PMC

Chemical structures of AA and its derivative.
Notes: Chemical structures of AA (A) and AA-PMe (B).
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester.

AA-PMe inhibits motility in SGC7901 and HGC27 cells.
Notes: Effects of AA-PMe on the invasion potential of SGC7901 (A, C) and HGC27 (B, D) cells were examined by manual counting and measuring absorbence at 570 nm, respectively. Cell invasion inhibition percentage data shown are mean ± SD of three samples from each treatment. Significant increases are denoted by ^*P<0.05, ^**P<0.01, compared with their respective controls.
Abbreviations: AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; SD, standard deviation.

The morphological feature changes and apoptosis induced by SGC7901 and HGC27 cells.
Notes: Gastric cancer cells were treated with 0.1% DMSO (control); 5 µM AA or AA-PMe; 10 µM AA or AA-PMe; 25 µM AA or AA-PMe and then double stained with Annexin V-FITC/PI kit. The green light shows early-stage apoptotic cells while the red light shows the late apoptotic cells (×100).
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; PI, propidium iodide.

Effect of AA-PMe and AA on viability of gastric epithelial cells GES-1.
Notes: Cells were exposed to AA-PMe or AA for 72 hours, and cell viability was determined by CCK-8 assay. The relative survival rate was determined in relation to that of untreated control cells, which was set 100%. Statistically significant differences between AA-PMe compared to AA are represented by ^*P<0.05 and ^**P<0.01.
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; CCK8, cell counting kit-8.

Effect of AA-PMe and AA on cell cycle distribution in SGC7901 and HGC27 cells.
Notes: Gastric cancer cells (2×10⁶/mL) were synchronized by incubation overnight in the absence of serum and then treated with different concentration of AA and AA-PMe for 24 hours, after which the cells were washed, fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. (A, C) analyzed for SGC7901 cells and (B, D) was analyzed for HGC27 cells.

AA-PMe inhibits the migration of SGC7901 and HGC27 cells.
Notes: Effect of AA-PMe on the migration of SGC7901 (A, C) and HGC27 (B, D) cells was analyzed using wound healing assay. Representative photomicrographs of initial and final wounds are shown at 100× magnification and the migration distance was measured using the formula: (initial wound size – final wound size)/2. Cell migration data shown are mean ± SD of three samples for each treatment. Significant increases are denoted by ^*P<0.05, ^**P<0.01, compared with their respective controls.
Abbreviations: AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; SD, standard deviation.

AA- and AA-PMe-induced gastric cancer cells apoptosis.
Notes: (A) SGC7901 cells were treated by different concentrations of AA-PMe or AA for 24 hours followed by Annexin V-FITC/PI assay. One of FACS graphs was shown in each group; (B) results of the same experiment in HGC27 cells. Columns: mean of three independent experiments; bars: SD. Significant increases are denoted by *P<0.05, **P<0.01, compared with their respective controls.
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; PI, propidium iodide; SD, standard deviation.

AA-PMe enhanced the expression of proapoptotic Bax gene and suppressed the rest – Bcl-2, c-Myc, cyclin D1, MMP-2, and MMP-9 genes.
Notes: SGC7901 and HGC27 cells (2×10⁶/mL) were treated with AA-PMe or AA at different concentrations for 24 hours, and the total RNA was extracted and examined for expression of Bax (A, B), cyclin D1 (C, D), Bcl-2 (E, F), MMP-2 (G, H), c-Myc (I, J), and MMP-9 (K, L) by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control to show equal RNA loading. Bars: SD between the triplicates.
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; SD, standard deviation.

AA-PMe reduced MMP-2 and MMP-9 protein levels in SGC9701 and HGC27 cells analyzed by Western blot.
Notes: Whole-cell extracts were prepared and analyzed using Western blot with anti-MMP-2 and MMP-9 antibodies. Equal protein loading was confirmed by probing the Western blots with an anti-GAPDH antibody. The density of each band was measured and normalized to that of GAPDH. (A) Western blots of MMP-2, MMP-9 in SGC7901 cells. (C) Densitometric analysis of the relative level of MMP-2 and MMP-9 in SGC7901 cells. (B, D) The same methods as (A) and (C) in HGC27 cells. Significant reductions are denoted by ^*P<0.05, ^**P<0.01, compared with their respective controls.
Abbreviations: AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; SD, standard deviation.

Inhibitory effect of AA and AA-PMe on the viability of SGC7901 and HGC27 cells.
Notes: (A) Viability of SGC7901 cells determined by CCK8 assay. Cells were exposed to AA or AA-PMe for 72 hours. The relative survival rate was determined in relation to that of untreated control cells, which was set as 100%. Data are mean ± SD of three triplicate experiments. (B and C) Growth curve of SGC7901 cells. Cells were incubated with different concentrations of AA or AA-PMe for 24, 48, and 72 hours, then harvested, stained with trypan blue and counted. (D–F) results of the same experiments performed in HGC27 cells as (A–C). ^*P<0.05, ^**P<0.01 vs control.
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester; CCK8, cell counting kit-8; SD, standard deviation.

Compared to AA, AA-PMe showed a stronger ability in enhancing the expression of proapoptotic protein Bax and suppressing the antiapoptotic protein Bcl-2, c-Myc, and Caspase 3.
Notes: SGC7901 and HGC27 cells (2×10⁶/mL) were treated with different concentrations of AA-PMe or AA for 24 hours, and then the whole-cell extracts were prepared and analyzed using Western blot using the indicated antibodies. Equal protein loading was confirmed by probing the Western blots with an anti-GAPDH antibody. The density of each band was measured and normalized to that of GAPDH. (A) Western blots of Bcl-2, Bax, c-Myc and caspase 3 in SGC7901 cells. (C) Densitometric analysis of the relative level of Bcl-2, Bax, c-Myc, and caspase 3 in SGC7901 cells. (B and D) The same methods as A and C in HGC27 cells. Significant increases are denoted by ^*P<0.05, ^**P<0.01, compared with their respective controls.
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester.

Changes in the expression levels of cell-cycle regulatory proteins induced by AA-PMe and AA.
Notes: Lysates were prepared from the cells after treatment with 0, 1, 5, 10, 25, and 50 µM AA-PMe or AA for 24 hours. ^*P<0.05 and ^**P<0.01 represent the difference of AA and AA-PMe at same concentration. Gastric cancer cells were then subjected to Western blot analysis using the indicated antibodies. Equal protein loading was confirmed by probing the Western blots with an anti-GAPDH antibody. The density of each band was measured and normalized to that of GAPDH. (A) Western blots of cyclin D1, CDK4, CDK6, p15, and pRb in SGC7901 cells. (C) Densitometric analysis of the relative level of cyclin D1, CDK4, CDK6, p15, and pRb in SGC7901 cells. (B, D) The same methods as A and C in HGC27 cells. Error bars represent standard errors of the mean (SEM).
Abbreviations: AA, Asiatic acid; AA-PMe, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-l-proline methyl ester.