3.1 Resveratrol inhibits epithelial-mesenchymal transition in lung cancer cells.
There were many studies shown epithelial-mesenchymal transition (EMT) plays an important role in regulating invasion and migration of cancer cells. To elucidate whether resveratrol inhibited cell mobility of lung cancer cells, CL1-5 and A549 lung carcinoma cells were treated with various concentrations of resveratrol, and cell mobility were determined by migration and invasion assay. Treatment with the indicated concentrations of resveratrol decreased the migration and invasion ability of lung cancer cells in a dose-dependent manner (Fig.1). During EMT transition, cancer cells lose the expression of E-cadherin which regulating cell-cell contact and acquired mesenchymal markers such as vimentin, fibronectin, and N-cadherin [21,31,119]. To examine that the effects of resveratrol in lung cancer cells would abolish EMT transition, lung cancer cells treated with 10PM resveratrol for 48h and then analyzed E-cadherin and mesenchymal markers expression by western blotting analysis.
Resveratrol increased the expression of E-cadherin and decreased the expression of mesenchymal markers such as fibronectin, N-cadherin and vimentin in lung cancer cells (Fig.2). To confirm resveratrol-inhibited EMT with up-regulation of the expression of E-cadherin, immunnofluorescence staining was preformed. The expression of E-cadherin was induced in resveratrol-treated A549, but not in control cells (Fig.3). The cell morphology of CL1-5 cells changed from spindle-shaped mesenchymal type to less-elongated epithelial cell morphology after treatment with resveratrol (Fig.4). The effect of resveratrol on cell viability of CL1-5 cells was down by MTS assay. Treatment with resveratrol for the indicated time had no effect on cell viability (Fig.5). These data indicated that treatment with resveratrol decreased cell mobility through down-regulation EMT and changed cell morphology from spindle-shaped to epithelial characteristics of lung cancer cells.!
3.2 Resveratrol significantly inhibits EMT-inducing transcription factor, FOXC2, at transcriptional level in lung cancer cells.
To elucidated how resveratrol-inhibited EMT, EMT-inducing transcription factors, FOXC2, twist, slug and snail were analysis by western blot analysis in lung cancer cell lines. Resveratrol significantly decreased the expression of FOXC2 in CL1-5 and A549 cells, but had less effect on twist and slug (Fig.6). Inhibition the expression of FOXC2 by resveratrol had the same result in other lung cancer cell lines (Fig.7). To examine that resveratrol-mediated inhibition of FOXC2 was through down-regulation of gene expression, RT-PCR was performed. Fig.7 revealed that resveratrol inhibited the expression of FOXC2 was at transcriptional level in lung cancer cells. The inhibition ability of FOXC2 in response to resveratrol was for 48h and resveratrol enhanced E-cadherin expression was at time-dependent manner (Fig.8).
To determine the regulatory mechanism underlying the transcription of FOXC2 gene by resveratrol, we analyzed the promoter region of human FOXC2 gene. Within the ~1.9-kb fragment of upstream the start codon, there were several transcription factor binding sites. To identify the resveratrol-response elements, a series of 5’
promoter deletion mutants of the FOXC2 gene (F1-F6) were constructed. The F1, F3, F5 and F6 reporter constructs were down-regulated by resveratrol about 40-50%
which indicating that resveratrol-response elements lie between -215 and +6 (F6) (Fig.9). These observations suggest that resveratrol-inhibition EMT were through down-regulated the expression of FOXC2 in human lung cancer cells.
3.3 Ectopic expression of FOXC2 reverses resveratrol-mediated cell mobility and EMT.
To investigate whether the expression of FOXC2 involved in resveratrol-mediated repression of cell mobility in lung cancer cells, FOXC2 was
ectopic expressed in A549 lung cancer cells. Overexpression of FOXC2 in A549 decreased the levels of E-cadherin and up-regulated mesenchymal markers, fibronectin, N-cadherin and vimentin expression (Fig.10). It was therefore of interest to determine whether resveratrol-inhibited EMT transition were exerted through the regulation of forkhead protein. Western blot and immunofluorescence staining experiments were preformed. Stable expressed cells with FOXC2 increased the migration and invasion ability in vitro study, and rescued resveratrol-reduced cell mobility and resveratrol-inhibited the expression of EMT markers (Fig.11-13).
