Evaluation of the effect of cordyspin on the expression pattern of H3K9 methyltransferase (G9a) in chronic leukemia cell line K562

Abstract

Chronic myeloid leukemia (CML) is a commonly occurring myeloproliferative disorder, accounting for 15-20% of leukemia cases. While tyrosine kinase inhibitors have greatly improved the treatment of CML, only a few patients can sustain a therapy-free remission without relapse and many of them still experience disease recurrence and progression into advanced stages. This problem can be attributed to the persistence of leukemic stem cells, which can self- renew independently of the BCR-ABL oncogene. Therefore, alternative pathways need to be identified to address this issue. Epigenetic changes have been recognized as significant contributors to the development and spread of leukemia. Specifically, elevated levels of the enzyme G9a have been implicated in the pathophysiology of various hematological diseases, including CML. However, the clinical and preclinical application of existing G9a inhibitors is limited due to their high toxicity and low bioavailability. Consequently, there is a need to explore alternative agents, such as natural metabolites with anticancer properties. The purpose of this study is to investigate the impact of Cordycepin, a natural metabolite, on the expression pattern of the histone methyltransferase H3K9 or G9a gene in the K562 cell line. Additionally, the study aims to analyze the effects of Cordycepin on miRNAs that regulate G9a (miR-1, miR-122) and G9a target genes (mTOR, RARRES3). Materials and methods: After determining the optimum number of cells for the MTT assay, cells were subjected to increasing doses of cordycepin in order to determine the cytotoxic effects and the IC50 value of cordycepin. Subsequently, K562 cells were divided into four groups of 12 and 24 hour treated and control groups for further analysis. To investigate the expression levels of G9a, mTOR, RARRES3 genes, as well as mir-1 and mir-122 microRNAs, quantitative real-time PCR (qRT-PCR) was employed. And t-test and one way Anova were used for statistical analysis. Results: The results obtained from the MTT analysis revealed that the growth and proliferation rate of k562 cells treated with cordycepin were significantly reduced. Additionally, the expression of the G9a gene in the k562 cell line exhibited a significant five-fold decrease after 12 hours of cordycepin treatment compared to the control group. However, there were no significant changes observed in the G9a gene expression pattern over the course of 24 hours of cordycepin treatment compared to its control group. Moreover, cordycepin treatment following G9a inhibition resulted in the suppression of mTOR gene expression in both the 12-hour and 24-hour treatment groups compared to their respective control groups. Importantly, the decrease in expression observed after 24 hours of treatment was significantly lower than that seen after 12 hours of treatment. Additionally, a significant time-dependent increase in the expression of miRNAs miR-1 and miR-122 was observed in the cordycepin-treated groups compared to their respective control groups. Notably, RARRES3 expression was not detected in K562 cells in this study.