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Malvidin-3-galactoside from blueberry suppresses the growth and metastasis potential of hepatocellular carcinoma cell Huh-7 by regulating apoptosis and metastases pathways

2020-05-22JiLinJinlongTinChiShuZhnChngYunnLiuWishngWngRuihiLiuBinLiYuhuWng

食品科学与人类健康(英文) 2020年2期

Ji Lin,Jinlong Tin,b,Chi Shu,b,Zhn Chng,Yunn Liu,Wishng Wng,Ruihi Liu,Bin Li,b,,*,Yuhu Wng,b,,*

a National R&D Professional Center For Berry Processing,Shenyang Agricultural University,Liaoning,PR China

b Key Laborotary of Healthy Food Nutrition and Innovative Manufacturing,Liaoning,PR China

c Emergency Medicine Department of General Hospital of Shenyang Military Command,Laboratory of Rescue Center of Severe Trauma PLA,Liaoning,PR China

d Shenyang Blueberry Industry co.LTD.,Liaoning,PR China

e Department of Food Science,Cornell University,Ithaca,NY,USA

ABSTRACT Malvidin-3-galactoside (Mv-3-gal) is the major anthocyanin monomer in blueberry anthocyanins.The compound is well-characterized by its anti-oncogenesis function in multiple organs including liver.In the current study,the mechanism driving the anti-hepatocellular carcinoma(HCC)function of Mv-3-gal was further explored by focusing on apoptosis and metastases pathways.HCC cell line Huh-7 was firstly administrated with Mv-3-gal of different concentrations and the effect of Mv-3-gal on cell proliferation,apoptosis,colony formation ability,metastasis potential as well as the activity of Akt/PTEN and MAPK pathways was assessed.The results showed that Mv-3-gal inhibited the cell proliferation and colony formation ability,induced cell cycle arrest and apoptosis in a dose-dependent manner.Regarding the metastasis potential,Mv-3-gal suppressed the migration and invasion potential of Huh-7 cells by regulating MMPs expression.Taken together,the findings highlighted the anti-HCC potent of Mv-3-gal,which was associated with the inhibition of Akt/PTEN,MAPK and MMP pathways.

Keywords:Malvidin-3-galactoside Hepatocellular carcinoma Apoptosis MAPK signaling

1.Introduction

Blueberry is a widely distributed fruit type rich in nutrients including sugars,proteins,vitamins,minerals,and unsaturated fatty acids and is highly recommended by the World Food Organization(WFO)as one of the healthy fruits.Except for the conventional nutrient substances,blueberry also contains abundant flavonoids[1-3],which forms the basis of blueberry’s physiological activities in human bodies [4,5].Currently,the anti-aging,cholesterol elimination,and cardiovascular prevention functions of blueberries are well recognized by numerous studies[6,7].With emerging attention being paid to the function of blueberries,recent studies also report the anti-carcinoma function of the fruits and indicate that the effect of blueberries might be attributed to the function of anthocyanins.As a major function flavonoid type in blueberry,anthocyanins have shown the potential to inhibit the growth of multiple tumor types,including on hepatocellular carcinoma cells,esophageal cancer cells,and colon cancer cells [8,9].There are several anthocyanins in blueberries and based on the previous qualitative analysis,Mv-3-gal is conceived to be the most common anthocyanins shared by different blueberry varieties[10,11].Thus,it was reasonable to hypothesize that Mv-3-gal might play a central role in the anti-tumor function of anthocyanins derived from blueberries.

Of distinct cancer types,hepatocellular carcinoma ranks second in the cancer-related death factors.In China,the cancer type has become a major threat to people’s health and a burden to public health system with the rapid development of life quality in the recent years[12].Typical HCC is characterized by malignant tumors forming in the liver and can be further classified into liver cancer and metastatic liver cancer[13].The global incidence of HCC is over 626000/year and the mortality is close to 600000/year [14].Currently,with the development in conventional treatment strategy including resection surgeries or chemo radiotherapies,the survival rate of HCC patients has been considerably improved.However,HCC is seriously threatening the health and life of our people,it shows a high rate of recurrence as a result of invasion and metastasis [15,16].Given the obstacles in the current management of HCC,the exploration of candidates for the future development of mild but effective anti-HCC therapies is not only necessary,but also prompting.

