Propyl gallate induces human pulmonary fibroblast cell death through the regulation of Bax and caspase-3

Abstract

Propyl gallate (PG) has been found to exert an inhibitory effect on the growth of different cell types, including lung cancer cells. However, little is known about the cytotoxicological effects of PG specifically on normal primary lung cells. The current study examined the cellular effects and cell death resulting from PG treatment in human pulmonary fibroblast (HPF) cells. DNA flow cytometry results demonstrated that PG (100-1,600 μM) had a significant impact on the cell cycle, leading to G1 phase arrest. Notably, 1,600 μM PG slightly increased the number of sub-G1 cells. Additionally, PG (400-1,600 μM) resulted in the initiation of cell death, a process that coincided with a loss of mitochondrial membrane potential (MMP; ΔΨm). This loss of MMP (ΔΨm) was evaluated using a FACS cytometer. In PG-treated HPF cells, inhibitors targeting pan-caspase, caspase-3, caspase-8, and caspase-9 showed no significant impact on the quantity of annexin V-positive and MMP (ΔΨm) loss cells. The administration of siRNA targeting Bax or caspase-3 demonstrated a significant attenuation of PG-induced cell death in HPF cells. However, the use of siRNAs targeting p53, Bcl-2, or caspase-8 did not exhibit any notable effect on cell death. Furthermore, none of the tested MAPK inhibitors, including MEK, c-Jun N-terminal kinase (JNK), and p38, showed any impact on PG-induced cell death or the loss of MMP (ΔΨm) in HPF cells. In conclusion, PG induces G1 phase arrest of the cell cycle and cell death in HPF cells through apoptosis and/or necrosis. The observed HPF cell death is mediated by the modulation of Bax and caspase-3. These findings offer insights into the cytotoxic and molecular effects of PG on normal HPF cells.

Keywords: Human pulmonary fibroblast; caspase; cell cycle; cell death; mitogen-activated protein kinase; propyl gallate.

Figure 1.

Effects of PG on cell cycle phase distributions in HPF cells. Cells in the exponential growth phase were incubated in the presence of the designated concentrations of PG for 24 h. Cell cycle phase distributions were evaluated by DNA flow cytometry. A: Each histogram shows the cell cycle distributions in PG-treated HPF cells. M1 indicates sub-G1 cells. G1, S, and G2 represent the phases of the cell cycle. B: Graph displaying the proportions of each cell cycle phase derived from A. C: Graph displaying the proportions of sub-G1 cells derived from A. *p < 0.05 as compared with untreated control cells.

Figure 2.

Effects of PG on cell death and MMP (ΔΨm) in HPF cells. Exponentially growing cells were incubated in the presence of the designated concentrations of PG for 24 h. Annexin V-FITC and rhodamine staining were performed in HPF cells and were measured using a FACStar flow cytometer. A and B: Representative histograms for annexin V-FITC (A) and rhodamine staining in HPF cells (B). M1 indicates annexin V-FITC-positive (A) and rhodamine 123-negative [MMP (ΔΨ_m) loss] HPF cells (B). M2 indicates cells without MMP (ΔΨm) loss. C and D: Graphs of the percentages of M1 regions in A (C) and B (D). E: Graph displaying the proportions of MMP (ΔΨm) levels in HPF cells derived from M2 regions in B. *p < 0.05 as compared with untreated control cells.

Figure 3.

Effects of caspase inhibitors on cell death and MMP (ΔΨm) in PG-treated HPF cells. Exponentially growing cells were pretreated with each caspase inhibitor (15 µM) for 1 h and then treated with 800 µM PG for 24 h. Annexin V-FITC and rhodamine staining were measured in HPF cells using a FACStar flow cytometer. A and B: Representative histograms for annexin V-FITC (A) and rhodamine staining in HPF cells (B). M1 indicates annexin V-FITC-positive (A) and rhodamine 123-negative [MMP (ΔΨm) loss] HPF cells (B). C and D: Graphs show the percentages of M1 regions in A (C) and B (D). *p < 0.05 as compared with untreated control cells.

Figure 4.

Effects of apoptosis-related siRNAs on cell death in PG-treated HPF cells. HPF cells (at approximately 30%–40% confluence) were transfected with either a nontargeting control siRNA or the indicated apoptosis-related siRNAs. One day later, cells were treated with 800 µM PG for an additional 24 (A) or 48 h (B). A and B: Annexin V-FITC and PI staining in HPF cells were measured using a FACStar flow cytometer. The percentages shown in each figure represent annexin V-FITC-positive cells, regardless of PI-negative and PI-positive cells.

Figure 5.

Effects of MAPK inhibitors on cell death and MMP (ΔΨm) in PG-treated HPF cells. Cells undergoing exponential growth were pretreated with each MAPK inhibitor (10 µM) for 30 min and then treated with 800 µM PG for 24 h. Annexin V-FITC and rhodamine staining in HPF cells were measured using a FACStar flow cytometer. A and B: Representative histograms for annexin V-FITC (A) and rhodamine staining in HPF cells (B). M1 indicates annexin V-FITC-positive (A) and rhodamine 123-negative [MMP (ΔΨ_m) loss] HPF cells (B). C and D: Graphs show the percentages of M1 regions in A (C) and B (D). *p < 0.05 as compared with PG-untreated control cells.