Resveratrol (RES), a polyphenolic compound present in grapes and red wine, has potential anticancer properties. The present study aimed to examine the effects of resveratrol and its underlying mechanism on hepatocellular carcinoma (HCC) cell lines HepG2, Bel-7402 and SMMC-7721. It was demonstrated that resveratrol inhibited the viability and proliferation of HCC cells assessed by MTT and EdU assays. TUNEL assay revealed that resveratrol induced cell apoptosis by increasing HCC apoptosis rate from 3±0.78% to 16±1.12% with upregulation of B-cell lymphoma (Bcl)-2 associated X, apoptosis regulator and cleaved-poly (ADP-Ribose) polymerase 1 (PARP), and downregulation of Bcl-2, caspase-3, caspase-7 and PARP. As a sirtuin (SIRT) 1 activator, resveratrol elevated SIRT1 protein expression and its enzyme activity and decreased expression levels of phosphorylated (p)-phosphoinositide-3-kinase (PI3K), p-AKT Serine/Threonine Kinase 1 (AKT), and its downstream target p-Forkhead Box O3a in HepG2 cells. Furthermore, inhibition of SIRT1 enzymatic activity by EX527 resulted in increased phosphorylation levels of PI3K and AKT. This demonstrated that resveratrol inhibited the PI3K/AKT pathway by SIRT1 activation. In addition to inhibition of cancer cell migration, tumor suppressor gene DLC1 Rho GTPase activating protein level was upregulated and its phosphorylation was enhanced by AKT with resveratrol treatment. These findings suggested that resveratrol inhibits proliferation and migration through SIRT1 mediated post-translational modification of PI3K/AKT pathway in HCC cells.
Hepatocellular carcinoma (HCC) is one of the major leading causes of tumor-associated deaths, with high rates of incidence and disease-related mortality and morbidity in the world (
Resveratrol (RES, trans-3,5,4′-trihydroxystilbene) is a polyphenol compound derived from grapes, berries, peanuts and other sources, and it has inhibitory effects on several types of cancer cell lines such as colon, lung and prostate and affects diverse molecular targets (
The phosphatidylinositol 3′-kinase (PI3K)/AKT pathway plays an important role in cell survival and PI3K activity has been linked to a variety of human cancers (
Deleted in liver cancer 1 (DLC1), a focal adhesion protein, is identified as a putative tumor suppressor in HCC in 1998 (
The purposes of the present study were to determine the molecular mechanism of resveratrol affected proliferation and migration through SIRT1 mediated post-translational modification of PI3K/AKT signaling pathway in HCC cells.
The human hepatocellular carcinoma (HCC) cell lines Bel-7402, SMMC-7721, hepatoblastoma cells HepG2 (The HepG2 cell line was originally thought to be a hepatocellular carcinoma cell line but was later shown to be from an hepatoblastoma, PubMed=19751877), and human liver normal cell line HL-7702 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). HepG2 cells were cultured in DMEM, other cells in RPMI 1640 medium. All the experiments were performed in medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, maintained at 37°C in humidified atmosphere with 5% CO2.
MTT assay was used to assess cell viability. The cells were seeded in 96-well plates at a density of 1×104/well overnight and treated without or with resveratrol (Sigma, St. Louis, MO, USA) dissolved in 0.1% (v/v) DMSO at various concentrations for 24 h. Then cells were incubated with MTT solution for 4 h. The formazan crystals dissolved by 150 µl DMSO, the solution was absorbed at 492 nm using enzyme-linked immunosorbent assay reader (Awareness, Palm City, FL, USA).
Cell proliferation was tested by EdU (5-ethynyl-2-deoxyuridine) incorporation assay kit (Ribobio, Guangzhou, China). Briefly, cells cultured in 96-well plates exposed to 50 µM EdU for 2 h at 37°C, and fixed in 4% formaldehyde. After permeabilization with 0.5% Triton-X, the cells were reacted with 1xApollo reaction cocktail for 30 min, the DNA contents were stained with Hoechst 33342 and visualized under fluorescent microscope. Cells were counted in five selected arbitrarily fields, at least 300 cells were counted per well. EdU positive cells were calculated with (EdU incorporated-in cells/Hoechst stained cells) ×100%.