Emerging evidence infers that bioactive natural products are a good source for development of novel cancer preventive and therapeutic drugs [17].For instance,curcumin,resveratrol,and arachidonic acid are well known cancer chemopreventive agents which act by targeting multiple pathways[12,13].Regarding anthocyanins fromLonicera caerulea Beileifruit is reported to antagonize HCC in multi-pronged mechanisms [18]and anthocyanins from Mulberry fruit had a potent protection effect on CCl4-induced liver fibrosis[19].However,although anthocyanins with diverse origins have shown their potential to prevent or attenuate HCC,the underlying mechanism driving the function remains partially revealed.Based on the above information,the major purpose of the current study was to determine the effect of Mv-3-gal from blueberries on the progression of HCC.Moreover,the current study also attempted to provide some information on the anti-HCC mechanism of anthocyanins by focusing on the influence of Mv-3-gal on the activity of Akt/PTEN and MAPK signaling transductions.

To fulfill the research purposes,the human HCC cell line Huh-7 was administrated with Mv-3-gal of different concentrations.Then the signaling pathways associate with the cell proliferation(Akt/PTEN),apoptosis(MAPK)and metastasis(MMP)potential was assessed to elucidate the effect and associated mechanism of Mv-3-gal on HCC progression.The findings provide a theory basis for the anti-HCC function of Mv-3-gal,and highlight the potential value of blueberry Mv-3-gal for development as liver-protecting products.

2.Materials and methods

2.1.Reagents

Malvidin-3-galactoside (with a purity of >95%) was purchased from Sigma-Aldrich.Dulbecco’s modified eagle medium (DMEM)was purchased from Hyclone (SH30021.01,Utah,US).Australian fetal bovine serum was purchased from Gibco (10099-141,California,US).MTT kit (C0009) and HE staining kit (C0105) were purchased from Beyotime biology technology (Shanghai,China).RNaseA(10109142001),propidium iodide(PI)(P4170-10MG),flow cytometry kit (APOAF-20TST),and 2,3-dimethoxynaphthalene-1,4-dione (D5439-5MG) were purchased from Sigma (St.Louis,US).Transwell chamber (353503) and Matrigel (3542334) were purchased from BD (Lake Franklin,New Jersey,US).BCA protein concentration determination kit was purchased from wanleibio(WLA004,Shenyang,China).Rapid preparation kit of SDS-PAGE Gel (TGXTMFastCastTM),Immun-Star WesternC chemiluminescence Kit(1705062),Molecular weight standard of predyed protein(161-0394),and PVDF(1620264)membrane were purchased from Bio-Rad (Hercules,California,US).Dried skimmed milk was purchased from Erie(Q/NYLB 0039S,Beijing,China).Antibodies against cyclin B1 (10205-2-AP),PARP (55004-1-AP),p38 (14064-1-AP)were purchased from Proteintech Wuhan.Cyclin E (ab52189),anti-cleaved-caspase-3 (ab13847),cleaved-PARP (ab32064),pp38 (ab47363),ERK1/2 (ab32081),p-ERK1/2 (ab76299),and Akt(ab32505) were purchased from Abcam (Cambridge Science Park,UK).Antibodies against CyclinD1 (#2978-1978S),p-JNK (#9255-9255S),PTEN (#9188-9188S),and p-Akt (4051S) were purchased from Cell signaling(Boston,US).Antibodies agaisnt caspase-3(sc-7148),Bax (sc-526),JNK (sc-7345),and GAPDH (sc-32233) were purchased from Santa(Dallas,Texas,US).

2.2.Cell culture and treatment

Human HCC cell line Huh-7 was obtained from the Type Culture Collection Center at the Chinese Academy of Sciences (Shanghai,China).The cells were cultured in DEME supplemented with 10%fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2and 95% air.For experimental treatment,Huh-7 cells were cultured with fresh substrate containing 0,50,100,and 200 μg/mL Mv-3-gal (stored at 2-8°C until use,with a purity of >95%)dissolved in distilled water.

