalienable nature of dignity and universality of certain values, the Constitution of the Republic of Poland, introduces to the foundations of Polish legal system some elements of natural law which may be used for application of the Basic Law. Constitutional recognition of these elements only makes sense on the assumption of their cognizability. Therefore, as an important element of constitutional concept of natural law is taken the recognition of the argument of cognitivism according to which moral assessments may have the nature of judgments and truth qualification (they may be true or false). In the course of application of the constitution, norms of natural-law character and natural-law justification. Since dignity and the essence of freedoms and rights based on dignity are the only inviolable values recognized by the constitution, the arguments of natural law lead to a far-going reinterpretation of constitutional norms. The norm of natural law protecting inviolable values will have precedence in the event of collision with norms protecting other values, also with constitutional norms. Even if such a norm is formulated on the basis of the provisions of the constitution, in fact natural law is given higher rank than elements based only on enacted law. Despite that, reliability of a legally established order does not seem to be radically endangered. Konstytucja RP uznając przyrodzoność i niezbywalność godności oraz uniwersalność niektórych wartości, wprowadza do podstaw całego polskiego systemu prawnego elementy prawnonaturalne, które mogą być wykorzystane w stosowaniu konstytucji. Konstytucyjne uznanie takich elementów ma sens przy założeniu ich poznawalności, stąd istotnym elementem konstytucyjnej koncepcji prawa naturalnego jest uznanie tezy kognitywizmu głoszącej, że oceny moralne mogą mieć charakter sądów i kwalifikację prawdziwościową – mogą być prawdziwe lub fałszywe. W procesie stosowania konstytucji mogą być formułowane normy o charakterze prawnonaturalnym oraz uzasadnienia prawnonaturalne. Ponieważ jedynymi wartościami uznanymi w konstytucji za nienaruszalne jest godność oraz istota wolności i praw, których godność jest źródłem, argumentacja prawnonaturalna może prowadzić do daleko idącej reinterpretacji norm konstytucyjnych. Norma prawnonaturalna chroniąca warunki konieczne poszanowania godności lub istoty wolności i praw będzie miała pierwszeństwo w razie kolizji z normami chroniącymi inne wartości, także z normami konstytucyjnymi. Choć norma taka będzie formułowana na podstawie przepisów konstytucji, to jednak faktycznie prawo naturalne uzyskuje wyższą rangę od elementów opartych jedynie na stanowieniu. Mimo tego bezpieczeństwo prawne nie wydaje się być radykalnie zagrożone. O prawnonaturalnym charakterze normy i uzasadnienia decyduje oparcie w ocenach moralnych, które pretendują do bycia sądami, do bycia prawdziwymi lub fałszywymi. Z punktu widzenia stosowania konstytucji i wymogu intersubiektywnej komunikowalności i kontrolowalności dookreślania treści prawnonaturalnych, dla nadania ocenom charakteru sądów istotny jest kształt procedur i argumentacji prowadzących do tego dookreślenia. Powinny to być procedury i argumentacja typowe dla dyskursu mającego na celu sformułowanie prawdziwych sądów. Maksymalizowana powinna być dyskursywność takich procedur, a podstawą rozstrzygnięcia powinien być, jeśli to tylko możliwe, konsens. Nie mogą być uważane za rozstrzygające argumenty odwołujące się do woli indywidualnej lub zbiorowej, reakcji emocjonalnych, stopnia rozpowszechnienia danej oceny czy do tradycji kulturowej

Quantitative Analysis of the Anti-Proliferative Activity of Combinations of Selected Iron-Chelating Agents and Clinically Used Anti-Neoplastic Drugs

<div><p>Recent studies have demonstrated that several chelators possess marked potential as potent anti-neoplastic drugs and as agents that can ameliorate some of the adverse effects associated with standard chemotherapy. Anti-cancer treatment employs combinations of several drugs that have different mechanisms of action. However, data regarding the potential interactions between iron chelators and established chemotherapeutics are lacking. Using estrogen receptor-positive MCF-7 breast cancer cells, we explored the combined anti-proliferative potential of four iron chelators, namely: desferrioxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), (<i>E</i>)-<i>N′</i>-[1-(2-hydroxy-5-nitrophenyl)ethyliden] isonicotinoyl hydrazone (NHAPI), and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), plus six selected anti-neoplastic drugs. These six agents are used for breast cancer treatment and include: paclitaxel, 5-fluorouracil, doxorubicin, methotrexate, tamoxifen and 4-hydroperoxycyclophosphamide (an active metabolite of cyclophosphamide). Our quantitative chelator-drug analyses were designed according to the Chou-Talalay method for drug combination assessment. All combinations of these agents yielded concentration-dependent, anti-proliferative effects. The hydrophilic siderophore, DFO, imposed antagonism when used in combination with all six anti-tumor agents and this antagonistic effect increased with increasing dose. Conversely, synergistic interactions were observed with combinations of the lipophilic chelators, NHAPI or Dp44mT, with doxorubicin and also the combinations of SIH, NHAPI or Dp44mT with tamoxifen. The combination of Dp44mT with anti-neoplastic agents was further enhanced following formation of its redox-active iron and especially copper complexes. The most potent combinations of Dp44mT and NHAPI with tamoxifen were confirmed as synergistic using another estrogen receptor-expressing breast cancer cell line, T47D, but not estrogen receptor-negative MDA-MB-231 cells. Furthermore, the synergy of NHAPI and tamoxifen was confirmed using MCF-7 cells by electrical impedance data, a mitochondrial inner membrane potential assay and cell cycle analyses. This is the first systematic investigation to quantitatively assess interactions between Fe chelators and standard chemotherapies using breast cancer cells. These studies are vital for their future clinical development.</p></div

Chelator and anti-cancer drug interactions in MCF-7 cells as a function of activity.

