The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line

Figure 1 The effects of different concentration of AFP on the proliferation of cells. Bel 7402 cells were incubated with different concentrations of AFP for 48 h and the cell proliferation was measured by MTT assay. The data represented the mean values of six independent experiments performed each in triplicate. ^aP < 0.05 and ^bP < 0.01 vs control (0 mol·L^-1).

Figure 2 The blockage of anti-AFP to the effect of AFP on the proliferation of cells. A. The data of MTT assay. B. The data of ³H-TdR incorporation. The cells were respectively treated with either AFP (20 mg·L^-1), anti-AFP (40 mg·L^-1), AFP (20 mg·L^-1) + anti-AFP (40 mg·L^-1) or HSA (20 mg·L^-1) for 48 h. (MTT assay) or 18 h (³H-TdR incorporation). The data represented as the mean value of four independent experiments performed each in triplicate. ^aP < 0.05 and ^bP < 0.01 vs control (0 mol·L^-1).

Figure 3 Scatchard analysis of ¹²⁵I-AFP binding to Bel 7402 cells. The properties of AFP receptor in Bel 7402 cells was detected with receptor binding assay and analyzed by a program of Radioligand Binding Assay of Receptor. The data were selected from three independent experiments.

Figure 4 The effects of AFP on the cAMP concentration in cytosol of human hepatoma Bel 7402 cells. 4 × 10⁴ cells were respectively treated with AFP (20 mg·L^-1), anti-AFP (40 mg·L^-1), AFP (20 mg·L^-1) + anti-AFP (40 mg·L^-1) or HSA (20 mg·L^-1). The data represented the mean values of four independent experiments performed each in triplicate. ^bP < 0.01 vs control (0 mol·L^-1).

Figure 5 The effects of AFP on the activity of PKA in Bel 7402 cells. 4 × 10⁵ cells per ml were respectively treated with either AFP (20 mg·L^-1), anti-AFP (40 mg·L^-1), AFP (20 mg·L^-1) + anti-AFP (40 mg·L^-1) or HAS (20 mg·L^-1) for differ-ent time and the activities of intracellular PKA were then detected. The data represented the mean values of four independent experiments performed each in triplicate. ^aP < 0.05 and ^bP < 0.01 vs control (0 mol·L^-1).

Figure 6 A: The change of Ca²⁺ concentration in human hepatoma Bel7402 cells was measured by confocal microscopic scanning. Cells were incubated with 5 μmol·L^-1 fluo-3/AM at 37 °C for 30 min and then stimulated with Hank’s. (1) AFP (20 mg·L^-1); (2) HSA (20 mg·L^-1); (3) Anti-AFP; (40 mg·L^-1); (4) or AFP (20 mg·L^-1) + Anti-AFP (40 mg·L^-1); (5) The arrow indicate the stimulated time point. B: The graph shows the scanning results. The data represented as the mean value of 10 cells ± s. ^aP < 0.05 and ^bP < 0.01 vs control (0 mol·L^-1)

Figure 7 The effects of AFP on the expression of p53 (A) and p21 (B) proteins in Bel 7402 cells. 1 × 10⁵ cells were respectively treated with AFP (20 mg·L^-1), anti-AFP (40 mg·L^-1), AFP (20 mg·L^-1) + anti-AFP (40 mg·L^-1) or HSA (20 mg·L^-1) for 24 hours and the expression of p53 and p21protein were detected by Western blot assay. Lane 1: control group; lane 2: HSA treated group; lane 3: AFP treated group; lane 4: anti-AFP treated group; lane 5: AFP plus anti-AFP treated group. The data was selected from 3 indepen-dent experiments. A:p53; B:p21. The columns represent the means of triplicate determinations ± s.

Figure 8 The effects of AFP on the expression of N-ras mRNA in Bel 7402 cells. 1 × 10⁵ cells were respectively treated with AFP (20 mg·L^-1), anti-AFP (40 mg·L^-1), AFP (20 mg·L^-1) + anti-AFP (40 mg·L^-1) or HSA (20 mg·L^-1) for 12 hours and expression of N-ras mRNA was detected by Northern blot assay. Lane 1: control group; Lane 2: HSA treated group; Lane 3: AFP treated group; Lane 4: anti-AFP treated group; Lane 5: AFP plus anti-AFP treated group. The data was selected from 3 independent experiments. A: Auto-radiograph of Northern blot. B: Quantitated by densitometric scanning of N-ras mRNA expression blot in Bel7402 cells (relative IOD units). The columns represent the means of triplicate determinations ± s.