Reactive oxygen species-induced activation of Yes-associated protein-1 through the c-Myc pathway is a therapeutic target in hepatocellular carcinoma

BACKGROUND The Hippo signaling pathway regulates organ size by regulating cell proliferation and apoptosis with terminal effectors including Yes-associated protein-1(YAP-1).Dysregulation in Hippo pathway has been proposed as one of the therapeutic targets in hepatocarcinogenesis.The levels of reactive oxygen species(ROS)increase during the progression from early to advanced hepatocellular carcinoma(HCC).AIM To study the activation of YAP-1 by ROS-induced damage in HCC and the involved signaling pathway.METHODS The expression of YAP-1 in HCC cells(Huh-7,HepG2,and SNU-761)was quantified using real-time polymerase chain reaction and immunoblotting.Human HCC cells were treated with H2O2,which is a major component of ROS in living organisms,and with either YAP-1 small interfering RNA(siRNA)or control siRNA.To investigate the role of YAP-1 in HCC cells under oxidative stress,MTS assays were performed.Immunoblotting was performed to evaluate the signaling pathway responsible for the activation of YAP-1.Eighty-eight surgically resected frozen HCC tissue samples and 88 nontumor liver tissue samples were used for gene expression analyses.RESULTS H2O2 treatment increased the mRNA and protein expression of YAP-1 in HCC cells(Huh-7,HepG2,and SNU-761).Suppression of YAP-1 using siRNA transfection resulted in a significant decrease in tumor proliferation during H2O2 treatment both in vitro and in vivo(both P<0.05).The oncogenic action of YAP-1 occurred via the activation of the c-Myc pathway,leading to the upregulation of components of the unfolded protein response(UPR),including 78-kDa glucoseregulated protein and activating transcription factor-6(ATF-6).The YAP-1 mRNA levels in human HCC tissues were upregulated by 2.6-fold compared with those in nontumor tissues(P<0.05)and were positively correlated with the ATF-6 Levels(Pearson’s coefficient=0.299;P<0.05).CONCLUSION This study shows a novel connection between YAP-1 and the UPR through the c-Myc pathway during oxidative stress in HCC.The ROS-induced activation of YAP-1 via the c-Myc pathway,which leads to the activation of the UPR pathway,might be a therapeutic target in HCC.

Engineered 3D liver-tissue model with minispheroids formed by a bioprinting process supported with in situ electrical stimulation

Three-dimensional(3D)bioprinting,an effective technique for building cell-laden structures providing native extracellular matrix environments,presents challenges,including inadequate cellular interactions.To address these issues,cell spheroids offer a promising solution for improving their biological functions.Particularly,minispheroids with 50-100μm diameters exhibit enhanced cellular maturation.We propose a one-step minispheroid-forming bioprinting process incorporating electrical stimulation(E-MS-printing).By stimulating the cells,minispheroids with controlled diameters were generated by manipulating the bioink viscosity and stimulation intensity.To validate its feasibility,E-MS-printing process was applied to fabricate an engineered liver model designed to mimic the hepatic lobule unit.E-MS-printing was employed to print the hepatocyte region,followed by bioprinting the central vein using a core-shell nozzle.The resulting constructs displayed native liver-mimetic structures containing minispheroids,which facilitated improved hepatic cell maturation,functional attributes,and vessel formation.Our results demonstrate a new potential 3D liver model that can replicate native liver tissues.