Contributed equally
Resveratrol (RSV) is a polyphenolic compound that naturally occurs in grapes, peanuts and berries. Considerable research has been conducted to determine the benefits of RSV against various human cancer types. Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability and has decreased expression in human cancer. The present study investigated the biological effect of RSV on TTP gene regulation in colon cancer cells. RSV inhibited the proliferation and invasion/metastasis of HCT116 and SNU81 colon cancer cells. Furthermore, RSV induced a dose-dependent increase in TTP expression in HCT116 and SNU81 cells. The microarray experiment revealed that RSV significantly increased TTP expression by downregulating E2F transcription factor 1 (E2F1), a downstream target gene of TTP and regulated genes associated with inflammation, cell proliferation, cell death, angiogenesis and metastasis. Although TTP silencing inhibited TTP mRNA expression, the expression was subsequently restored by RSV. Small interfering RNA-induced TTP inhibition attenuated the effects of RSV on cell growth. In addition, RSV induced the mRNA-decaying activity of TTP and inhibited the relative luciferase activity of baculoviral IAP repeat containing 3 (cIAP2), large tumor suppressor kinase 2 (LATS2), E2F1, and lin-28 homolog A (Lin28) in HCT116 and SNU81 cells. Therefore, RSV enhanced the inhibitory activity of TTP in HCT116 and SNU81 cells by negatively regulating cIAP2, E2F1, LATS2, and Lin28 expression. In conclusion, RSV suppressed the proliferation and invasion/metastasis of colon cancer cells by activating TTP.
Colorectal cancer is one of the most common cancer types worldwide and the second most lethal cancer in the USA (
Resveratrol (3,5,4′-trihydroxystilbene; RSV) is a polyphe-nolic compound that naturally occurs in grapes, peanuts and berries (
Tristetraprolin (TTP) is an AU-rich element (Res)-binding protein that regulates mRNA stability (
Colorectal cancer cells (HCT116 and SNU81) were purchased from the Korean Cell Line Bank (Seoul, Korea) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all Capricorn Scientific GmbH, Ebsdorfergrund, Germany) at 37°C in a humidified 5% CO2 atmosphere. RSV was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany; cat. no. 274666) and suspended in dimethyl sulfoxide (DMSO; cat. no. D2650; Sigma-Aldrich; Merck KGaA). Colorectal cancer cells were treated with RSV in medium containing 2.5% FBS for 24 h at 37°C.
The pGL3/TTPp-1411 promoter construct (
The total protein was extracted from HCT116 and SNU81 cells using ice-cold radio-immunoprecipitation assay buffer [50 mM Tris HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% (v/v) Triton X-100; 0.1% (w/v) SDS] and a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Protein concentrations were determined using the bicinchoninic acid assay, according to the manufacturer's protocol (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The protein samples (10
RNA isolation was performed using TRIzol reagent (Thermo Fisher Scientific, Inc.) and was synthesized to cDNA using Moloney murine leukemia virus reverse transcriptase kit (cat. no. 3201; Beams Biotechnology, Seongnam, Korea) and Oligo-dT primer (cat. no. 79237, Qiagen, Hilden, Germany), according to manufacturer's protocol for 60 min at 37°C. For the PCR (5
For the MTT assay, 1×104 cells/well were seeded in 96-well culture plates with complete RPMI-1640 medium. The cells were subsequently incubated with different RSV concentrations (10, 20 or 50
The effect of RSV on the invasive properties of HCT116 and SNU81 cells was assessed using Boyden chambers (Neuro Probe, Inc., Gaithersburg, MD, USA) that were precoated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 1 h at room temperature. The bottom wells were filled with 28
HCT116 and SNU81 cells were seeded into 12-well plates at 1×104 cells/well and incubated for 24 h at 37°C and 5% CO2. The cells were subsequently treated with RSV in a dose-dependent manner and incubated for 10 days at 37°C and 5% CO2. Fresh medium containing RSV (5, 10 and 20
TTP small interfering RNA (siRNA; cat. no. sc-36760) or control siRNA (cat. no. sc-37007; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was transfected (100 nM) into HCT116 and SNU81 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After 24 h, cells were treated with RSV (20
The mirVana™ miRNA isolation labeling kit (Ambion; Thermo Fisher Scientific, Inc.) was used for isolating total RNA from the HCT116 cells at 90% confluence, according to the manufacturer's protocol. The Illumina Total Prep RNA amplification kit (Illumina, Inc., San Diego, CA, USA) was used for hybridization with biotin-labeled cRNA. Illumina Human-12 BeadChip V.4 microarray (Illumina, Inc.) was used for hybridizing samples, and data were extracted using the Genome Studio (Illumina, Inc.). R software (R-3.50;
GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all statistical analyses. Data are presented as the mean ± standard deviation. Comparisons among the groups were performed by paired Student's t-test and a two-way analysis of variance with Duncan's multiple range test. P<0.05 was considered to indicate a statistically significant difference.
