Studies Of Alpha-fetoprotein Specific Transfer Factors Induce Apoptosis In Bel7402 Alpha-fetoprotein Positive Hepatocarcinoma Cells

Background and PurposeHepatocellular carcinoma (HCC) is one of the most common causes of malignant death in China. Though HCC therapy has made large progress, the effects of traditional therapy methods haven't been satisfying. Recently, many HCC-associated self-antigens have been investigated as the target for HCC therapy, one of the antigens are alpha-fetoprotein (AFP). AFP is an oncoprotein that is produced during pregnancy by the fetal yolk sac or fetal liver. It is hardly detectable in normal adults, but often elevated significantly in most of the HCCs. Therefore, AFP has been a serological marker for HCC in clinical diagnosis.Researchers have found that there are specific membrane AFP receptors in many hepatocarcinoma cells that can secrete AFP themselves and it is important for the binding of AFP with AFP receptors to keep HCC growth. On the other hand, AFP as a kind of suppressive factor can inhibit immune response of many immunocytes including T lymphocytes, B lymphocytes, Dendritic cells and NK cells. Therefore, when HCC produces, AFP of high expression can not only directly enhance the growth of HCC, but also help hepatocarcinoma cells escape from immune surveillance.Specific transfer factors (TF), which are obtained from antigen-sensitized lymphcytes, are ribonucleopeptides with low molecular weight(≤6 kDa).They carry immune information from antigen-specific sensitized immunocytes of donors , can transfer the specific immune information to recipients of negative immune response and eventually induce recipients to antigen-specific immune response. Furthermore. specific TF can specifically bind with the antigen that sensitize lymphcytes and the binding of antigen with antigen-specific TF will cause specific TF losing their original biological activity. As a kind of regulator of cell immunity, specific TF has been broadly applied in the therapy of diseases including immunodeficiency and infectious diseases.Since AFP as a marker for HCC is a kind of suppressive factor to immune system, while antigen-specific TF as a kind of regulator of cell immunity can induce recipients to antigen-specific immune response. Therefore, we are interested in using AFP as the antigen to prepare AFP-specific transfer factors (AFP-TF) to investigate whether they have specific active effects. In our report, we found for the first time that AFP-TF could inhibit the growth of Bel7402 and HepG2 AFP positive hepatocarcinoma cells, and induce their apoptosis, but have no obvious effect on Sk-Hep-1 AFP negative hepatocarcinoma cells and Changliver normal liver cells. We further investigate the approach of AFP-TF induced AFP positive Bel7402 cells apoptosis and the function of AFP that is secreted by Bel7402 cells in the process of apoptosis.Methods1. Preparation and activity detection of AFP-TF1) AFP (Biodesign, USA) as the antigen immunized swines and AFP-TF were prepared from sensitized spleens of swines. The preparation was performed as described by Kirkpatrick.2) The activity of AFP-TF was detected in vitro by leucocyte adherence inhibitionassay.3) The activity of AFP-TF was detected in vivo by footpad swelling response assay ofmice4) The specific binding assay of AFP-TF with AFP2. Studies of AFP-TF induced AFP positive Bel7402 and HepG2 hepatocarcinoma cells apoptosis 1) Cell viability was detected by MTT assay.2) Cell morphological change was observed by a light microscope.3) Nuclear change was detected by Giemsa staining.4) Nuclear condensation was detected by Hochest 33258 staining.5) DNA fragmentation was detected by TUNEL assay.6) Cell cycle change was analyzed by FACSCalibur.7) Ultrastructural change of Bel7402 cell was detected by TEM.3. Studies of approaches of AFP-TF induced Bel7402 cells apoptosis1) Changes of intracellular protein expression (Bcl-2, Bax, Cytochrome C) was detected by western blot.2) Intracellular Ca²⁺concentration was detected by Fluo-3 AM fluorescent probe using spectrofluorometer.3) Intracellular mitochondrial membrane potential was measured by Rh123 fluorescent probe using spectrofluorometer.4) The activity of Caspase-9 and Caspase-3 was detected by caspase-3 and caspase-9 assay kit (colorimetric analysis).4. Studies of interaction between AFP-TF and intracellular AFP in the apoptosis of Bel7402 cells.1) The expression of intracellular AFP mRNA was detected by RT-PCR.2) The expression of intracellular AFP was detected by western blot.3) The distribution and expression of AFP were detected by immunocytochemistry.4) The construction of pEGFP-AFP vector.5) pEGFP-AFP vector was transfected in Bel7402 cells using Lipofectamine TM 2000.6) After exogenous AFP was directly added to culture in advance or AFP expression carrier was transfected in Bel7402 cells, cell viability was detected using AFP-TF by MTT assay. ResultsThe first partThe activity detection of AFP-TFThe activity detection in vitro showed that AFP-TF had fine activity of