Distribution and localization of microfilament cytoskeleton is regulated by EBP50

Objective:To explore the influence of EBP50(ezrin-radixin-moesin-binding phospho-protein-50) on microfilament cytoskeleton content and distribution in cultured Hela cells, and to investigate the relationship between the changes in microfilament cytoskeleton localization and EBP50 after PDGF(platelet-derived growth factor) stimulation, and to further clarify the molecular mechanism by which EBP50 suppresses tumor cell proliferation and migration.Methods:pBK-CMV-HAEBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were transfected into Hela cells.G418 at 350 mg/L was used to screen for cell clones stably expressing EBP50.Western blot was carried out to detect EBP50 expression.Similarities and differences in microfilament cytoskeleton content and distribution in Hela cells transfected with pBK-CMV-HA-EBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were also treated with PDGF(10 ng/mL and 20 ng/mL, 37 ℃, 15 min) and stained by rhodamine-labeled phalloidin to observe the distribution of microfilament cytoskeleton in the two groups.EBP50 protein distribution in PDGF-stimulated Hela cells was detected by immunofluorescence.Results:Western blot results confirmed that the EBP50 cDNA fragment could express EBP50 in cultured Hela cell lines and that cell lines stably expressing EBP50 were successfully obtained.Western blot and fluorescence results showed that in the cell line transfected with empty vector, the microfilament cytoskeleton was thick, loose, multidirectional and displayed crossing arrangements.The content of microfilament cytoskeleton in the cell line transfected with pBK-CMV-HA-EBP50 was different from that found in the cell line transfected with empty vector.EBP50 expression enhanced microfilament cytoskeleton polymerization into compact thin filaments.Under the stimulation of PDGF, EBP50 migrated to the cell membrane from the cytosol together with microfilament cytoskeleton and co-localized there.Conclusion:EBP50 can change the distribution of microfilament cytoskeleton in cultured Hela cells and can also bind the microfilament cytoskeleton to the cell membrane under the stimulation of PDGF.EBP50 may play a role in the proliferation and migration of tumor cells by influencing the distribution and localization of microfilament cytoskeleton.

Nitrate increases cisplatin chemosensitivity of oral squamous cell carcinoma via REDD1/AKT signaling pathway

Although cisplatin is one of the chemotherapeutics most frequently used in oral squamous cell carcinoma(OSCC)treatment,it exerts multiple side effects and poor chemosensitivity.Nitrate reportedly demonstrates several beneficial biological functions,and synthesized nitrates enhance the therapeutic efficacy of chemotherapy.However,the role of inorganic nitrate in cisplatin chemotherapy remains unclear.We therefore investigated the effect of inorganic nitrate exerted on cisplatin sensitivity in OSCC.We found that nitrate did not affect OSCC cell growth and apoptosis in OSCC cells and OSCC xenograft tumor animal studies.Cisplatin induced REDD1 expression and AKT activation in OSCC.However,nitrate could increase cisplatin chemosensitivity,reduce its REDD1 expression,and attenuate AKT signaling activation in OSCC cells.Dysregulation of high levels of REDD1,which could enhance AKT activation,was positively associated with poor prognosis in OSCC patients.Thus,reduced REDD1 expression and retarded AKT activation induced by inorganic nitrate might be a new potential approach to the sensitization of oral cancer to cisplatin treatment in the future.