2.3.MTT cell viability

To investigate the influence of Mv-3-gal on cancer cell viability,Huh-7 cells were seeded in 96-well tissue culture plates at a density of 5×103cells per well,and then treated with 0,50,100,and 200 μg/mL Mv-3-gal.Thereafter,20 mL MTT solution was added into each well,and incubated for 4 h.The blue formazan products in the Huh-7 cells were dissolved in DMSO and the absorbance value at 490 nm was measured using a microplate reader.

2.4.Colony formation assays

Huh-7 cells (1 × 103/well) were inoculated into wells and cultured overnight at 37°C.Then the cultures were supplemented with 0,50,100,and 200 μg/mL Mv-3-gal and incubated for 3 weeks until visible colonies appeared.Thereafter,the colonies were fixed with 4% paraformaldehyde and stained with crystal violet.The colony number of each group was counted under a microscope reader(iMark,Bio-Rad,Hercules,CA,U.S.A.).

2.5.Flow cytometry

Flow cytometry was used to determine the effect of Mv-3-gal administrations on the cell cycle distribution and cell apoptosis in Huh-7 cells.For the detection of cell cycle distribution,Huh-7 cells were inoculated at 1×105cells per well,then the cells were treated with 0,50,100,and 200 μg/mL Mv-3-gal for 48 h and then washed twice with cold PBS.Afterwards,the cells were fixed in 800 μL methanol for 30 min at 37°C and stained with 800 μL of crystal violet for 30 min.Cell cycle distribution was investigated using DNA flow cytometry analysis (BD C6,BD,China) and the results were represented as a percentage of the cells in each phase of the cell cycle.

For the detection of cell apoptosis,Huh-7 cells were treated with 0,50,100,and 200 μg/mL Mv-3-gal for 72 h,then washed twice with PBS,digested using trypsin enzyme,and centrifuged at 1000 r/min for 5 min,then the cells were fixed with 99.7% ethanol for 2 h at 4°C.The fixed cells were collected by centrifugation (1000 r/min for 5 min),cleaned with PBS,and centrifuged again.RNaseA(100 μL)was added to incubate the cell precipitates for 30 min in a 37°C water bath,after which Annexin V/PI solutions were added,followed by incubation without light for 30 min at room temperature.The proportion of apoptotic cells was then measured using flow cytometry analysis(BD C6,BD,China).

2.6.Western blotting

The cells were cultured with 0,50,100,and 200 μg/mL Mv-3-gal for 6,12,24,48,and 72 h at 37°C.Adherent and floating cells were harvested for western blotting analysis.After washing twice with PBS,the cells were lysed in whole cell radio immunoprecipitation assay(RIPA)lysis buffer(Beyotime,Haimen,Jiangsu,China) containing phenylmethylsulfonyl fluoride (PMSF,20 μL).The supernatants were obtained by centrifuging at 4°C and 12,000 r/min for 5 min.The protein concentrations in the supernatants were determined using BCA Protein Assay Kit(WLA004,Wanleibio,China).GAPDH was employed as internal reference protein.20 μg protein from each sample was separated by polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane(1620264,Bio-Rad,America).Then the membranes were blocked with 5%skim milk.Afterwards,primary antibodies against cyclin B (1:1000),cyclin E (1:1000),cyclinD1 (1:500),cleaved-caspase-3 (1:500),caspase-3 (1:1000),Bax(1:1000),PARP(1:2000),cleaved-PARP(1:1000),JNK(1:2000),p-JNK (1:1000),p38 (1:1000),p-p38 (1:1000),ERK1/2 (1:2000),p-ERK1/2 (1:1000),Akt (1:2000),p-Akt (1:1000),PTEN (1:2000),and GAPDH (1:4000) were added onto the membranes and incubated at 4°C overnight.After washing with PBST,the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies cyclinB (1:2000),cyclin E (1:2000),cyclinD1(1:2000),anti-cleaved-caspase-3(1:2000),caspase-3(1:2000),Bax(1:2000),PARP (1:2000),cleaved-PARP (1:1000),JNK (1:2000),p-JNK(1:2000),p38(1:2000),p-p38(1:2000),ERK1/2(1:2000),p-ERK1/2(1:1000),Akt(1:2000),p-Akt(1:2000),PTEN(1:2000),and GAPDH (1:3000).Chemiluminescence detection was performed with Immun-Star WesternC Chemiluminescence Kit(1705062,Bio-Rad,China)and the results were detected using Image J analysis.

