Bilirubin impairs the sorafenib sensitivity in Huh7 cells through fatty acid oxidation by up-regulating PPARα/CPT1A

To observe the effect of bilirubin (Bil) on sorafenib (Sor) in the treatment of liver cancer and explore the involved molecular mechanisms.

In vitro, Huh7 cells were treated with different concentration of Sor (0, 2, 4, 8, 16, 32 μmol/L), of Bil (0, 2.5, 5, 10, 20, 40 μmol/L) and of Sor (0, 2, 4, 8, 16, 32 μmol/L) +Bil (20 μmol/L) for 24, 48 and 72 h. Cell viability was detected by CCK-8 assay. Huh7 cells were treated with Sor (4 μmol/L), Bil (20 μmol/L) and Sor (4 μmol/L)+Bil (20 μmol/L), cell cycle was detected by flow cytometry after 48 h, and the effects of drugs on cell proliferation were detected by clone formation assay after 14 d. In vivo, 16 mice bearing xenografts of Huh7 cells were divided into Con group, Bil group (25 mg·kg^-1·d^-1), Sor group (15 mg·kg^-1·d^-1) and Bil (25 mg·kg^-1·d^-1) +Sor (15 mg·kg^-1·d^-1) group (n=4), according to the S type sampling method. The expression levels of peroxisome proliferator-activated receptor (PPARα) and carnitine palmityl transferase 1A (CPT1A) were detected by Western blotting and real time PCR. After etomoxir (Eto), the inhibitor of CPT1A, was added, CCK-8 and clone formation assay were used to verify the mechanisms correlated with PPARα/CPT1A.

CCK-8 results showed that the inhibitory effect of Sor on Huh7 cells was in a dose and time-dependent manner (P<0.05), and Bil had no significant effect on Huh7 cells (P>0.05). Compared with Sor group, the cell proliferation in Bil+Sor group was increased (P<0.05), the relative cell colony formation rate was grown (P<0.05), and the number of cells in S phase was decreased (P<0.05). In vivo, results showed that the tumor volume and weight of Sor group were lower than that of Con group (P<0.05), and the tumor volume and weight of Bil+Sor group were higher than those of Sor group (P<0.05). Western blotting and RT-qPCR results showed that PPARα and CPT1A expression levels were increased in Bil+Sor group compared with Sor group (P<0.05). After Eto was added, the cell proliferation and the relative cell clone formation rate of Bil+Sor+Eto group were decreased compared with Bil+Sor group (P<0.05).

Bil impairs the antitumor effects of sorafenib treatment in hepatocellular carcinoma through fatty oxidation by up-regulating PPARα/CPT1A.