The cell pellet was lysed in 3 mL lysis buffer (50 mM Tris-HCl pH 7.5, 0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, protease inhibitor cocktail (Roche)) for 1.5 h at 4 °C, followed by centrifugation at 1000×g for 3 min. The pellet was washed once in 1.2× restriction buffer and resuspended again in 500 μL of 1.2× restriction buffer. Camfrog Video Chat is a video chat client that provides access to a wide variety of live streaming video chat rooms. Meet thousands of other users and chat with them live via webcam! Search the built-in live photo webcam directory to meet new people to chat with or have private video chats with your friends and family! Search the world's information, including webpages, images, videos and more. Google has many special features to help you find exactly what you're looking for.
- Rongfei Chai
- Huiling Fu
- Zhaodi Zheng
- Tingting Liu
- Shuhua Ji
- Guorong Li
Affiliations: Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan, Shandong 250014, P.R. China
- Published online on:September 26, 2017https://doi.org/10.3892/mmr.2017.7612
- Pages: 8037-8044
Copyright: © Chai et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
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Hepatocellular carcinoma (HCC) is one of the majorleading causes of tumor-associated deaths, with high rates ofincidence and disease-related mortality and morbidity in the world(1). As it is still difficult tomake an early diagnosis for HCC, most of the patients are diagnosedat advanced stages. Despite the improvement of conventionaltherapies for HCC, including surgery, chemotherapy andradiotherapy, the length or quality of life of patients with HCC isstill poor. Therefore, it is urgent to develop a new preventivestrategy for liver cancer.
Resveratrol (RES, trans-3,5,4′-trihydroxystilbene)is a polyphenol compound derived from grapes, berries, peanuts andother sources, and it has inhibitory effects on several types ofcancer cell lines such as colon, lung and prostate and affectsdiverse molecular targets (2).Sirtuin 1 (SIRT1) has been reported to be a key target ofresveratrol in several tumor models (3,4).Whether SIRT1 as a tumor promoter or tumor suppressor remainscontroversial, it might depend on tumor type (5). Resveratrol suppresses tumor cellgrowth and metastasis in colorectal cancer cells by targeting SIRT1protein and regulating NF-κB signaling pathway (6). The forkhead box O transcriptionfactors (FoxOs) have emerged as critical transcriptional factors inregulating metabolism and stress responses and been considered asdownstream targets of SIRT1. FoxO1 translocated into nucleusincreases FoxO1-DNA binding and expression of proapoptotic gene Bim(7). SIRT1 might regulate cellapoptosis by deacetylating FoxOs protein.
The phosphatidylinositol 3′-kinase (PI3K)/AKTpathway plays an important role in cell survival and PI3K activityhas been linked to a variety of human cancers (8). AKT, a downstream kinase of PI3K,regulates many cellular proteins including metabolism, apoptosisand proliferation (9). PI3Kpathway phosphorylates FoxOs via activation of its downstreamkinase AKT (10). Inhibition ofPI3K pathway leads to dephosphorylation and nuclear translocationof active FoxOs, which induce cell cycle arrest and apoptosis(11). These indicate that FoxOsare important downstream effectors of PI3K/AKT pathway. Resveratrolhas been shown to inhibit activation of multiple survival pathwaysincluding PI3K/AKT pathway to induce apoptosis in various cancercells (9,12).
Deleted in liver cancer 1 (DLC1), a focal adhesionprotein, is identified as a putative tumor suppressor in HCC in1998 (13). It functions as aRhoGTPase activating protein (RhoGAP) (14). Activated protein kinase C (PKC) andprotein kinase D (PKD) stimulate the association between DLC1 and14-3-3 protein, which blocks DLC1 nucleocytoplasmic shuttling andinhibits RhoGAP activity of DLC1 (15). DLC1 activity could be regulated bypost-translational modification and it might be a substrate of AKT.Expression of DLC1 suppresses cell proliferation,anchorage-independent growth, tumorigenicity and invasiveness inHCC cells (16). DLC1 inhibitsRho-dependent stress fiber formation in fibroblasts and serves as atumor suppressor gene in human non-small cell lung carcinomas(17). Thus, we hypothesized thatAKT involved in regulation of DLC1 mediated cell motilityinhibition in HCC.