2.7.2,3-Dichloro-1,4-naphthoquinone staining

ROS level was detected by 2,3-Dichloro-1,4-naphthoquinone staining:briefly,the cells were washed twice with 1 mL PBS,and PBS was discarded.Pancreatic enzyme was added to digest cells in 37°C cell incubator.After digestion was complete,complete culture medium with 6 times the volume of pancreatic enzyme was used to stop digestion and the cells were transferred into the centrifuge tube.Centrifugation was performed at 1000 r/min and 5 min,and the supernatant was discarded.About 5×104cells were added to per well.Add 0,50,100,and 200 ug/mL Mv-3-gal.Then 2,3-Dichloro-1,4-naphthoquinone staining(diluted 100 times with PBS)was added into mixture and incubated with cells for 15 min.After being washed with PBS twice,a drop of glycerin was added on the slide and the images were captured using a fluorescence microscope analysis (Olympus,China).The fluorescent-emitting cells,indicating the production of ROS,were observed from images taken under a fluorescence microscope analysis(Olympus,China).

2.8.Transwell

The effect of Mv-3-gal on the migration and invasion potential was assessed using transwell assays.The upper chamber was precoated with matrigel(354234,BD,US)and 100 μL cell suspension along with 0,50,100,and 200 μg/mL Mv-3-gal were innoculated on the upper chamber.Simultaneously,600 μL medium supplemented with 10% serum was added on the lower chamber (24 wells).After allowing the cells to growth in the chamber for 24 h,the cells left on the upper chamber were completely removed and the cells invading to the lower surface of the membrane were fixed in methanol for 30 min at 37°C and stained with 800 μL crystal violet for 30 min.The numbers of cells penetrating to the lower chamber were quantified by fluorescence microscope analysis.The migration potential of Huh-7 cells was detected as described above with matrigel coated.

Table1 Oligonucleotide primer sets for real-time PCR.

2.9.Real-time PCR

After digestion with pancreatic enzyme,the cells were centrifuged at 1000 rpm for 5 min,and then cultured with 0,50,100,and 200 ug/mL Mv-3-gal for 72 h.Total RNA in cells of different groups was extracted using a total RNA extraction kit (RP1201,BioTeke,China)according to the manufacturer’s instructions.cDNA was synthesized from extracted RNA(1 μg)using 1 μL of super MMLV reverse transcriptase (PR6502,BioTeke,China).The levels of mRNA expression were quantified using an RT-PCR system(Exicycler 96,BIONEER,Korea).The pcr primer details used in this study are presented in Table1.

2.10.Statistical analysis

All the data was expressed as mean±standard deviation(SD).The overall effect of Mv-3-gal on different parameters was evaluated using one-way analysis of variance(ANOVA)and difference among groups was analyzed using multiple comparison with Tukey method.All the analyses were performed using SPSS v.16.0.

3.Results

3.1.Mv-3-gal inhibited the growth potential in Huh-7 cells

To examine whether Mv-3-gal has an influence on cells growthin vitro,MTT assay was performed to detect the growth of Huh-7 cells.As shown in Fig.1A,compared to control group (0 μg/mL),the cell viability was dramatically inhibited in 50,100,and 200 μg/mL Mv-3-gal-treated for 24 h.Treated cells for 48 h,50 and 100 μg/mL Mv-3-gal has no significant effect on Huh-7 cells,whereas,200 μg/mL Mv-3-gal displays obvious cytotoxicity(P<0.01).Moreover,50,100 and 200 μg/mL Mv-3-gal has a notable cytotoxic effect against Huh-7 cells at 72 h.The highest inhibitory rate was at the dose of 200 μg/mL Mv-3-gal at 72 h (P<0.001).This indicated that an appropriate concentration of Mv-3-gal and appropriate treatment time have a notable cytotoxic effect against Huh-7 cells.Except for MTT assays,colony formation assay was performed to confirm the inhibition effect of Mv-3-gal on HCC cells.As shown in Fig.1B,compared to control group,50,100,and 200 μg/mL Mv-3-gal dramatically reduced the number of colonies (stained purple)in a dose-dependent manner.Taken together we could concluded that the growth of Huh-7 cells were suppressed by Mv-3-gal.