TUNEL staining was performed using an EdUTP TUNEL cell detection kit (Ribobio, Gangzhou, China) according to the manufacturer's instructions. The cells cultured in 96-well plates were treated without or with 100 µM resveratrol, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, washed twice, incubated with TUNEL detecting liquid for 1 h at 37°C and observed by a fluorescent microscope (Olympus, Tokyo, Japan) at 488 nm excitation and 530 nm emission. TUNEL positive cells were calculated as the number of apoptotic cells/DAPI stained cells ×100%.
The cells were lysed by RIPA (Beyotime, Shanghai, China). The inhibitor of SIRT1, EX-527 (Selleck Chemicals, Houston, TX, USA), was used to PI3K/AKT pathway. Proteins were separated by SDS-PAGE and transferred on membranes were incubated in primary antibodies against SIRT1, p-AKT, AKT, p-PI3K, PI3K, PARP, Cleaved-PARP, p-FoxO3a, Caspase-3/-7, Bax, Bcl-2 and p53 (CST, Danvers, MA, USA) and FoxO1, FoxO3a (Santa Cruz Biotechnology, San Diego, CA, USA) overnight at 4°C, followed by incubation with HRP-conjugated rabbit/mouse secondary antibodies (ZSGB-BIO, Beijing, China). Protein expressions were visualized ECL detection system (Beyotime, Shanghai, China).
Immunoprecipitation (IP) was carried out using Pierce Classic Magnetic IP/Co-IP Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The protein lysates were incubated with DLC1 antibody (BD Biosciences, San Jose, CA, USA), and precipitated with Protein A/G Magnetic Agarose at 4°C. The immunocomplex collected was washed, and the immunoprecipitates were subjected to western blotting and phosphorylation signals were determined using phospho-AKT substrate (PAS) antibody (CST, Danvers, MA, USA).
Cells were seeded into 24-well plates (1.0×105 cells/well). Sterile pipette tip was used to produce a wound line between cells after the cells grew to 80–90% confluence and allowed the cells migrated for 24 h. Images were captured and the relative distance traveled by the leading edge from 0 to 24 h was assessed using Image Pro Plus 6.0 software (n=5).
SIRT1 activity was quantified with a SIRT1 Fluorometric Assay Kit (Sigma, St. Louis, MO, USA) according to the manufacturer's protocol. Fluorescence intensities were measured with a microplate fluorometer (excitation wavelength=360 nm, emission wavelength=450 nm). Experimental values are represented as a percentage of control.
All of the assays were performed three times independently at least. Value presented as the means ± standard deviation (SD) by GraphPad Prism software (GraphPad Software, CA, USA). Statistical analyses were performed using one-way ANOVA and Student's t-test, *P<0.05, **P<0.01 were considered to indicate a statistically significant difference.
Three HCC cell viability was determined by MTT assay. The results showed that resveratrol inhibited cell viability when its concentrations were higher than 80 µM compared to normal HL-7702 cell (
To understand the molecular basis of proliferation inhibition caused by resveratrol, proliferation regulation protein PCNA (proliferating cell nuclear antigen) was evaluated in HepG2 cells. The level of PCNA reduced after 100 µM resveratrol treatment (
TUNEL assay was assessed whether anti-proliferative effects of resveratrol against HCC cells are mediated via apoptosis. Results showed that resveratrol increased apoptosis from 4±0.83% to 13±1.32%, 3±0.78% to 14±0.72%, 5±0.33% to 16±1.12% in HepG2, Bel-7402 and SMMC-7721 cells (
Studies have reported that Bcl-2 could be a crucial target gene of PI3K/AKT signaling, whereas AKT has been shown to negatively regulate the activity of proapoptotic members of the Bcl-2 family (
To investigate effects of resveratrol on the pathway of PI3K/AKT/FoxO3a to induce cell apoptosis, western blotting was performed for their phosphorylation levels. Resveratrol inhibited phosphorylation of PI3K and AKT without effect on total levels of PI3K and AKT in HepG2 cells (
We then examined whether resveratrol stimulates expression of SIRT1. Resveratrol up-regulated protein expression of SIRT1 in HCC cells but not in normal liver HL-7702 cells (
