Piperine Induces Apoptosis and Autophagy in HSC-3 Human Oral Cancer Cells by Regulating PI3K Signaling Pathway

Abstract

Currently, therapies for treating oral cancer have various side effects; therefore, research on treatment methods employing natural substances is being conducted. This study aimed to investigate piperine-induced apoptosis and autophagy in HSC-3 human oral cancer cells and their effects on tumor growth in vivo. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated that piperine reduced the viability of HSC-3 cells and 4',6-diamidino-2-phenylindole staining, annexin-V/propidium iodide staining, and analysis of apoptosis-related protein expression confirmed that piperine induces apoptosis in HSC-3 cells. Additionally, piperine-induced autophagy was confirmed by the observation of increased acidic vesicular organelles and autophagy marker proteins, demonstrating that autophagy in HSC-3 cells induces apoptosis. Mechanistically, piperine induced apoptosis and autophagy by inhibiting the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin pathway in HSC-3 cells. We also confirmed that piperine inhibits oral cancer tumor growth in vivo via antitumor effects related to apoptosis and PI3K signaling pathway inhibition. Therefore, we suggest that piperine can be considered a natural anticancer agent for human oral cancer.

Keywords: PI3K/Akt/mTOR pathway; anticancer effects; apoptosis; autophagy; oral cancer; piperine.

Figure 1

Effects of piperine on inhibition of HSC-3 cell viability. (A) The chemical structure of piperine. (B) Cell viability of HSC-3 cells treated with piperine (0, 50, 100, 150, and 200 µM for 24 h). Data are presented as mean and standard deviation (SD) for three samples. * p < 0.05, compared with the control group.

Figure 2

Effects of piperine on induction of apoptosis in HSC-3 cells. (A,B) Fluorescence microscopic images of HSC-3 cells treated with piperine (100 and 150 µM for 24 h) and stained with DAPI. The arrows indicate DAPI-positive HSC-3 cell. (C,D) FACS results of HSC-3 cells treated with piperine (100 and 150 µM for 24 h) and were double stained with annexin-V/PI. (E,F) Expression levels of PARP, Bax and Bcl-2 in HSC-3 cells after piperine (100 and 150 µM for 24 h) treatment. β-actin was used as loading control, and the quantification was performed using ImageJ. Data are presented as mean and SD for three samples. * p < 0.05, compared with the control group.

Figure 3

Effects of piperine on the induction of autophagy in HSC-3 cells. (A) Fluorescence microscopic images of HSC-3 cells treated with piperine (100 and 150 µM for 24 h) and stained with AO. (B,C) Expression levels of Beclin-1 and LC3 in HSC-3 cells after piperine (100 and 150 µM for 24 h) treatment. Cell viability of HSC-3 cells pretreated with (D) 3-MA (2 mM) or (E) HCQ (25 μM) for 2 h, followed by treatment with piperine (100 µM for 24 h). (F,G) Expression levels of Bax and Bcl-2 proteins in HSC-3 cells pretreated with 3-MA (2 mM for 2 h), followed by treatment with piperine (100 µM for 24 h). β-actin was used as loading control, and the quantification was performed using ImageJ. Data are presented as mean and SD for three samples. * p < 0.05, compared with the control group; ^# p < 0.05, compared with the piperine treatment group.

Figure 4

Effects of piperine on the induction of apoptosis and autophagy through the PI3K/Akt/mTOR pathway in HSC-3 cells. (A,B) Expression levels of p-PI3K, p-Akt and p-mTOR proteins in HSC-3 cells after piperine (100 and 150 µM for 24 h) treatment. (C) Cell viability of HSC-3 cells pretreated with LY294002 (10 μM for 2 h) followed by treatment with piperine (100 μM for 24 h). (D,E) Expression levels of Bax, Bcl-2, p-mTOR, Beclin-1 and LC3 proteins in HSC-3 cells pretreated with LY294002 (10 µM for 2 h), followed by treatment with piperine (100 µM for 24 h). β-actin was used as loading control, and the quantification was performed using ImageJ. Data are presented as mean and SD for three samples. * p < 0.05, compared with the control group; ^# p < 0.05, compared with the piperine treatment group.

Figure 5

Effects of piperine on tumor growth inhibition in vivo. Nude mice bearing HSC-3 cells as xenograft models were treated with piperine (0, 50 mg/kg daily for 4 weeks), and (A) tumor volume, (B) tumor weight and (C) body weight were measured. (D) H&E staining performed for the liver and kidney.

Figure 6

Effects of piperine-induced apoptosis through the PI3K signaling pathway in oral cancer tumor. Expression levels of (A,B) Bax, Bcl-2, (C,D) p-PI3K, p-Akt, and p-mTOR proteins in oral cancer tumors after piperine (0, 50 mg/kg daily for 4 weeks) treatment. β-actin was used as loading control, and the quantification was performed using ImageJ. Data are presented as mean and SD for three samples. * p < 0.05, compared with the control group.