The purposes of the present study were to determinethe molecular mechanism of resveratrol affected proliferation andmigration through SIRT1 mediated post-translational modification ofPI3K/AKT signaling pathway in HCC cells.
Materials and methods
The human hepatocellular carcinoma (HCC) cell linesBel-7402, SMMC-7721, hepatoblastoma cells HepG2 (The HepG2 cellline was originally thought to be a hepatocellular carcinoma cellline but was later shown to be from an hepatoblastoma,PubMed=19751877), and human liver normal cell line HL-7702 wereobtained from the Cell Bank of Type Culture Collection of ChineseAcademy of Science (Shanghai, China). HepG2 cells were cultured inDMEM, other cells in RPMI 1640 medium. All the experiments wereperformed in medium containing 10% fetal bovine serum, 100 U/mlpenicillin and 100 µg/ml streptomycin, maintained at 37°C inhumidified atmosphere with 5% CO2.
Proliferation assay by MTT andEdU
MTT assay was used to assess cell viability. Thecells were seeded in 96-well plates at a density of1×104/well overnight and treated without or withresveratrol (Sigma, St. Louis, MO, USA) dissolved in 0.1% (v/v)DMSO at various concentrations for 24 h. Then cells were incubatedwith MTT solution for 4 h. The formazan crystals dissolved by 150µl DMSO, the solution was absorbed at 492 nm using enzyme-linkedimmunosorbent 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 platesexposed 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 reactedwith 1xApollo reaction cocktail for 30 min, the DNA contents werestained with Hoechst 33342 and visualized under fluorescentmicroscope. Cells were counted in five selected arbitrarily fields,at least 300 cells were counted per well. EdU positive cells werecalculated with (EdU incorporated-in cells/Hoechst stained cells)×100%.
Apoptosis detection by TUNELassay
TUNEL staining was performed using an EdUTP TUNELcell detection kit (Ribobio, Gangzhou, China) according to themanufacturer's instructions. The cells cultured in 96-well plateswere treated without or with 100 µM resveratrol, fixed in 4%paraformaldehyde, permeabilized with 0.1% Triton X-100, washedtwice, incubated with TUNEL detecting liquid for 1 h at 37°C andobserved by a fluorescent microscope (Olympus, Tokyo, Japan) at 488nm excitation and 530 nm emission. TUNEL positive cells werecalculated as the number of apoptotic cells/DAPI stained cells×100%.
Western blotting andCo-immunoprecipitation analysis
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 bySDS-PAGE and transferred on membranes were incubated in primaryantibodies 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, SanDiego, CA, USA) overnight at 4°C, followed by incubation withHRP-conjugated rabbit/mouse secondary antibodies (ZSGB-BIO,Beijing, China). Protein expressions were visualized ECL detectionsystem (Beyotime, Shanghai, China).
Immunoprecipitation (IP) was carried out usingPierce Classic Magnetic IP/Co-IP Kit (Thermo Scientific, Waltham,MA, USA) according to the manufacturer's protocol. The proteinlysates were incubated with DLC1 antibody (BD Biosciences, SanJose, CA, USA), and precipitated with Protein A/G Magnetic Agaroseat 4°C. The immunocomplex collected was washed, and theimmunoprecipitates were subjected to western blotting andphosphorylation signals were determined using phospho-AKT substrate(PAS) antibody (CST, Danvers, MA, USA).
Wound healing assay
Cells were seeded into 24-well plates(1.0×105 cells/well). Sterile pipette tip was used toproduce a wound line between cells after the cells grew to 80–90%confluence and allowed the cells migrated for 24 h. Images werecaptured and the relative distance traveled by the leading edgefrom 0 to 24 h was assessed using Image Pro Plus 6.0 software(n=5).
