21α-MMD and its mechanistic potential against lung cancer cell growth, migration, and invasion in vitro.

(A) The chemical structure of 21α-Methylmelianodiol (21α-MMD), a natural triterpenoid isolated from the fruit of Poncirus trifoliata. (B) MRC-5 and L132 human normal lung cell lines and A549, H460, H358, H1299, and H292 human lung cancer cell lines were plated on 96-well plate and were treated with varying concentrations of 21α-MMD for 24 h and cell growth was analyzed by MTT assay and plotted as percentage of viable cells. Values are compared to the corresponding control value. (C) Clonal formation growth of indicated lung cancer cells was conducted after a 7-day growth period after a single administration of various concentrations of 21α-MMD. Images on the left displayed are crystal violet stained colonies while on the right are graphs representing count measurements of the colonies. (D and E) Phase-contrast microscopy was conducted on cells after exposure to 25 µM 21α-MMD to identify changes in the morphology and the DNA was observed by DAPI (lower left) and by PI (right) staining observed by confocal microscope. (F) H1299 and A549 cells were incubated with 5 μM 21α-MMD for 24 h followed by cell invasion analysis. Matrigel was diluted with serum-free culture medium and applied on the insert in the upper chambers of the multiwell and the cells were incubated to invade. Invaded cells were stained with crystal violet for apparent detection with a phase-contrast microscope. (G) Migration of cells was analyzed with the same method as in F but except without matrigel inclusion. H1299 and A549 cells were incubated with 6 μM 21α-MMD for 24 h. Columns indicate mean ± SD. (*p<0.05; **p<0.01)