Background
Colorectal cancer (CRC) is one of the leading causes of cancer-associated death in the worldwide [
1]. Many patients diagnosis of CRC are at advanced stages, and the prognosis of these patients remains very poor. Generally, when the oncogenes associated with cell proliferation were up-regulated, or the tumor suppressor genes were down-regulated, the tumor cells would evade immune system and form tumors in distal locations/organs through the pathway of invasion and metastases [
2,
3].
Epithelial-to-Mesenchymal Transition (EMT), a biological process occurs in various types of epithelial cancers including CRC, is associated largely with increased invasion and metastases [
4-
6]. EMT mainly experiences the following steps: dissociation of adhesions between epithelial cells, loss of the apical-basolateral polarity, reorganization of the actin cytoskeleton, and increases of cell motility. Recently, numerous studies demonstrated that, some cytokines like TGF-β, HGF and EGF can induce the process of EMT [
7-
9]. Meanwhile, various signaling pathways associated with EMT were activated, such as TGF-β/Smads signaling pathway we focused on.
Traditional Chinese Medicine (TCM), whether the formula or the extracted monomer, have been identified as effective anticancer drugs in various cancers. Long-term basic research and clinical application suggested that, resveratrol has been an ideal alternative drug in the therapy of different diseases. Recently, anti-cancer activity of resveratrol has been explored in various types of cancer including pancreatic cancer, myeloma, ovarian cancer, breast cancer via the regulation of multiple pathways [
10-
13]. Several experimental studies have demonstrated that, resveratrol played an effective anti-cancer activity in the treatment of colorectal cancer including our own completed research [
14-
16].
However, the underlying molecular mechanisms through which resveratrol inhibits migration and invasion of CRC cells have not been fully elucidated, and since EMT is a key process associated with the progression of CRC, herein we aimed to investigate the potential mechanism of resveratrol on the inhibition of TGF-β1-induced EMT in CRC cells.
Methods
Materials
Recombinant human TGF-β1 was purchased from R&D. Resveratorl was purchased from Sigma-Aldrich and dissolved at a concentration of 100 mM in DMSO as a stock solution. Rabbit monoclonal antibodies against human E-cadherin, Vimentin, Slug, Snail, ZEB1, Twist1, MMP-2, MMP-9, β-actin were purchased from Cell Signaling Technology. Matrigel was purchased from BD Biosciences, and 24-well transwells was purchased from Corning.
Cell culture
Human colorectal cancer cell line LoVo (ATCC, USA) was maintained in F12K medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and incubated in a humidified, 5% CO2 atmosphere, at 37°C.
CCK assay for cell proliferation
Cell Counting Kit-8 (CCK-8) was selected to determine cell proliferation. Briefly, LoVo cells were seeded in 96-well plates at 1 × 104 cells/well, when the cells reached 60% confluence, the medium was replaced with fresh medium containing different concentrations of resveratrol, and incubated for 48 h and 72 h. The medium was then discarded, and the cells were incubated with medium containing CCK-8 reagent for 4 hours. The absorbance was determined at 450 nm using a microplate reader (Biorad, USA). All the experiments were repeated three times.