SIRT1 activity assays
SIRT1 activity was quantified with a SIRT1Fluorometric Assay Kit (Sigma, St. Louis, MO, USA) according to themanufacturer's protocol. Fluorescence intensities were measuredwith a microplate fluorometer (excitation wavelength=360 nm,emission wavelength=450 nm). Experimental values are represented asa percentage of control.
All of the assays were performed three timesindependently at least. Value presented as the means ± standarddeviation (SD) by GraphPad Prism software (GraphPad Software, CA,USA). Statistical analyses were performed using one-way ANOVA andStudent's t-test, *P<0.05, **P<0.01 were considered toindicate a statistically significant difference.
Effect of resveratrol on cellviability and proliferation
Three HCC cell viability was determined by MTTassay. The results showed that resveratrol inhibited cell viabilitywhen its concentrations were higher than 80 µM compared to normalHL-7702 cell (Fig. 1A). From80–200 µM, 100 µM resveratrol was selected as IC50 (half maximalinhibitory concentration) in the subsequent experiments. Itimplicated that resveratrol was able to reduce cancer cellviability in a dose-dependent manner. The percentage of EdUpositive cells was markedly reduced in HCC cells with 100 µMresveratrol treatment compared to the controls, whilenon-tumorigenic cell line HL-7702 had a slight reduction (Fig. 1B and C).
To understand the molecular basis of proliferationinhibition caused by resveratrol, proliferation regulation proteinPCNA (proliferating cell nuclear antigen) was evaluated in HepG2cells. The level of PCNA reduced after 100 µM resveratrol treatment(Fig. 1D). It was consistent withabove results of EdU assay. These findings demonstrate theanti-proliferative effect of resveratrol on HCC cells.
Resveratrol induced apoptosis viadecreasing phosphorylation of FoxO3a with suppressing PI3K/AKTpathway
TUNEL assay was assessed whether anti-proliferativeeffects of resveratrol against HCC cells are mediated viaapoptosis. Results showed that resveratrol increased apoptosis from4±0.83% to 13±1.32%, 3±0.78% to 14±0.72%, 5±0.33% to 16±1.12% inHepG2, Bel-7402 and SMMC-7721 cells (Fig. 2A).
Studies have reported that Bcl-2 could be a crucialtarget gene of PI3K/AKT signaling, whereas AKT has been shown tonegatively regulate the activity of proapoptotic members of theBcl-2 family (8). Next effects ofresveratrol on apoptosis-related proteins were further detected. Asshowed by Fig. 2B, resveratrolinhibited Bcl-2 expression and concomitant up-regulatedproapoptotic protein Bax, causing a significant decrease inBcl-2/Bax ratio. The apoptosis regulators were further detected andthe precursor forms of caspase-3/7 induced by resveratrol weredown-regulated obviously in HCC cells (Fig. 2B). The activation of caspases werealso related to another marker of apoptosis, proteolysis of the DNArepair enzyme PARP (18). Theresults indicated that precursor form PARP decreased as active formcleavage-PARP significantly enhanced (Fig. 2B).
Resveratrol activated SIRT1 andinhibited SIRT1-mediated post-translational modification ofPI3K/AKT signaling
To investigate effects of resveratrol on the pathwayof PI3K/AKT/FoxO3a to induce cell apoptosis, western blotting wasperformed for their phosphorylation levels. Resveratrol inhibitedphosphorylation of PI3K and AKT without effect on total levels ofPI3K and AKT in HepG2 cells (Fig.3D), and inhibited FoxO3a phosphorylation in HCC cells with nototal FoxO3a change (Fig. 3D).These data indicated that resveratrol down-regulated p-FoxO3a levelwith reduction of phosphorylation level of PI3K/AKT.
