Molecular mechanisms of colorectal cells migration in antitumor action of new-synthetized platina(IV) complex and natural bioactive substances

Kolorektalni karcinom predstavlja drugu najčešći maligni tumor digestivnog trakta i uzrok smrtnosti kod ljudi, tako da su istraživanja antikancerogenih bioaktivnih supstanci od velikog značaja za terapiju ove bolesti. Razumevanje mehanizama migracije, invazije, metastaze i ispitivanje novih bioaktivnih supstanci sa antimigratornim delovanjem, predstavlja jednu od strategija u terapiji karcinoma. Reaktivne vrste kiseonika i azota predstavljaju bazu regulacije niza molekularno-fizioloških funkcija u organizmu, uključujući genezu i progresiju karcinoma. Stoga je poznavanje međuzavisnosti molekularnih mehanizama redoks statusa i signalnih puteva migracije ćelija karcinoma kolona od velikog značaja. Kompleksi platine(IV) imaju efikasnije antitumorsko delovanje u odnosu na cispaltinu, sa manjom toksičnošću a većom efikasnošću u tretmanu rezistentih tumora. Testirani ekstrakti gljiva i lišajeva se koriste u tradicionalnoj medicini i njihovi efekti na migraciju ćelija kolorektalnog karcinoma nisu dvoljno ispitani. Primena kotretmana, prirodnih bioaktivnih supstanca i hemioterapeutika (cisplatina), kao i novosintetisanih kompleksa platina(IV), kroz sinergističko antikancerogeno delovanje, doprinosi smanjenju negativnih efekta hemioterapeutika. Cilj disertacije je da se ispita potencijalno antikancerogeno delovanje (citotoksičnost i antimigratorni potencijal) novosintetisanih kompleksa platine(IV) i bioaktivnih supstanci iz prirodnih izvora (ekstrakti lekovitih gljiva i lišajeva), sa akcentom na molekularne mehanizme i ključne proteine signalnih puteva migracije, kao i na parametre redoks statusa u ćelijama kolorektalnog karcinoma. Specifični ciljevi i metodologija: (1) testirati antikancerogene efekte bioaktivnih supstanci, hemijski novosintetisanih i ekstrahovanih iz prirodnih izvora na ćelijskim linijama karcinoma debelog creva (HCT-116, SW-480) u tretmanima (a) Pt(IV) kompleksi: [PtCl4(dbu-S,S-eddp)] - Pt(IV) kompleks1, [PtCl4(dpe-S,S-eddp)] - Pt(IV) kompleks2, odgovarajući ligandi (Ligand 1 i Ligand 2), cisplatina (pozitivna kontrola); (b) ekstrakti gljiva (Phellinus linteus, Cordyceps sinensis, Lentinus edodes, Coprinus comatus, Ganoderma lucidum) i lišajeva (Platismatia glauca, Pseudoevernia furfuraceae); (v) kotretman Pt(IV) kompleksi sa ekstraktom Phellinus linteus, sa akcentom na tip inetrreakcije komponeneti u kotretmanu; (2) ispitati citotoksičnost tretmana (MTT test) i tip ćelijske smrti (akridin oranž/etidijum bromid mikroskopska metoda) ćelija kancera i zdravih ćelija fibroblasta (MRC-5); (3) ispitati promene migratornog potencijala HCT-116 i SW-480 ćelija, Transwell migratornim testom i definisanjem lokalizacije i kvantifikacijom ključnih proteina signalnih puteva migracije i međućelijske komunikacije, pomoću imunoflorescentne mikroskopske metode (E-kadherin i citoplazmatski β-katenin, kao markeri međućelijskih veza; Frizzled-7-receptor i nuklearni β-katenin, kao markeri Wnt signalnog puta; N-kadherin i vimentin, kao komponente citoskeleta migratornih ćelija); (4) odrediti koncentraciju metaloproteinaze 9, kolorimetrijskom metodom (ELISA); (5) odrediti promene parametara redoks statusa (spektrofotometrijsko određivanje koncentracije superoksid anjon radikala, nitrita i redukovanog glutationa).Colorectal carcinoma is the second most common malignant tumor of the digestive tract and cause of mortality in humans, so research of anti-cancer bioactive substances is of great importance for the treatment of this disease. Understanding the mechanisms of migration, invasion, metastasis, and the testing of new bioactive substances with anti-migratory activity is one of the strategies in the treatment of cancer. Reactive oxygen and nitrogen species represent the basis for regulation of molecularphysiological functions in the body, including genesis and progression of the cancer. Therefore, the knowledge of the interdependence of molecular mechanisms of redox status and signaling pathways of migration of colon carcinoma cells is of great importance. Platinum(IV) complexes have more effective anti-tumor activity compared to cisplatin, with lower toxicity and greater efficacy in the treatment of resistant tumors. Tested extracts of mushrooms and lichen are used in traditional medicine and their effects on the migration of colorectal carcinoma cells have not been particularly tested. The use of natural bioactive substances and chemotherapeutics (cisplatin, as well as newly synthesized platinum(IV) complexes) in co-treatment, through synergistic anticancer activity, contributes to the reduction of the negative effects of chemotherapeutics. The aim of the dissertation is to investigate the possible anticancer activity (cytotoxicity and antimigratory potential) of newly synthesized platinum(IV) complexes and bioactive substances from natural sources (extracts of medicinal mushrooms and lichen), with emphasis on molecular mechanisms and key proteins for migration signaling pathways, as well as on parameters of redox status in colorectal carcinoma cells. Specific objectives and methodology: (1) to test the anticancer effects of bioactive substances, chemically newly synthesized substances and those extracted from natural sources on colon carcinoma cell lines (HCT-116, SW-480) in treatments with (a) Pt(IV) complexes: [PtCl4(dbu-S,S-eddp)] - Pt(IV) complex 1, [PtCl4(dpe-S,S-eddp)] - Pt(IV) complex 2, corresponding ligands (Ligand 1 and Ligand 2), cisplatin (positive control); (b) mushrooms extracts (Phellinus linteus, Cordyceps sinensis, Lentinus edodes, Coprinus comatus, Ganoderma lucidum) and lichens (Platismatia glauca, Pseudoevernia furfuraceae); (v) co-treatment of Pt(IV) complexes with the extract of Phellinus linteus, with an emphasis on the type of interreaction; (2) to examine the cytotoxicity of the treatments (MTT assay) and the type of cell death (acridine orange/ethidium bromide microscopic method) for cancer cells and healthy fibroblast cells (MRC-5); (3) to examine changes in the migratory potential of HCT-116 and SW-480 cells, the Transwell migration assay, and the definition of localization and the quantification of key proteins of migration signaling pathways and intercellular communication using an immunofluorescence microscopic method (E-cadherin and cytoplasmic β-catenin as markers of intercellular connections; Frizzled-7 receptor and nuclear β-catenin, as markers of Wnt signaling pathway; N-cadherin and vimentin, as components of cytoskeleton of migratory cells); (4) to determine the concentration of metalloproteinase 9 by colorimetric method (ELISA); (5) to determine the changes in redox status parameters (spectrophotometric determination of the concentration of superoxide anion radical, nitrites and reduced glutathione)