Fructus phyllanthi tannin fraction induces apoptosis and inhibits migration and invasion of human lung squamous carcinoma cells in vitro via MAPK/MMP pathways

Abstract

Aim: Fructus phyllanthi tannin fraction (PTF) from the traditional Tibetan medicine Fructus phyllanthi has been found to inhibit lung and liver carcinoma in mice. In this study we investigated the anticancer mechanisms of PTF in human lung squamous carcinoma cells in vitro.

Methods: Human lung squamous carcinoma cell line (NCI-H1703), human large-cell lung cancer cell line (NCI-H460), human lung adenocarcinoma cell line (A549) and human fibrosarcoma cell line (HT1080) were tested. Cell viability was detected with MTT assay. Cell migration and invasion were assessed using a wound healing assay and a transwell chemotaxis chambers assay, respectively. Cell apoptosis was analyzed with flow cytometric analysis. The levels of apoptosis-related and metastasis-related proteins were detected by Western blot and immunofluorescence.

Results: PTF dose-dependently inhibited the viability of the 3 human lung cancer cells. The IC50 values of PTF in inhibition of NCI-H1703, NCI-H460, and A549 cells were 33, 203, and 94 mg/L, respectively. PTF (15, 30, and 60 mg/L) dose-dependently induced apoptosis of NCI-H1703 cells. Treatment of NCI-H1703 and HT1080 cells with PTF significantly inhibited cell migration, and reduced the number of invasive cells through Matrigel. Furthermore, PTF dose-dependently down-regulated the expression of phosphor-ERK1/2, MMP-2 and MMP-9, up-regulated the expression of phosphor-JNK, but had no significant effect on the expression of ERK1/2 or JNK.

Conclusion: PTF induces cell apoptosis and inhibits the migration and invasion of NCI-H1703 cells by decreasing MPPs expression through regulation of the MAPK pathway.

Figure 1

Chemical structures of gallic acid, ellagic acid, corilagin, and chebulagic acid, four main components in PTF.

Figure 2

Inhibitory effects of PTF on the migration of NCI-H1703 and HT1080 cells in vitro. The confluent NCI-H1703 and HT1080 monolayers were wounded by scraping; NCI-H1703 were subsequently treated with PTF at a dose of 7.5 or 15.0 mg/L, whereas HT1080 were treated with PTF at a dose of 25.5, 50.0, or 100.0 mg/L. (A) PTF inhibited NCI-H1703 cell migration to the wound surface was monitored at different time points. (B) PTF inhibited HT1080 cell migration (magnification ×200). (C) and (D) The migratory areas were measured and analyzed using Bioflux Montage software for NCI-H1703 and HT1080 respectively. Mean±SD. n=5. ^cP<0.01 vs the control group.

Figure 3

Inhibitory effects of PTF on the invasion of NCI-H1703 and HT1080 cells. NCI-H1703 were treated with PTF at a dose of 7.5 or 15.0 mg/L, and HT1080 were treated with PTF at a dose of 25.5, 50.0, or 100.0 mg/L. Forty-eight hours later, the cells were harvested and seeded into the upper chamber coated with Matrigel. The number of cells that invaded the lower chamber represented the invasion capabilities. (A) PTF inhibited NCI-H1703 invasion, and (B) PTF inhibited HT1080 cell invasion (magnification ×200). (C) and (D) Quantification of the invaded NCI-H1703 and HT1080 cells. Mean±SD. n=6. ^bP<0.05, ^cP<0.01 vs the control group.

Figure 4

The effects of PTF on apoptosis induction in NCI-H1703 cells. (A) NCI-H1703 cells were exposed to PTF at a dose of 15.0, 30.0, or 60.0 mg/L and the procaspase-activating compound 1 (PAC-1, 32 μmol/L) for 24 h. Apoptotic morphological changes, such as cytoplasm shrinkage and plasma membrane blebbing, were observed using a light microscope (×200). (B) NCI-H1703 cells were treated with different doses of PTF in complete culture medium for 24 h, and apoptosis was measured by annexin-V and propidium iodide staining followed by flow cytometry detection. Three populations of cells were observed: viable cells (negatively stained, lower left quadrants), early apoptotic cells (annexin-V positive and propidium iodide negative, lower right quadrant) and cells in the late stages of apoptosis (annexin-V and propidium iodide positive, upper right quadrants). (C) Quantitative analysis of early and late apoptosis in NCI-H1703 cells induced by different doses of PTF. The data are provided as the mean±SD. n=4. ^bp<0.05, ^cp<0.01 vs the control.

Figure 5

The effects of PTF on ERK1/2 and JNK expression in NCI-H1703 cells. (A) Total expression and phosphorylation of ERK and JNK were analyzed by Western blotting in NCI-H1703 cells treated with PTF for 24 h. β-Actin was used as a loading control. (B) The ratios of p-ERK/ERK and p-JNK/JNK were analyzed. Results are represented as the mean±SD from three independent experiments. n=3, ^bP<0.05, ^cP<0.01 vs the control.

Figure 6

Inhibitory effects of PTF on MMP-2 and MMP-9 expression in NCI-H1703 cells. (A) Changes in the protein expression of metalloproteinases MMP-9 and MMP-2 in NCI-H1703 treated by PTF. β-Actin was used as a loading control. (B) PTF decreased MMP-2 and MMP-9 expression in NCI-H1703 cells, which was assessed via quantitative densitometry analysis. Results are represented as the mean±SD from three independent experiments. n=3, ^cP<0.01 vs the control.

Figure 7

Fluorescence images of MMP-2 and MMP-9 expression in NCI-H1703 cells treated with PTF. (A–D) MMP-9 stain shows the responses to 0, 15.0, 30.0, and 60.0 mg/L PTF treatments, respectively. (E–H) Images A–D with a bright field overlay, respectively. (I–L) MMP-2 stain shows the responses to 0, 15.0, 30.0, and 60.0 mg/L PTF treatments, respectively. (M–P) Images I–L with a bright field overlay, respectively. A 100 μm scale bar is shown.