Piperine Inhibits Cell Proliferation and Induces Apoptosis of Human Gastric Cancer Cells by Downregulating Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway

Abstract

BACKGROUND Piperine has been reported to inhibit proliferation and induce apoptosis in various cancer cells. This study aimed to explore the efficacy and underlying mechanism of piperine in human gastric cancer. MATERIAL AND METHODS MTT assay was performed to examine the effect of piperine (concentrations of 0-300 μM) on the proliferation of human gastric cancer SNU-16 cells and normal human gastric epithelial GES-1 cells. Flow cytometry and Western blot were used to determine cell apoptosis and the expression level of protein (Cyto C, cleaved PARP, cleaved caspase-3, Bax, Bcl-2, Bad, Bcl-xl, PI3K, pPI3K, Akt, and pAkt), respectively. To further investigate the anti-tumor mechanism of piperine in SNU-16 cells, we used a small-molecule Akt activator SC79 in this study. The in vivo mechanism of piperine against gastric cancer was evaluated using a xenograft tumor model. RESULTS The results showed that piperine inhibited proliferation and induced apoptosis of SNU-16 cells. Piperine upregulated the protein expression of Bax, Bad, Cyto C, cleaved PARP, and cleaved caspase-3, but downregulated the protein expression of Bcl-2, Bcl-xl, pPI3k, and pAkt. However, SC79 reversed the function of piperine on the apoptosis-related proteins. An in vivo study revealed that, compared with the control group, the tumor volume of mice treated with piperine was significantly reduced. Piperine enhanced cleaved caspase-3 expression but decreased Ki-67 expression in a dose-dependent manner. Moreover, the nontoxicity effect of piperine was confirmed by H&E staining analysis in kidney and heart tissues of mice. CONCLUSIONS Our findings suggest that piperine inhibits proliferation and induces apoptosis of human gastric cancer cells through inhibition of the PI3K/Akt signaling pathway.

Figure 1

Inhibitory effect of piperine on the growth of human gastric cancer SNU-16 cells. (A) The structure of piperine. (B) Morphological view of live and dead SNU-16 cells treated with 50–150 μM of piperine. Photomicrographs were taken by inverted phase-contrast microscope (scale bar=50 μm). (C) Growth inhibition rates of SNU-16 cells and normal human gastric epithelial GES-1 cells treated with piperine at indicated concentrations for 24 h. Data are presented as the mean±SD of at least 3 independent experiments, * P<0.05, ** P<0.01, and *** P<0.001 compared with control SNU-16 cells. ^# P<0.05 and ^## P<0.01 compared with control GES-1 cells.

Figure 2

Piperine induces the apoptosis of SNU-16 cells. (A) SNU-16 cells were exposed to piperine (50, 100, 150 μM) for 24 h. Percentage of cell apoptosis was determined by annexin V/PI staining and flow cytometry. (B) The apoptotic rates of SNU-16 cells induced by piperine. Data are presented as the mean±SD of at least 3 independent experiments, * P<0.05, ** P<0.01 and *** P<0.001 compared with control cells.

Figure 3

Effect of piperine on changes of apoptosis-related proteins. SNU-16 cells were treated with piperine (0, 50, 100, and 150 μM) for 18 h before analysis. GAPDH was used as loading control. (A) Western blot bands for Cyto C, cleaved caspase-3, and cleaved PARP proteins. (B) Quantification of Cyto C, cleaved caspase-3, and cleaved PARP proteins. (C) Expression of Bcl-2, Bax, Bad, and Bcl-xl proteins was determined by western blot. (D) Quantification of Bcl-2, Bax, Bad, and Bcl-xl proteins. The Bcl-2/Bax and Bcl-xl/Bad ratio were downregulated by piperine treatment in SNU-16 cells. (E) Expression of pPI3K, PI3K, Akt, and pAkt proteins after piperine treatment was determined by western blot. (F) Quantification of pPI3K, PI3K, Akt and pAkt proteins. Data was presented as the mean ±SD of at least 3 independent experiments, * P<0.05, ** P<0.01 and *** P<0.001 compared with control cells.

Figure 4

The efficacy of Akt activator (SC79) on piperine-induced apoptosis. SNU-16 cells were determined after treatment with piperine for 24 h in the presence or absence of SC79 (10 μM). (A) Apoptosis detection with annexin V/PI double staining in different groups by flow cytometry. (B) Column bar graph of mean cell florescence for early apoptotic, late apoptotic, and total apoptotic cells. (C) Expression of pAkt, Akt, cleaved caspase3, and cleaved PARP proteins in the presence or absence of SC79. (D) Quantification of pAkt, Akt, cleaved caspase3, and cleaved PARP proteins. (E) Expression of Bcl-2 and Bax proteins after piperine and/or SC79 treatment. (F) Quantification of Bcl-2 and Bax proteins. The Bcl-2/Bax ratio was significantly upregulated in the presence of SC79. Data are presented as the mean±SD of at least 3 independent experiments, * P<0.05, ** P<0.01 and *** P<0.001 compared with control cells; ^# P<0.05, ^## P<0.01 and ^### P<0.001 compared with the piperine alone group.

Figure 5

Piperine inhibited SNU-16 xenograft tumor growth in vivo. Gastric cancer SNU-16 (A) tumor volume and (B) harvested tumor in mice after treatment with piperine. (C) Weight change curves of animals in different groups. (D) The histology of heart and kidney in vivo (scar bar=50 μm). (E) The values of relative hepatic indexes. (F) Expression of caspase-3 and Ki-67 after piperine treatment in vivo (scar bar=40 μm). Data are presented as the mean±SD, * P<0.05 and ** P<0.01 compared with control group.