The CASP8 Knockout Huh-7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from the human Huh-7 hepatocellular carcinoma cell line, engineered to disrupt the CASP8 gene. This loss-of-function model eliminates caspase-8, the initiator caspase essential for death receptor-mediated apoptosis, enabling precise dissection of cell death and survival pathways in hepatic cancer cells.
Huh-7 is a well-characterized hepatocellular carcinoma line established from a 57-year-old Japanese male and widely used in liver biology, HCV research, and drug metabolism studies. As adherent hepatic epithelial cells, they retain key hepatocyte functions, providing a clinically relevant model for investigating apoptosis resistance, oncogenic signaling, and metabolic interactions in liver cancer.
CASP8 encodes caspase-8, an apical caspase activated by death receptors such as Fas (CD95), TNFR1, and TRAIL receptors (DR4/DR5) upon ligand binding (FasL, TNF-??, TRAIL). Via the adaptor FADD, caspase-8 is recruited to the death-inducing signaling complex (DISC), where it autoproteolytically activates and cleaves downstream executioner caspases-3 and -7, as well as Bid, to execute apoptosis. Caspase-8 also negatively regulates necroptosis through cleavage of RIPK1 and can modulate NF-??B signaling and inflammasome activity. Its activity is antagonized by c-FLIP at the DISC and by IAPs that inhibit caspase cascades.
In Huh-7 cells, CASP8 ablation confers resistance to apoptosis triggered by FasL, TNF-??, or TRAIL, recapitulating a common cancer evasion mechanism. Notably, under caspase-8-deficient conditions, TNF-?? stimulation can switch cell fate to RIPK1-dependent necroptosis, detectable by phosphorylated RIPK1 and MLKL oligomerization. This dual phenotype makes the model instrumental for studying the apoptosis-necroptosis switch and screening for sensitizers that restore death receptor-mediated killing in hepatocellular carcinoma.
These polyclonal knockout cells are suitable for diverse functional studies. Typical assays include western blot analysis of caspase-8 and cleaved caspase-3, Bid cleavage; RT-qPCR for CASP8 mRNA; flow cytometric apoptosis measurements (annexin V/PI) following death ligand treatment; cell viability assays (MTT, CellTiter-Glo) with TNF-?? plus cycloheximide; co-immunoprecipitation of DISC components such as FADD and caspase-8; and necroptosis analysis via RIPK1 phosphorylation and MLKL oligomerization. They additionally enable investigation of liver cancer biology, tumor microenvironment crosstalk, and high-throughput drug screening for sensitizers of death receptor-mediated apoptosis. For additional information, please contact Ascent Research.
