Abstract
Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms.Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were evaluated by Transwell and Transwell with matrigel assays, respectively. Furthermore, cell cycle and apoptosis were assessed by flow cytometry, while the mechanisms of action were determined by Western blotting.Results: PMF exhibited significant anti-proliferative effect on A549 cells in concentration-dependent and time-dependent manners, with half maximal inhibitory concentration (IC50) of 54.51 μM. Treatment with PMF (10, 20 and 40 μM) for 48 h resulted in significantly decreased migration and invasion in A549 cells. In addition, PMF at concentrations of 25, 50 and 75 μM induced cell cycle arrest in G0/G1phase and enhanced cell apoptosis in A549 cells. Furthermore, caspase-3, caspase-9 and Bax protein expressions were up-regulated while Bacl-2 and COX-2 protein expressions were significantly downregulated at 10, 20 and 40 μM concentrations of PMF.Conclusion: PMF suppresses A549 cell growth, migration and invasion. The mechanism may be related to the induction of mitochondria-mediated apoptosis pathway via regulation of caspase-3, caspase-9, Bcl-2 and Bax expressions, and inhibition of PGE2 synthesis by reducing COX-2 expression.Keywords: Phlomisoside F, Lung cancer, Cell mobility, Apoptosis, PGE2, COX-2 expression, Caspase, Cell cycle arrest
Highlights
Lung cancer is the most common cancer in males with increasing incidence and mortality, and is the leading cause of cancer-related deaths in China [1]
The effect of phlomisoside F (PMF) on A549 cell viability was detected by the CCK-8 assay
We confirmed the inhibitory effect of PMF on A549 cells invasion and migration by the matrigel invasion assay and the transwell migration assay in vitro, respectively
Summary
Lung cancer is the most common cancer in males with increasing incidence and mortality, and is the leading cause of cancer-related deaths in China [1]. Phlomisoside F (PMF), a furanolabdane diterpene glycoside, is from the ethyl acetate extract of P. younghusbandii root, which has been reported in previous studies [10,11]. 2 × 103 cells per well were seeded in 96-well plates and incubated with either vehicle (DMSO) or increasing concentrations of PMF. Annexin V - FITC Apoptosis Detection Kit (Beyotime, Jiangsu, China) was used to determine the effect of PMF on A549 cells apoptosis. After treatment with or without various concentrations of PMF (25, 50 and 75 μM) for 48 h, cells were harvested and were stained by annexin-V-fluorescein isothiocyanate (FITC) and PI according to the manufacturer’s protocol. 0.5 mL of A549 cells with serum-free RPMI 1640 (1 × 105 cells/well) were seeded in the upper chamber and treated with or without indicated PMF (10, 20 and 40 μM). Data analysis was carried out using SPSS 19.0 software package and one way analysis of variance (ANOVA) with Dunnett’s test was used to compare the means between two groups
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