Carnosic Acid Induces Apoptosis and Inhibits Akt/mTOR Signaling in Human Gastric Cancer Cell Lines

Abstract

Gastric cancer is among the most common malignancies worldwide. Due to limited availability of therapeutic options, there is a constant need to find new therapies that could target advanced, recurrent, and metastatic gastric cancer. Carnosic acid is a naturally occurring polyphenolic abietane diterpene derived from Rosmarinus officinalis and reported to have numerous pharmacological effects. In this study, the cytotoxicity assay, Annexin V-FITC/PI, caspases 3, 8, and 9, cell cycle analysis, and Western blotting were used to assess the effect of carnosic acid on the growth and survival of human gastric cancer cell lines (AGS and MKN-45). Our findings showed that carnosic acid inhibited human gastric cancer cell proliferation and survival in a dose-dependent manner. Additionally, carnosic acid is found to inhibit the phosphorylation/activation of Akt and mTOR. Moreover, carnosic acid enhanced the cleavage of PARP and downregulated survivin expression, both being known markers of apoptosis. In conclusion, carnosic acid exhibits antitumor activity against human gastric cancer cells via modulating the Akt-mTOR signaling pathway that plays a crucial role in gastric cancer cell proliferation and survival.

Keywords: Akt; PARP; apoptosis; carnosic acid; caspase; gastric cancer; mTOR; survivin.

Figure 1

The chemical structure of carnosic acid.

Figure 2

Effects of the carnosic acid on (A) AGS and (B) MKN-45 cells’ viability at three different incubation times (24, 48, and 72 h).

Figure 3

The level of apoptosis and necrosis was assessed by flow cytometry in (A) AGS and (B) MKN-45 cells, using Annexin V/PI staining. The control groups were without any treatment, and the treated group was treated with 20 and 25 μg/mL carnosic acid in AGS and MKN-45 cell lines, respectively for 24 h. Q1, Q2, Q3, and Q4 represent necrotic cells, late apoptotic, early apoptotic, and living cells, respectively. * p < 0.001.

Figure 4

(A) Caspases 3, 8, and 9 activity on AGS cells. The control group received no treatment, and the treated group was treated with 20 µg/mL of carnosic acid for 24 h, * p < 0.001. (B) The effect of carnosic acid on the cleavage of PARP analyzed by Western blotting. The cells were treated with vehicle (0.1% DMSO) or treated with carnosic acid at 20 µg/mL for 24 h.

Figure 5

Carnosic acid induces cell cycle arrest in AGS cells. (A) AGS cells treated with DMSO (negative control) and (B) AGS cells treated with the carnosic acid (20 µg/mL) for 24 h. The cell-cycle analysis was performed using flow cytometry, and propidium was used to evaluate the DNA content.

Figure 6

The effect of the carnosic acid on protein expression of AKT1, Phospho-AKT-S473, mTOR, Phospho-mTOR-S2448, and survivin analyzed by Western blotting. The cells were treated with vehicle (0.1% DMSO) or treated with carnosic acid at 20 µg/mL for 24 h. The quantitative analysis of each band density after normalization to the control is represented by QR.