In vivo imaging by tail vein injection
Experimental lung metastases were achieved by injections of a single-cell suspension of LoVo cells containing green fluorescent protein (GFP) into the lateral tail vein. One week later, the mice were randomized into four groups of 8 animals each. Resveratrol with a dose of 0, 50, 100, 150 mg/kg [
17] was interfered in distinct groups via intragastric administration every day for 3 weeks. Seven weeks later, prior to in vivo imaging, the mice were anesthetized with penobarbital sodium, and the images of established lung metastases were observed by LB983 NIGHTOWL II system (Berthold Technologies GmbH, Germany). Afterwards, both of the lung organs were excised, fixed with 10% neutral buffered formalin, and paraffin-embedded. The lung sections were fully cut, and each section was set to 6 μm. All the lung sections were stained with hemaoxylin-eosin (HE), following by counting the number of lung metastases, and assessing comprehensively the extent of metastasis. All experimental protocols were reviewed and approved by the animal ethics committee of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
Anti-tumor effect of resveratrol on mice with orthotopic transplantation tumor
Single-cell suspensions of LoVo cells (2 × 10
6 in 100 μL) were injected into the subcutaneous area of female BALB/c nude mice (4–6 weeks old) obtained from SLAC (SLAC Laboratory Lab, Shanghai, China). After the tumors reached the size of 100 mm
3, the tumors were excised, fractionated, and transplanted into the appendix of the nude mice. After two weeks, the mice were randomized into four groups of 8 animals each. Resveratrol with a dose of 0, 50, 100, 150 mg/kg [
17] was interfered in distinct groups via intragastric administration every day for 3 weeks. After 42 days, animals were sacrificed by cervical dislocation in deep CO
2 anesthesia, primary tumors were surgically removed and weighted (g). Then, a part of the removed tumors, the lung and liver of the mice were investigated by HE staining. All experimental protocols were reviewed and approved by the animal ethics committee of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
Western blot
Whole cell proteins were prepared according to the instructions of ProteoJET Cytoplasmic Kit (Fermentas, USA). The extracted protein was quantified by BCA protein assay. Proteins were loaded onto the SDS-PAGE gels for electrophoresis, transferred to PVDF membranes, blocked in 5% milk, and incubated with the primary antibodies following by the HRP-conjugated secondary antibodies. All the resulting immunocomplexes were visualized by enhanced chemiluminescence. Each experiment was repeated independently three times.
Immunofluorescence microscopy
LoVo cells (2.5 × 105) were fixed for 40 minutes with 4% paraformaldehyde in PBS at room temperature, blocked with 5% non-fat dry milk, and permealized with solution containing 1% BSA, 0.5% Triton X-100. The cells were first stained with E-cadherin rabbit antibody followed by Cy3-conjugated goat anti-rabbit IgG or first stained with the Vimentin rabbit antibody followed by FITC-conjugated goat anti-rabbit IgG. DAPI was applied for nuclear staining. Immunofluorescence images were taken with a DMI3000B inverted microscope (Leica, Germany). All the experiments were repeated three times.
Transwell assay for migration and invasion
LoVo cells (5 × 105, in F12K medium with 0.5% FBS) pretreated with or without different concentration of resveratrol were seeded into the upper part of a transwell chamber. For migration analysis, 600 μl F12K medium with 10 μg/ml fibronectin and 15% FBS was added in the lower part of the chamber, and the assay was performed. Migrated cells were analyzed by crystal violet staining, followed by observing under a DMI3000 B inverted microscope (Leica, Germany). Five random views were selected to count the migrated cells. For invasion analysis, 100 μl matrigel (BD, USA) was firstly added onto the bottom of the upper transwell chamber before LoVo cells were seeded, and the following procedures were as same as migration analysis, except that the invasive cells were analyzed after co-culture for 48 hours. Each experiment was repeated independently three times.
RT-PCR
For RT-PCR of Snail gene, the primers were designed as follows: 5-CAATCGGAAGCCTAACTA-3, 5-CAGATGAGCATTGGCAGCG-3, with control GAPDH: 5-GAAGGCTGGGGCTCATTTG-3, 5-GGGCCATCCACAGTCTTC-3, and the product were confirmed with agarose electrophoresis. All assays were performed in triplicate and independently repeated three times.
Plasmid constructions
Human Snail gene (Gene ID: 6615) was amplified by RT-PCR from LoVo cells, using the forward and reverse primers: 5-CCGCTCGAGATGCCGCGCTCTTT-3, 5-CGGGATCCTCAGCGGGGACATCC-3, sequenced by the Sangon Biotech company (Shanghai, China), and the right Snail fragments (795 bp) were sub-cloned into the expression vector of pcDNA3.1, named pcDNA3.1-Snail.
Analysis of the E-cadherin promoter
To test E-cadherin promoter activity, LoVo cells were co-transfected with either the recombinant plasmid pGL3-basic-E-cadherin or -basic-mut-E-cadherin with a control positive plasmid pRL-SV40. The promoter activity was measured using a dual-luciferase assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions.
Statistical analysis
All the data were presented as mean ± standard deviation (± S) X ) and analyzed with SPSS18 Software. The mean values of two groups were compared by Student’s t test. P < 0.05 was considered as statistically significant, and P < 0.01 was considered as statistically highly significant.
