IDH mutant tip glioma’ da, Temozolomid’ in Chk1/2’ nin inhibitörü AZD7762 ve BER yolağı komponenti APE1 inhibitörü ile kombinasyonunun anti-kanser etkilerinin incelenmesi ve bu iki kombinasyon ile hedeflemenin diğer tamir yolaklarını düzenleyen genlerin ekspresyonu üzerine etkilerinin değerlendirilmesi
Project no. 20840 · Status: Kapanmış (Yayın bekleniyor) · 2019
Summary
Despite the multiple therapeutic modulus such as radiation and DNA alkylating agent Temozolomide (TMZ), the survival rate of malignant gliomas remains at 6,8%. The 90% of gliomas (stages II and III) contain a single mutant isocitrate dehydrogenase 1 (IDH1) allele. Transformation caused by IDH1 mutation is associated with TMZ resistance or decreased TMZ activity. However, there is no clinically available strategy for sensitizing IDH1 mutant tumors to TMZ yet. In this study, in order to increase the efficiency of TMZ, it was aimed to examine cell death and cell cycle progression and the gene expression changes regulating DNA repair pathways by targeting with TMZ in combination with APE1 inhibitor targeting BER pathway and AZD7762 which is an inhibitor of Chk1/2 proteins in IDH1 mutant brain cancer cells. The efficacy of inhibitors in combination with TMZ in IDH mutant U87-mg cells was determined via isobologram analysis using WST-1 solution. DNA double-strand break after damage caused by the combination of TMZ with individual inhibitors was examined by using “Alexa Fluor 488 Mouse anti-H2AX Kit" in Flow cytometer. The number of AP sites was evaluated using the “DNA Damage Assay Kit (AP sites)" in the microplate reader. The effect of two different combinations on the genes regulating DNA repair pathways was performed using “RT² Profiler™ PCR Array Human DNA Damage Signaling Pathway” in real time qRT-PCR and analysed by 2-??CT method. The regulator effect of mono-drug and combination treatment on the cell cycle was assessed using the “Cycletest™ Plus DNA Reagent” in the Flow cytometer. The combination of TMZ and AZD7762 in IDH mutant U87-mg cells showed a synergistic effect at 24, 48 and 72nd hour (h), and TMZ exhibited a dose reduction index (DRI) of 3.37, 6.31, 10.26, respectively. This dual combination caused 1.14-fold, 2.56-fold, and 3.78-fold apoptosis at 24, 48, and 72nd h, respectively. When the effect of the combination on the cell cycle was evaluated, compared to the untreated control group, G2/M arrest was observed to increase 1.29-fold at the 24th h, 2.83-fold at the 48th h, and 1.73-fold at the 72nd h, when polyploidy increased 5.67- fold. When double-strand break formation was evaluated after G2/M arrest, H2AX phosphorylation was found to increase 16.66-fold at the 24th h, 14.7-fold at the 48th h, and 9.3-fold at the 72nd h compared to the untreated group. It was determined that the expression of the damage response proteins in the downstream region of ATM/Chk1 and ATM/Chk2 decreased, and the genes regulating apoptosis were overexpressed. In IDH mutant U87-mg cells, the combination of TMZ and APE1 inhibitor showed an additive effect at 24th h and synergistic effect at 48th and 72nd h. The DRI of the APE1 inhibitor on the TMZ was 1.55, 1.79, 6.23 at 24, 48 and 72nd h, respectively. When combined with the APE1 inhibitor with TMZ, apoptosis was determined to be 3.35-fold at the 24th h, 3.18-fold at the 48th h, and 2.05-fold at the 72nd h. Then, investigating the effect of the combination on the cell cycle, 1.11-fold G2/M arrest at the 24th h, 3.98-fold S phase and 1.49-fold G2/M arrest at the 48th h, and 2.21-fold G2/M arrest and 4.32-fold polyploidy at the 72nd h was determined. The formation of the double-strand break as a result of G2/M arrest was analyzed, it was found that H2AX phosphorylation increased 2.33 -fold at the 24th h, and there was no significant change at the 48th and 72nd h. In addition, a 1.77-fold increase was observed in the accumulation of the AP site at the 24th h compared to the control group, while a 1.58-fold increase was observed at the 72nd h. In line with all these results, AZD7762 by increasing the efficiency of TMZ, decreasing the expression of genes associated with resistance against the alkylated agent and inducing apoptosis by driving the cell into mitotic chaos by shows that it has a great therapeutic potential that can be used in cancer treatment. Further, AZD7762 needs in vivo experiments to evaluate its activity. A striking anticancer activity was observed in the first 24th h of the combination of TMZ and APE1 inhibitor III, and the strong effect of this dual combination in the first 24th h is promising in reducing the exposure time of patients to TMZ. As a continuation of this study, we are planning to focus on more specific targets with the data we found in future studies, to become the groundwork for in vivo studies and to contribute to the literature.