We then examined whether resveratrol stimulatesexpression of SIRT1. Resveratrol up-regulated protein expression ofSIRT1 in HCC cells but not in normal liver HL-7702 cells (Fig. 3A). Deacetylation of FoxO proteinshas been shown to result from the activity of SIRT1 (19). It has been shown that SIRT1promotes transcription of FoxO target genes involved in stressresistance, while decreasing transcription of genes involved inapoptosis (20). Our result showedthat protein levels of FoxO1 and Ac-FoxO1 were significantlydecreased with resveratrol treatment compared with control(Fig. 3D). Up-regulation of SIRT1activated by resveratrol involved in deacetylation of FoxO1.
To determine the relationship between SIRT1 activityand PI3K/AKT signaling pathway, the activity of intracellular SIRT1was analyzed after SIRT1 inhibitor EX527 was used. Consistent withits protein level, SIRT1 activity increased by resveratrol anddecreased after exposure to EX527 in HepG2 cells (Fig. 3B). Treatment of 1 µM EX527 enhancedp-PI3K, p-AKT and p-FoxO3a levels while slight effect on total PI3K(Fig. 3C). These results showedthat resveratrol suppressed post-translational modification ofSIRT1 mediated PI3K/AKT signaling.
Resveratrol enhanced phosphorylationof DLC1 by AKT and inhibited cell migration
As PI3K/AKT pathway is an important cell survivalcascade, DLC1 might be a substrate of AKT. In order to explorewhether AKT involved in regulation of tumor suppressor DLC1,phosphorylation on the biological activities of DLC1 wasdemonstrated in HCC cells. DLC1 protein expression elevated inthree kinds of HCC cells and no effect on HL-7702 cells byresveratrol (Fig. 4A). An antibodyagainst PAS (phospho-AKT substrate) was employed to detectphosphorylation of DLC1. Immunoprecipitation result demonstratedthat DLC1 phosphorylation level was enhanced by resveratrol inHepG2 cells (Fig. 4B). Therelevance and functional effect of AKT mediated phosphorylation ofDLC1 remain unclear and await further investigation.
Wound healing assay measures the ability of cells tomigrate into an area of a cell culture plate denuded of cells(wound). Our result showed that resveratrol inhibited the woundclosures from 32.5 to 11.5% in HepG2 cells by 24 h treatment(Fig. 4C). The findings revealedthat up-regulation of DLC1 and its phosphorylation level viaresveratrol treatment might cause motility inhibition in cancercells.
Although the capacity of resveratrol to preventcancer development has been studied for many years, its mechanismunderlying remains to be fully elucidated. Proliferating cellnuclear antigen (PCNA) is a critical event in growth regulation ofcancer cells (21). Theanti-metastatic effect of resveratrol was associated withrestriction of invasion, mobility, adhesion, and MMP expression incolon carcinoma (22). Here, wefound that resveratrol inhibited the viability and proliferation byMTT and EdU assays and suppressed expression of PCNA accompanyingproliferation inhibition in HCC cells.
The PI3K/AKT signaling is a critical pathway in cellproliferation, survival, neovascularization and tumor growth(23). AKT is an importantdownstream target kinase of PI3K signaling pathway. Activated AKTcan inhibit release of cytochrome c and apoptosis factor, therebyinhibiting apoptosis and promote the growth of cancer cells(8). Resveratrol has been shown toinhibit constitutive activation of PI3K/AKT pathway to induceapoptosis in several types of cancer cells (12,24).FoxO3a is the downstream targets of AKT and AKT can promote FoxO3aphosphorylation, leading to FoxO3a translocation from the nucleusto the cytoplasm, which de-activates FoxO3a; conversely, inhibitionof AKT promotes de-phosphorylation of FoxO3a, resulting in nucleartranslocation of FoxO3a (25). Ourresults showed that resveratrol resulted in significant inhibitionin constitutively elevated levels of phosphorylated PI3K/AKT andreduced phosphorylated FoxO3a significantly in HepG2 cells. AKTinhibits apoptosis through multiple targets, including Bcl-2 familyand caspase proteases (8). Bcl-2members are well characterized as regulators of apoptosis, such asBax and Bim. The ratio of Bcl-2/Bax protein regarded as a drivingforce for apoptosis in cancer cells (26). Caspases are a family of cell deathproteases triggered in response to proapoptotic signals and play anessential role in the execution phase of apoptosis (27). TUNEL assays are used to detect DNAfragmentation from apoptosis. In the present study, resveratrolresulted in an increase of green fluorescence signal which wasindicative of apoptosis. Also, resveratrol caused a significantdown-modulation of Bcl-2/Bax ratio and activated caspase-3,caspase-7, PARP and induced the cleavage-PARP in HCC cells. Itsuggested that the apoptosis of HCC cells induced by resveratrolmight act through the mitochondrial pathways.
