results

Outline of the results - 2012

In this phase of the project contributions have been made to the study of geometric and electronic structure, and of the reactivity of the complexes of metals with potential biomedical utility (Pt, Pd, Re, Mo, Fe), as well as of organic compounds whose biological relevance is either direct or implied by their metabolites or precursors (complex structures based on phenothiazines and on other heterocycles, sulfur- nitrogen-, phosphorus-, and arsenic-based ligands). Also studied were metal centers from metalloenzymes and their models, which by their roles in signaling and oxidative stress are involved in biological mechanisms of action relevant for the project. These have included reactions involving sulfur compoundsof the class responsible for sulfhemoglibenemia, and involving cobalamins, aiming at coordination chemistry issues, linkage isomerism, electronic structures, bond breaking and formation via homo-and heterolytic mechanisms. From the methodological point of view, in terms of molecular modeling techniques were employed, that had not been previously explored in such detail in our country, especially ab initio dynamics in complex transition metal systems and QM / MM techniques on higher spin states. Also studied was the effect of the reference platinum-based compounds and of newly-synthesized substances on the structure and reactivity of metalloproteins involved in oxidative stress, and on intact red cells. Identified were also new ways by which these drugs may induce side effects, and ways to counter them. For the organic compounds containing phenothiazine units, radicals formed in the interaction with hemoglobin were highlighted, whose study by ESR spectroscopy revealed atypical relationship with the radicals already known in literature for phenothiazines, with this opportunity developping a new protocol for preparing the phenothiazine radical, involving oxidation-type enzymes under mild conditions, followed by rapid freezing of the sample and measuring at liquid nitrogen temperature. Also, protein derivatives were synthesized with potential biological activities. Redox processes studied in this setting enabled validation through a set of experiments yet unpublished, of three new methods for evaluation of reactivity involving free radicals in synthetic compounds and natural extracts. These involve the use the ESR technique directly on the ethanolic extracts, a UV-vis method for measuring antioxidant capacity through competitive inhibition of the ascorbate peroxidase reactivity of hemoglobin, and a method for measuring of prooxidant reactivity using hemoglobin and laccase. Compared to the classes compounds whose synthesis and characterization has been reported in the past years, research has been extended in the direction of ferrocene compounds units in order to obtain systems with biological relevance (with several tested on different cell lines), such as 2-arilideneferoceno [e] cyclohexanone derivatives, 3-aryl-3, 3a ,4,5-tetrahidroferoceno [g] indazole and complexes of platinum and palladium of 10-ethyl-4 -diphenylphosphinous-phenothiazine. Besides the direct study of metals with potential biological activity, a new phase of the study metallomics was engaged, that relates to the phenomenon of up-take of metal by cellular mechanisms against drug chemoresistance, and elucidating signaling pathways appeared in tumor and normal cells treated with complexes of platinum, palladium and gallium. Reducing the toxicity of the organometallic compounds displaying cytostatic activity can be achieved via the concomitant use o of cyclodextrins and compounds with regenarative activity, such as flavonoids, which could partially counteract some of the toxic effects of the organometallic compounds. Analytical methods were developed for determining the contents of Pt and Ga from tissues, cultures and cell extracts or fractions and analyses were performed on samples collected by biopsy from tumor tissue and from blood by venopunctionin patients with cervical carcinoma stages I and II under chemotherapy including cisplatin infusion. The amount of Pt was determined in biological samples in order to highlight whether after treatment Pt had been taken up into the cells within the tumor tissue, and the amount of Pt in the cells at 24 hours after infusion was reported. Also assessed was the platinum in the peripheral blood at the same time, reflecting the clearance of the drug from the body. The data were then correlated with the expression of molecules involved in tumor angiogenesis, to determine possible connections between magnitude of Pt insertion in tumor cells and the inhibition of neoangiogenesis, which would be an important prognostic factor of treatment outcome. One will also seek to correlate the Pt concentrations with the general� clinical evolution of the patients. Also followed was the in vitro biological effect on tumor cells of two complexes of gallium. In order to investigate their mechanisms of action, the intracellular levels of gallium were measured for A2780 and A2780cis tumora cell cultures. It was found that the incorporation