Contributed equally
Colon cancer is a common type of cancer worldwide and accounts for a significant number of cancer-related deaths. Although surgical techniques and treatment strategies for colon cancer have advanced over the past two decades, the prognosis has not improved considerably. Resveratrol, a natural stilbene compound, possesses antioxidant, cardioprotective and anticancer properties. However, the role of resveratrol in colon cancer has not been fully elucidated. The present study demonstrated that resveratrol inhibited cell proliferation and colony growth in DLD1 and HCT15 colon cancer cells, but did not affect normal colon epithelial cells. The resveratrol-mediated inhibition of cell proliferation correlated with an induction of apoptosis and with G1 phase cell cycle arrest in colon cancer cells. Additionally, resveratrol treatment decreased the protein expression levels of cyclin D1, cyclin E2 and BCL2 apoptosis regulator, while it increased BCL2 associated X and tumor protein p53, all of which are involved in the regulation of cell cycle and apoptosis. Notably, the results obtained from
Colon cancer is the third most common malignancy and the fourth leading cause of cancer-related deaths worldwide (
Over recent years, there has been increasing interest in natural compounds derived from plants that show potential preventative effects against cancer development but have minor side effects on normal cells or organs (
AKT is a proto-oncogene belonging to the serine/threonine kinase family that regulates a large number of downstream mediators and ultimately controls critical cell survival and metabolic processes (
Signal transducer and activator of transcription (STAT)3 is an important transcription factor that translocates to the nucleus to regulate the expression of essential pro-invasive factors, such as matrix metallopeptidases, heat shock protein (HSP)70 and HSP90, and it is also downstream of various tyrosine kinase receptor signaling pathways that are involved in angiogenesis (
The present study investigated the anticancer activity of resveratrol in colon cancer cells. The results demonstrated that resveratrol inhibited cell growth of colorectal cancer by inhibiting AKT and its downstream signaling targets, and that AKT served as an upstream regulator of STAT3.
Resveratrol was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against phosphorylated (p-) STAT3 (Tyr705; cat. no. 9145), STAT3 (cat. no. 9139), p53 (cat. no. 48818), Bcl2 (cat. no. 15071), Bax (cat. no. 5023), AKT1 (cat. no. 75692), AKT2 (cat. no. 2964), cyclin D1 (cat. no. 2978) and cyclin E2 (cat. no. 4132) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). An AKT1/2 (cat. no. sc-1619) antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Active AKT1/2 recombinant protein was purchased from SignalChem (Richmond, BC, USA). RPMI-1640, fetal bovine serum (FBS) and Basal Medium Eagle (BME) were obtained from Biological Industries (Kibbutz Beit-Haemek, Israel). F-12K medium was purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
The human colon cancer cell lines DLD1 and HCT15 were purchased from the American Type Culture Collection (Manassas, VA, USA). The human colonic epithelial cell (HCEC) line was kindly provided by Dr Jerry W Shay, University of Texas Southwestern Medical Center, Dallas, Texas (
Cells at a density of 1×103 or 1×105 per well were seeded in 96-well plates in a final volume of 100
DLD1 or HCT15 cells (8×103 per well) were suspended in 1 ml of BME/10% FBS, and 0.33% agar with various concentrations of resveratrol and plated on a layer of solidified BME, 10% FBS, and 0.5% agar with the same concentration of resveratrol as the suspension. The cultures were maintained at 37°C in an incubator with 5% CO2 for 1-2 weeks. The colonies were photographed and counted with Image-Pro Plus software (v.6.2; Media Cybernetics, Rockville, MD, USA).
