Crude extract of Desmodium gangeticum process anticancer activity via arresting cell cycle in G1 and modulating cell cycle-related protein expression in A549 human lung carcinoma cells

Abstract

Background: Desmodium gangeticum (L.)DC., which belongs to the Leguminosae family, has been used in Taiwan and other subtropical countries as an external medicine to remove blood stasis, activate blood circulation, and reduce inflammation. It has been reported to have antioxidant effects and improve inflammatory responses in rats stimulated by pro-inflammatory agents and induced gastric ulcers in experimental animals over the past few decades. This plant has also been used to treat parasitic infections, but there are no reports regarding its effects on lung cancer. Therefore, this study attempted to investigate its water crude extract (in abbreviation DG) on lung cancer cells.

Methods: A549 human lung cancer cells were tested for survival using MTT, trypan blue, and propidium iodide. The effects of various concentrations of the crude extract of D. gangeticum (DG) (0.125~1 mg/ml) on the cell cycle and apoptosis of A549 cells were analyzed by flow cytometry and Western blotting methods.

Results: DG can inhibit the growth of A549 human lung cancer cells in a concentration- and time-dependent manner. DG arrested A549 cells in the G1 phase by increasing the proteins expression of p21, p27, cyclin D1, and cyclin E. Additionally, DG decreased the expression of cyclin A, B1, and Cdc 2 (CDK1) proteins.

Conclusions: DG demonstrated the anti-lung cancer activity by arresting the cell cycle in G1 via increasing the p21, p27, cyclin D1, cyclin E, and decreasing Cdc2, cyclin A, and B1 proteins expression in A549 human lung cancer cells.

Keywords: A549 human lung cancer cells; Anti-lung cancer; Apoptosis; Cell cycle arrest; Desmodium gangeticum (L.) DC; G1.

Fig. 1

After DG treatment, the morphology and viability change of A549 human lung cancer cells. (A) The morphology changes of A549 cells after treatment with various DG concentrations (0.5 and 1 mg/ml) for 24, 48, and 72 h, respectively. (B) The viability of A549 was treated with various concentrations of DG (0.125~1 mg/ml) for different time courses (24, 48, and 72 h) by MTT viability assay. DG showed a concentration-dependent and time-dependent inhibition on the morphology and cell viability of A549 cells. (C) The IC 50 of DG at different time of treatment. Results represented as mean ± S.E. *P < 0.05, **P < 0.01 was considered statistically significant compared to the control group.

Fig. 2

The cell viability of A549 cells treated with various concentrations of DG (0.125–2.0 mg/ml) for different time lengths. (A) The viability of A549 cells treated with various concentrations of DG for 24, 48, 72h, respectively, by flow cytometry. (B) The viability change and time course of A549 cells treated with various concentrations of DG for 24 to 72h. A549 Cell viability was detected by propidium iodide staining and expressed as % of control. DG inhibited the cell viability in a concentration-dependent manner by flow cytometry analysis. Data were expressed as mean ± S.E. *P < 0.05, **P < 0.01, and ***P < 0.001 was considered statistically significant compared to the control group.

Fig. 3

DG treatment-induced A549 lung cancer cells apoptosis. (A) A549 cells were treated with various concentrations of DG (0.5, 1 mg/ml) for different time courses (24h, 48h, and 72 h). After DG treatment, A549 cells were stained with FITC conjugated Annexin-V/PI. The percentage of Annexin-V/PI stained cells was determined by flow cytometry. It was divided into a quadrant: (Q1) necrotic cells, (Q2) late apoptosis cells, (Q3) living cells, (Q4) early apoptotic cells. (B) Represented the FITC conjugated Annexin-V/PI positive cells in each quadrant. DG showed a concentration- and time-dependent-induction of A549 cells apoptosis. Values are expressed as mean ± S.E. *P < 0.05, **P < 0.01 was considered statistically significant compared to the control group.

Fig. 4

Flow cytometric analysis of cell cycle in A549 lung cancer cells after various concentrations of DG (0.125~1 mg/ml) treatment for different time courses (24~48 h). (A) The flow cytometry charts of DG-treated A549 cells. DG increased the G1 phase and reduced the S phase concentration and time-dependent. (B) Represented cell cycle distribution of control and DG-treated A549 cells as measured by propidium iodide staining of DNA at 24h, 48h, and 72 h, respectively. After treatment with various concentrations of DG (0.25~1 mg/ml) in A549 cells, DG increased the cell population in G1. When *P < 0.05 or **P < 0.01 is regarded as statistical significance in the proportion of cells in the G1 phase versus control cells.

Fig. 5

DG treatment for 48 h affected the protein expression of various cell cycle-related kinases in A549 lung cancer cells. Various concentration of DG (0.25~1 mg/ml) was used. The expression of cyclin A, cyclin B1, and Cdc2 were decreased after DG treatment; however, the expression of cyclin D1, cyclin E, p21, and p27 was increased after DG treatment.

Fig. 6

The anti-lung cancer mechanism of DG on A549 human lung cancer cells is proposed in this diagram. DG arrests cell cycle in the G1 phase via increasing protein expression of p21, p27, cyclin D1, and cyclin E; whereas DG decreases the expression of cyclin A, cyclinB1, and Cdc2, leading to apoptosis of A549 cells.