To investigate the effect of RSV on colorectal cancer progression, MTT and clonogenic assays were performed. The MTT assay demonstrated that treatment with RSV had a dose-dependent inhibitory effect on HCT116 and SNU81 cell viability (
The effect of RSV on the migration and invasion of colorectal cancer cells was also investigated. RSV-treated HCT116 and SNU81 cells were assessed using Matrigel invasion and collagen migration assays. RSV significantly inhibited the invasive ability of colorectal cancer cells in a dose-dependent manner (
To investigate the effect of RSV on gene expression levels in colorectal cancer cells, the microarray experiment was performed. RSV administration significantly regulated several genes associated with inflammation, proliferation, cell death, angiogenesis and metastasis (
The present study further determined the endogenous mRNA and protein expression levels of TTP in HCT116 and SNU81 cells using RT-qPCR and western blot analysis (
The present study further assessed whether RSV increased TTP promoter activity in a reporter assay. HCT116 and SNU81 cells were transiently transfected with a pGL3/hTTPp-1411 construct, followed by treatment with RSV. Following treatment with RSV for 24 h, the luciferase activity was measured, revealing significantly increased TTP promoter activity in HCT116 and SNU81 cells (
Despite the fact that RSV has been studied in various human cancers, its biological effects on colorectal cancer have not been fully elucidated. The present study aimed to investigate the effects of RSV in human colorectal cancer cells and to elucidate its effect on regulating TTP expression. Although TTP serves key roles in cancer cells, TTP regulation by RSV in colorectal cancer cells is yet to be clearly investigated. The present study demonstrated that RSV inhibited cell proliferation and invasion/metastasis by activating TTP in human colorectal cancer cells.
Phytochemicals are promising therapeutic agents for cancer treatment. RSV, a natural compound occurring in grapes, peanuts and berries, has anticancer properties against a number of types of cancer, including colorectal cancer (
An increasing number of studies have validated the importance of TTP expression in human cancer (
Given that TTP gene silencing triggers cancer development, it may be hypothesized that the loss of TTP function in cancer cells may induce transcriptional silencing through TTP promoter regulation. The present study indicated that RSV increased the mRNA and protein expression levels of TTP in human colorectal cancer cells. Further studies regarding whether RSV regulates TTP promoter activity in colorectal cancer cells demonstrated that RSV significantly increased TTP promoter activity in HCT116 and SNU81 cells. Furthermore, RSV significantly inhibited the promoter activities of cIAP2, E2F1, LATS2 and Lin28, which are downstream target genes of TTP in HCT116 and SNU81 cells. RSV enhanced the TTP inhibitory activity in HCT116 and SNU81 cells by negatively regulating cIAP2, E2F1, LATS2 and Lin28 expression. These findings are in accordance with earlier studies wherein TTP overexpression suppressed the stability of E2F1 and Lin28 mRNA (
Not applicable.
The present study was supported by the 'Research Base Construction Fund Support Program' funded by Chonbuk National University in 2017; by grants from the Medical Research Center Program (grant no. NRF-2017R1A5A2015061) through the National Research Foundation, which is funded by the Korean government (MSIP); by the Basic Science Research Program (grant no. 2015R1C1A2A01054054); and by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant no. HI16C1866).
