Aged black garlic extract inhibits HT29 colon cancer cell growth via the PI3K/Akt signaling pathway

Abstract

Accumulating evidence indicates that aged black garlic extract (ABGE) may prove beneficial in preventing or inhibiting oncogenesis; however, the underlying mechanisms have not been fully elucidated. The present study aimed to investigate the effects of ABGE on the proliferation and apoptosis of HT29 colon cancer cells. Our results demonstrated that ABGE inhibited HT29 cell growth via the induction of apoptosis and cell cycle arrest. We further investigated the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signal transduction pathway and the molecular mechanisms underlying the ABGE-induced inhibition of HT29 cell proliferation. We observed that ABGE may regulate the function of the PI3K/Akt pathway through upregulating PTEN and downregulating Akt and p-Akt expression, as well as suppressing its downstream target, 70-kDa ribosomal protein S6 kinase 1, at the mRNA and protein levels. In conclusion, these findings suggest that the PI3K/Akt signal transduction pathway is crucial for the development of colon cancer. ABGE inhibited the growth and induced apoptosis in HT29 cells through the inhibition of the PI3K/Akt pathway, suggesting that ABGE may be effective in the prevention and treatment of colon cancer in humans.

Keywords: HT29 colon cancer cells; PI3K/Akt signaling pathway; aged black garlic extract; growth.

Figure 1

Effects of ABGE on the growth of HT29 cells in a time- and dose-dependent manner. HT29 cells were cultured for 24, 48 and 72 h in DMEM containing ABGE. The effects of ABGE on cell growth were determined by the MTT assay. The results are expressed as the means ± standard deviation from three independent experiments. Data were analyzed using the Student’s t-test. The values between the control group and the cells treated with ABGE differed significantly (P<0.05). ABGE, aged black garlic extract.

Figure 2

Effects of ABGE on the cell cycle and apoptosis of HT29 cells in vitro. The HT29 cells were treated with varied concentrations of ABGE (20, 50 and 100 mg/ml, with 0/0.1% DMSO vehicle as the control) for 24 h. The cells were harvested, stained with PI and analyzed by flow cytometry. (A) Representative results of the alteration of the cell cycle and apoptosis assessment by Annexin V/PI single-staining assay. ABGE (B) induced a statistically significant increase in the apoptosis of HT29 cells and (C) interfered with the G2/M+S phase of the cell cycle. The results are expressed as the means ± standard deviation from three independent experiments. Data were analyzed using the Student’s t-test. ^*P<0.05, compared to the control group. ABGE, aged black garlic extract.

Figure 3

Effects of ABGE on (A) Akt protein expression and (B) PTEN protein expression in HT29 cells treated with various concentrations of ABGE (20, 50 and 100 mg/ml, with 0/0.1% DMSO vehicle as the control) for 24 h. The cells were harvested and analyzed by western blotting. Anti-β-actin was used as the loading control. The results are expressed as the means ± standard deviation from three independent experiments. Data were analyzed using the Student’s t-test. ^*P<0.05, compared to the control group. ABGE, aged black garlic extract.

Figure 4

Effects of ABGE on p-Akt and p70S6K1 protein expression in HT29 cells. The values are expressed as means ± standard deviation. Data were compared with the control group and analyzed using the Student’s t-test. Significant decreases in the mRNA expression levels were observed for p-Akt (n=3) and p70S6K1 (experiments were repeated in triplicate). ^*P<0.05 compared to the control group. ABGE, aged black garlic extract.