Annonaceous acetogenins mediated up-regulation of Notch2 exerts growth inhibition in human gastric cancer cells in vitro

Abstract

Background: Gastric cancer (GC) is a global health problem because of limited treatments and poor prognosis. Annonaceous acetogenins (ACGs) has been reported to exert anti-tumorigenic effects in cancer, yet the mechanism underlying its effects on GC remains largely unknown. Notch signaling plays a critical role in cell proliferation, differentiation and apoptosis. Therefore, it may contribute to the development of GC. This study aims to explore the role of Notch2 in ACGs' activities in GC cells.

Results: ACGs inhibited GC cells' viability in a dose dependent manner and led to cell apoptosis and cell cycle arrest in G0/G1 phase with an increased Notch2 expression. Additionally, Notch2 siRNA reduced ACGs-induced cell growth inhibition while Notch2 cDNA transfection did the opposite.

Materials and methods: ACGs were administrated in GC cells and cell proliferation was assayed by MTS, cell apoptosis and cell cycle were detected by flow cytometry. Additionally, the expression of Notch2 and the downstream target Hes1 were identified by Western blot. Furthermore, Notch2-siRNA transfection and Notch2-cDNA were performed to investigate the role of Notch2 in the antitumor effect of ACGs.

Conclusions: Up-regulation of Notch2 by ACGs is a potential therapeutic strategy for GC.

Keywords: Notch2; annonaceous acetogenins; apoptosis; gastric cancer cell; proliferation.

Conflict of interest statement

CONFLICTS OF INTEREST

These authors declare that they have no competing financial interests.

Figures

Figure 1

(A) Comparison of Notch2 expression level at mRNA and protein level among GC cell lines. Left: Expression of Notch2 gene was detected by real-time fluorescence quantitative-PCR (RFQ-PCR), n = 3. Right: Expression of Notch2 protein was detected by western blot, n = 3. (B) The inhibition rate was calculated as the following equation: inhibition rate (%)=(1-OD of ACGs treatment group/ OD of control group) ×100%. The half maximal inhibitory concentration (IC 50) is a measure. The solvent control was 0.1% DMSO. The results are expressed as the means ± SEM, n = 6.

Figure 2

(A) ACGs inhibited AGS and MKN-45 cells growth in a dose and time-dependent manner. AGS and MKN-45 cells were treated with 2.5 μg/ml,5 μg/ml, and 10 μg/ml ACGs for 12 h, 24 h, and 36 h respectively. Cell proliferation was tested by MTS assay. Data represented mean± SEM, n = 6. The statistical significant was confirmed compared with control group. *P < 0.05, **P < 0.01. (B) Effects of ACGs administration on GC cell morphology. Cells were treated with ACGs at the concentrations 2.5, 5 and 10 μg/ml for 36 respectively. Cell morphology was observed under an inverted phase contrast microscope and images were obtained. Significant cell shrinkage and a decreased cellular attachment rate were observed in the ACGs-treated group.

Figure 3. Effects of ACGs administration on the apoptosis in AGS and MKN-45 cells

(A) GC cells were treated with 5 μg/mL ACGs for 12 h, 24 h, 36 h respectively, stained with Annexin V/PI and analyzed by flow cytometry. (B) GC cells were treated with 2.5, 5 and 10 μg/ml ACGs for 36 h respectively, stained with Annexin V/PI and analyzed by flow cytometry. Representative flow cytometric analyses of apoptosis are shown. Four subpopulations and their fractions are indicated: normal cells (lower left), dead cells (upper left), early apoptotic cells (lower right), and late apoptotic cells (upper right). The apoptotic indices are expressed as the number of apoptotic cells/the total number of counted cells ×100%.

Figure 4. Cell cycle analyses of ACGs-treated AGS and MKN-45 cells by flow cytometric

GC cells were treated with the concentration of 2.5, 5 and 10 μg/ml ACGs for 36 h, respectively. Then cells were harvested, stained with PI and subjected to flow cytometry for analyzing cell distribution at each phase of the cell cycle.

Figure 5. The expression of Notch2 and Hes1 in AGS and MKN-45 cells after ACGs treatment

GC cells were treated with the concentration of 2.5, 5 and 10 μg/ml ACGs for 36 h, respectively. The expression of Notch2 and Hes-1 protein was quantified by Western blot and compared with internal control β-actin. Control cells were treated with 0.1% DMSO. Each experiment was repeated three times. (A) The expression of Notch2. (B) The expression of Hes-1.

Figure 6. GC cells were transfected by Notch2-siRNA for 24 h and then treated with 5 μg/ml ACGs for 24 h

The control cells were transfected by control Notch2-siRNA. Each experiment was repeated three times. (A) Top: The expression of Notch2 was detected by Western blot compared with internal control β-actin; Each histogram indicates the relative band intensity (B) The cell growth was detected at 12, 24 and 36 h by MTS after ACGs treatment.

Figure 7. GC cells were transfected by Notch2-cDNA for 24 h and then treated with 5 μg/ml ACGs for 36 h

The cells were transfected by pCMV-Tag4 vector as control. Each experiment was repeated three times. (A) Top: The expression of Notch2 was measured by western blot compared with internal control β-actin; each histogram indicates the relative band intensity. (B) The cell growth was detected at 12, 24 and 36 h by MTS after ACGs treatment.

Figure 8

(A) HPLC fingerprint chromatograms determined at 210 nm for five batches of total annonaceous acetogenins. (B) The structure of Bullatacin.