Molecular Role of EGFR-MAPK Pathway in Patchouli Alcohol-Induced Apoptosis and Cell Cycle Arrest on A549 Cells In Vitro and In Vivo

Abstract

Nowadays, chemotherapy is still the main effective treatment for cancer. Herb prescriptions containing Pogostemon cablin Benth (also known as "Guang-Huo-Xiang") have been widely used in Chinese medicine today. In our research, we found that patchouli alcohol, a compound isolated from the oil of Pogostemon cablin Benth, exerted antitumor ability against human lung cancer A549 cells ability both in vitro and in vivo. MTT assay was used to assess cell viability. Hoechst 33342 staining and TUNEL cover glass staining provided the visual evidence of apoptosis. Caspase activity measurement showed that patchouli alcohol activated caspase 9 and caspase 3 of mitochondria-mediated apoptosis. Consistently, patchouli alcohol inhibited the xenograft tumor in vivo. Further investigation of the underlying molecular mechanism showed that MAPK and EGFR pathway might contribute to the antitumor effect of patchouli alcohol. Our study proved that patchouli alcohol might be able to serve as a novel antitumor compound in the clinical treatment of lung cancer.

Figure 1

Growth inhibition and apoptosis effect of PA. (a) The chemical structure of PA. (b) Inhibition effect of PA on A549 cells using MTT assay. (c) Effect of PA on HEK293, L02, and HFL-1 cells for 48 h using MTT assay. (d) The increasing tendency of apoptotic ratio induced by increasing PA concentrations of 0, 50, 75, and 100 μg/mL and 75 μg/mL combined with EGF. (e) Mechanism of PA induced apoptosis on A549 cells. A549 cells were exposed for 48 h with increasing PA. Proteins levels were probed by western blotting assay. Statistical differences were compared between PA treatment group and control group and considered significant at the levels of ^∗∗ P < 0.01 or ^∗∗∗ P < 0.001.

Figure 2

The apoptosis effect and involved signaling pathway activity of PA on A549 cells. The A549 cells were exposed for 48 h with increasing PA. (a) PA induced MMP collapse for 48 h treatment. (b) PA exposure induced caspase 9 activation in A549 cells for 48 h. (c) PA exposure induced caspase 3 activation in A549 cells for 48 h. (d) The Hoechst staining immunofluorescence microscopy image of A549 cells for Hoechst in vitro; Scale Bar: 10 μm. (e) The TUNEL staining immunofluorescence microscopy image of A549 cells for DAPI (blue), TUNEL FITC (green), and their merge in vitro; Scale Bar: 50 μm. (f) Effect of PA on EGFR and MAPK signaling pathways in A549 cells. Statistical differences were compared between PA treatment group and control group and considered significant at the levels of ^∗ P < 0.05, ^∗∗ P < 0.01, or ^∗∗∗ P < 0.001.

Figure 3

Typical G₁/S cell cycle arrest on A549 cells induced by increasing PA exposure of 0, 50, 75, and 100 μg/mL. (a) PA induced G₁/S cell cycle arrest. (b) The involved proteins activities. Statistical differences were compared between PA treatment group and control group and considered significant at the levels of ^∗ P < 0.05, ^∗∗ P < 0.01, or ^∗∗∗ P < 0.001.

Figure 4

The antiproliferation effect of PA on A549 xenograft model. (a) The body weights of nude A549 models in vivo. (b) The tumor sizes of A549 nude models in vivo. (c) The tumor of each group. (d) Effect of PA on the expression levels of cleaved-caspase 3 and Ki67 in A549 nude model; Scale Bar: 100 μm. Statistical differences were compared between PA treatment group and control group and considered significant at the levels of ^∗ P < 0.05, ^∗∗ P < 0.01, or ^∗∗∗ P < 0.001.

Figure 5

A549 cells were exposed for 48 h combined with EGF. (a) Mechanism certification of PA induced apoptosis and cell cycle arrest on A549 cells. (b) The graphical abstract of PA's action on A549 cells. Statistical differences were compared between PA treatment group and control group and considered significant at the levels of ^∗∗∗ P < 0.001.