SIRT1 plays a key role in both cell death andsurvival with p53 family members, FoxOs and the nuclear factor-κBfamily (28). Furthermore,resveratrol suppresses the proliferation of gastric cancer cells ina SIRT1-dependent manner in vitro and in vivo(29). We showed that resveratrolsignificantly increased SIRT1 expression in HCC cells. As anicotinamide adenine dinucleotide-dependent protein deactylase,SIRT1 is known to be directly involved in the acetylation of FoxOsand expression of proapoptotic protein Bim (19). FoxO1 has emerged as an importantprotein that modulates the expression of apoptosis-related genes incancer cells (7). SIRT1 knockdownenhanced Ac-FoxO1 expression to block reactive oxygenspecies-induced apoptosis in mouse embryonic stem cells (30). Our results showed that resveratrolsignificantly decreased expressions of FoxO1 and Ac-FoxO1 withactivation of SIRT1 by resveratrol.
SIRT1 has been also implicated as a negativeregulator for the PI3K/AKT pathway by deacetylating the tumorsuppressor PTEN (31) and bydown-regulation of both AKT and phosphorylation levels to inhibitthe PI3K/AKT pathway in glioblastoma cell (32). The regulation of PI3K/AKT pathwayby SIRT1 may provide a potential mechanism in tumorigenesis, andSIRT1 inhibitor EX527 was used to evaluate the underlyingmechanism. We found that resveratrol up-regulated SIRT1 level todecrease PI3K and AKT phosphorylation and the phosphorylation ofPI3K and AKT became significantly higher when SIRT1 was inhibitedin HepG2 cells. It indicated that the inhibition of PI3K/AKTpathway by resveratrol is mediated by up-regulation of SIRT1.
DLC1 is a Rho GTPase-activating protein (RhoGAP) andfrequently deleted and underexpressed in cancers (14). Restoration of DLC1 gene expressioninduces apoptosis and inhibits both cell growth and tumorigenicityin HCC cells (33). Our previousresults has been shown that DLC1 is a multifunctional protein whichinteracts with tensin, talin, FAK in focal adhesion (34,35).DLC1 expression could significantly suppress Rho-dependent actinstress fiber formation in hepatocellular carcinoma and fibroblastcell lines (16). Cell migrationis tightly regulated by the activity of Rho proteins through actincytoskeletal rearrangements (36).In addition, DLC1 overexpression inhibited cell migration byinduced disassembly of stress fibers and extensive membraneprotrusions around cells on laminin-1 in HCC (37). Our result showed that resveratrolsignificantly up-regulated expression of DLC1 protein and inhibitedthe migration ratio from 32.5 to 11.5% in HCC cells, indicatingthat induced DLC1 level was associated with tumor suppressioneffect. The post-translational modification of DLC1 has garneredmuch attention as the important regulatory mechanism of DLC1activity, and kinases such as AKT, PKC and PKD have been shown tophosphorylate DLC1 at different residues and regulate itsbiological activities via RhoGAP-dependent as well asRhoGAP-independent pathways (15,38).Phosphorylation of DLC1 by PKA contributes to enhance RhoGAPactivity and promotes activation of DLC1, which suppresses hepatomacell growth, motility and metastasis both in vitro and invivo models (39). Toelucidate whether AKT could phosphorylate DLC1, an antibody againstPAS (phospho-AKT substrate) was employed to detect phosphorylationof DLC1. Our findings showed that DLC1 was directly phosphorylatedby AKT in HepG2 cells. These results suggested that DLC1 as a tumorsuppressor was up-regulated by resveratrol and itspost-translational modification was mediated by PI3K/AKT signaling.Although previous studies have characterized functional effects ofthe identified phosphorylated residues of DLC1 (40), the physiological stimuli of thesephosphorylations remain unclear. Future work are warrant to clarifyhow DLC1 regulated by its domains and phosphorylation as well asprecise downstream mechanisms through post-translationalmodification of DLC1 acts as a tumor suppressor.