All data generated or analyzed during this study are included in this published article.
SRL and HJ conceived the study and drafted the manuscript. WTK and WZK performed the immunoassays and molecular studies. SZK performed the molecular studies and SHL participated in the statistical analysis and helped to draft the manuscript. SMK designed the research and drafted the manuscript. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
RSV suppresses the proliferation of colorectal cancer cells. (A) Regulation of TTP expression by RSV and suppression of tumor progression in cancer cells. MTT assays demonstrated a decline in cancer cell viability in HCT116 and SNU81 cells treated with RSV, in a dose-dependent manner. Data are presented as the mean ± SD of three different experiments. ***P<0.001. (B) A clonogenic assay was performed on HCT116 and SNU81 cells treated with RSV at increasing doses in order to assess proliferation. Colonies were counted under a light microscope and were photographed. Data are presented as the mean ± SD of three different experiments. **P<0.01; ***P<0.001 vs. DMSO. RSV, resveratrol; TTP, tristetraprolin; DMSO, dimethyl sulfoxide; SD, standard deviation.
RSV inhibits the invasion and metastasis of colorectal cancer cells. (A) Invasion assay. HCT116 and SNU81 cells were treated with RSV, following which the percentage of invasive and migratory cells was determined. TTP suppressed the invasion and migration of colon cancer cells following treatment with RSV in a dose-dependent manner, as observed in the Boyden chamber assay. Graphical representations of the results for HCT116 and SNU81 cells are presented. (B) Migration assay. The
Microarray heat map demonstrating the effect of RSV on genes associated with inflammation, proliferation, cell death, angiogenesis and metastasis in HCT116 cells. Data are presented in matrix format with rows indicating the individual gene and columns indicating each sample. Red and green represent upregulated and downregulated gene expression levels, respectively, as indicated in the scale bar (log 2 transformed). RSV, resveratrol; Cont, control; ZFP36, ZFP36 ring finger protein; TNF, tumor necrosis factor; IL, interleukin; MIP, major intrinsic protein of lens fiber; PTGS, prostaglandin-endoperoxide synthase; CCND1, cyclin D1; MYC, Myc proto-oncogene; KRAS, KRAS proto-oncogene GTPase; FOS, Fos proto-oncogene AP-1 transcription factor subunit; E2F transcription factor 1; AHRR, aryl-hydrocarbon receptor repressor; BCL2, apoptosis regulator Bcl-2; TRAF2, TNF receptor associated factor 2; VEGF, vascular endothelial growth factor; NOS1, nitric oxide synthase 1; LIN28, lin-28 homolog A; LIN28B, lin-28 homolog B.
RSV induces TTP expression in colorectal cancer cells. (A) Endogenous mRNA and protein expression levels of TTP in colon cancer cells (HCT116 and SNU81). *P<0.05. (B) RSV induced the mRNA and protein expression of TTP in a dose-dependent manner in HCT116 and SNU81 cells. β-actin was detected as the loading control for RT-qPCR and western blotting. The relative protein expression level was calculated as a ratio of the control (DMSO), using ImageJ software to detect the intensity of the protein band. **P<0.01 vs. DMSO. (C) RSV restored TTP expression in cell lines whose TTP expression was reduced by TTP siRNA. HCT116 and SNU81 cells were transfected with scRNA or TTP siRNA (100 nM). After 24 h, DMSO or RSV (20
RSV controls the downstream target genes of TTP. (A) HCT116 and SNU81 cells were transfected with pGL3/TTPp-1411 containing the TTP promoter (−1343 to +68) for 24 h and treated with RSV at increasing doses for 24 h. Following treatment with RSV for 24 h, the luciferase activity was measured. The expression levels obtained from pGL3-basic (empty vector)-transfected cells without treatment with RSV were set to 1. Each bar represents the mean ± SD of three independent experiments. ***P<0.001. (B) RSV induced the mRNA-decaying activity of TTP. HCT116 and SNU81 cells were transfected with psiCHECK2-cIAP2, LATS2, Lin28 and E2F1 3′UTRs. At 24 h post-transfection, cells were treated with 20