The Role And Mechanism Of JNK1/HIF-1? And 14-3-3?/ERK Pathway In Caffeic Acid-inhibited The Progression Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC),a highly vascularized tumor,is the second leading cause of tumor-related mortality in China.Current standard practices for treatment of HCC,surgical resection and liver transplantation,are less than satisfactory because of intra-hepatic and extra-hepatic metastasis and post-surgical recurrence.In addition,the therapeutic effects of non-operative treatments,such as transcatheter arterial chemoembolization(TACE)and sorafenib,are also unsatisfactory because of toxic/side-effects and drug resistance.For the potential mechanisms,studies have revealed that,the angiogenesis,invasion,and self-renewal are possibly involved.Accordingly,the continued searches for novel agents,which exhibit less cytotoxic and have effectively repressive effects on the above-mentioned three aspects in HCC are urgently needed.Caffeic acid(3,4-dihydroxycinnamic acid,CaA),is a naturally occurring hydroxycinnamic acid derivatives.Due to its extensive sources,hypotoxicity,and diverse biological functions,CaA has been intensively investigated in many physiological or pathological processes,including anti-cancer.In HCC,CaA has been identified as a matrix metalloproteinase-9(MMP-9)inhibitor,leading to the attenuations of tumor growth and metastasis.Our previously studies reveal that,Caffeic acid attenuates the angiogenic function of HCC cells via reduction in Jun N-terminal kinase 1(JNK1)-mediated hypoxia-inducible factor-1?(HIF-1?)stabilization;also,CaA can attenuate the cancer stem cells(CSCs)-like properties in HCC cells via demethylation-activating microRNA-148a,causing the blockage of transforming growth factor ?(TGF(?)signaling.Collectively,these findings suggest that,the anti-cancer effects of CaA in HCC are positive.So the further searches for novel involved molecular mechanisms are also urgently needed.Methods1.Cell culture and CaA treatmentAfter HepG2,MHCC97H,or HuH7 cells were conventional cultured for 24 h,they were treated with 0 or 40?M of CaA in the presence or absence of 100 ?M CoCl2for another 24 h.Then such cells were used to determine the abilities of angiogenesis,invasion,and CSCs-like porperties.2.Determination of cell viabilityTreated cells were incubated with 20.0?l of WST-8 solution(Beyotime Co.Ltd)for 4 h.The absorbance at 450 nm was measured with a multimode microplate reader.The values for medium control cells were used to determine the 100%level.3.siRNA transfectionCells were transfected with such siRNAs by using Lipofectamine 2000 reagent for 12 h.Then,such cells were conventional cultured for another 24 h before being used for other experiments.4.Quantitative real-time polymerase chain reaction(qRT-PCR)Total RNA was transcribed into cDNA using AMV Reverse Transcriptase.The qRT-PCR was performed using an ABI 7300 real-time PCR detection system.5.Western blotsProteins were transferred to polyvinylidene fluoride(PVDF)membranes,which were then probed with primary antibody at 4? overnight.Then membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h.The immune complexes were detected by the enhanced chemiluminescence.6.Co-ImmunoblotEqual amount of total proteins were separated by 10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis,and transferred to PVDF membranes.Then they were incubated with immunoprecipitates antibody and subsequently with A+G sepharose beads at 4 ? overnight.To determine protein-protein interaction,the immunoprecipitates were analyzed by Western blots.7.Enzyme-linked immunosorbent assay(ELISA)Samples or standard protein were added to the wells for 2 h at 37 ?.Then biotinylated antibody was added for 2 h,followed by incubating with streptavidin-HRP for another 1 h,follow by adding the substrate,the colorimetric reaction was determined by measuring the absorbance at 450 nm with a multi-well plate reader.8.Tube formation assayHUVECs cells were seeded in a 96-well plate on matrigel-After then,such cells were incubated in the HuH7 conditioned mediums for 6 h,respectively.Capillary morphogenesis was evaluated by using a phase-contrast microscope.Quantification of tube formation was assisted by S.CORE.9.HUVECs recruitment assayHUVECs were seeded in the upper compartments of the chambers,and HuH7 cells were placed in the lower compartments.Migrated cells were viewed under a phase-contrast microscope,and counted by Image-Pro-Plus 6.0 software in five randomly chosen fields.10.Chromatin-Immunoprecipitation(ChIP)assay.To crosslink proteins to DNA,such cells were treated with 1%formaldehyde in PBS by gentle agitation.Next,cells were washed,resuspended in lysis buffer,and sonicated.Then we used the immunoprecipitation for the protein-DNA complexes.The specific sequences from immunoprecipitated and input DNA were determined by PCR analysis.Amplifications of the VEGF gene sequences among the pull of DNA were performed with specific primers.11.Gelatin zymographyWithout prior boiling,proteins were separated,by 10%sodium dodecyl sulfate-polyacrylamide gel containing 1 mg/ml gelatin under non-reducing conditions.After electrophoresis,the gels were washed twice in 2.5%Triton X-100/50 mM Tris-HC1,and incubated at 37 'C in the absence of CO2 for 2 h,followed by staining with coomassie blue for 3 h,and de-stained with acetic acid and