Taken together, our findings suggested thatresveratrol activated SIRT1 to induce liver cancer cell apoptosisand to inhibit migration through SIRT1 mediated post-translationalregulation of PI3K/AKT signaling and phosphorylation level ofFoxO3a and DLC1 and deacetylation of FoxO1 leading to tumorsuppression in HCC cells.
This work was supported by the National NaturalScience Foundation of China (Grant No. 31672377), the Major KeyScience and Technology Project of Shandong Province(2015ZDJS04003), the Key Program of Shandong Provincial NaturalScience Foundation of China (ZR2013CZ002), Science and TechnologyProgram of Jinan (201202033).
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On the left side of this page, you’ll find a list of all the updates released for this version of Windows. You can also find more information about releases and any known issues. Installing the most recent update ensures that you also get any previous updates you might have missed, including any important security fixes.
For more information about the update and how to get it, see:
What's new for Windows 10, version 21H1, Windows 10, version 20H2, and Windows 10, version 2004 release notes
Windows 10, versions 21H1, 20H2, and 2004 share a common core operating system and an identical set of system files. As a result, the release notes for Windows 10, versions 21H1, 20H2, and 2004 will share an update history page. Each release page will contain a list of addressed issues for 21H1, 20H2, and 2004 versions. Note that the 21H1 and 20H2 versions will always contain the fixes for 2004; however, 2004 will not contain the fixes for 21H1 and 20H2. The update history page will provide you with the build numbers for 21H1, 20H2, and 2004 versions so that it will be easier for support to assist you if you encounter issues.
Current status of Windows 10, version 21H1
For the most up-to-date information about known issues for Windows and Windows Server, please go to the Windows release health dashboard.
Certain Japanese half-width Katakana and full-width Katakana characters that have a consonant mark aren’t interpreted as the same character. When you use the CompareStringEx() function with the NORM_IGNOREWIDTH flag to compare them, these characters are evaluated as different because of an issue in the sorting rule. This issue affects all the updates starting on June 9, 2020 for Windows 10, version 2004.
Important If you have not installedKB4586853 or later on the computer, setting an invalid value in this registry might prevent the computer from starting up.
This workaround reverts the National Language Support (NLS) sorting rule to version 6.2, which is used in Windows 10, version 1909 and earlier. When sharing data between systems, consider applying the workaround consistently. If you use this workaround, conduct sufficient testing and evaluations to mitigate problems caused by different sorting rule versions on multiple systems.
When using the Microsoft Japanese Input Method Editor (IME), applications might not be able to automatically change Roman/Kana input modes.
Note for developers Affected apps are using the ImmSetConversionStatus function or VK_KANA key emulation.
To mitigate this issue, do one of the following:
We are working on a resolution and will provide an update in an upcoming release.
Notes and messages
If you have questions or need help activating or troubleshooting Windows, see our help topics below:
For information about how to update, see Update Windows 10.
If you have questions about manually installing or removing an update, see Windows Update: FAQ.
Getting an error message when updating? See Troubleshoot problems updating Windows 10.
If you need to activate Windows, see Activation in Windows 10. If you’re having trouble with activation, see Get help with Windows activation errors.
To get the latest major update to Windows 10, see Get the Windows 10 May 2021 Update.