Antitumor effect of low-dose of rapamycin in a transgenic mouse model of liver cancer

Abstract

Objectives: Rapamycin has been shown to exhibit potent immunosuppressive activity while simultaneously providing an antitumor effect. However, it remains debatable whether rapamycin is effective in the prevention and treatment of hepatocellular carcinoma (HCC). Thus, the current study aimed to investigate whether low-dose rapamycin is effective in preventing HCC growth and treating HCC after tumor development in transgenic mice. The mechanism of the antitumor effects of rapamycin were also evaluated. Methods: We produced non-germline transgenic mouse models using hydrodynamics-based transfection to co-express HrasG12V and a short-hairpin RNA down-regulating p53 in combination in the liver. Transgenic mice were randomly assigned to five experimental groups: negative control, positive control, tacrolimus only (1.5 mg/kg), rapamycin only (1.5 mg/kg), and tacrolimus plus rapamycin (1.5 mg/kg tacrolimus + 1.5 mg/kg rapamycin). Rapamycin and tacrolimus were given orally, once daily by gavage. The mice were further divided into two groups according to the time to commencement of immunosuppressant. Rapamycin and/or tacrolimus were given 1 day after transfection in the de novo treatment group, and 2 weeks after transfection in the post-tumor development group. All mice were sacrificed 4 weeks after transfection. Results: Of 44 mice in total, six were in the negative control group, six were in the positive control group, 17 were in the de novo treatment group, and 15 were in the post-tumor development group. In the de novo treatment group, liver tumors successfully developed in all mice with the exception of the rapamycin only group, which displayed the marked suppression of tumor growth. In the post-tumor development group, liver tumors successfully developed in all mice, and the rapamycin only group displayed no significant suppression of tumor growth compared to the positive control group. The liver tumors were characterized as HCC using immunohistochemical staining, which revealed significantly higher alpha-fetoprotein levels in all groups than in the negative control group. In the T lymphocyte subset analysis, the number of CD4+ effector T cells and CD4+ regulatory T cells was significantly decreased in the positive control, tacrolimus only, and tacrolimus plus rapamycin groups compared to that in the negative control group. However, the ratio of CD4+ regulatory T cells/CD4+ T cells was slightly increased in the rapamycin only group compared with that in the negative control group (29.8 ± 4.6 % vs. 24.5 ± 5.6 %, P = 0.065). Immunohistochemical analysis revealed significantly higher expression of phosphorylated-mTOR, 4E-BP1, and S6K1 in the positive control group than in the rapamycin only group. Additionally, the percentage of tumors with positive staining for 4E-BP1 and S6K1 was significantly lower in the rapamycin only group than that in the positive control group. Conclusion: The current study demonstrated that low-dose rapamycin might be effective to prevent HCC growth, but may be ineffective as treatment after HCC development. Thus, the current study implies that the clinical use of low-dose rapamycin may play a role to prevent HCC recurrence after an R0 treatment, such as liver transplantation.