of gallium is dependent on the level and duration of treatment, is cumulative, is dependent on the nature of substituents within the complexes, and is significantly different for the cisplatin-resistant cells, where gallium is taken up into the cells one order of magnitude more efficiently. This phenomenon may allow for efficient counteraction of chemoresistance using new compounds with a central Ga metal. The higher take-up of gallium upon treatment with compound 2 leads to more significant DNA lesions, located at 8 oxoguanine, oxidized pyrimidines, and methyladenine. PCR has revealed the ability of the two substances to modulate the expression of genes involved in drug resistance: TGF-beta- 1, Bcl-xL and FasLigand. This line of research was followed up with the in vivo study of the effects of administration of gallium complexes. Thus followed were, in laboratory animals (Wistar Rats). changes induced after administration by intraperitoneal injection on the red cell homeostasis, white cells and platelets. Clinical chemistry parameters such as protein and albumin, some enzymes (GOT,GGT, alkaline phosphatase) - with interest for the liver function, as well as uric acid, creatinine and protein nitrogen compounds of interest for the renal function. Also determined were the concentrations of Ca and Mg in blood. Special investigations were aimed at metallograms from organs: liver, kidney, brain. TheMorning and Vesper changes were monitored, in a regime of intraperitoneal administration of the compounds, as observed under in vitro research. Comparative evaluation of the effects of in vitro AND in vivo is under way, including comparison o the metallograms of liver, kidney, brain after experiments with cis-platinum versus the data obtained using the Ga compounds. Also studied was the action of natural compounds in combination with irradiation and treatment with standard platinum. On the basis of assessing DNA damage treated cells, reactive oxygen species and early apoptosis cells appeared following irradiation, it was concluded that extracts natural have photoprotection effects and influence radiochemotherapy treatment. The teams involved in this interdisciplinary project include a significant proportion of young researchers. The original research results of the doctoral students participating in the project, is illustrated among others in the seven PhD theses publicly defended.

Outline of the results - 2011

A number of new meso-bis (phenothiazine-3-yl) porphyrins and their metal coordination compounds, potentially active in photodynamic therapy, were tested in vitro on A431 epidermoid type Keratinocyte and HaCaT cell lines and by irradiation with red ( 615-630 nm) and blue (400-505 nm) light. The biologically tested porphyrins were investigated by electrochemical methods - cyclic voltammetry measurements and square wave (SW) voltammetry - in order to also correlate the reduction and oxidation potentials with their behavior towards myoglobin, hemoglobin, and their intercalation in DNA pTZ57R. Other outstanding results were obtained after DNA intercalation tests in pTZ57R, with phenothiazine amines and Troger bases (BT) with phenothiazine units. Gel electrophoresis has revealed that the two classes of substances are inserted in plasmid DNA causing modification of the macromolecular geometry of DNA. Interaction between DNA and BT was demonstrated by changing the value of the optical rotation diastereoisomers of BT after the interaction with the DNA. Ga complexes having as ligands phosphino-arylthiol derivatives synthesized in the group have promising antiproliferative activity; studies and will be extended to their arsenic counterparts. A series of difosfaalchenilgermylenes and difosfaalchenilstanylenes were synthesized and characterized, with multiple sites of coordination - the divalent metal center Ge (II) or Sn (II),the P = C double bonds, and the phosphorus atoms. Coordination of (NHC) Ge (Mes * P = CCl) 2 is carried out in the bidentate manner for W and Mo, via the Ge atom(s) and the one of the two phosphorus atoms, with the remaining phosphorus atom pendant; the coordination to Au is monodentate through Ge . Studies at the HF/6-31G level on (d) thio-calix [n] arenes, n = 4.6, in the presence and absence of the ions Li +, Na +, Cs +, Ca2 + and Ba2 +, targeted the manner in which thecavity size and the electronic properties of host / guest calixarenes influence transport capacity. Contributions were made to the study of geometric and electronic structure, as well as the reactivity, of metallic, organometallic and organic structures whose biological relevance is direct or implied by their metabolites or precursors. Also studied were metal centers relevant for signaling and oxidative stress. Such systems included centers of Fe, Cu, Co, Mo, Pt, Pd, Au, and amino acid type ligands (proteins, peptides, ex. bleomycin), heterocycles (e.g .porphyrins, phthalocyanines and porphyrazines), derivatives of boron. The effect of reference compounds of platinum-based (cisplatin, carboplatin, oxaliplatin) on the structure and reactivity of metalloproteins with involvement in