DLD1 and HCT15 cells were seeded in 6-well plates and treated with different concentrations of resveratrol (10, 20, 30 or 40
DLD1 and HCT15 cells were treated with various concentrations of resveratrol for 72 h and were then lysed with RIPA buffer (50 mM Tris base, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA and 0.1% SDS; dissolved in 400 ml water and adjusted to pH 7.4) supplemented with 1 mM PMSF. Protein concentration was determined with a bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein was loaded (50
To further confirm that resveratrol can bind AKT1 and AKT2, a
Resveratrol-Sepharose 4B beads (Amersham Pharmacia Biotech; GE Healthcare, Chicago, IL, USA) were prepared following the manufacturer’s instructions. DLD1 and HCT15 cell lysates (500
The viral vectors and the packaging vectors (pMD2G, psPAX2, Mock, shAKT1 and shAKT2) were obtained from Sigma-Aldrich (Merck KGaA). Several shRNA sequences targeting AKT1 and AKT2 were tested. Then, all plasmids were transfected into 293T cells, and viral supernatant fractions were collected after 48 h. DLD1 and HCT15 cells were infected with mock or shAKT1 and shAKT2 virus particles for 24 h. Cells were selected with puromycin to obtain both AKT1- and AKT2-silenced cell lines (shAKT1/2). The appropriate experiments were performed with these cells until the control cells (without infection) completely died (usually 2-3 days) in the puromycin medium.
All quantitative data are presented as the mean values ± standard deviation. Each experiment was repeated at least three times. Data were analyzed with SPSS 19.0 software (IBM Corporation, Armonk, NY, USA). Statistically significant differences were determined using one-way analysis of variance, and multiple comparisons between groups were conducted using the Dunnett’s test. P<0.05 was considered to indicate a statistically significant difference.
The chemical structure of resveratrol is shown in
Next, further analyses were conducted to determine whether resveratrol could lead to the inhibition of cancer cell growth by regulating cell cycle progression and apoptosis. The results of Annexin V staining revealed that resveratrol treatment resulted in a significant increase in the apoptosis rate in both cell lines, DLD1 and HCT15 (
To elucidate the underlying mechanism of resveratrol’s effects, potential targets of resveratrol were screened by Schrödinger software (release 2017) (
Because of the crucial role of AKT in colon cancer, the present study examined whether knocking down AKT expression would produce an antitumor effect in colon cancer cells. After AKT1 and AKT2 were knocked down with short hairpin RNA (shRNA) in DLD1 and HCT15 colon cancer cells, the expression of AKT1 and AKT2 was markedly decreased (
AKT1/2 knockdown exhibited a significant inhibitory effect on anchorage-independent colony growth by decreasing the number and size of colonies compared to the number and size of the colonies in the mock group (
Next, it was examined whether silencing AKT1/2 could affect cancer cell apoptosis or cell cycle progression. Upon AKT1/2 knockdown, the number of cells in the G1 phase of the cell cycle increased, indicating that the cells suffered cell cycle arrest at the G1 phase (
Colorectal cancer (CRC) is the most common gastrointestinal tract cancer worldwide. Approximately 50% of those diagnosed will succumb to colorectal cancer, making it the second leading cause of cancer-related deaths in both sexes. Despite decades of research and some promising discoveries, the mainstay of colorectal cancer treatment remains based on cytotoxic chemotherapy agents, such as irinotecan or oxaliplatin combined with fluoropyrimidine and leucovorin (FOLFIRI or FOLFOX6 regimens). Both the FOLFIRI and FOLFOX6 regimens have shown modest outcomes when used as first-line therapies. For colon cancer therapy, 5-fluo-rouracil (5-FU) and leucovorin have been the only options. The addition of irinotecan or oxaliplatin increases overall survival (OS) to 18 months. The present study demonstrated that resveratrol effectively inhibited the proliferation and colony formation of human colon cancer cells DLD1 and HCT15, and also induced cell apoptosis and G1 phase cell cycle arrest. These findings suggested that resveratrol might be a promising cancer prevention agent or therapeutic agent. As a natural compound, resveratrol may have fewer side effects and lower toxicity to normal colon epithelial cells. Indeed, the present results demonstrated that resveratrol treatment had no effect in the viability of the normal HCEC cell line.
