Epithelial-mesenchymal transition (EMT) is a pivotal event in the invasion and metastasis of cancer cells.
The epithelial-mesenchymal transition (EMT), an important morphological event in which polarized epithelial cells convert to contractile and motile mesenchymal cells, is recognized as an important process during embryonic development and tissue organization (
In the present study, we hypothesized that PVAE, a typical medicine for decoction, may have a role as an inhibitor of EMT in cancer progression and thus could serve as a dietary chemopreventive agent against malignant tumors. We report that PVAE significantly inhibited the invasion and migration of lipopolysaccharide (LPS)-induced EMT in two metastatic cancer cell lines through downregulation of the NF-κB/Snail signaling pathway. We propose that PVAE is a good candidate for use as a dietary chemopreventive agent with antimetastatic activity against malignant tumors.
The PV used in the present study was kindly supplied by Professor Jung-Hye Choi (Kyung Hee University). Aqueous extraction procedures were performed by boiling 100 g PV in 500 ml distilled water for 30 min and then filtering through Whatman filter paper No. 2 (Advantec, Tokyo, Japan). Subsquently, the filtrates were combined and evaporated under a vacuum and then lyophilized with a freeze dryer (Ilshine Lab, Suwon, Korea). The dry residue was stored at −20°C. MDA-MB-231 and SKOV-3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/stereptomycin antibiotics. The antibodies NF-κB p65 subunit and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Snail was purchased from Cell Signalling Technology (Beverly, MA, USA), vimentin and β-catenin were purchased from Abcam (Cambridge, MA, USA), and N-cadherin were purchased from BD Biosciences (San Jose, CA, USA).
All proliferation assays were based on the 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method. Cells were seeded in a 96-well plate, 1×104 cells/well. After overnight culture, PVAE was added to the cells and further cultured for 24 h. The media was removed and DMSO was added at MTT solubilization solution. Absorbance was measured at 550 nm.
Single-cell suspensions of 5×103 cells were seeded into 6-well plate and allowed to attach for 24 h at 37°C in culture medium. Cells were then treated with 100
MDA-MB-231 cells were grown in 4-chamber slides in serum-free media, and were treated with LPS (5
Migration was assessed by a wound healing assay. Cells were seeded at 2×104 MDA-MB-231 and SKOV-3 cells/well and were cultured for 24 h. After scraping the cell monolayer with a sterile micropipette tip, the wells were washed with PBS, and treated with LPS (5
The invasion of tumor cells was assessed in Transwell chambers equipped with 8-
MDA-MB-231 and SKOV-3 cells were treated with with LPS (5
The results are presented as mean ± SE, and statistical comparisons between groups were carried out using one-way ANOVA followed by the Student's t-test.
We initially examined the effect of PVAE on the proliferation of the human metastatic cancer cell lines MDA-MB-231 (breast cancer cells) and SKOV-3 (ovarian cancer cells). Cells were treated for 24 h with different concentrations of PVAE, and cell viability was measured by MTT assay. As shown in
LPS may act as an independent factor to trigger the EMT process, as has previously been reported by Chen and colleagues (
To further investigate the effect of PVAE on LPS-induced EMT, we monitored the expression of the EMT-related proteins, N-cadherin, β-catenin and vimentin by western blotting (
Numerous studies have reported that many drugs may inhibit the invasion and migration of cancer cells by suppressing NF-κB activation and Snail induction, suggesting that the NF-κB signaling pathway is critically involved in the acquisition of EMT through its downstream target, the transcription factor Snail. To determine whether the effects of PVAE on LPS-induced changes in EMT marker expression involve inhibition of NF-κB/Snail activation, we monitored expression of the NF-κB p65 subunit and Snail by western blotting. As shown in
Cancer cell metastasis is frequently associated with activation of EMT, which results in a loss of the cells' epithelial traits and acquisition of many properties of mesenchymal cells. The profound changes in cytoskeletal architecture that occur during EMT, together with a reduction in intercellular adhesion and an increase in motility, are fundamental to the metastatic process, enabling these cells to break through the basal membrane and migrate over long distances (
As previously reported, LPS induces EMT in breast cancer cells, increasing their invasion and migration and resulting in enhanced lung metastasis (
PV, a Labiate plant, is a traditional fever remedy, and more recently has been used to treat tuberculosis, thyroid gland swelling, jaundice, infectious hepatitis, bacillary dysentery, pleuritis, hypertension and cancer (
In the present study, we showed that PVAE inhibited LPS-induced EMT, determined by monitoring changes in cell migration and invasion, and expression of cell-cell adhesion proteins and EMT-related proteins like N-cadherin. Cadherins are transmembrane glycoproteins that mediate Ca2+-dependent cell-cell adhesion (
Our data also demonstrated that the mechanism of action of PV may involve suppression of NF-κB/Snail signaling. NF-κB is a structurally conserved family of dimeric transcription factors that plays pivotal roles in maintaining an invasive phenotype as well as promoting carcinogenesis (
In conclusion, we demonstrated that PVAE inhibition of tumor invasion and migration is associated with the EMT process during tumor progression, and is possibly mediated by suppressing NF-κB/Snail signaling and regulating the expression of N-cadherin, β-catenin and vimentin-important downstream EMT markers. Although further
The present study was supported by the Traditional Korean Medicine R&D program funded by the Ministry of Health & Welfare through the Korea Health Industry Development Institute (HI14C05830000) and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2057861).
PVAE inhibits the growth of human cancer cells. (A) Antiproliferative effects of PVAE after 24 h in MDA-MB-231 and SKOV-3 cells. Cell proliferation was determined by MTT assay. Results are expressed as the mean ± SD of three replicate measurements from a single representative experiment of three separate experiments. (B and C) The inhibitory effect of PVAE on colony formation by (B) MDA-MB-231 and (C) SKOV-3 cells. Representative photographs demonstrating colony formation are shown.
PVAE inhibits LPS-induced migration of MDA-MB-231 and SKOV-3 cells. (A) Representative images of wound healing were captured at the time of the scratch and 24 h later. (B) Cell migration into the wounded area was quantified as the fold ratio of wound healing (n=5; *P<0.05, **P<0.01 vs. LPS-treated cells).
PVAE inhibits LPS-induced cell invasion. (A) Cell invasion was determined by Transwell assays. Representative photomicrographs of membrane-associated cells stained with crystal violet. (B) Cell invasion values were quantified as the fold ratio (n=5; *P<0.05, **P<0.01 vs. LPS-treated cells).
PVAE regulates the expression of EMT-related proteins. (A) N-cadherin, β-catenin, and vimentin protein expression in MDA-MB-231 and SKOV-3 cells was determined using western blot analysis. (B) MDA-MB-231 cells were immunostained with anti-N-cadherin antibody (green), and nuclei were counterstained with DAPI (blue) after PVAE treatment.
LPS-induced NF-κB/Snail signaling is inhibited by PVAE. Western blot analysis of the NF-κB p65 subunit and Snail in MDA-MB-231 and SKOV3 cells after treatment for 24 h with LPS, with and without PVAE.