Investigation of the effect of CA IX enzyme inhibition on the EZH2 gene and histone 3 modifications

Abstract

Abstract Objectives Colon cancer is the most common gastrointestinal cancer worldwide with high morbidity and mortality rates. The main purpose of our study is to elucidate the interaction mechanism of the H+ ion concentration effect in the CO2/HCO3 − buffer system of tumor-associated carbonic anhydrase IX (CA IX) enzyme inhibition in the HT-29 colon cancer cell line on cell epigenetic modifications. Methods Cell culture was performed using the human colon cancer cell line HT-29. CA IX enzyme inhibitor Acetazolamide (AZA) was administered. The results of the cell viability test and inhibition were evaluated. Extracellular pH measurements were performed. Total histone protein isolation was performed and Histone H3 modifications were analyzed by ELISA method. After RNA isolation, complementary DNA (cDNA) synthesis was carried out. RT-PCR was performed to determine the gene expression levels of hypoxia-inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and CA IX. Results CA IX enzyme inhibition in the HT-29 cell line decreased the expression of CA IX (p<0.05) and HIF1A (p<0.01) genes and increased the expression of the EZH2 (p<0.05). There was a significant decrease in the expression of CA IX (p<0.05) and HIF1A genes as a result of inhibition with AZA performed under hypoxic conditions. It was observed that CA IX enzyme inhibition increases the expression of the EZH2 gene by more than 3 times (p<0.01). As a result of AZA inhibition, methylation levels were observed to increase in normoxic conditions, while methylation levels were observed to decrease in hypoxic conditions. Conclusions Observing the changes in the H3 modifications and changes in the expression of CA IX, HIF1A and EZH2 genes in this study supports that CA IX enzyme inhibition plays an active role in epigenetic modifications.