Inter-regulation Of IGFBP1 And FOXO3a Unveils Novel Mechanism In Ursolic Acid-inhibited Growth Of Hepatocellular Carcinoma Cells

ObjectiveHuman Bel-7402 and HepG2 hepatocellular cancer cell line were used to investigate the inhibition of proliferation and underlying molecular mechanism of ursolic acid.MethodsCell viability was measured by MTT assays. Western blot was performed to measure the phosphorylation and protein expression of p38, transcription factors FOX03a and IGFBPl. Quantitative real-time PCR(qRT-PCR) was used to examine the mRNA levels of IGFBP1 gene. Exogenous expression of FOX03a and IGFBPl genes was carried out by transient trasfection assays. IGFBP1 promoter activity was measured by Dual Luciferase Reporter Kit.ResultsWe showed that UA stimulated phosphorylation of p38 MAPK. In addition, UA increased the protein, mRNA expression, and promoter activity of insulin-like growth factor (IGF) binding protein 1 (IGFBP1), which was abrogated by the specific inhibitor of p38 MAPK (SB203580). Intriguingly, we showed that UA increased the expression of forkhead box 03A (FOX03a) and overexpressed FOX03a enhanced phosphorylation of p38 MAPK, all of which was not observed in cells silencing of endogenous IGFBP1 gene. Moreover, exogenous expressed IGFBP1 strengthened UA-induced phosphorylation of p38 MAPK and FOX03a protein expression. Consistent with these, UA inhibited tumor growth and increased phosphorylation of p38 MAPK, protein expression of IGFBP1 and FOX03a in vivo. Collectively, our results show that UA inhibits growth of HCC cells through p38 MAPK-mediated induced expression of IGFBP1 and FOX03a. The interactions between IGFBP1 and FOX03a, and feedback regulatory loop of p38 MAPK by IGFBP1 and FOX03a resulting in reciprocal pathways, contribute to the overall effects of UA.ConclusionOur results show that ursolic acid inhibits the growth of human hepotacelluar cancer cells through activation of p38MAPK signaling, followed by increase of FOX03a and IGFBP1 protein expression. This results in the increase of IGFBP1 gene expression.The interactions between IGFBP1 and FOX03a, and feedback regulatory loop of p38 MAPK by IGFBP1 and FOX03a resulting in reciprocal pathways, contribute to the overall effects of UA. This study unveils a novel mechanism by which ursolic acid inhibits growth of human hepotacellular cancer cells. This study corroborates a potential novel mechanism by which UA controls HCC growth and implies that the rationally target IGFBP1 and FOX03a can be potential for the therapeutic strategy against HCC.