Objectives:Hepatocellular carcinoma(HCC)has limited systemic options with substantial toxicity.Gquadruplex(G4)structures in oncogene promoters are attractive but challenging drug targets.This study aimed to determine ...Objectives:Hepatocellular carcinoma(HCC)has limited systemic options with substantial toxicity.Gquadruplex(G4)structures in oncogene promoters are attractive but challenging drug targets.This study aimed to determine whether glutamic acid-chelated cobalt(GACC)is a G4-active scaffold with anti-HCC efficacy and favorable in vivo safety,and whether an AI-guided phenotypic response surface(PRS)can optimize less toxic combinations.Methods:Anticancer activity was tested in HCC cell lines(PLC/PRF/5,Hep3B,HepG2)and non-transformed THLE-2 hepatocytes(CCK-8,IC_(50)).In vivo safety/efficacy were assessed in zebrafish embryo toxicity assays,a Hep3B xenograft model,and a tert-overexpressing transgenic zebrafish model,with hepatotoxicity monitored in a liver-fluorescent reporter line.Target engagement was examined by docking,native PAGE,a KRAS promoter G4 DNA polymerase stop assay,BG4 immunofluorescence,and KRAS qPCR.PRS was used to optimize GACC-metformin-regorafenib combinations.Results:GACC reduced HCC viability(IC_(50)~86-115μM)and showed low embryotoxicity(IC_(50)6.87 mM).In zebrafish xenografts,GACC(50μM)reduced Hep3B tumor fluorescence by~90%without detectable hepatotoxicity,whereas sorafenib decreased liver size/fluorescence.In tert-overexpressing zebrafish,GACC suppressed proliferation and Wnt/β-catenin-associated transcripts and reduced mitotic figures and nuclear atypia.Mechanistically,GACC increased KRAS promoter polymerase stalling,enhanced nuclear G4 signal,and reduced KRAS transcripts.PRS identified an off-grid triple combination that reduced PLC/PRF/5 viability to 19%while maintaining THLE-2 viability at 52%and preserving zebrafish development.Conclusion:GACC is a G4-active cobalt-glutamate scaffold with anti-HCC activity and favorable zebrafish safety,and a zebrafish-plus-PRS workflow enables rational,less toxic combination design.展开更多
基金Support from the National Science and Technology Council(NSTC),Taiwan(111-2320-B-400-018-MY3,114-2320-B-400-022-MY3)by the National Health Research Institutes(NHRI)intramural program(MG-113-PP-06)awarded to Chiou-Hwa Yuh is gratefully acknowledgedCore facility services and instrumentation support were supported by NSTC(112-2740-B-400-001,Taiwan Zebrafish Core Facility at NTHU-NHRI)and NSTC(111-2731-M-002-001,XRD000200,Instrumentation Center,National Taiwan University).
文摘Objectives:Hepatocellular carcinoma(HCC)has limited systemic options with substantial toxicity.Gquadruplex(G4)structures in oncogene promoters are attractive but challenging drug targets.This study aimed to determine whether glutamic acid-chelated cobalt(GACC)is a G4-active scaffold with anti-HCC efficacy and favorable in vivo safety,and whether an AI-guided phenotypic response surface(PRS)can optimize less toxic combinations.Methods:Anticancer activity was tested in HCC cell lines(PLC/PRF/5,Hep3B,HepG2)and non-transformed THLE-2 hepatocytes(CCK-8,IC_(50)).In vivo safety/efficacy were assessed in zebrafish embryo toxicity assays,a Hep3B xenograft model,and a tert-overexpressing transgenic zebrafish model,with hepatotoxicity monitored in a liver-fluorescent reporter line.Target engagement was examined by docking,native PAGE,a KRAS promoter G4 DNA polymerase stop assay,BG4 immunofluorescence,and KRAS qPCR.PRS was used to optimize GACC-metformin-regorafenib combinations.Results:GACC reduced HCC viability(IC_(50)~86-115μM)and showed low embryotoxicity(IC_(50)6.87 mM).In zebrafish xenografts,GACC(50μM)reduced Hep3B tumor fluorescence by~90%without detectable hepatotoxicity,whereas sorafenib decreased liver size/fluorescence.In tert-overexpressing zebrafish,GACC suppressed proliferation and Wnt/β-catenin-associated transcripts and reduced mitotic figures and nuclear atypia.Mechanistically,GACC increased KRAS promoter polymerase stalling,enhanced nuclear G4 signal,and reduced KRAS transcripts.PRS identified an off-grid triple combination that reduced PLC/PRF/5 viability to 19%while maintaining THLE-2 viability at 52%and preserving zebrafish development.Conclusion:GACC is a G4-active cobalt-glutamate scaffold with anti-HCC activity and favorable zebrafish safety,and a zebrafish-plus-PRS workflow enables rational,less toxic combination design.
