Molekularni mehanizmi apoptzoze u ćelijama karcinoma kolona nakon in vitro tretmana ekstraktima lekovitih bilja

Kolorektalni karcinom predstavlja značajan medicinski problem pošto su učestalost oboljevanja i smrtnost od ove bolesti u stalnom porastu. Supstance prirodnog porekla koje imaju sposobnost da interaguju sa signalnim putevima uključenim u ćelijske funkcije i modifikuju signalne puteve tokom procesa kancerogeneze usporavajući ili blokirajući ovaj proces, poseduju brojne prednosti u odnosu na sintetske. Zbog toga se javlja sve veća potreba ka pronalasku potencijalnih antitumorskih supstanci poreklom iz prirodnih izvora, kao i ispitivanjima efekata ovih supstanci i biljnih ekstrakata na izazivanje apoptoze, kao jedne od vodećih strategija u terapiji malignih bolesti. Ciljevi istraživanja doktorske disertacije su: ispitivanje antitumorskog potencijala ekstrakata lekovitih biljaka flore Srbije, tradicionalno korišćenih u tretmanima različitih digestivnih poremećaja (Teucrium chamaedrys L., Allium flavum L., Gentiana punctata L., Ligustrum vulgare L. i Centaurium erythrea Rafn.) na HCT-116 i SW480 ćelijama kolorektalnog karcinoma i zdravim fibroblastima kože; ispitivanje potencijalnih sinergističkih efekata ekstrakata biljaka A. flavum i G. punctata i novosintetisanog Pd(II) kompleksa (Paladijum(II) kompleks sa ligandom 3-[(2-hidroksibenziliden) amino]-2-tioksoimidazo lidin-4-on); ispitivanje molekularnih mehanizama proapoptotske aktivnosti izabranih vrsta biljaka u pojedinačnim I kombinovanim tretmanima na HCT-116 i SW480 ćelijama, uključujući i mehanizme redoks zavisne modulacije procesa apoptoze i iRNK ekspresiju gena čiji proteinski produkti učestvuju u metabolizmu antitumorskih supstanci i pojavi rezistencije malignih ćelija. Ekstrakti ispitivanih vrsta lekovitih biljaka deluju citotoksično na ćelijskim linijama kolorektalnog karcinoma, dok na zdravim fibroblastima kože ne pokazuju ovakve efekte, što potvrđuje njihova antitumorska svojstva. Intenzitet citotoksičnosti zavisi od vrste biljke, kvantitativnog i kvalitativnog sastava fenolnih jedinjenja prisutnih u njima I korišćenog rastvarača u ekstrakcionoj proceduri. Među ispitivanim vrstama ekstrakata, metanolni i acetonski ekstrakt pokazuju podjednako dobru citotksičnu aktivnost, a etilacetatni ekstrakt najslabiju, što je u korelaciji sa sadržajem ukupnih fenola i flavonoida u ekstraktima. Citotoksični efekti ispitivanih vrsta biljaka su posledica indukovane apoptoze, kao dominantno prisutnog tipa ćelijske smrti. Tretmani ekstraktima biljaka izazivaju povećanu ekspresiju Fas receptora na membrani i aktivnost kaspaze 8 u spoljašnjem apoptotskom putu u obe ćelijske linije. Povećana aktivnost kaspaze 9 potvrđuje ulogu mitohondrija u apoptozi, dominantno u HCT-116 ćelijama u odnosu na SW480. Tretmani biljnim ekstraktima utiču na redoks status u ćelijama i dovode do redoks zavisne modulacije procesa apoptoze, kao mogućeg uzroka proapototskog delovanja biljaka. Rezultati ispitivanja iRNK ekspresije uglavnom pokazuju inhibitorne efekte biljnih ekstrakata na iRNK ekspresiju gena (CYP1A1 (citohrom P450), GSTP1 (glutation-S-transferaza) i MRP2 (Multidrug Resistance-associated Protein 2)). Pd(II) kompleks izaziva značajnu citotoksičnost na ćelijama kolorektalnog karcinoma, ali i visoke procente nekroze i citotoksične efekte na zdravim fibroblastima kože. Tretmani Pd(II) komplekom izazivaju najveću aktivnost kaspaze 9, što ukazuje na značajnu ulogu mitohondrija u procesu apoptoze u HCT-116 ćelijama. U zavisnosti od aplicirane doze indukuje dvojake efekte na parametre signalnih puteva apoptoze i redoks statusa. Visoke koncentracije Pd(II) kompleksa izazivaju oksidacioni stres u ćelijama karcinoma kolona, što je moguć uzrok indukovane nekroze, dok niske koncentracije uvode ćelije u apoptozu. Ekspresija iRNK ispitivanih gena za metaboličke enzime je nepromenjena ili negde povećana, što ukazuje na metabolizam Pd(II) kompleksa ovim enzimima. Kombinovani tretmani Pd(II) kompleksa i ekstrakata biljaka A. flavum i G. punctata smanjuju toksičnost Pd(II) kompleksa na zdravim ćelijama, a suprotno povećavaju citotoksičnost na malignim ćelijama, pri čemu dolazi do međusobnih sinergističkih efekata. U kombinovanim tretmanima smanjuje se procenat nekroze u odnosu na pojedinačni tretman Pd(II) kompleksom, a povećava citotoksičnost izazvana apoptozom. Ekspresija iRNK gena čiji su proteinski produkti uključeni u metabolizam antitumorskih supstanci generalno je smanjena u kombinovanim tretmanima u odnosu na pojedinačne u obe ćelijske linije, što rezultira povećanom citotoksičnošću Pd(II) kompleksa zbog njegovog slabijeg metabolisanja i transporta kroz membranu u prisustvu biljaka koje inhibiraju ekspresiju ovih proteina. Obzirom na aktuelnost teme, rezultati doktorske disertacije imaju veliki značaj u ispitivanju novih antitumorskih supstanci poreklom iz prirodnih izvora, povećavaju značaj korišćenja biljaka kao izvora medikamenata, doprinose razvoju adekvatne terapije i time daju doprinos disertacije kako u naučnom, tako i u praktičnom smislu. Aktivne komponente ispitivanih vrsta biljaka, sa posebnim naglaskom na biljku A. flavum, neophodno je dodatno izolovati, testirati i odrediti način apliciranja prilikom terapije u budućim in vitro i in vivo eksperimentalnim istraživanjima. Obzirom na inhibitornu aktivnost ekspresije metaboličkih enzima, neophodno je istaći mogućnost kombinovanog apliciranja ovih biljaka ili njihovih prečišćenih komponenti sa odgovarajućim citostaticima u cilju smanjenja