Investıgatıon of The In Vıtro And In Vıvo Effects of Telomere-Targeted New Drug Candıdate Compounds on Dıfferent Cancer Cell Lınes
Özet
Cancer, characterized by its heterogeneity, metastatic character, and limited treatability, stands as a leading global cause of mortality. Cancer cells elongate their telomeres through the activity of the telomerase enzyme, thereby gaining immortality by evading control checkpoints in the cell cycle. The expression of telomerase is present in approximately 95% of cancer cells, while its activity is absent in somatic cells. Consequently, in recent years telomerase/telomere-targeted therapies have shown significant promise. Due to the heterogeneity of cancer cells and their varying telomere lengths, long-term anti-telomerase therapies can lead to adverse effects. Therefore, there is a need for novel molecules that can target telomeres, regardless of their telomeric lengths, and exhibit rapid efficacy. Within the scope of this doctoral thesis research, the objective was to investigate molecules added to telomeric structures by telomerase, which may induce genomic instability and cell death through telomeric DNA damage, both in vitro and in vivo. These molecules were synthesized by the MAIA biotechnology company (Chicago, USA) and delivered to the Department of Medical Biochemistry. The candidate drug molecules were tested on different cancer cell lines [breast cancer (MCF-7), non-small cell lung cancer (A549), cervical cancer (HeLa), and colon cancer (HT29)] at nine different concentrations for 96 hours, and cytotoxicity was assessed using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. The obtained EC50 results were compared with 6-thio-dG in each cell line, and a molecule named “L6” was identified as a new potential drug candidate with a lower EC50 value compared to 6-thio-dG. The EC50 values of different cell lines to the L6 molecule were evaluated, and HT29 colon cancer cells, which exhibited the highest sensitivity to the L6 molecule, were selected as the target cell line. The DNA damage induced by these molecules at telomeric ends was assessed using the Telomere Induced Foci (TIF) method with confocal microscopy. In this method, co-localization of telomeric probes and staining with the γH2AX antibody specific to DNA damage was used to demonstrate TIF. The results obtained from the TIF method indicate that the telomeric DNA damage caused by the L6 molecule is statistically significant compared to the control group and 6-thio-dG. Additionally, an assessment of global genomic damage reveals that the L6 molecule and 6-thio-dG induce DNA damage to an equal extent. Furthermore, the effects of the candidate molecules on the base excision repair (BER) pathway proteins Apurinic/Apyrimidinic Endonuclease-1 (APE1) and Poly(ADP-ribose) polymerase-1 (PARP1), as well as the quantities of DNA damage product 8-Hydroxy-2'-deoxyguanosine (8-OH-dG) was investigated liquid chromatography-high resolution mass spectrometry (LC-HR/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. L6 promotes a significant downregulation in the expression levels of both PARP1 and APE1 in HT29 cells. The in vivo efficacy of L6 was evaluated both in CD1 nude mice and BALB/c mice. In the xenograft model established with the HT29 cell line, the optimal dosage of L6 was determined to be 3 mg/kg, administered in a total of 4 doses, with a treatment frequency of twice per week. A syngeneic animal model was generated using the CT26 cell line, followed by the administration of sequential therapy with the L6 molecule and anti-PD-L1. After the euthanize the animals, immunophenotyping was performed by flow-cytometry on excised tumor tissues. The results obtained indicate that the L6 molecule led to a reduction in tumor size, decrease in Treg cell count, and an increase in the activated CD8+ cells.
