Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Abstract

Background: Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results: In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions: The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells.

Figure 1

Effects of HCT on cell viability and morphological changes in human lung cancer A549 cells. (A) The A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml) for 24 and 48 h, the viable cells were determined by MTT assay as described in Materials and Methods. The experiments were performed in triplicate (n = 3). ***p < 0.001 was considered significantly different in comparison with the control. (B) The cells’ morphological changes were examined and photographed by phase-contrast microscopy.

Figure 2

Effects of HCT on cell-cycle transition in human lung cancer A549 cells. (A) The A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml) for 24 h, and then were harvested for determination the distribution of cell cycle by flow cytometry and (B) quantitative results were expressed as described in Materials and Methods. The experiments were performed in triplicate (n = 3). Data represents mean ± S.D. of three experiments. ***p < 0.001 was considered significantly different in comparison with the control.

Figure 3

Effects of HCT on DNA condensation, damage and apoptosis in human lung cancer A549 cells. (A) A549 cells stained with DAPI to observe DNA condensation after 24 h of treatment with 500 μg/ml of HCT. (B) A549 cells were examined DNA damage by Comet assay after 24 h of treatment with 500 μg/ml of HCT. Cells were photographed under fluoresce microscopy (x200) as described in Materials and Methods. (C) A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml) for 24 h, and then were harvested for determination the sub-G1 (apoptosis) cell population by flow cytometry as described in Materials and Methods. (D) The bar diagram of the length of Comet tail. The experiments were performed in triplicate (n = 3). *p < 0.05, ***p < 0.001 was considered significantly different in comparison with the control.

Figure 4

Effects of HCT on G₀/G₁ relative protein levels in A549 cells. A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml) for 24 h and then performed western blotting analysis for Cyclin D1, Cyclin A, CDK4, CDK2, and p27 in HCT treated cells as described in Materials and Methods. The experiments were performed in triplicate (n = 3).

Figure 5

Effects of HCT on caspases-8 and caspase-3 activities in human lung cancer A549 cells. The A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml) for 24 h, and then total cell extracts were incubated with (A) caspases-8 specific substrate (Ac-IETD-pNA) and (B) caspase-3 specific substrate (Ac-DEVE-pNA) respectively. The release of pNA was measured at 405 nm by a spectrophotometer as described in Materials and Methods. The experiments were done in triplicate (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 was considered significantly different in comparison with the control.

Figure 6

Effects of HCT on A549 cells in the Fas/CD95-dependent apoptotic pathway. The A549 cells were treated with HCT (0, 125, 250 and 500 μg/ml) for 12 h, and (A) Fas/CD95 protein expression levels was detected by immune staining and analyzed by flow cytometry as described in Materials and Methods. The experiments were performed in triplicate (n = 3). **p < 0.01, ***p < 0.001 was considered significantly different in comparison with the control. (B) Caspase-8 and caspase-3 protein expression levels in HCT-examined cells were analyzed by Western blotting as described in Materials and Methods.

Figure 7

Effects of caspases-3 and caspase-8 specific inhibitor on cell viability in HCT- treated human lung cancer A549 cells. Cells were pretreated with (A) the caspase-3 inhibitor (z-DEVE-fmk) and (B) the caspase-8 inhibitor (z-IETD-fmk) for 1 h after exposure to HCT (0, 125, 250 and 500 μg/ml) for 24 h exposure, viable cells were determined by MTT assay as described in Materials and Methods. The experiments were performed in triplicate (n = 3). The experiments were performed in triplicate. **p < 0.01, ***p < 0.001 was considered significantly different in comparison with the control.

Figure 8

A proposed model of HCT modulates G₀/G₁ arrest and Fas/CD95- mediated death receptor apoptotic cell death on human lung cancer A549 cells.