3.2.Mv-3-gal induced cell cycle arrest in Huh-7 cells

Abnormal cell cycle distribution can lead to decrease in slower growth and lower survival in cancer cells[20].As shown in Fig.2,compared to control group,50 and 100 μg/mL Mv-3-gal arrested the cells in the S phase,accompanied by the cells in the G1 phase decreased (P<0.05),and the cells in the S phase increased (P<0.05).At this time,Mv-3-gal promotes cells from G1 phase to S phase and inhibits DNA replication.200 μg/mL Mv-3-gal increased the number of cells in the G1 phase,no significant changes in the S and G2 phases,the cells arrested in the G1 phase,and Mv-3-gal inhibited cells from the G1 phase to the S phase.All in all,these results indicate that the growth of hepatocellular carcinoma cells was significantly inhibited after being cultured with different concentrations of Mv-3-gal.

Fig.1.Effects of Mv-3-gal on cell proliferation on Huh-7 cells.(A)Effects of Mv-3-gal on Huh-7 cell viability.(B)Cell clone assay to detected cell proliferation.Huh-7 cells were exposed to 0-200 μg/mL Mv-3-gal detected using crystal violet staining.Compare the viability of 0,50,100,200 μg/mL at 24 h,we use“*”,when compare the viability of 0,50,100,200 μg/mL at 48 h,we use“#”,when compare the viability of 0,50,100,200 μg/mL at 72 h,we use“△”.(*)P <0.05 vs group of 0 μg/mL.(**)P <0.01 vs.group of 0 μg/mL.(***)P <0.001 vs.group of 0 μg/mL.

Fig.2.Modulation effects of Mv-3-gal on Huh-7 cell cycle distribution.(A)Representative images of cell cycle distribution of each group.(*)P <0.05 vs group of 0 μg/mL.(**)P <0.01 vs.group of 0 μg/mL.(***)P <0.001 vs.group of 0 μg/mL.

3.3.Mv-3-gal induced cell apoptosis in Huh-7 cells

To investigate the apoptosis-promoting effect of Mv-3-gal on Huh-7 cells,the proportion of apoptotic cells and morphology were analyzed by flow cytometry analysis.The Q3 corner corresponds to normal cells,the Q4 corner to early apoptotic cells,the Q1 corner to the death of the autophagic and non-specific cells,and the Q2 corner to late apoptotic cells and necrotic cells.As shown in Fig.3A,the apoptosis rate of control group(0 μg/mL)was 8.7%.After 50,100,and 200 μg/mL Mv-3-gal treatment for 72 h,the apoptosis rates were 17.8%,20.5%,and 24.2%respectively.The cells apoptosis rate dramatically increased in a dose-dependent manner,we concluded that Mv-3-gal could promote Huh-7 cell apoptosis.

In order to further clarify the molecular mechanism driving the function of Mv-3-gal,the levles of apoptosis-related proteins including cleaved caspase-3,caspase-3,PARP,cleaved-PARP,and bax were detected using western blotting analysis.The results showed that 50,100,and 200 μg/mL Mv-3-gal markedly increased the levels of caspase-3,cleaved-caspase-3 and cleaved-PARP in a dose-dependent manner compared to control group(0 μg/mL,P<0.05) (Fig.3B).For pro-apoptotic protein bax,100 and 200 μg/mL Mv-3-gal obviously increased the expression of bax (P <0.05),which demonstrated Mv-3-gal could induce Huh-7 cells apoptosis via the modulation of caspase-3,PARP,and bax levels.

Fig.3.Modulation effect of Mv-3-gal on Huh-7 cell apoptosis(n=3).(A)Representative images of apoptotic rates determined by flow cytometry.Statistical analysis results apoptosis rates.(B)Levels of cleaved caspase-3,caspase-3,Bax,PARP,and p-PARP proteins in Huh-7 cells.(*)P <0.05 vs group of 0 μg/mL.(**)P <0.01 vs.group of 0 μg/mL.(***)P <0.001 vs.group of 0 μg/mL.

