An Investigation of the Relationship between Paraoxonase and PI3K/Akt Pathway in HepG2 Cells and Their Exosomes in Normoxia and Hypoxia

Hepatocellular carcinoma (HCC) is characterized by a hypoxic tumor microenvironment and oxidative stress. Paraoxonase (PON), an enzymatic antioxidant, is associated with critical mechanisms during carcinogenesis. The PI3K/Akt pathway is effective in ROS detoxification, redox homeostasis, and the regulation of PON. Recent studies have highlighted the potential of non-invasively obtained exosomal contents in early cancer detection, therapeutic monitoring, and the assessment of drug resistance. Under hypoxic and oxidative stress conditions, exosome release is increased, and their content is altered. This study investigated the relationship between the PON enzyme family and PI3K/Akt pathway proteins in the HCC cell line (HepG2) and their exosomes under normoxic and hypoxic conditions.In this study, exosomes were isolated from HepG2 using the MACS system and characterized via Bradford assay, SEM, and NTA. Lysates from cells and exosomes were obtained from HepG2 cells cultured under both normoxic and hypoxic conditions. Paraoxonase and lactonase activities were measured spectrophotometrically. The expression levels of the PON family (PON1, PON2, PON3) and PI3K/Akt pathway proteins (Akt, p-Akt, mTOR, p-mTOR) were detected using western blot.Our results demonstrated the successful isolation of exosomes from HepG2. Under hypoxia, a reduction in paraoxonase and lactonase activity was observed compared to normoxia, despite an increase in the protein levels of the PONs (p < 0.05). While hypoxia increased Akt, p-Akt, mTOR, and p-mTOR levels in HepG2, it caused a decrease in exosomes.Under hypoxia, the decreased PON activity contributes to the carcinogenesis of HCC. The increased expression levels of PONs are thought to be involved in activating the PI3K/Akt pathway by acting as an oncogene. Importantly, the observed alterations in exosomal content may be effective in the activation of the PI3K/Akt pathway.