Elevated serum alpha fetoprotein levels promote pathological progression of hepatocellular carcinoma

Abstract

Aim: To investigate the biological role of alpha fetoprotein (AFP) and its clinical significance in carcinogenesis of hepatocellular carcinoma (HCC).

Methods: Clinical analysis of HCC patients and immunohistochemical examination were conducted to evaluate the relationship between serum AFP level and patient mortality. Confocal microscopy, Western blotting, dimethylthiahzolyl-2,5-diphenyl-tetrazolium bromide, Cell Counting Kit-8 assays and flow cytometry were performed to explore the possible mechanism.

Results: Among the 160 HCC patients enrolled in this study, 130 patients survived 2 years (81.25%), with a survival rate of 86.8% in AFP < 2 0 μg/L group, 88.9% in AFP 20-250 μg/L group, and 69.6% in AFP > 250 μg/L group, demonstrating a higher mortality rate in HCC patients with higher AFP levels. Surgical treatment was beneficial only in patients with low AFP levels. The mortality rate of HCC patients with high AFP levels who were treated surgically was apparently higher than those treated with conservative management. The results of immunohistochemistry showed that AFP and AFP receptor were merely expressed in tissues of HCC patients with positive serum AFP. Consistently, in vitro analysis showed that AFP and AFPS were expressed in HepG2 but not in HLE cells. AFP showed a capability to promote cell growth, and this was more apparent in HepG2 cells, in which the proliferation was increased by 3.5 folds. Cell cycle analysis showed that the percentage of HepG2 cells in S phase after exposure to AFP was modestly increased.

Conclusion: HCC patients with higher AFP levels show a higher mortality rate, which appears to be attributable to the growth promoting properties of AFP.

Keywords: Alpha fetoprotein; Hepatocellular carcinoma; Mortality; Receptor; Survival.

Figure 1

Survival rates of hepatocellular carcinoma patients and expression of alpha fetoprotein and alpha fetoprotein receptor in tumor tissues. A: Comparison of survival rates in hepatocellular carcinoma (HCC) patients with various alpha fetoprotein (AFP) levels who were treated surgically or non-surgically. Serum AFP levels of these HCC patients were divided into three groups: < 20 μg/L, 20-250 μg/L and > 250 μg/L; B: Immunohistochemical analysis of AFP and AFP receptor (AFPR) expressions in HCC tumor tissues. Membranous expression of AFPR is indicated with arrows. HE: Hematoxylin-eosin staining; AFP: Alpha fetoprotein; AFPR: Alpha fetoprotein receptor; HCC: Hepatocellular carcinoma.

Figure 2

Expression of alpha fetoprotein and alpha fetoprotein receptor in HepG2 and HLE cells. A: Western blotting; B and C: Confocal microscopy. The immunoblots and images captured by cofocal microscopy are representative of experiments that were repeated at least three times. AFP: Alpha fetoprotein; AFPR: Alpha fetoprotein receptor.

Figure 3

Effects of alpha fetoprotein in proliferation of HepG2 and HLE cells. Different concentrations (0, 0.01, 0.1, 1, 10 and 100 mg/L) of alpha fetoprotein (AFP) were tested in cell culture. The proliferation of HepG2 (A) and HLE (B) cells was evaluated with the dimethylthiahzolyl-2,5-diphenyl-tetrazolium bromide (MTT) assay at 24 and 48 h. HepG2 and HLE cells were further tested with 0, 50, 100, 200 and 400 μg/L AFP and proliferation was evaluated by MTT (C) and a cell counting kit (cck)-8 assay (D) at 24 h. The differences in proliferation between HepG2 and HLE cells were statistically analyzed with SPSS16 software. Data are representative of experiments that were repeated three times and are presented as mean ± SD for 6-9 samples.

Figure 4

Effects of alpha fetoprotein on cell cycle progression of HepG2 and HLE cells. The distribution of cells in different cell cycle phases was determined according to DNA content. The data are representative of 3 independent experiments. Each panel represents a different treatment group. A: HepG2 cells without treatment; B: HepG2 cells treated with 400 μg/L alpha fetoprotein (AFP) for 24 h; C: HLE cells without treatment; D: HLE cells treated with 400 μg/L AFP for 24 h. In each panel, the number represents cell percentage in S phase. Data are presented as mean ± SD of 3 samples. AFP: Alpha fetoprotein.