Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Cancer Immunol Immunother. 2004 May;53(5):461-70. doi: 10.1007/s00262-003-0461-0. Epub 2003 Nov 28.


Purpose: Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h (51)Cr-release assay and a 2-h [(3)H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.

Methods: Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h (51)Cr release assay.

Results: The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [(3)H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h (51)Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [(3)H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.

Conclusion: This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Apoptosis Regulatory Proteins
  • Carcinoma, Hepatocellular / immunology*
  • Carcinoma, Hepatocellular / pathology
  • Caspase 3
  • Caspases / metabolism
  • Cell Division
  • Cytotoxicity, Immunologic*
  • Enzyme Activation
  • Fas Ligand Protein
  • Flow Cytometry
  • Humans
  • Killer Cells, Natural / immunology*
  • Leukemia / immunology
  • Leukemia / pathology
  • Liver Neoplasms / immunology*
  • Liver Neoplasms / pathology
  • Membrane Glycoproteins / metabolism*
  • Necrosis
  • Receptors, TNF-Related Apoptosis-Inducing Ligand
  • Receptors, Tumor Necrosis Factor / genetics
  • Receptors, Tumor Necrosis Factor / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • TNF-Related Apoptosis-Inducing Ligand
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / metabolism*


  • Apoptosis Regulatory Proteins
  • FASLG protein, human
  • Fas Ligand Protein
  • Membrane Glycoproteins
  • Receptors, TNF-Related Apoptosis-Inducing Ligand
  • Receptors, Tumor Necrosis Factor
  • TNF-Related Apoptosis-Inducing Ligand
  • TNFRSF10A protein, human
  • TNFSF10 protein, human
  • Tumor Necrosis Factor-alpha
  • CASP3 protein, human
  • Caspase 3
  • Caspases