To determine the relationship between SIRT1 activity and PI3K/AKT signaling pathway, the activity of intracellular SIRT1 was analyzed after SIRT1 inhibitor EX527 was used. Consistent with its protein level, SIRT1 activity increased by resveratrol and decreased after exposure to EX527 in HepG2 cells (
As PI3K/AKT pathway is an important cell survival cascade, DLC1 might be a substrate of AKT. In order to explore whether AKT involved in regulation of tumor suppressor DLC1, phosphorylation on the biological activities of DLC1 was demonstrated in HCC cells. DLC1 protein expression elevated in three kinds of HCC cells and no effect on HL-7702 cells by resveratrol (
Wound healing assay measures the ability of cells to migrate into an area of a cell culture plate denuded of cells (wound). Our result showed that resveratrol inhibited the wound closures from 32.5 to 11.5% in HepG2 cells by 24 h treatment (
Although the capacity of resveratrol to prevent cancer development has been studied for many years, its mechanism underlying remains to be fully elucidated. Proliferating cell nuclear antigen (PCNA) is a critical event in growth regulation of cancer cells (
The PI3K/AKT signaling is a critical pathway in cell proliferation, survival, neovascularization and tumor growth (
SIRT1 plays a key role in both cell death and survival with p53 family members, FoxOs and the nuclear factor-κB family (
SIRT1 has been also implicated as a negative regulator for the PI3K/AKT pathway by deacetylating the tumor suppressor PTEN (
DLC1 is a Rho GTPase-activating protein (RhoGAP) and frequently deleted and underexpressed in cancers (
Taken together, our findings suggested that resveratrol activated SIRT1 to induce liver cancer cell apoptosis and to inhibit migration through SIRT1 mediated post-translational regulation of PI3K/AKT signaling and phosphorylation level of FoxO3a and DLC1 and deacetylation of FoxO1 leading to tumor suppression in HCC cells.
This work was supported by the National Natural Science Foundation of China (Grant No. 31672377), the Major Key Science and Technology Project of Shandong Province (2015ZDJS04003), the Key Program of Shandong Provincial Natural Science Foundation of China (ZR2013CZ002), Science and Technology Program of Jinan (201202033).
Inhibition of resveratrol on cell viability and proliferation. (A) Selection of resveratrol doses. (B) EdU assay with resveratrol treatment. EdU stain showed red light while Hoechst33342 blue light. Scale bar is 100 µm. (C) Quantitative analysis of EdU positive cells. (D) Expression of PCNA and relative expression level. *P<0.05 and **P<0.01 vs. control.
Resveratrol induced apoptosis and regulated apoptosis-related protein levels in liver cancer cells. (A) Fluorescent microscopy images of TUNEL assay. DAPI staining (blue) was used to identify cell nuclei, TUNEL-positive cells are by green. Scale bar represents 100 µm. Bar graph showed the percentage of apoptotic cells. (B) Protein levels of PARP, cleaved PARP, caspase-3, caspase-7, Bcl-2 and Bax. Bar graphs showed their relative levels to β-actin, and the ratio of Bcl-2/Bax. *P<0.05 and **P<0.01 vs. control.
(A) Resveratrol significantly activated protein expression of SIRT1 in liver cancer cells. (B) EX527 inhibited deacetylase activity of SIRT1 in HepG2 cells. (C) Protein levels of PI3K, p-PI3K, p-AKT and p-FoxO3a after treated with EX527. (D) Protein levels of PI3K, p-PI3K, AKT, p-AKT, FoxO1, Ac-FoxO1, FoxO3a and p-FoxO3a. The bar graphs and representative images are shown. *P<0.05 and **P<0.01 vs. control.
Resveratrol inhibited migration and enhanced phosphorylation of DLC1 by AKT in HepG2 cell. (A) Expression of DLC1 with resveratrol treatment in liver cancer cells and HL-7702. Bar graph showed the ratio of DLC1 protein to GAPDH. (B) DLC1 was immunoprecipitated by DLC1 antibody and subjected to western blotting using PAS antibody. Phosphorylation was elevated with resveratrol treatment. (C) Scratch images and quantitation of wound closure. **P<0.01 vs. control.