Ursolic acid induces apoptosis and anoikis in colorectal carcinoma RKO cells

Abstract

Background: Ursolic acid (UA) is an anti-cancer herbal compound. In the present study, we observed the effects of UA on anchorage-dependent and -independent growth of human colorectal cancer (CRC) RKO cells.

Methods: RKO cells were cultured in conventional and detached condition and treated with UA. Cell viability was evaluated by CCK-8 assay. Cell cycle was analyzed by flow cytometry. Apoptosis was identified by Hoechst 33258 staining and flow cytometry analysis. Activities of caspases were measured by commercial kits. Reactive oxygen species (ROS) was recognized by DCFH-DA fluorescent staining. Anoikis was identified by EthD-1 fluorescent staining and flow cytometry analysis. Expression and phosphorylation of proteins were analyzed by western blot.

Results: UA inhibited RKO cell viability in both a dose- and time-dependent manner. UA arrested the cell cycle at the G0/G1 phase, and induced caspase-dependent apoptosis. UA inhibited Bcl-2 expression and increased Bax expression. In addition, UA up-regulated the level of ROS that contributed to UA activated caspase-3, - 8 and - 9, and induced apoptosis. Furthermore, UA inhibited cell growth in a detached condition and induced anoikis in RKO cells that was accompanied by dampened phosphorylation of FAK, PI3K and AKT. UA also inhibited epithelial-mesenchymal transition (EMT) as indicated by the down-regulation of N-Cad expression and up-regulation of E-Cad expression.

Conclusions: UA induced caspase-dependent apoptosis, and FAK/PI3K/AKT singling and EMT related anoikis in RKO cells. UA was an effective anti-cancer compound against both anchorage-dependent and -independent growth of RKO cells.

Keywords: AKT; Anoikis; Apoptosis; Caspases; Colorectal cancer; Epithelial-mesenchymal transition; FAK; PI3K; Reactive oxygen species; Ursolic acid.

Fig. 1

The chemical structure of UA

Fig. 2

Effects of UA on RKO cells growth. a and b, RKO cells were dose-and time-dependently treated with UA, and cell vitality were evaluated by CCK-8 assay. c, RKO cells were treated with UA (14–20 μM) for 48 h, and observed by microscopy (× 200 magnification). ^*p < 0.05, ^**p < 0.01, versus control group

Fig. 3

UA arrests cell cycle. RKO cells were treated with UA (14–20 μM) for 48 h and cell cycle distribution were analyzed by flow cytometry (a) and the results were expressed as mean ± SD (b). ^**p < 0.01, versus control group

Fig. 4

UA induces apoptosis. UA treated RKO cells were subjected to Hoechst 33258 staining (a), flow cytometry analysis for apoptosis detection (b) and expressed by mean ± SD (c). ^**p < 0.01, versus control group

Fig. 5

UA activates Caspases. Caspase-3 (a), − 8 (b) and − 9 (c) activities in UA treated RKO cells. d, After pre-incubated with Z-VAD-FMK for 2 h, RKO cells were treated with UA and subjected to apoptosis detection. Bcl-2 and Bax expression in UA treated RKO cells were evaluated by Western blot (e), and analyzed by the Quantity One software (f). ^*p < 0.05, ^**p < 0.01, versus control group; ^#p < 0.05, ^##p < 0.01, versus UA group

Fig. 6

UA increases ROS. RKO cells were treated with UA, stained with DCFH-DA, observed under a microscope (× 200) (a), and the fluorescence were detected by plate reader (b). After pre-incubated with NAC for 2 h, RKO cells were treated with UA, and subjected to detection of Caspase-3 (c), − 8 (d) and − 9 (e) activities and apoptosis (f). ^**p < 0.01, versus control group; ^#p < 0.05, ^##p < 0.01, versus UA group

Fig. 7

UA induces anoikis. RKO cells grown in ploy-HEMA coated plates were treated with UA for 48 h and subjected to cell viability assay (a), EthD-1 staining (× 200) (b), and the red fluorescence detected by plate reader (c), stained with FITC Annexin V and PI and analyzed by flow cytometry (d). ^**p < 0.01, versus control group

Fig. 8

UA regulates anoikis-related proteins. Proteins expression and phosphorylation in UA treated RKO cells were evaluated by Western blot with indicated antibodies (a, c, e and g), and analyzed by the Quantity One software (b, d, f and h). ^*p < 0.05, ^**p < 0.01, versus control group