Multiple critical protein-encoding genes and pathways are believed to be responsible for tumorigenesis. Colorectal tumors contain a median of 76 mutations, and 15 of these affect candidate cancer genes. Increased understanding of the genetic and genomic changes in colorectal cancer has helped to direct therapies and predict responses. Genetic and epigenetic errors in signal transduction pathways result in malignant transformations and have thus emerged as key candidates for targeted molecular therapies. Over the past 10 years, the number of targeted agents used in various malignancies has increased dramatically. Currently, there are seven FDA-approved targeted agents for colorectal cancer, with many more in development and in clinical trials. The addition of targeted therapies over the past 10 years has improved OS in colorectal cancer by between 20 and 24 months. Resveratrol is a naturally occurring phytochemical that is produced by plants. Resveratrol has been demonstrated to have diverse biological properties, including anti-inflammatory, antioxidant, antiviral, neuroprotective, antifungal and anticancer properties (
In summary, the present study revealed that resveratrol inhibited colon cancer cell proliferation and colony formation, and induced cell apoptosis and G1 phase arrest. The mechanism of the anticancer effects of resveratrol was demonstrated to occur, at least partially, via inhibiting the AKT/STAT3 signaling pathway (
Not applicable.
This study was supported by the Medical Science Research projects of Henan Province (grant no. 201702248), the Science and Technology Research Projects of Henan Province (grant no. 182102310376) and the Science and Technology Research Projects of Henan Province (grant no. 182102310125).
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.
DL, SL and ZG designed the study. GW and GJ performed the kinase and pull down assays. KY, ZZ, LB and YG analyzed the data. NL, WD, BC, YL and XC performed all other experiments. DL and GJ wrote the paper. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effects of resveratrol on colon cancer cell proliferation and colony formation. (A) Chemical structure of resveratrol. (B) Toxicity of resveratrol in normal colon cells. HCECs were treated with various doses (10, 20, 30 or 40
Resveratrol induces apoptosis and G1 phase cell cycle arrest in colon cancer cells. (A) Effects of resveratrol on cell apoptosis in the colon cancer cell lines DLD1 and HCT15. Cells treated with different concentrations of resveratrol for 72 h were analyzed with an Annexin V staining assay. (B) Cells treated with resveratrol had increased cell cycle arrest at the G1 phase. **P<0.01 and ***P<0.001 compared with untreated cells. (C) Representative images from western blot analysis showing the effects of resveratrol on the expression of cyclin D1 and E2, and (D) on p53, Bax and Bcl2. The cells were treated with vehicle or resveratrol (10, 20, 30 or 40
Resveratrol binds to AKT1 and AKT2. (A) Computational docking model of the binding between resveratrol and AKT1/2. (A-a) The binding sites of AKT1 are Ala230, Glu228 and Glu234 and (b) an enlarged view of the binding. (A-c) The binding sites of AKT2 are Ala232, Glu279, Glu230 and Asp293 and (d) an enlarged view of the binding. (B)
Roles of AKT1/2 in colon cancer cell growth. (A) Efficiency of AKT1/2 shRNA in DLD1 and HCT15 cells. (B) Effect of AKT1/2 knockdown on DLD1 and HCT15 cell proliferation and (C) colony formation (scale bar, 100
Effects of AKT1/2 knockdown on cellular processes. (A) Effect of AKT1/2 knockdown on DLD1 and HCT15 cell cycle progression and (B) apop-tosis. *P<0.05 and ***P<0.001 compared with mock-treated cells. (C) Representative images from western blot analysis showing changes in the levels of cyclin D1 and cyclin E2 (cell cycle markers), (D) Bax and Bcl2 (apoptosis markers), and (E) p-STAT3 and STAT3 (downstream signaling targets), following AKT1/2 knockdown. AKT, AKT serine/threonine kinase; Bax, BCL2 associated X; Bcl2, BCL2 apoptosis regulator; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated; shRNA, short hairpin RNA.
A proposed scheme illustrating the roles of AKT in colon cancer and its regulation by resveratrol. AKT upregulates Bcl2 and cyclin D1, promotes STAT3 phosphorylation, and downregulates p53 and Bax. Subsequently, AKT promotes cell cycle progression, prevents apoptosis and increases cell proliferation. Resveratrol inhibits the function of AKT and its downstream targets, therefore inducing cell cycle arrest and apoptosis, as well as inhibiting cell proliferation. AKT, AKT serine/threonine kinase; Bcl2, BCL2 apoptosis regulator; STAT3, signal transducer and activator of transcription 3; p53, tumor protein p53; Bax, BCL2 associated X; CDK, cyclin-dependent kinase.