An investigation of the relationship between paraoxonase enzyme family and PI3K/AKT signaling pathway in hepatocellular carcinoma cells and their exosomes under normoxic and hypoxic conditions

Hepatocellular carcinoma (HCC), characterized by hypoxic tumor microenvironment and oxidative stress, is the one of the leading cause of cancer deaths worldwide. Paraoxonase (PON) (EC 3.1.1.2) is an enzymatic antioxidant and plays a role in protecting cells from oxidative stress. The PON enzyme family is associated with important mechanism such as inhibition of apoptosis and cancer cell survival during carcinogenesis. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is effective in ROS detoxification, redox homeostasis and PON regulation. Exosomes that mediate signal transduction are also closely related to the carcinogenesis process. The use of non-invasively obtained exosome contents for early screening, early diagnosis, monitoring therapeutic effects and detecting drug resistance has attracted attention recently. Under hypoxia and oxidative stress conditions, exosome release increases and exosome content changes. The aim of our study is to determine the relationship between PON enzyme family and the PI3K/Akt signaling pathway proteins in hepatocellular carcinoma cell line (HepG2) and exosomes under normoxic and hypoxic conditions. To this end, we isolated exosomes using MACS system and characterized exosomes using Bradford, SEM, and NTA analyzes in the HepG2 cell line. HIF-1α protein levels were determined by ELISA assay and western blot under both normoxic and hypoxic (2, 4, 6, 12, and 24 hours) conditions to determine the appropriate hypoxia exposure time for the HepG2 cell line. Cell and exosome lysates were obtained from HepG2 cells cultured under these two conditions. The levels of paraoxonase and lactonase enzymes were measured spectrophotometrically. In addition, the enzyme family PON (PON1, PON2, PON3) and the proteins of the PI3K/Akt pathway (Akt, p-Akt, mTOR, p-mTOR) were analyzed by western blot. Based on the results, we suggest an appropriate hypoxia exposure time of 4 hours for the HepG2 cell line. In addition, our results show that exosomes were successfully isolated from the HepG2 cell line. Although there was a significant increase in protein levels of the PON enzyme family (PON1, PON2, PON3) under hypoxic conditions compared to normoxic conditions, a decrease in paraoxonase and lactonase enzyme activities was observed (p˂0.05). While hypoxic conditions increased Akt, p-Akt, mTOR and p-mTOR levels in HepG2 cells, it caused a decrease in exosomes. Our results suggest that decreased PON enzyme activity under hypoxic conditions has been shown to contribute to the carcinogenesis process of HCC. The increased expression levels of PON is thought to be involved in activating the PI3K/Akt signalling pathway by acting as an oncogone. In this study the results of exosomes status was also crucial to play a role in the relationship between PI3K/Akt and PON. Keywords: Exosome, Hepatocellular carcinoma, Hypoxia, Paraoxonase, PI3K/Akt pathway.