Investigation by which signal way cathecol stimulates apoptosis in parental and epurubic-resistant lung cancers

In this study, cytotoxic, antiproliferative, cell cycle blocking and apoptotic effect of catechol, a benzene derivative, on parental lung cancer (P-H1299), epirubicin-HCl resistant lung cancer (R-H1299) and healthy fibroblast (Bj) cells were investigated. The mechanism by which the apoptotic signaling pathway is most stimulated in each cell has been revealed. Cytotoxic effects of catechol on cells were determined by Cell Titer BlueR Cell Viability. IC50 values (concentration that kills 50% of cells) of catechol in P H1299, R-H1299 ve Bj were calculated respectively for catechol 58 µg/ml, 90 µg/ml ve 207 µg/ml. The cytotoxic effect of catechol on P-H1299 cells was higher than on both R-H1299 and Bj cells. In our study, caspase 3/7 activity and mRNA expressions were studied in cells treated with IC50 concentration of catechol for 24 hours. Caspase-3/7 activity, one of the key enzymes of the apoptotic pathway in cells, was measured using the ApoTox-Glo Triplex Assay (Promega) kit. Caspase 3/7 activity in cells was 2,8 times higher in P-H1299 cells, 2,3 times higher in R-H1299 cells and 1,6 times higher in Bj cells compared to control cells. Caspase 3/7 activity of P-H1299 cells was found 1,2 times higher than R-H1299 cells and 1,75 times higher than Bj cells. In order to investigate which signal pathway catechol stimulates apoptosis in cells, mRNA expressions of Bax (Bcl-2 Associated X Protein), Bcl-2 (B-Cell Lymphoma 2), ATM (Ataxia telangiectasia mutated kinase), ATR (Ataxia telangiectasia and Rad3-related), cyclin D1, PCNA (proliferating cell nuclear antigen) genes were investigated by RT-PCR (reverse transcriptase polymerase polymerase chain reaction). When all cells treated with catechol were compared, PCNA, which shows the antiproliferative effect, and cyclin D1 mRNA, which is a cell cycle marker, were found the lowest in P-H1299 cells. The apoptotic effect of catechol in cells was demonstrated by looking at the mRNA expression rates of Bax, a pro-apoptotic gene, and Bcl-2, an antiapoptotic gene. Bax/Bcl-2 ratios in P-H1299, R-H1299 and Bj cells exposed to catechol for 24 hours were calculated as 1,5 times 2,0 times and 0,8 times respectively. The Bax/Bcl-2 signaling pathway was most stimulated in R-H1299 cells. Catechol showed an apoptotic effect by increasing the Bax/Bcl-2 ratio in P-H1299 and R-H1299 cells. However, it showed an antiapoptotic effect by causing a decrease in the Bax/Bcl-2 ratio in Bj cells. In order to reveal the DNA damage caused by catechol in cells, mRNA expression levels of ATM and ATR genes were measured. Both ATM and ATR mRNA expression were increased in P-H1299, R-H1299 and Bj cells treated with catechol, indicating that catechol causes DNA damage on cells. When compared to healthy cells, ATM and ATR mRNA expression was lower in R-H1299 cells than in P-H1299 cells. However, when PCNA, cyclin D1, Bax, Bcl-2, ATM and ATR mRNA expression levels in all cells were compared with control cells, it was observed that mRNA expression of ATM and ATR genes increased the most. As a result, although it is seen that catechol causes apoptotic effect through multiple signaling pathways, it has been revealed that the most effective signaling pathway is ATM and ATR signaling pathway, which is explained by the presence of single and double strand breaks in DNA.