Magnolol, a natural compound, induces apoptosis of SGC-7901 human gastric adenocarcinoma cells via the mitochondrial and PI3K/Akt signaling pathways

Abstract

Gastric cancer is the fourth most commonly diagnosed cancer with the second highest mortality rate worldwide. Surgery, chemotherapy and radiation therapy are generally used for the treatment of stomach cancer but only limited clinical response is shown by these therapies and still no effectual therapy for advanced gastric adenocarcinoma patients is available. Therefore, there is a need to identify other therapeutic agents against this life-threatening disease. Plants are considered as one of the most important sources for the development of anticancer drugs. Magnolol, a natural compound possesses anticancer properties. However, effects of Magnolol on human gastric cancer remain unexplored. The effects of Magnolol on the viability of SGC-7901 cells were determined by the MTT assay. Apoptosis, mitochondrial membrane potential and cell cycle were evaluated by flow cytometry. Protein expression of Bcl-2, Bax, caspase-3 and PI3K/Akt was analysed by Western blotting. Magnolol induced morphological changes in SGC-7901 cells and its cytotoxic effects were linked with DNA damage, apoptosis and S-phase arrest in a dose-dependent manner. Magnolol triggered the mitochondrial-mediated apoptosis pathway as shown by an increased ratio of Bax/Bcl-2, dissipation of mitochondrial membrane potential (ΔΨm), and sequential activation of caspase-3 and inhibition of PI3K/Akt. Additionally, Magnolol induced autophagy in SGC-7901 cells at high concentration but was not involved in cell death. Magnolol-induced apoptosis of SGC-7901 cells involves mitochondria and PI3K/Akt-dependent pathways. These findings provide evidence that Magnolol is a promising natural compound for the treatment of gastric cancer and may represent a candidate for in vivo studies of monotherapies or combination antitumor therapies.

Figure 1

Effects of Magnolol on morphological characteristics and viability of SGC-7901 cells. (A) Chemical structure of Magnolol. (B) SGC-7901 cells were treated with various concentrations of Magnolol for 48 h. Cell death was measured by using MTT assay. Data shown are means ± SD (n=3). (C) Morphological changes of SGC-7901 and FRT cells were observed under the phase-contrast microscopy after treating without (control) and with 40 and 80 μM of Magnolol for 48 h.

Figure 2

Effect of Magnolol on cell cycle distribution. SGC-7901 cells were treated with: (a) 0 μM, (b) 40 μM, (c) 60 μM, and (d) 80 μM of Magnolol for 48 h and then they were stained with PI for flow cytometric analysis. Histograms show number of cells/channel (y-axis) vs. DNA content (x-axis). The values indicate the percentage of cells in the indicated phases of the cell cycle. The data shown are representative of three independent experiments with the similar results. ^*p<0.05; and ^**p<0.01 compared with the control.

Figure 3

Apoptosis induced by Magnolol in SGC-7901 cells. SGC-7901 cells were treated with (A) 0, (B) 40, (C) 60 and (D) 80 μM of Magnolol for 48 h. Then they were stained with FITC-conjugated Annexin V and PI for flow cytometric analysis. The flow cytometry profile represents Annexin V-FITC staining in x-axis and PI in y-axis. The number represents the percentages of apoptotic cells in each condition. As shown, the cell populations in the lower right (Annexin V⁺/PI⁻) represents early apoptotic cells, upper right (Annexin V⁺/PI⁺) represents late apoptotic cells.

Figure 4

The effects of Magnolol on mitochondrial transmembrane potential and expression levels of apoptosis-related proteins. (A) The values indicate the percentage of Rho-123 fluorescence in the SGC-7901 cells treated without (control) and with (40, 60, and 80 μM) Magnolol for 24 h. The data shown are representative of three independent experiments with the similar results. ^*p<0.05 and ^**p<0.01 compared with the control. (B-D) Expression levels of Caspase-3, Bcl-2, Bax, pPI3K, pAkt, and Akt in SGC-7901 cells treated without (control) and with Magnolol (40, 60, and 80 μM) for 48 h were monitored by Western blot assay. β-actin was used as loading control. Western blots are representative of three independent experiments.

Figure 5

Formation of acidic vesicular organelles (AVOs) was observed by fluorescence microscopy and quantified by flow cytometry using the AO staining. (A) Cells were treated with Magnolol for 48 h before stained with acridine orange. Cells were examined by fluorescence microscopy. Representative images of cells from three independent experiments are shown. (B) The number represents the percentage of AVOs formation in SGC-7901 cells in each profile after treating cells without (control) and with Magnolol (80 μM) and rapamycin (positive control group) for 48 h. Three independent experiments were performed.

Figure 6

Effect of Magnolol-induced autophagy on cell death of SGC-7901 cells. (A) Cells were treated with Magnolol (80 μM) in the presence or absence of 3-MA. The number represents the percentage of dead cells in each profile after treating cells with 3-MA alone, Magnolol (80 μM) alone, Magnolol (80 μM) together with 3-MA with negative control group for 48 h. Three independent experiments were done. (B) Effect of Magnolol on intracellular ATP. Cells were treated with 40, 60 and 80 μM Magnolol for 48 h before the measurement of ATP by luminometric assay. Results shown are means ± SEM from three independent experiments.

Figure 7

Hypothetical model of cytotoxic mechanism of Magnolol in human gastric adenocarcinoma SGC-7901 cells.