Abstract
An acidic extracellular pH (pHe) is a feature of the solid tumor microenvironment, which contributes to cell migration and invasion of tumor cells. Our previous study has demonstrated that acidic pHe disrupts the integrity of adherens junction of hepatocellular carcinoma (HepG2) by activation of Src, and promotes migration by cell dispersion. In this study, we investigated whether reactive oxygen species (ROS), a signaling messenger, initiated the signaling for acidic pHe-induced cell migration. Our results showed that incubation of HepG2 cells in pH 6.6 culture medium induced HepG2 cells from tight cluster to a dispersed and flattened cell profile, and this change was accompanied by an increase in cell migration ability. By flow cytometry, pH 6.6 treatment induced higher ROS levels compared to those at pH 7.4. Furthermore, ROS production mainly came from mitochondria by flow cytometry with MitosoxTM , mitochondria superoxide indicator. pH 6.6- induced-ROS increased cell migration, since an antioxidant dithiothreitol (DTT) prevented this effect. Extracellulr signal-regulated kinase (ERK) phosphorylation increased at 30 minutes after pH 6.6 stimulation, and sustained up to 90 minutes. Inhibition of ERK by a mitogen-activated protein kinase (MEK) inhibitor, U0126, or by siERK abrogated pH 6.6- induced cell migration of HepG2 cells. In addition, DTT blocked pH 6.6-induced phosophorylation of ERK. This suggests that ROS is the upstream event of ERK. In response to pH 6.6 treatment, focal adhesions clustered at peripheral cell membrane and randomly-distributed microfilaments were rearranged into parallel – oriented stress fibers. Western blotting analyses demonstrated that FAK was phosphorylated by pH 6.6 treatment. Furthermore, FAK played a key role in cell migration, since inhibition of FAK by a FAK inhibitor , PF573228, or by siFAK effectively prevented pH 6.6-induced cell migration. DTT pretreatment abrogated pH 6.6-induced phosophorylation of ERK and FAK. It implicates that ROS is the upstream event of both ERK and FAK. Inhibition of ERK by U0126 and siERK blocked pH 6.6- induced phosphorylation of FAK and retarded cell migration, indicating the presence of the ERK-FAK pathway. Besides, Src and p130CAS were activated by pH 6.6 treatment, and PP2 pretreatment disturbed pH 6.6-induced stress fiber formation, suggesting the involvement of the Src-p130CAS pathway in this event. In conclusion, this study characterize a novel signaling way responsible for enhancing the migratory behavior of HepG2 cells via the ROS- ERK- FAK signaling in an acidic microenvironment.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