leukocyte adherence inhibition. Besides, the activity detection of AFP-TF in vivo showed that the difference of footpad swelling between experiment group and control group was significant. Furthermore, after AFP-TF specifically bound with AFP, the activity of AFP-TF disappeared.The second part Cell viability detectionAFP-TF had obvious inhibition effects on Bel7402 and HepG2 AFP positive hepatocarcinoma cells, but not Sk-Hep-1 AFP negative hepatocarcinoma cells and Changliver normal liver cells.Analysis of the controlAFP could enhance growth of Bel7402 cells, while AFP antibody could slightly inhibit growth of the cells. After AFP-TF specifically bound with AFP, the Supernatants with no AFP-TF activity had no obvious effect on Bel7402 cells, so was the HBsAg-TF.Cell apoptosis detectionAfter Bel7402 and HepG2 cells were treated with AFP-TF, the cells exhibited special morphologies of apoptosis, such as the shrinkage and turning small of cells, the vesicle heaving on the cell surface, and the appearance of apoptotic bodies. Giemsa and Hochest33258 staining indicated that the nucleus condensed and the chromatin cleaved. TUNEL assay showed the nucleus exhibited puce (TUNEL positive), which indicated DNA fragmentation. However. AFP-TF-treated SK-Hep-1 and Changliver cells had no obvious apoptotic character. Cell cycle analysisAfter treatment with AFP-TF, the percentage of Bel7402 cells obviously increased in G0/G1 phase and slightly increased in G₂/M phase, while the percentage of HepG2 cells also obviously increased in G0/G1 phase. However, the percentage of SK-Hep-1 and Changliver cells in different phases changed little.TEM observingTEM obserbing showed the condensation and margination of chromatin, the disappearance of microvillus on cell surface, the blurry organ structure in cytoplasm. However, the cell profile was intact.The third part Western blot analysisAfter Bel7402 cells were treated with AFP-TF, Bcl-2 expression was upregulated whereas Bax expression was downregulated, and cytochrome c expression in cytoplasm increased while cytochrome c expression in mitochondria decreased.Mitochondrial membrane potentialIn AFP-TF-treated Bel7402 cells, mitochondrial membrane potential increased immediately within 1 h, and the decline was time-dependent within 12 h. Though mitochondrial membrane potential had a slight raise in the 24th h, it was much lower than that of the control.Ca²⁺ concentrationIn AFP-TF-treated Bel7402 cells, Ca²⁺ concentration increased immediately within 0.5 h, and then gradually declined with the increase of time. Besides, the decline was time-dependent within 24 h.The activity of Caspase-9 and Caspase-3In AFP-TF-treated Bel7402 cells,the mean level of caspase-9 and caspase-3 activity was significantly higher than that in the untreated ones. The fourth part RT-PCR analysisRT-PCR showed the level of AFP mRNA changed little in AFP-TF-treated apoptotic Bel7402 cellsWestern blot analysisWestern blotting showed AFP expression was decreased in AFP-TF-treated apoptotic Bel7402 cells and the decrease was dose-dependent.ImmunocytochemistryThe untreated cells showed a distinct immunoprecipitate staining in the cell membrane and cytoplasm, and the staining boundaries were clear. However, AFP-TF treatment induced a diffuse and light immunoprecipitate staining of AFP,and the staining boundaries became blurred.Cell viability detection (Addition of AFP in the culture or AFP transfection in Bel7402 cells in advance)After addition of exogenous AFP in the culture or transfected with AFP in Bel7402 cells in advance, the inhibition effects of AFP-TF on the cells reduced compared with AFP-TF-treated normal Bel7402 cells.Conclusion1. AFP-TF can specifically inhibit the growth of Bel7402 and HepG2 AFP positive hepatocarcinoma cells, but not Sk-Hep-1 AFP negative hepatocarcinoma cells and Changliver normal liver cells.2. AFP-TF can induce the apoptosis in Bel7402 and HepG2 AFP positive hepatocarcinoma cells.3. AFP-TF can block Bel7402 cells mostly in G₀/G-1 phase and partly in C₂/M phase, while block HepG2 cells mostly in G₀/G₁ phase. 4. AFP-TF can induce the disruption of mitochondrial membrane potential, the immediate elevation of Ca²⁺ concentration in cytoplasm, a prominently decreased ratio of bcl-2 to bax, the release of cytochrome c from mitochondria to cytosol, and ultimately an activation of caspase-9 and caspase-3.Therefore, AFP-TF induced Bel7402 apoptosis is mitochondria-dependent.5. AFP-TF downregulate AFP expressin of Bel7402 cells at post-transcription level,and AFP over-expression can antagonize the inhibition effect of AFP-TF on Bel7402 cells, which indicates that intracellular AFP participates in AFP-TF induced Bel7402 cells apoptosis.6. The growth inhibition of AFP-TF on Bel7402 cells may be that the binding of superfluous AFP-TF with intracellular AFP causes intracellular AFP losing its original function, and eventually induces its degeneration and downregulation, which can't maintain the basic growth of Bel7402 cells, and ultimately result in Bel7402 cells apoptosis.