Discussions
Various plant or fruit-derived agents with few side effects have been accepted as potential alternatives for the therapy of colorectal cancer. Resveratrol extracted from grape or Polygonum cuspidatum is a natural antioxidant, which can reduce blood viscosity, maintain the blood flow, and inhibit the platelet aggregation [
18-
22]. In addition, resveratrol has anti-cancer activity in a great number of malignant tumors like prostate, skin, ovarian, breast and colon cancers [
10-
13,
16].
Our previous studies have revealed the potential therapeutic effect of resveratrol against invasion and metastasis of colorectal cancer cells [
16]. In that study, we found resveratrol inhibited invasion and metastasis through MALAT1 mediated β-catenin signaling pathway. Here, we observed a previously unknown mechanism, in which resveratrol could inhibit invasion and migration via reversing Epithelial-to-Mesenchymal Transition induced by TGFβ1.
EMT is characterized by the loss of cell-cell adhesion and the increase of cell motility, and it is a key process in cancer progression and metastasis [
5,
6], which making the inhibition of EMT process an attractive therapeutic strategy. EMT could be triggered by many growth factors like TGF-β, EGF, HGF [
7-
9,
13,
22,
23]. Our present studies demonstrated that TGF-β1-induced LoVo cells undergo morphological alterations characteristic of EMT characterized by up-regulated expression of mesenchymal markers Vimentin and down-regulated expression of E-cadherin epithelial markers including and increased metastasis and invasion, up-regulated expression of mesenchymal markers Vimentin and down-regulated expression of E-cadherin epithelial markers. TGF-β1 also enhances enhanced the expression of zinc-finger transcriptional factors Snail, which then repressed the E-cadherin transcription. These transcriptional repressors of E-cadherin are required during EMT development [
5]. Our study showed that resveratrol reduced migration and invasion in a concentration-dependent manner and inhibited TGF-β1-induced EMT in LoVo cells, as proved by the increase of the expression of E-cadherin and the decrease of Vimentin. During EMT development, TGF-β induced the Snail expression, and the increased Snail transcriptional factor would inhibit the promoter activity of E-cadherin, leading to the decreased E-cadherin transcription. In addition, our results showed that, the expression of EMT inducing transcription factors Snail could be inhibited by resveratrol effectively.
Although EMT is a coordinated, organized program involving interaction between different cells and tissue types, the EMT program could be activated in response to alterations of microenvironment, which would contribute to occurrence of the diseases including cancer progression [
24,
25]. We observed that, TGF-β1-induced EMT promoted the invasion and migration ability of LoVo cells, but reseveratrol could inhibit the promoting effect in a concentration-dependent manner. Expression analysis also demonstrated that treatment of resveratrol significantly down-regulated MMP2 and MMP9 induced by TGF-β1. Therefore, resveratrol might inhibit the invasion and metastasis of CRC cells by suppressing TGF-β1-induced EMT.
TGF-β/Smad signaling pathway is a classical pathway associated closely with the proliferation, differentiation, migration, and so on. In this system, TGF-β1 regulates cellular processes by binding and phosphorylating cell-surface receptors (TGF-βRI/TGF-βRII), and the activated TGF-βRI phosphorylates Smad2 or Smad3, will bind to Smad4 [
26,
27]. The resulting Smad complex then moves into the nucleus, where it interacts in a cell-specific manner with various transcription factors to regulate the transcription of many genes [
23,
28]. Snail was one of TGF-β/Smad signaling pathway mediated gene [
29,
30], which repressed the E-cadherin expression, promoted the EMT process, and finally increased the ability of invasion and metastasis of CRC cells in our study. However, resveratrol could inhibit the invasion and metastasis by preventing the continuation of EMT process.
Acknowledgements
This work was supported by National Natural Science Foundation of China (81303102, 81473478, 81303103, 81473628), Program of Shanghai Municipal Education Commission (12YZ058), Shanghai Municipal Health Bureau (2011ZJ030, 20114037), Chen Guang project of Shanghai Municipal Education Commission and Shanghai Education Development Foundation (13CG47).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: QL QJ JR JC. Performed the experiments: QJ XL ZH LZ LY HJ. Analyzed the data: QJ XL ZH HS JR JC. Contributed reagents/materials/analysis tools: QL QJ JR JC. Wrote the paper: QJ XL QL JR JC. All authors have read and approved the final manuscript.