Ugotavljanje občutljivosti humanih tumorskih celic za elektrokemoterapijo s spojinami platine (II) in vitro

Elektroporacija (EP) je fizikalna metoda s katero z dovajanjem električnih pulzov v celični membrani za kratek čas ustvarimo pore, kar olajša prehod v celice določenim molekulam. Za zdravljenje rakavih obolenj se vse bolj uporablja EP v kombinaciji s cisplatinom (CDDP) in bleomicinom, čemur rečemo elektrokemoterapija (EKT). Cisplatin je platinova (II) spojina, ki s kovalentnimi povezavami povzroči spremembe v strukturi DNA in vodi v smrt rakave celice. Kljub temu, da je CDDP eden najpogosteje uporabljenih kemoterapevtikov, pa povzroča hude stranske učinke in tudi odpornost rakavih celic. Zaradi tega se raziskave usmerjajo v razvoj novih platinovih (II) analogov s protitumorskim delovanjem, kamor spadata tudi [PtCl (5,7-dibromo-8-hidroksikinolinato)(S-dmso)] (spojina A) in njen analog, [PtCl (5,7-dibromo-8-hidroksikinolinato)(pta)] (spojina B). Citotoksičnost obeh spojin v kombinaciji z EP smo preverili na humani celični liniji raka debelega črevesja (HT29) in raka dojke (MCF7). S testom klonogenosti smo izračunali vrednosti IC50 in jih primerjali z vrednostmi IC50 CDDP, s pripravo citospinov in barvanjem po Giemsi pa smo na podlagi morfoloških značilnosti ugotovili delež apoptotičnih in nekrotičnih celic. Ugotovili smo, da spojini A in B delujeta citotoksično na tumorske celice in povzročata predvsem apoptozo ter deloma nekrozo. Elektroporacija statistično značilno poveča citotoksično delovanje vseh treh platinovih (II) spojin in je učinkovita metoda za izboljšanje vnosa ne samo CDDP, pač pa tudi spojine A in B. Spojina B se je v kombinaciji z in brez EP izkazala za bolj toksično od spojine A in hkrati tudi enako ali celo bolj citotoksično kot CDDP pri obeh celičnih linijah in je zato primerna za nadaljne testiranje, kot potencialni kemoterapevtik. Electroporation (EP) is a physical technique in which an electric field is applied to cells in order to increase the permeability of cell membrane for poorly permeating molecules. Electroporation in combination with cisplatin (CDDP), also called electrochemotherapy (ECT), is used for local treatment of several malignant tumors. Cisplatin is a well-known platinum (II) antitumor drug which antitumor activity has been linked to its ability to form DNA adducts, which subsequently lead to apoptosis of cancer cells. Although CDDP is broadly used, its main limitations are severe side effects and tumor cells’ resistance. Because of this, researchers are focusing on developing new platinum (II) complexes with antitumor activity, including [PtCl (5,7-dibromo-8-hydroxyquinolinato)(S-dmso)] (compound A) and it's analog, [PtCl (5,7-dibromo-8-hydroxyquinolinato)(pta)] (compound B). In the thesis, we analysed the cytotoxicity of compound A and B in combination with EP in human colon cancer (HT29) and human breast cancer cell line (MCF7). We determined IC50 values with clonogenic assay and compared it to IC50 values of CDDP. We used Giemsa staining to determine the percentage of apoptotic and necrotic cells. Results show that all compounds, A, B and CDDP have a cytotoxic effect on both tumor cell lines and cause primarily apoptotic cell death. Furthermore, EP has proved to be a very effective method for drug uptake as it has significantly improved cytotoxicity of all three platinum (II) complexes. Compound B has turned out to be more cytotoxic with and without EP than compound A and showed similar or better cytotoxicity than CDDP in both cell lines. Based on our results, compound B is very promising potential therapeutic agent.