oxidative stress was examined, moving then to validate the results by confirming these effects and the cells intact (red blood cells, and E. coli). These have allowed for identification of new ways by which these drugs may induce side effects, and ways to counter them. A comparison of the effects of the newly synthesized compounds on the same system where the reference compounds yielded results, highlights differences in reactivity that may partly correlate with structural elements, such as hydrophobicity and steric. Protein derivatives of possible biological activity were also produced and investigated. In the context of attempting to elucidate the mechanisms of action of organometallic and coordination derivatives, the biological activity of Pd- curcumin complexes was evaluated; based on the assessment of cytotoxicity it was determined that they have a selective action against the cells studied: statistically significantly, they were more toxic in vitro to the tumor lines than to the normal ones. A comparison of the antiproliferative effect of curcumin and of two Pd curcumin complexes was made on uterine HeLa cervix tumor cells, colorectal carcinoma Colo cells, liver tumor stem CCS, highly malignant melanoma metastasis M1/15, normal keratinocytes, HaCaT, and normal human lymphocytes. It was found that due to the dual selective inhibition of tumor proliferation and stimulation of the immune system antitumor response, the curcumine-Pd complexes have potential for adjunctive therapy in the treatment of malignant casuistry. In order to elucidate the cellular and molecular mechanisms of resistance to treatment chemotherapeutic substances , investigated were colorectal adenocarcinoma cells HT-29R and Colo320R who acquired resistance to oxaliplatin. Microscopic images of the lines Colo320 (a) Colo320R (b), HT-29 (c) and HT-29R show morphological changes occurring as a result of acquisition of resistance to platinum: the resistant ones become spindly, we see the transition from epithelial phenotype to the mesenchymal , as indicatd by pseudopodes and cell polarity loss, leading to decreased intercellular adhesion and increased distance in culture. Compared to the platinum-sensitive cells, the resistant ones show a distinctive cellular and molecular picture - different morphology, distinctly different in terms of cytotoxicity, the interactions formed by cellular DNA with platinum, and most importantly the expression profile of specific genes, especially in terms of genes responsible for inhibition of apoptosis and cell proliferation. DNA transcription and cell replication is impaired. In vivo studies were aimed at the effect of platinum drugs taking as reference two major targets of their action in humans: incorporating primary tumor cell surface markers and action on tumor and normal cells. In order to determine the level of Pt in tumor tissue and circulating blood a bank of biological material was constituted. The expression of genes and receptors on lymphocytes of patients treated with platinum drugs was examined, using semi-quantitative PCR methods. Chosen as target was the GITR gene of the TNF family, tumor necrosis factors, which has two variants involved in anti-tumor immune response: version 1 and 3. The primers used for PCR were designed so as to exclusively avoid to amplify the genomic DNA and thereby to permit amplification of different variants of RNA. Several variants in genes of the GITR var1 and var3 were identified by this method. Lymphocytes collected from subjects undergoing chemotherapy with cisplatin expressed the GITR gene in the majority of cases; in all cases variant 1 was present, in one case being expressed variant 3 as well. For human lymphocytes and macrophages treated with platinum, the modulation of the expression of surface markers of macrophages in tumor infiltrates under the action clinical-use platinum compounds as well as of newly-synthesized metal compounds. On infiltrated macrophages isolated from ascitic fluid derived from a case of colon carcinoma, treated in vitro with cisplatin, oxaliplatin and carboplatin, determinations were made on CD40 Ligand/TNFSF5 and CXCL10/IP-10, as molecules involved in the antitumor immune response and inflammation. The effect of standard Pt drugs on lymphocyte subpopulations was investigated by quantitative assessment of metalloproteinase MMP2 and MMP9m, involved in cell signaling. The results show a strong ability of tumor cells to secrete metalloproteases 2, involved in inflammation following treatment with cisplatin, while metalloprotease 9 is poorly expressed. Other in vivo studies performed on a group of Wistar rats showed side effects of organometallic substances such as impaired gastrointestinal area, kidneys, hypomagnesemia, hypokalemia AND hypocalcemia. Involved in this interdisciplinary project are teams including graduate students (master and PhD) and PhDs in chemistry, whose contribution is important, both from a scientific standpoint, in their training as specialists, and in creating a different perception of the social and economic value of research activity and of the importance of teamwork.