Investigation of molecular systems that trabectedin (ET-743) affects in breast cancer cell cultures

Trabectedin (ET-743) is an anti-cancer molecule that was originally isolated from Ecteinascidia turbinata (sea squirt) at 1969. It has been approved for the treatment of patients with advanced soft tissue sarcoma and ovarian cancer either alone or in combination with present chemotherapeutics and now being prepared synthetically. However, it is still waiting to discover which signaling pathways are involved in the effect of Trabectedin in cancer cells. The aim of this study is to investigate (1) the anti-proliferative effect of Trabectedin, possible (2) apoptotic and (3) anti-angiogenic molecules that interact with Trabectedin, (4) molecules that regulate the cellular reduction/oxidation balance, (5) molecules that play critical roles in epigenetic regulation on breast cancer cell lines (MDA-MB-453 ve MCF-7) and normal breast epithelial cells (MCF-10A) by using current molecular methods. Anti-proliferative effect of Trabectedin was determined by XTT cell viability assay kit and real time cell analyze system (xCELLigence). To show whether the anti-proliferative effect of Trabectedin is through apoptosis, DNA fragmentation and mitochondrial membrane potential were measured. A preliminary study was done to produce hypotheses by an ELISA based apoptosis array (multple western blotting) including 35 molecules associated with apoptosis. Caspase-8 and Caspase-9 inhibitors were used to see the changes in DNA fragmentation to determine that apoptosis occurs via death receptors (extrinsic pathway) or mitochondrial pathway (intrinsic pathway). Experiments were confirmed by western blotting and qRT-PCR methods. An ELISA based apoptosis array including 55 molecules associated with angiogenesis was performed to investigate the anti-angiogenic effect of Trabectedin. Reactive oxygen species and Glutathione S Transferase (GST) activities were measured to investigate whether Trabectedin affects reduction/oxidation balance of the breast cancer cells. The effect of Trabectedin on epigenetic system of cells was shown by measuring Histone Deacetylase (HDAC) and DNA Methyltransferase (DNMT) activities. Trabectedin was cytotoxic to breast cancer cell cultures (MCF-7 and MDA-MB-453) in a dose (10-6 -10-12 M) and time (24., 48. and 72. h) dependent manner. IC50 values of Trabectedin in MCF-7 and MDA-MB-453 cell cultures were 4.8±0.8 and 2.5±0.4 nM respectively, at 48 h. It was determined that dose and time dependent cytotoxic effect of Trabectedin was less in human MCF-10A normal breast epithelial cells (IC50= 9.55x10-9 nM). There was a dose and time dependent increase in DNA fragmentation in Trabectedin treated breast cancer cells, however in MCF-10A cells, DNA fragmentation was observed only at higher doses of Trabectedin (?10-7 M). Trabectedin caused a dose-dependent decrease in mitochondrial membrane potential in MCF-7 and MDA-MB-453 cells. Levels of proapoptotic molecules such as; TRAIL R1/DR4 (2.6 fold), TRAIL R1/DR5 (3.1 fold), Fas/TNFRSF6 (1.7 fold), TNF RI/TNFRSF1A (11.2 fold) and FADD (4.0 fold) were increased in MCF-7 cells treated with Trabectedin for 48 h as compared to control cells (p?0.05). Levels of inhibitors of apoptosis family proteins (AIP) such as; CIAP-1, XIAP, Livin and Survivin were decreased by 5.5, 2.5, 2.6, 3.0 fold, respectively (p?0.05). Levels of pro-apoptotic molecules such as Bad, Bax, Active caspase-3, Cytochrome-c, SMAC/DIABLO, Htra2/Omi were increased by 3.6, 4.2, 4.4, 4.8, 4.5 and 5.2 fold respectively, in 48 h Trabectedin treated MDA-MB-453 cells (p?0.05). Levels of anti-apoptotic molecules such as Bcl-2, Bcl-XL, pro-caspase-3 were decreased by 4.8, 5.2 and 6.0 fold (p?0.05). Levels of AIP family proteins CIAP-1 and Survivin were decreased by 5.0 and 2.7 fold, respectively (p?0.05). mRNA levels of pro-apoptotic molecules such as TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD and Fosfo-p53 were increased by 2.9, 2.1, 8.4, 2.1 and 3.5 fold, respectively, however anti-apoptotic molecules such as CIAP-1 and XIAP were decreased by 4.2 and 3.8 fold in Trabectedin treated MCF-7 cells (p?0.05). mRNA levels of pro-apoptotic molecules such as Bax, Bad, Cytochrome c and SMAC/Diablo were increased by 2.6, 2.5, 3.4, 10.5 fold respectively, however, mRNA level of anti-apoptotic Bcl-2 molecule was decreased by 1.8 fold in MDA-MB-453 cells (p?0.05). It was found that Trabectedin induced apoptosis through death receptor pathway (extrinsic pathway) in MCF-7 cells but mitochondrial pathway (intrinsic pathway) in MDA-MB-453 cells by using Caspase-8 and Caspase-9 specific inhibitors. There was no significant change in DNA fragmentation levels of MDA-MB-453 cells pretreated with Caspase-3 inhibitor, as compared to Trabectedin alone treated cells (p>0.05). The levels of anti-angiogenic proteins such as Serpin E1/PAI-1 and TIMP-1 were increased by 3.5 and 4.2 fold in Trabectedin treated MCF-7 cells (p?0.05). However, anti-angiogenic proteins such as Tissue Factor/Factor III, DPPIV/CD26, uPA, VEGF were decreased by 8.4, 6.2, 6.2, 6.8 fold, respectively (p?0.05). In MDA-MB-453 cells, anti-angiogenic proteins such as ENDOGLIN, PF4, TIMP-1 and TIMP-2 were increased by 26.0, 3.5, 3.0, 2.2 fold respectively, however pro-angiogenic proteins such as IGFBP-2, MMP-8, MMP-9 and VEGF were decreased by 4.2, 6.2, 2.1, 8.4 fold respectively by Trabectedin treatment (p?0.05). While Trabectedin increased the generation of reactive oxygen species and GST activity in a dose dependent manner (10-12 -10-6 M), it decreased DNMT and HDAC activities in 48 h Trabectedin treated (10-12 -10-6 M) MCF-7 and MDA-MB-453 cells (p?0.05). This thesis is the first in vitro study indicating that Trabectedin has cytotoxic, apoptotic and anti-angiogenic effects in breast cancer cell cultures. These data reveal that after completing in vivo and phase studies Trabectedin may be an effective chemotherapeutic molecule for breast cancer treatment.