Fig.4.Modulation effect Mv-3-gal on ROS formation in Huh-7 cells.Huh-7 cells were treated with 0-200 μg/mL Mv-3-gal for 12 h and stained cells using 2,3-Dichloro-1,4-naphthoquinone staining.

Fig.5.Modulation effect of Mv-3-gal on MAPK-related protein expressions in Huh-7 cells.Huh-7 cells were treated with 0-200 μg/mL Mvl-3-gal for 12 h and the levels of JNK,p-JNK,p38,p-p38,ERK1,ERK2,and p-ERK in Huh-7 cells were detected.(*)P <0.05 vs group of 0 μg/mL.(**)P <0.01 vs.group of 0 μg/mL.(***)P <0.001 vs.group of 0 μg/mL.

3.4.Mv-3-gal inhibited the production of ROS

The effect of Mv-3-gal on the production of ROS was explored using 2,3-Dichloro-1,4-naphthoquinone staining.The amount of red cells represented the number of ROS generation.As shown in Fig.4,Mv-3-gal-treated group(50,100,and 200 μg/mL Mv-3-gal)reduced the number of red cells in a dose-dependent manner compared to control group (0 μg/mL).Thus,our results suggest that Mv-3-gal inhibited ROS accumulation.After Mv-3-gal treatment,the change of ROS accumulation likely related to the reduction of cell proliferation and apoptosis-promoting in Huh-7 cells.

3.5.Mv-3-gal promoted cell apoptosis through the p38/JNK/ERK MAPK pathways in Huh-7 cells

p38/JNK/ERK MAPK signaling pathways are the key members of the MAPK family and involve in inducing apoptosis,and their role is related to p38,JNK,and ERK activity.To investigate whether MAPK signaling pathway contributes to the effect of Mv-3-gal on Huh-7 cells,we analyzed the levels of MAPK-related proteins(JNK,p-JNK,p38,p-p38,ERK1,ERK2,and p-ERK1/2)by western blotting analysis.As shown in Fig.5,compared to control group,the phosphorylation level of p38 and JNK(p-p38 and p-JNK)were markedly increased at 200 μg/mL Mv-3-gal at 12 h(P<0.05),while 50,100,and 200 μg/mL Mv-3-gal dramatically reduced the level of phosphorylation ERK (p-ERK) in a dose-dependent manner (P<0.05).Therefore,these results suggest that Mv-3-gal could interfere with activation of the p38/JNK/ERK MAPK pathways in Huh-7 cells.

Fig.6.Modulation effect of Mv-3-gal on Akt/PTEN signaling.Huh-7 cells were with 0-200 μg/mL Mvl-3-gal for 12 h and the levels of Akt,p-Akt,and PTEN in Huh-7 cells were detected.(*)P <0.05 vs group of 0 μg/mL.(**)P <0.01 vs.group of 0 μg/mL.(***)P <0.001 vs.group of 0 μg/mL.

3.6.Mv-3-gal modulated Akt/PTEN signal pathway

As shown in Fig.6,Mv-3-gal obviously increased the level of PTEN in Mv-3-gal-treated group (P<0.05).100 and 200 μg/mL Mv-3-gal markedly increased the level of Akt(P<0.05).Whereas,the expression of Akt phosphorylation decreased (P<0.05).Thus,we could infer that the antitumor effect of Mv-3-gal seems to be dose-dependent because treatment of a higher dose (200 μg/mL)was more effective compared to that of the lower doses.Mv-3-gal may inhibit the proliferation of hepatocellular carcinoma cells by regulating Akt/PTEN signal pathway.

