Inhibition of colorectal cancer by ursolic acid via noncanonical Hedgehog signaling pathway

OBJECTIVE To identify the inhibitory effect of ursolic acid on the colorectal cancer HCT116 cells in vitro and in vivo, and to explore the underlying mechanism. METHODS The smoothened（SMO） gene-silenced human colorectal cancer HCT116;cell line was established by transfection with the lentivirus carrying SMO shRNA. The cytotoxic effect of ursolic acid on HCT116;cells was determined by MTT assay. The effect of ursolic acid on the migration of HCT116;cells was studied by wound healing assay. The effect of ursolic acid on apoptosis of HCT116;cells was explored by Hoechst33342/PI double staining and flow cytometry. The effects of ursolic acid on the expressions of apoptotic marker gene Bcl-2, Bax, caspase-3 and caspase-9 were measured by real-time quantitative RT-PCR（RT-qPCR） and Western blotting（WB） analysis. RT-qPCR and WB were used to examine the relationship between GLI1, c-Myc expression and PI3 K/Akt pathway to further investigate the mechanism of GLI1 activation in HCT116;cells. The effects of ursolic acid on the expressions of GLI1, p-Akt, Akt, c-Myc, SHH and SUFU of noncanonical Hedgehog pathway were evaluated by RT-qPCR and WB assays. Xenograft nude mouse model bearing HCT116;cells was established and intraperitoneally treated with ursolic acid to investigate the effect on tumor growth in vivo. The body weight and tumor size of mice were assessed regularly every 2 d. The effect of ursolic acid on the apoptosis of tumor tissue was determined by TUNEL assay. The expressions of Bcl-2, Bax, GLI1, p-Akt, Akt, c-Myc,SHH, SUFU mRNA and proteins were measured by RT-qPCR and WB. The levels of Bcl-2, Bax, GLI1, p-Akt, c-Myc and SHH proteins in tumor tissues were also evaluated by immunohistochemistry. RESULTS Ursolic acid significantly inhibited the growth and migration of HCT116;cells in vitro, compared with the control（P<0.05）. Meanwhile, ursolic acid also induced apoptosis of HCT116;cells in vitro（P<0.05）. Furthermore, SC79（Akt activator） enhanced the expressions of p-Akt, GLI1 and c-Myc, which could be abolished by ursolic acid, and the effect was equal to Akt inhibitor LY294002.The expressions of Bcl-2, GLI1, p-Akt, c-Myc, SHH mRNA and proteins were reduced by ursolic acid, while the levels of Bax and SUFU were increased. Ursolic acid could inhibit the growth and induce the apoptosis of colorectal cancer xenograft in vivo. Similarly, lower levels of Bcl-2, GLI1, p-Akt, c-Myc and SHH, and higher expression of Bax and SUFU were noted in ursolic acid-treated mice. CONCLUSION Ursolic acid can inhibit the growth and induce apoptosis of HCT116;cells both in vitro and in vivo. And the mechanism is related to the suppression of PI3 K/Akt-mediated noncanonical Hedgehog signaling pathway.