isopropanol.12.Transwell assayTreated-MHCC97H cells were seeded onto the upper chamber of the transwells in supplements-free medium.The lower chambers of the transwells were filled with the medium containing 100 ng/ml epidermal growth factor.The chambers were incubated at 37 ? for 24 h.Then,the cells migrating through the filter were stained,viewed,and counted as described above in 10.13.Sphere assaysIn costar non-adherent 24 well dishes,treated HCC cells were suspended in defined,serum-free medium composed of DMEM-F12,10 ng/ml of human recombinant basic fibroblast growth factor and 10 ng/ml of EGF.Cells were grown for 10 days and fed every 48 h.Spheres were then counted under a microscope.14.Anchorage-independent growthSoft agar plates were prepared in 10 cm(diameter)dishes with under-layers of 0.70%agarose in DMEM medium supplemented with 10%FBS.Cells were plated in2 ml of 0.35%agarose over the agar base.Cultures were fed every 3 d,and after 20 d,colonies were counted under a microscope.15.Animals,xenografts,and immunohistochemistryThe BALB/c nude mice were kept in a temperature-controlled environment(20-22 ?)with a 12 hour light dark cycle and with free access to drinking water and chow.For xenograft studies MHCC97H cells were injected subcutaneously into the right armpit of the mice.Three weeks later,CaA(0 or 10 mg/kg·BW)was administered intraperitoneally(i.p)twice per week.Tumor volumes were measured weekly and tumor size was calculated using the formula:V= 1/2(width2× length).After 11 weeks,the mice were sacrificed,and tumor tissues were removed for further investigation.For immunohistochemistry(IHC),The quantitation of immunostaining was performed by two independent researchers who were blinded regardingexperimental details with Q-scores.16.Statistical analysisData were presented as the means ± SD.A Student's t test,and a one-way analysis of variance(ANOVA)were used to assess significant differences between groups.In our present study,p values<0.05 were considered statistically significant.Results1.Effects of CaA on the angiogenesis under hypoxia Application of 20 M CaA treatment of HepG2 and MHCC97H cell lines,in the presence of CoCl2 for 24 h can inhibit the expression and secretion of VEGF and the angiogenic ability effectively.2.Effects of CaA on HIF-1? under hypoxiaCoCl2 treatment induced a time-dependent increased levels of HIF-la mRNA and protein.Interestingly,co-treated with CaA attenuated the CoCl2-induced increased expression of HIF-la protein,however,there was no significant effects of CaA on HIF-1? mRNA expression under CoCl2 treatment.3.Mechanisms of CaA on HIF-1?/VEGF under hypoxiaCoCl2 treatment increased the expressions of HIF-1? and phosphorylated signal transducers and activators of transcription-3(p-STAT-3).Then,by directly binding to the promoter of VEGF gene,HIF-1? effectively activated VEGF.However,CaA attenuated the CoCl2-induced activation of HIF-1? likely by reducing JNK1 activation and reducing HIF-1? stabilization.Moreover,CaA decreased the CoCl2-induced increased expression of p-STAT-3.These two functions resulted in an attenuated recruiting of the HIF-1? to the VEGF promoter.4.In vivo functinos of CaA on HCC growth and angiogenesisCompared with the vehicle control(dimethyl sulfoxide,DMSO),CaA treatment resulted in a significant reduction in tumor weight;in addition,CaA significantly attenuated the angiogenesis in the xenograft tumor as determined by the CD31 staining and MVD.Compared with vehicle control,reductions of p-JNK1,HIF-1?,STAT-3,and VEGF expression,and attenuations of HIF-1? and STAT-3 nuclear location were observed in CaA-treated group.5.Effects of CaA on angiogenesis,invasion,and CSC-like properties under normoxiaIn NC-siRNA-transfected cells,CaA blocked the angiogenic ability,invasion,and CSCs-like properties effectively.6.Effects of 14-3-3? of CaA-attenuated angiogenesis,invasion,and CSC-like properties under normoxiaThe 14-3-3?-siRNA-transfection dramatically reduced the tube formation,invasion,and CSCs-like propertie.Meanwhile,in NC-siRNA-transfected cells,CaA treatment obviously attenuated the HuH7 cells-enhanced HUVECs recruitment,hyper-viability,tube formation,and significantly inhibited the invasion and CSCs-like properties;however,in 14-3-3?-siRNA-transfected group,these extents of inhibitions were markedly attenuated.7.Potential roles of CaA in regulating the 14-3-3?In medium control MHCC97H and HuH7 cells,CaA treatment obviously inhibited the expressions of 14-3-3T? mRNA and protein;however,in U0126-pre-treated group,CaA only slightly attenuated the expression of 14-3-3r? In addition,we also found that,compared with medium control group,CaA treatment increased the extents of 14-3-3? ubiquitination.Conclusions1.In the presence of CoCl2(a hypoxia mimic),CaA attenuated the angiogenic function of HCC cells via reduction in JNK-1-mediated HIF-la stabilization;2.JNKl/HIF-la signaling plays an important role in CaA-inhibited angiogenesis;3.CaA attenuated angiogenesis,invasion,and CSC-like properties under normoxia4.CaA transcriptionally and post-transcriptionally regulated 14-3-3? via an ERK-and ubiquitination-dependent manner;5.14-3-3? played an important role in the maintenance of angiogenesis,invasion,and CSCs-like properties in HCC cells;6.14-3-3? played an important role in the CaA-attenuated angiogenesis,invasion,and CSCs-like properties in HCC cells.