Introduction : Calcineurin inhibitors (CNI) such as tacrolimus and cyclosporine A are potent immunosuppressive agents, but they also increase the risk of hepatocellular carcinoma (HCC) recurrence. Epithelial-mesenchymal transition (EMT) is a key process in cancer cell progression and metastasis. In this study, we aim to observe the effect of adding sirolimus with other immunosuppressive agents on reducing EMT.

Methods : HCC cell lines SK-Hep1 were used in both in vivo and in vitro studies. The effects of various immunosuppressant agents, using different dosages and combination therapies, on HCC cell proliferation were assessed. Cell migration assay, western blot analysis and immunofluorescent staining were performed.

Results : In the in vitro study, the use of CNI increases cell migration, inducing EMT in a dose-dependent manner. The use of sirolimus, in isolation in combination with other CNI, showed a dose-dependent attenuation of cell viability and cell migration. In the high dose tacrolimus subgroup, the use of low dose sirolimus reduces cell migration from 29.6% to 22%, and to 11.8% when given high dose sirolimus (p< 0.05), but this is still higher than control. In the in vitro Western blot analysis, adding sirolimus to tacrolimus showed a general decreased expression of pro-EMT proteins. In the in vivo Western blot analysis, the same combination showed increased E-Cadherin expression, and decreased pro-EMT protein expression. Using immunofluorescent staining in vitro, TGF-β is decreased.

Conclusions : CNI induces EMT in a dose-dependent manner. This effect is more potent in tacrolimus compared to cyclosporin A. Sirolimus together with CNI attenuates CNI-induced EMT, but not completely.