Effect Of Fragile Histidine Triad Gene Transduction On Proliferation And Apoptosis Of Human Hepatocellular Carcinoma Cells Hep3B

Objective: Fragile histidine triad(FHIT) gene, a tumor suppressor gene, correlates with tumorigenesis of live cancer. And the FHIT gene is altered in Hep3B cell line. We designed this study to evaluate the effects of human fragile histidine triad gene on cell proliferation and apoptosis of human hepatocellular carcinoma lines Hep3B.Materials and Methods: 1. Immunohistochemical(S-P) method was used to detect the expression of FHIT in the 46 patients with HCC and 10 cases with normal control. Among these specimen there were 34 males and 12 females with the ages ranging from 18-72, averaged 56.3; The average tumor size was (6.4±3.1) cm . All 46 hepatocellular carcinoma (HCC) specimens were pathologically confirmed. All the specimens were further identified in terms of pathological grading on the basis of WHO criteria, including 14 cases of well-differentiated liver cancer(n=14), moderately-differentiated(n=20) and poorly-differentiated(n=12). Ten benign liver specimens were served as controls. Immunohistochemical (S-P) and image analysis methods were used to detect the expression of FHIT, PTEN in the 46 patients with HCC and 10 cases with normal control. To investigate the expression of fragile histidine triad (FHIT) and PTEN in primary hepatocellular carcinoma(HCC)and their relationship to clinicopathological factors.2. Construct a recombinant pcDNA3.1(+)/FHIT including functional region of human fragile histidine triad gene(FHIT) to transfer into the cells of human hepatocellular carcinoma in vitro. The mRNA and proteinexpression of the gene in the transfected cells was detected by RT-PCR and Western blot respectively. MTT assay was used to assess the effect of FHIT on proliferation. The change of cell cycle and apoptosis was measured by flow cytometry.3. Plasmid pcDNA3.1 (+)/FHIT was transfected into hepatocellular cell line Hep3B(Hep3B-FHIT); pcDNA3.1(+) was transfected into Hep3B cell as control(Hep3B-C). These cells were selected using G418 for 14 days. Finally cells were harvested and injected hypodermically at back with either Hep3B-FHIT or Hep3B-C, 1 × 10⁶/mouse. The size of tumors was monitored every 3 days. Twenty-one days after injection, the tumor tissue at injection site was taken. Then the size and weight was measured .All data were used to analyze statistics.Results: 1. In the patients with HCC, the expressive rate of FHIT in the tumoral tissue was 56.52%, which was significantly lower than that in adjacent non-tumorous tissue and normal tissue. The absence of FHIT protein was correlated neither with the size, tumor capsula, serum AFP level nor with chronic hepatitis B virus infection and cirrhosis of liver. There was significant relationship between the expression of FHIT and differention (x²=6.619;P=0.045) and thrombus in the portal vein (x² =4. 336;P=0.037). In the patients with HCC, average optical density of FHIT and PTEN in the tumoral tissue were (0.1339 ± 0.864, 0.1534 ± 0.1562), which was significantly lower than that in adjacent non-tumorous tissue and normal tissue(P<0.05). Both of the expression of FHIT and PTEN were related to the tumor differention and thrombus in the portal vein.2. We cloned normal human FHIT cDNA into pcDNA3.1(+). Then plasmid pcDNA3.1(+)/FHIT was transfected into hepatocellular cell line Hep3B(Hep3B-FHIT); pcDNA3. 1(+) was transfected into Hep3B cell as control(Hep3B-C). RT-PCR and Western blot analysis showed that Hep3B cells expressed high level of FHIT-mRNA and FHIT protein after infectionwith pcDNA3.1(+)/FHIT. The growth of Hep3B cells treated with pcDNA3.1(+)/FHIT was significantly inhibited. Compared with Hep3B and Hep3B-C, the number of Hep3B-FHIT cells in S-phase and G₂/M-phase decreased significantly [(12.57 ± 0.42)%, (15.12±1.31)%]. Meanwhile, the number of Hep3B-FHIT cells in G₀/G₁-phase increased significantly (72.23 ± 0.84)%.3. The tumorigenic capacity of Hep3B cells was decreased after transfection of pcDNA3.1(+)/FHIT in nude mice. The tumor weight and volume were (0.398 ± 0.244)gå’Œ(408±268 Win Hep3B-FHIT group, and were (0.763 ±0.193)gå’Œ(829±271) mm² in Hep3B-C group(p<0.05).Conclusions: 1. The loss of FHIT gene protein is a frequent event in HCC. Further studies will be required to elucidate the relation between tumor prognosis and FHIT loss.2. Transfer of FHIT gene could inhibit the growth of human hepatocellular carcinoma cells and induce cell apoptosis.3. pcDNA3.1(+)/FHIT can suppress tumorigenic ability of Hep3B cells and inhibit tumor growth in nude mice.