Induction of Apoptosis in Human Hepatoma Cells by Alpha-Fetoprotein

Abstract

We have investigated the effects of purified human α-fetoprotein (AFP) on the growth of the human hepatocarcinoma cells HepG2 in culture. Cancer-derived AFP (cAFP), isolated from the culture medium of AFP-secreting HepG2 cells and embryonal AFP (eAFP), isolated from human cord serum, were used for these studies. Both AFP preparations studied were shown to induce strong dose-dependent inhibition of HepG2 cell proliferation and complete growth arrest at high protein concentrations (more than 0.1 mg/ml). To test whether AFP may trigger an endogenous suicide program in hepatoma cells, we examined whether DNA fragmentation preceded cell death. After exposure of the cells to the high AFP dose (1.0 mg/ml), DNA fragmentation was detected as early as 2 h after treatment, and 70% of cells were apoptotic by 24 h. DNA fragmentation was shown to precede other signs of cell death for several hours. Typical morphological changes of apoptosis were observed after 4 h of exposure of cells to high AFP doses. Low concentrations of cAFP and eAFP (less than 0.1 mg/ml) failed to induce growth inhibition of HepG2 cells, rather showing a weak stimulative effect, demonstrating a biphasic AFP activity. Cell pretreatment with the transcriptional inhibitor actinomycin D had no measurable influence on AFP cytotoxicity. These findings demonstrate that protein synthesis is not required for this mechanism of cell death. The charcoal-treated ligand-free eAFP (eAFPp) had a dose-dependent growth-inhibitory activity, similar to intact protein, but slightly less intensive. The similar growth-inhibitory activities of cAFP, eAFP and eAFPp, which have a significant difference in bound-ligand content, provide evidence that the main role in cell growth regulation may be attributed to the protein moiety of the entire AFP molecule, but not to its ligands. These biologically active AFP ligands could, however, modulate AFP-growth-regulating activity. Growth factor deprivation distinctly enhanced the cytostatic activity of high AFP concentrations and also increased the mitogenic activity of low AFP levels, showing the interdependence of the growth-regulative activity of AFP and growth factors. The findings of this study demonstrated that AFP is directly introduced into the intracellular pathways of cell growth regulation and programmed cell death.

Growth-Regulative Activity of Human Alpha-Fetoprotein for Different Types of Tumor and Normal Cells

“…Prior to the addition of reagents, the culture medium from the adherent cells was removed by an automatic pipette, and 0.18 ml of fresh medium with or without FCS was added to the wells and left for 2 h. Thereafter, serially diluted AFP or human serum albumin (HSA, Sigma) in 0.02 ml of phosphate-buffered saline (PBS) was introduced to the wells for 24 h. For the last 4 h of incubation, 1 ÌCi of [ 3 H]-thymidine (1 Ci/mmol) was added to each well. Experimental data were expressed as a percentage of [ 3 H]-thymidine incorporation relative to the medium control [39,42].…”

“…On the contrary, growth-suppressive properties of pure AFP and of the AFP/estradiol reaction product were conclusively demonstrated in vivo and in vitro for both human and rat breast tumors [34][35][36][37][38]. Our recent data showed that human embryonal AFP, derived from human cord serum, and cancerassociated AFP, purified from the culture medium of AFP-secreting human hepatoma cells, can induce apoptotic cell death in sensi- Dudich/Semenkova/Gorbatova/ Dudich/Khromykh/Tatulov/Grechko/ Sukhikh tive HepG2 cells in a dose-dependent manner [39].…”

Hemoglobin-associated proteins isolated from blood serum of Ehrlich carcinoma-bearing mice

“…Another mechanism independent from the apoptosis-inducing effects is the stimulation of anti-tumor immune response. AFP enhances the dendritic-cell-mediated immune reaction of T-lymphocytes against tumor cells (31,32). It is intriguing that AFP isolated from human cord serum has been shown to inhibit the growth of human hepatocarcinoma cells (33).…”

Villin 1 is a predictive factor for the recurrence of high serum alpha‐fetoprotein‐associated hepatocellular carcinoma after hepatectomy

“…AFP might also function as an apoptotic factor. (15)(16)(17)(18)(19) However, the molecular background of HCC associated with increased AFP levels, the mechanisms responsible for the recurrence of HCC are presently unclear.Gene expression analysis by cDNA microarray offers a systematic approach to acquire comprehensive information regarding gene transcription profiles. It is now possible to perform a large-scale expression survey to identify candidate target genes and to develop a novel molecular-targeted therapy for HCC.…”

“…AFP might also function as an apoptotic factor. (15)(16)(17)(18)(19) However, the molecular background of HCC associated with increased AFP levels, the mechanisms responsible for the recurrence of HCC are presently unclear.…”