Objective : Transforming growth factor beta 1 (TGF-β1) and hypoxia inducible factor 1 alpha (HIF-1α) are known to be associated with tumor recurrence and poor prognosis in patients with hepatoma. However, effects of hepatic artery embolization (HAE) on the local expressions of TGF-β1 and HIF-1α have not been well investigated. The objective of this study was to evaluate the effects of HAE on the local expressions of TGF-β1 and HIF-1α in a rat hepatoma model. Further in vitro experiments were performed to assess the mechanism of TGF-β1 and HIF-1α expression.
Methods : Sprague Dawley rats bearing N1S1 hepatoma cells on the livers were randomly assigned to receive HAE (HAE group, n=5) with microsphere (75um) or sham treatment (sham group, n=4). Animals were euthanized at 48 hours after HAE, and tumors with surrounding liver tissues were harvested. Then, quantitative polymerase chain reaction (qPCR) and immunohistochemistry staining were performed and the differences of the expressions for both TGF-β1 and HIF-1α between the HAE group and sham group were compared. As for the in vitro study, 1 x 106 N1S1 hepatoma cells were incubated under either normoxic (21% O2) or hypoxic conditions (1% O2) for 48 hours, which imitate hypoxia in the liver after HAE. Additionally, some cells were cultured with HIF-1α inhibitors or exogeneous TGF-β1 to elucidate the relationship between TGF-β1 and HIF-1α expression. Western blotting was used to assess the expressions of TGF-β1 and HIF-1α in N1S1 cells. Enzyme-linked immunosorbent assay (ELISA) was used to measure TGF-β1 level secreted in the cell culture supernatant.
Results : qPCR of in vivo samples showed that local mRNA expression of TGF-β1 and HIF-1α were significantly higher in the HAE group than in sham groups (2.0-folds on TGF-β1 and 1.8-folds on HIF-1α, respectively; p<0.05). Immunohistochemistry mirrored the results of qPCR, showing a similar distribution of TGF-β1 and HIF-1α positive area. Western blotting of in vitro samples demonstrated that hypoxic conditions led both TGF-β1 and HIF-1α to increase compared with normoxic conditions (13.8-folds and 28.8-folds, respectively; p<0.01). TGF-β1 expression was significantly suppressed when treated with HIF-1α inhibitor using ELISA (1026 pg/ml and 1340 pg/ml, with and without HIF-1α inhibitor; p<0.01). HIF-1α expression significantly increased in Western blotting (2.0-folds, p<0.05) when exogenous TGF-β1 was added.
Conclusions : HAE enhances the local expressions of TGF-β1 and HIF-1α in a rat hepatoma model. Results of in vitro experiments suggested that hypoxic stress induced by HAE may trigger the interdependent expressions of both TGF-β1 and HIF-1α.