Uspostavljanje rezistentnih tumorskih ćelijskih linija kao modela za testiranje novih hemioterapeutika : molekularna karakterizacija rezistencije nastale dugotrajnim izlaganjem paklitakselu

Abstract

Glavni uzrok neuspeha hemioterapije u lečenju kancera je pojava višestruke (engl. „multi-drug“) rezistencije (MDR). Efikasnost paklitaksela (PTX) je često ograničena pojavom rezistencije. Cilj ove doktorske disertacije je bio ispitivanje molekularnih i fenotipskih promena u toku razvoja MDR-a indukovanih PTX-om kod ćelijskih linija humanog karcinoma debelog creva (DLD1) i glioblastoma (U87). Takođe je testirana upotrebljivost dobijenih MDR modela u evaluaciji četiri anti-kancer agensa. Kontinuirani tretman PTX-om doveo je do razvoja MDR-a kod obe ispitivane ćelijske linije koje su postale rezistentne na strukturno i funkcionalno različite hemioterapeutike. Nakon potvrde prisustva ukrštene rezistencije kod novouspostavljenih ćelijskih linija DLD1-TxR i U87-TxR, analizirana je ekspresija membranskih trasportera uključenih u razvoj MDR-a na nivou iRNK. Ćelije su imale povišen nivo ekspresije mdr1 gena i smanjen nivo ekspresije mrp1 gena. Prekomerna ekspresija P-glikoproteina (P-gp), koji kodira mdr1 gen, je uočena kod obe MDR ćelijske linije. Analiza na protočnom citofluorimetru je pokazala da je akumulacija Pgp supstrata (rodamina 123 i doksorubicina) kod DLD1-TxR i U87-TxR ćelija značajno manja u poređenju sa odgovarajućim parentalnim ćelijama, DLD1 i U87. Značajno smanjenje ekspresije gst-π gena i koncentracije glutationa (GSH) je uočeno kod U87- TxR ćelija. Sekrecija vaskularnog endotelijalnog faktora rasta (VEGF) je inhibirana u jednokratnom tretmanu kod ćelijskih linija karcinoma debelog creva i u kontinuiranom tretmanu kod ćelijske linije glioblastoma. Analiza ćelijskog ciklusa je pokazala da je jednokratni tretman PTX-om kod ćelijskih linija humanog karcinoma debelog creva praćen porastom subG0 faze, odnosno povećanjem procenta mrtvih ćelija, dok je kod ćelija glioblastoma došlo do umiranja tokom interfaze (G1, S ili G2). MDR tumorske ćelijske linije su stekle nove strukturne i numerićke hromozomske aberacije. Sticanje MDR fenotipa kod U87-TxR ćelija je praćeno smanjenem jednog nivoa ploidije. Takođe je uočen gubitak hromozomskog regiona 6q kod obe rezistentne ćelijske linije, kao i inaktivacija p53 tumor spresor gena kod U87-TxR ćelija i PTEN tumor supresor gena kod DLD1-TxR ćelija.

Multi-drug resistance (MDR) is a major obstacle to successful cancer treatment. The efficacy of paclitaxel (PTX) is often limited by appearance of drug resistance. The aim of this study was to explore molecular and phenotypic alterations during development of MDR induced by PTX in human colon carcinoma (DLD1) and glioblastoma (U87) cell lines. We also tested the usefulness of developed MDR models in the evaluation of four anti-cancer agents. Continuous treatment with PTX led to the development of MDR in both tested cancer cell lines that became resistant to structurally and functionally unrelated chemotherapeutics. After confirmation of the cross-resistance in newly established DLD1-TxR and U87-TxR, we analyzed the mRNA expression of membrane transporters involved in MDR. The cells had increased levels of mdr1 gene expression, while mrp1 was decreased. Over-expression of P-glycoprotein (P-gp), coded by mdr1, was observed in both MDR cancer cell lines. Flow cytometry analyzes showed that the accumulation of P-gp substrates (rhodamine 123 and doxorubicin) in DLD1-TxR and U87-TxR was significantly lower compared to DLD1 and U87, respectively. The significant depletion of gst-π gene expression and glutathione (GSH) concentration was observed in U87-TxR. Vascular Endothelial Growth Factor (VEGF) secretion was inhibited by single PTX treatment of colon cancer and in continuous treatment of glioblastoma cell lines. The analysis of cell cycle kinetics revealed extensive cell death in colon cancer cells that were accumulated in subG0 phase after PTX treatment, while glioblastoma cells died through interphase (G1, S or G2). The MDR cancer cell lines acquired novel structural or numerical chromosomal aberrations. Polyploidy reduction was observed after development of MDR in U87-TxR. Losses of 6q in both resistant cancer cell lines and inactivation of p53 in U87-TxR and PTEN in DLD1-TxR were also revealed. We evaluated the anti-cancer activities and MDR reversal potential of the Akt inhibitor (GSK690693), the Ras inhibitor (Tipifarnib) and two P-gp inhibitors (jatrophane diterpenoids Euphodendrophane H-Euph H and Euphodendrophane S -Euph S). Their effects vary due to the cell-type differences, existence of MDR phenotype or tumor suppressors’ alterations. Tipifarnib, Euph H and S, significantly sensitized MDR cancer cells to PTX.