<p>The computer simulations of the combination index (<i>CI</i>)-cellular proliferation (fraction affected - F<i>a</i>) dependency were obtained using CalcuSyn 2.0 software following a 72 h incubation of MCF-7 cells with combinations of the studied compounds at concentrations corresponding to their IC₅₀ values and IC₅₀ fractions and multiples (1/8, 1/4, 1/2, 1, 2, and 4). Results are means of <i>n</i>≥4 experiments.</p

Interactions of NHAPI and Dp44mT with tamoxifen in MCF-7, T47D and MDA-MB-231 breast cancer cells as a function of activity.

<p>The computer simulations of the combination index (<i>CI</i>)-cellular proliferation (fraction affected - F<i>a</i>) dependency were obtained using CalcuSyn 2.0 software. These studies were performed using 72 h incubations of MCF-7, T47D, or MDA-MB-231 cells with combinations of the studied compounds at concentrations corresponding to their IC₅₀ values and IC₅₀ fractions and multiples (1/8, 1/4, 1/2, 1, 2, and 4). Results are means of <i>n</i>≥4 experiments.</p

Anti-proliferative activity of the examined agents in MDA-MB-231 cells.

<p>The studied agents (NHAPI and Dp44mT and their fully coordinated iron (Fe) or copper (Cu) complexes and tamoxifen (TMX)) were incubated with MDA-MB-231 cells for 72 h at 37°C. Cell viability was determined using the neutral red uptake assay and the IC₅₀ values (the concentration inducing a 50% reduction in proliferation compared to the control) were calculated using CalcuSyn 2.0 software; <i>n</i>≥4 experiments. Statistical significance (ANOVA): *<i>p<</i>0.05, **<i>p<</i>0.01, ***<i>p</i><0.001 as compared to the same uncomplexed chelator.</p

Dissipation of the inner mitochondrial membrane potential (ΔΨ_m).

<p><b>(A)</b> Fluorescence microscopy of MCF-7 cells stained with the JC-1 probe after 72 h incubations of MCF-7 cells with combinations of NHAPI or its Fe complex with TMX. The scale bars represent 100 µm. Phase-contrast photomicrographs showing morphology of the same cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088754#pone.0088754.s011" target="_blank">Fig. S11</a>. <b>(B, C)</b> Flow cytometric analyses of MCF-7 cells treated with combinations of NHAPI or its Fe complex with TMX were stained with the JC-1 probe. Results in <b>(A)</b> are typical of <i>n</i>≥4 experiments; <b>(B, C)</b> Mean±SD (<i>n</i>≥4 experiments). Statistical significance (ANOVA): *<i>p<</i>0.05, **<i>p<</i>0.01, ***<i>p<</i>0.001 as compared to the control (untreated) group.</p

Combinations of NHAPI or its Fe complex with TMX in MCF-7 cells behave synergistically.

<p>The combination index-cellular proliferation dependency was calculated using CalcuSyn 2.0 software. These studies were performed using 72 h incubations of the studied agents at concentrations corresponding to the various IC₅₀ multiples and fractions; <i>CI</i> <1, ≈1, or >1 indicate synergism, an additive effect, or antagonism, respectively. Results are means of <i>n</i>≥4 experiments.</p

Combinations of NHAPI or its Fe complex with TMX caused decreases in proliferation and G₁-S cell cycle arrest.

<p>The proliferation dynamics were measured as the electrical impedance of MCF-7 cells <b>(A, B, C and D)</b> and were monitored using an xCELLigence System for 72 h. Cell cycle analyses <b>(E and F)</b> were performed following a 72 h/37°C incubation with the agent using flow cytometry following propidium iodide staining. Results are: <b>(A–D)</b> means of <i>n</i>  = 3 experiments and <b>(E, F)</b> mean±SD (<i>n</i>≥4 experiments). Statistical significance (ANOVA): *<i>p<</i>0.05, **<i>p<</i>0.01, ***<i>p<</i>0.001 as compared to the control (untreated) group.</p

Line drawings of the structures of the studied compounds.

<p>Metal chelators: desferrioxamine (DFO); salicylaldehyde isonicotinoyl hydrazone (SIH); (<i>E</i>)-<i>N′</i>-[1-(2-hydroxy-5-nitrophenyl)ethyliden] isonicotinoyl hydrazone (NHAPI); and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Anti-neoplastic agents: paclitaxel (PTX); 5-fluorouracil (5FU); doxorubicin (DOX); methotrexate (MTX); 4-hydroperoxycyclophosphamide (4HC); and tamoxifen (TMX).</p

Quantitative assessments of the anti-proliferative activity of (A) combinations of metal-chelating agents and (B) their iron (Fe) and copper (Cu) complexes that were formed at an metal binding equivalent of 1 in MCF-7 cells.

<p>All of the studied compounds and their combinations were incubated with MCF-7 cells for 72 h at 37°C at a concentration corresponding to their IC₅₀ value. The combination index (<i>CI</i>) values were calculated using the Chou and Talalay method from <i>n</i>≥4 experiments using CalcuSyn 2.0 software; <i>CI</i> <1, ≈1, or >1 indicate synergism, an additive effect, or antagonism, respectively. Statistical significance (ANOVA): *<i>p<</i>0.05, **<i>p<</i>0.01, ***<i>p</i><0.001 as compared to the same combination with uncomplexed parent chelator.</p