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3,4-Dihydroxybenzalactone Suppresses Human Non-Small Cell Lung Carcinoma Cells Metastasis via Suppression of Epithelial to Mesenchymal Transition, ROS-Mediated PI3K/AKT/MAPK/MMP and NFκB Signaling Pathways

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3,4-Dihydroxybenzalactone Suppresses Human Non-Small Cell Lung Carcinoma Cells Metastasis via Suppression of Epithelial to Mesenchymal Transition, ROS-Mediated PI3K/AKT/MAPK/MMP and NFκB Signaling Pathways

Wei Chao et al. Molecules.

Abstract

3,4-Dihydroxybenzalactone (DBL) was isolated from Phellinus linteus (PL), which is a folk medicine possessing various physiological effects. In this study, we used highly metastatic A549 cells to investigate efficacy of DBL inhibition of cancer metastasis and possible mechanisms. The results revealed DBL inhibited migratory and invasive abilities of cancer cells at noncytotoxic concentrations. We found DBL suppressed enzymatic activities, protein expression, and RNA levels of matrix metalloproteinase (MMP)-2 and MMP-9. Western blot results showed DBL decreased phosphoinositide 3-kinase (PI3K)/AKT, phosphorylation status of mitogen-activated protein kinases (MAPKs), and focal adhesion kinase (FAK)/paxillin, which correlated with cell migratory ability. DBL also affected epithelial to mesenchymal transition (EMT)-related biomarkers. In addition, DBL enhanced cytoprotective effects through elevated antioxidant enzymes including heme oxygenase 1 (HO-1), catalase, glutathione peroxidase (GPx), and superoxide dismutase (SOD). Moreover, DBL influenced the nuclear translocation of nuclear factor κB (NFκB), nuclear factor erythroid 2-related factor 2 (Nrf2), Snail, and Slug in A549 cells. Taken together, these results suggested that treatment with DBL may act as a potential candidate to inhibit lung cancer metastasis by inhibiting MMP-2 and -9 via affecting PI3K/AKT, MAPKs, FAK/paxillin, EMT/Snail and Slug, Nrf2/antioxidant enzymes, and NFκB signaling pathways.

Keywords: DBL; EMT; MMPs; Phellinus linteus; ROS; cancer metastasis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The chemical profile of dihydroxybenzalactone (DBL). (A) Chemical structure of DBL; (B) effects of DBL on cell viability in A549 cells for 24 and 48 h by MTT assay. A549 cells were treated with indicated concentrations (0, 6.25, 12.5, 25, 50 μM). Values represent mean ± SEM from three independent experiments.
Figure 2
Figure 2
The effects of DBL on migration, invasion, and adhesion of A549 cells. The migration and invasion assays were assessed by passing A549 cells through 6.5 mm polycarbonate filters of 8 μm pore size. (A) Migration assay: A549 cells were treated with various concentrations (0, 6.25, 12.5, 25, and 50 μM) of DBL for 8 h; (B) Invasion assay: the upper chambers were coated with Matrigel. A549 cells were treated with DBL for 24 h. All of the chambers were fixed, stained, and photographed in 200× microscopic power field; (C) Adhesion assay: the A549 cells were treated with DBL for 24 h, then seed cells in Matrigel-coated 96-well plates. The adhesion rate was evaluated by MTT assay. Data represent mean ± SEM from three independent experiments. Statistical significance was analyzed by one-way ANOVA and post hoc test was Scheffe test. (** p < 0.01 and *** p < 0.001).
Figure 2
Figure 2
The effects of DBL on migration, invasion, and adhesion of A549 cells. The migration and invasion assays were assessed by passing A549 cells through 6.5 mm polycarbonate filters of 8 μm pore size. (A) Migration assay: A549 cells were treated with various concentrations (0, 6.25, 12.5, 25, and 50 μM) of DBL for 8 h; (B) Invasion assay: the upper chambers were coated with Matrigel. A549 cells were treated with DBL for 24 h. All of the chambers were fixed, stained, and photographed in 200× microscopic power field; (C) Adhesion assay: the A549 cells were treated with DBL for 24 h, then seed cells in Matrigel-coated 96-well plates. The adhesion rate was evaluated by MTT assay. Data represent mean ± SEM from three independent experiments. Statistical significance was analyzed by one-way ANOVA and post hoc test was Scheffe test. (** p < 0.01 and *** p < 0.001).
Figure 3
Figure 3
DBL attenuates activities, protein expression, and mRNA level of MMPs in A549 cells. A549 cells were treated with various concentrations of DBL. (A) Collected supernatants and gelatin zymography were used to analyze the activities of MMP-2; (B) protein expression of MMP-9 and MMP-2 was determined by Western blot; (C) mRNA levels of MMP-9 and MMP-2 were examined by RT-PCR. The data are presented as the mean ± SEM for three different experiments performed in triplicate. Statistical significance was analyzed by one-way ANOVA and post hoc test was Scheffe test. (* p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control group.)
Figure 4
Figure 4
DBL-affected protein expression via Western blot in A549 cells. (A) The phosphorylation of mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38); (B) phosphoinositide 3-kinase (PI3K)/AKT; and (C) matrix metalloproteinase (MMP)-2 and MMP-9 protein expression treated with PI3K inhibitor (LY294002), ERK inhibitor (PD98059), JNK inhibitor (SP600125), and p38 inhibitor (SB203580), or co-treated with DBL; (D) Phosphorylation of focal adhesion kinase (FAK) and paxillin; (E) epithelial to mesenchymal transition (EMT)-related protein including mesenchymal biomarkers (N-cadherin, vimentin) and epithelial biomarker (E-cadherin). (F) Antioxidant enzymes: catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and heme oxygenase 1 (HO-1). A549 cells were treated with different concentrations of DBL for 24 h. Cell pellets were lysed with RIPA buffer. Quantitated proteins were separated by SDS-PAGE and conjugated with specific antibodies. The data are presented for three different experiments performed in triplicate.
Figure 5
Figure 5
DBL affected translocated protein expression in A549 cells. (A) The protein expression of nuclear factor κB (NFκB), nuclear factor erythroid 2-related factor 2 (Nrf2), Snail, and Slug in nucleus. The internal control is laminB.1 (B) The protein expression of NFκB, phosphorylated inhibitor of NFκB (p-IκB), IκB, Nrf2, and kelch-like ECH-associated protein 1 (Keap1) in cytosolic fractions. A549 cells were treated with different concentrations of DBL for the appropriate time. Separated nuclear and cytosolic fraction were used with commercial product (Pierce Biotechnology, Rockford IL, USA, 200 reactions). Quantitated proteins were separated by SDS-PAGE and conjugated with specific antibodies. The data are presented as the mean ± SEM for three different experiments performed in triplicate.
Figure 6
Figure 6
The mechanisms of DBL inhibit human non-small cell lung carcinoma cells metastasis.

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