Treatment with resveratrol had no effect on cell viability in A549 overexpression of FOXC2 cells (Fig.14-15). These results indicated that resveratrol-mediated repression of EMT transition through down-regulated the expression of FOXC2 in lung cancer cells.
3.4 PI-3K/Akt activity is required to increase the expression of FOXC2 in A549 cells.
Activation of FOXC2 is involved in phosphatidylinositol 3-kinase (PI3K) and ERK1/2 signaling pathways in adipocytes [120] and endothelial cells [121] but the role of FOXC2 in cell mobility and EMT in lung cancer cells is not clear. To address this issue, A549 cells were treated with PI-3K/Akt inhibitor, LY294002 and dual inhibitor of MEK1 and MEK2, U0126, and then FOXC2 expression were analyzed by western blot and by reporter assay. Fig.16 revealed that PI-3K/Akt inhibitor, LY294002, but not MEPK inhibitor, U0126, reduced the expression of FOXC2 and FOXC2 promoter activities (F1). To confirm this observation, we used another approach to test the role of Akt in regulating FOXC2 expression by transfected constitutively activate myristoylated Akt, myr-Akt, in A549 cells with or without resveratrol. These results revealed that constitutive activation of Akt induced up-regulation FOXC2 expression, and rescued resveratrol-mediated inhibition of
FOXC2. Overexpression of Akt in cancer cells led to EMT induction and rescued resveratrol-mediated EMT markers expression (Fig.17). These data suggest the possible involvement of PI-3K/Akt, rather than MAPK signaling pathway, in the regulation of FOXC2 expression and EMT transition in lung cancer cells.
3.5 Protein serine/threonine phosphatase 2A catalytic subunit C (PP2A/C) acts as an up-regulator of resveratrol-inhibited FOXC2 expression.
Many researchers have showed that PP2A, a serine/threonine phosphatase, interacts with Akt and thus down-regulates Akt activity by dephosphorylating Akt [122]. To examine that involvement of PP2A/C in resveratrol-mediated inhibition of PI-3K/Akt, A549 human lung cancer cells were treated with resveratrol with different concentrations or at different time and then analyzed the expression of PP2A/C and PP2A activity. Treatment with resveratrol increased the expression of PP2A/C and up-regulated the expression of PP2A/C in a time-dependent manner as well as PP2A activities (Fig.18). To determine the role of PP2A/C in regulating the expression of FOXC2 through dephosphorylated Akt, A549 cells were treated with resveratrol in the presence of okadaic acid (OA), a PP2A inhibitor. Data from western blot confirmed that decreased the expression of PP2A/C by PP2A inhibitor significantly increased FOXC2 and phospho-Akt expression and decreased resveratrol-induced PP2A activation (Fig.19). To further confirm this observation, we used another approach to test the role of PP2A/C by expression shLuc and shPP2A/C targeted knockdown control or PP2A/C by lentivirus infection, respectively. These results support those obtained above that down-regulation of PP2A/C increased the expression of FOXC2 and phospho-Akt by transient or stable infection (Fig.20). To ascertain the role of FOXC2 in resveratrol-mediated PP2A/C up-regulation, we knockdown PP2A/C and then treated with resveratrol. Down-regulated PP2A/C expression rescued resveratrol-inhibited FOXC2 and phosphorylated Akt expression and increased
resveratrol-inhibited cell mobility (Fig.21-22). These findings provided evidence that resveratrol-inhibited FOXC2 activation and cell mobility through down-regulation of Akt that inhibited by up-regulation the expression of PP2A/C.