Abstract
BackgroundSnail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE.MethodsARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments.ResultsSnail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells.ConclusionsThis study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.
Highlights
Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis
We found that Phorbol 12-myristate 13-acetate (PMA) induced superoxide, while less staining was observed when superoxide dismutase (SOD) antagonist was added (Figure 1B)
These results were confirmed by using dihydroethidium (DHE) staining which revealed elevated levels of superoxide in ARCaPE cells transfected with Snail (ARCaP-Snail low, −Snail high) in vitro as compared to the control (ARCaP-Neo) (Figure 1C)
Summary
Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE. Reactive Oxygen Species (ROS) that includes superoxide and hydrogen peroxide species has been associated with several disease states including cancer [6]. With regards to ROS and EMT; Transforming Growth FactorBeta (TGF-β-mediated EMT involved increased hydrogen peroxide and Mitogen-activated protein kinase/Extracelluar signal reduced kinase (MAPK/ERK) signaling in proximal tubular epithelial cells [11], while Matrix Metalloproteinase-3 (MMP-3) mediated EMT in mammary epithelial cells involved increase in ROS and Snail [12]. Overexpression of Snail in ARCaP prostate cancer cells has been shown to induce EMT and ROS (hydrogen peroxide and superoxide), possibly by regulating oxidative stress-responsive genes [13].
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