Investigation of the effects of proteosome inhibitor bortezomibe and topoisomerase inhibitors camptothesin and etoposide on nf-κb signaling pathway and apoptosis in the myeloid leukemia cell line

Nuclear factor kappa B (NF-κB) is a transcription factor which triggers more than 500 genes regulation that play an important role in many cases such as inflammation and immune system in addition to cell proliferation, apoptosis, angiogenesis, and metastasis. NF-κB has been shown to be constitutive activation in almost all hematologic malignities and solid tumors. In this case, the blocking agents of NF-κB signaling pathway have become a good target in the cancer therapy. However, combination treatments should be developed for the undesirable situations in which the blocking agents of NF-κB signaling pathway inhibit the mechanisms that play a role in tumor progression, in addition to inhibition of mechanisms such as inflammation and immune systems. In this study, it has been studied whether alone or combined usage of medicines which are bortezomibe (Bort), camptothecin (CPT), etoposide (Eto), which respectively inhibits proteasomes, topoisomerase I, topoisomerase II in clinics, affect chemo-therapeutically on chronic myeloid leukemia (K562) and acut-promyelocytic leukemia(HL-60). Another significant aim of this study is to find out whether bortezomib which gets NF-κB signaling pathway stopped by inhibiting its proteasome, causes a DNA damage or not. Additionally, we aimed to find whether the inhibitors of topoisomerase, which cause DNA damage, activate NF-κB signaling pathway or not, and to find the link between these mechanisms and apoptosis in molecular level. To achieve these aims, firstly the viability of cells was found by the application of alone or combined medicines in MTT test. The relation among apoptosis, DNA damages and the NF-κB signaling pathway was analyzed in gene level by real-time RT-PCR method. To determine the relation, the changes in mRNA level of NF-κB1 (p50), Rel-A (p65), ATM, ATR, p53 genes, and for apoptosis anti-apoptotic Bcl-2, pro-apoptotic Bax and Caspas-3 was found by 2-∆∆Ct method. As a result, we observed that Bort, CPT, and Eto cause a decrease in cell viability and apoptosis on both cell lines according to their dose by finding the changes of cell viability and the expression of apoptotic genes. Interestingly, we found that Bort cause an increase in ATM, ATR and p53 gene expression in mRNA level on both cell lines. Additionally, we have found that Bort triggers the pro-apoptotic function of NF-κB based on the increase of RelA expression in K562 and HL-60 cells rather than inhibiting NF-κB signaling pathway. We surprisingly found that Eto and CPT have an effect on Rel-A and NF-κB1 gene expression and they activate NF-κB signal pathway while confirming Eto and CPT cause DNA damage. All two-combined application of Bort, Eto and CPT caused an increase in cytotoxic activity. However, we determined that K562 cells had a better response(by %0-20 survivor rate) in cell vitality tests compared to HL-60 cells. All two combination of these medicines on both cell lines cause a decrease in bcl-2 and increase in Bax ve Caspaz 3 genes expression levels. The data confirmed that combined-therapy causes apoptosis. These results assert that all combinations effect NF-κB signal pathway which is related to Rel-A and eventually, these results support that Rel-A is an essential molecule for triggering the pro-apoptotic mechanism of NF-κB signal pathway. Keywors: NF-κB, Apoptosis, DNA Damage, Bortezomibe, Etoposide, Camptothecin, qRT-PCR