rezistencije malignih ćelija, kao jednog od najvećih problema u terapiji tumora.Colorectal cancer represents a major health problem, because the incidence of morbidity and mortality of this disease is constantly increasing. Substances from natural origin have the ability to interact with and modify signaling pathways involved in the cellular functions during the cancerogenesis, postponing or blocking this process and also have a number of advantages compared to synthetic compounds. Therefore, there is an emerging need for the investigation of potential anticancer substances from natural origin, mainly plant extracts, and studying their effects on the induction of apoptosis, since it is one of the major strategies in the cancer therapy. The aims of this doctoral dissertation are: investigation of anticancer potential of medicinal plants extracts from Serbian flora, traditionally used in treatments of different digestive disorders (Teucrium chamaedrys L., Allium flavum L., Gentiana punctata L., Ligustrum vulgare L. and Centaurium erythrea Rafn.) on HCT-116 and SW480 colon cancer cell lines and untransformed human skin fibroblasts; investigation of potential synergistic effects of A. flavum and G. punctata extracts co-treatment with newly synthesized Pd(II) complex (Pd(II) complex with 3-[(2-hydroxy-benzylidene)-amino]-2-thioxoimidazolidin- 4-one ligand); investigation of molecular mechanism of proapoptotic activity of investigated plant extracts in single and co-treatments on HCT-116 and SW480 cell lines, including mechanism of redox-dependant modulation of apoptosis and iRNK expression of genes responsible for anticancer substances metabolism and cell resistance on anticancer drugs.Extracts of the tested medicinal plants showed cytotoxic activity on colon cancer cell lines, whilest exhibited no effects on fibroblasts, which confirmed their anticancer properties. Effectiveness of cytotoxic activity depended of plant type, qualitative and quantitative composition of phenolic compounds and solvent used in the extraction procedure. Methanol and acetone plant extracts showed significant cytotoxic activity, while ethyl acetate extracts showed the lowest activity, which is in correlation with phenolic and flavonoid content in the extracts. Cytotoxic effects of the investigated plants were the outcome of the induction of apoptosis, as a dominant type of cell death. Plant extracts caused increasing of Fas receptor protein expression and activity of caspase 8 in extrinsic apoptotic pathway in the both cell lines. Increased caspase 9 activity confirm significance of mitochondria in apoptosis, more intensive in HCT-116 than in SW480 cells. Potential cause of proapoptotic activity of plant extracts could be explained by their effects on redox status and redox-dependant modulation of apoptosis. Results, in general, showed inhibitory activity on iRNK expression of gene CYP1А1 (Cytohrome P450), GSTP1 (Gluthation-S-Transferase) and MRP2 (Multidrug Resistance-associated Protein 2). Pd(II) complex induced significant cytotoxic effects on colon cancer cells, but at the same time induced high percent of necrosis and also exhibited cytotoxic effects on skin fibroblasts. Pd(II) complex caused the highest caspase 9 activity, sugesting that mitochondria play significant role in apoptosis in HCT-116 cells. Depending on the applied concentration, Pd(II) complex induced dual effects on apoptotic signaling pathway and parameters of redox status. Higher concentrations of Pd(II) complex induced oxidative stress in colon cancer cells, which could be a cause of the induction of necrosis, while lower concentrations induced apoptosis. Expression of iRNK of metabolic enzymes was unchanged or increased in some treatments, which suggests that Pd(II) complex was metabolized by these enzymes. Co-treatments of Pd(II) complex with A. flavum and G. punctata extracts decreased toxicity of Pd(II) complex on untransformed human skin fibroblasts and on contrary increased cytotoxicity on cancer cells, exhibiting synergistic effects. In co-treatments, percents of necrosis are decreased, compared to single treatments with Pd(II) complex, and cytotoxicity induced by apoptosis is increased. Expression of genes involved in anticancer drug metabolism, was generally decreased in co-treatment, compared to single treatment on both cell lines, which resulted in increased cytotoxicity of Pd(II) complex, because of its decreased metabolism and transport through cell membrane in the presence of plant extracts, since they inhibited expresion of these enzymes. Considering current trends in this research area, results of this doctoral dissertation have great impact on investigation of new anticancer substances from natural source, focus on the importance of using plants as the source of medicinal drugs, contribute to the development of the appropriate therapy and give contribution in both scientific and practical means. There is a need for the isolation of bioactive components of the investigated plants, further testing and development of application therapy procedure in future in vitro and in vivo experimental investigations, with special focus on A. flavum. With regards to inhibitory activity on gene expression of metabolic enzymes, it is necessary to highlight the possibility of co-treatments of tested plant extracts or their bioactive compounds with the appropriate chemotherapeutic, in order to reduce the resistance of malignant cells, as one of the greatest problems in the tumor therapy