Fig. 1. Ursolic acid (UA) plus 3,3’-diindolylmethane (DIM) inhibits cell growth and promotes apoptosis in ESCC.
(A) UA and DIM combination therapy inhibited cell viability in TE-8 and TE-12 cells. Cells were treated with UA (20 μM), DIM (50 μM), and combination treatment for 48 h. An EZ-CYTOX assay kit was used to assess the cell viability of esophageal cancer cells and to generate growth curves. (B) UA plus DIM combination reduced colony number and size in TE-8 and TE-12 cells. Colonies were cultured with UA (20 μM), DIM (50 μM), and the combination treatment of UA and DIM for 4 weeks in soft agar gel. The quantification of colony numbers was calculated by ImageJ. (C) UA plus DIM increased early-stage and late-stage apoptosis in TE-8 and TE-12 cells. Annexin V/PI staining was performed to detect cell apoptosis by flow cytometry. (D) UA plus DIM combination induced subG1 phase cell accumulation in TE-8 and TE-12 cells. Cells were treated with UA (20 μM), DIM (50 μM), and combination treatment for 48 h. SubG1 phase cells were indicated by apoptosis markers and detected by flow cytometry. (E) UA plus DIM enhanced apoptosis-related protein levels. Expression of apoptosis-regulatory factor proteins caspase-9/cleaved-caspase-9 and PRAP/cleaved-PARP were examined by Western blot analysis. The quantification of protein bands was estimated by ImageJ software. GAPDH was used as the internal control. Data are expressed as the mean ± standard error of the mean. CONT, control; DMSO, dimethyl sulfoxide; ESCC, esophageal squamous cell carcinoma; PI, propidium iodide; n.s., no significant. *, compared to the control; #, compare with UA plus DIM combination treatment. * or #, p < 0.05; ** or ##, p < 0.01.
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