3.7.Mv-3-gal inhibited the migration and invasion in Huh-7 cells

To evaluate the effect of Mv-3-gal on the migratory and invasive capacities of tumor associated mechanisms,the migration and invasion potential of Huh-7 cells was explored using trans well assays.The results showed that Mv-3-gal significantly reduced the number of cells (stained purple) in a dose-dependent manner(50,100,and 200 μg/mL) compare with control group (0 μg/mL,Fig.7A),this suggests that Mv-3-gal would inhibit the abilities of migration and invasion in Huh-7 cells.Moreover,the expressions of angiogenic factors,including VEGF,MMP-2,and MMP-9,were detected by quantitative real-time PCR.As shown in Fig.7B,Mv-3-gal remarkably inhibited the expressions of MMP-2 and MMP-9 at mRNA level at 200 μg/mL,but had no significant effect on VEGF compared with control group(0 μg/mL)in Huh-7 cells.

4.Discussion

Mv-3-gal characteriz ed as the most common anthocyanins shared by different blueberry varieties is attracting emerging attention around the world for their prominent bioactivity including anti-tumor effect [21,22].In this study,the experiments for the first time demonstrated that Mv-3-gal remarkably inhibited the proliferative capacity in Huh-7 cells,which can be utilized on the future development of blueberry as a functional food and auxiliary ingredient to prevent liver cancer.

As investigated in previous research that the protective effect and antioxidant mechanism of blueberry anthocyanins in a CC14-induced liver injury model in mice[23].Similarly,Lonicera caeruleaberry extract attenuates lipopolysaccharide induced hepatic function injury in BRL-3A cells [24].Moreover,bog bilberry extract contain abundant Mv-3-gal showed a dose-dependent cytotoxic on hepatocellular carcinoma cells[10].In our current study,the data showed that Mv-3-gal at different concentrations exhibit growth inhibitory effects on Huh-7 cells.Moreover,our results also showed that Mv-3-gal could induce G1/S phase cell arrest and apoptosis of Huh-7 cells,which has become the ideal strategies for the treatment of cancers

Fig.7.Modulation effect of Mv-3-gal on the migration and invasion activity of Huh-7 cells.(A) Transwell analysis of Huh-7 cells.(B) VEGF,MMP-2,and MMP-9 in mRNA levels in Huh-7 cells determined by real-time PCR.(*)P <0.05 vs group of 0 μg/mL.(**)P <0.01 vs.group of 0 μg/mL.(***)P <0.001 vs.group of 0 μg/mL.

The processes of cell cycle and apoptosis are regulated by various intracellular cytokines,including MAPK and Akt signal pathways[25,26].The MAPK signaling pathways are involved in the pathogenesis of many human diseases,such as Parkinson’s disease and cancers[27].The anthocyanins isolated fromVitis coignetiae Pulliatfor their potential anti-proliferative and apoptotic effects through activating p38-MAPK and suppressing Akt in human colon cancer HCT-116 cells[28].Phosphorylated p38,JNK and ERK are involved in processes such as inflammation,stress response,cell proliferation,and apoptosis,and played the transit station of many signal transduction pathways in body cells [29],and the p38/JNK/ERK signaling pathways play important regulatory target for cells in normal and disease states.Our study indicated that Mv-3-gal inhibited cell proliferation and promoted cell apoptosisviaregulating p38/JNK/ERK MAPK and Akt/PTEN signal pathways.Similarly,Wang et al.[30]reported that a new prenylated flavonoid isolated from Daphne giraldii nitsche could induce apoptosis by regulating p38 and JNK/MAPK pathways in Hep3B and HepG2 cells,and providing a new,safe,and effective agent for the treatment of HCC.

ROS production was inhibited by activating the p38 MAPK signaling pathway [31].Other studies indicate thatFusobacterium nucleatumcan affect ROS Generation,and Inflammatory Cytokine Production and promote apoptosis in Human Gingival Fibroblasts by activating the Akt/MAPK signaling pathway [32].Studies have shown that changes in ROS production levels play an important role in regulating cell proliferation,apoptosis and inflammation[33,34].Another study indicated thatFusobacterium nucleatumcan induce bladder cancer cell apoptosis by regulating ROS production[35].In our study,our results indicated that Mv-3-gal regulated the survival,apoptosis,and proliferation of Huh-7 cells by inducing the activation of p38 and JNK,which may be associated with ROS production.However,the activity of ERK was suppressed simultaneously,which needed further exploration.

PTEN is essential for normal cell maintenance and is one of the most frequently mutated tumor suppressor genes in human cancer[36].A research reported that PTEN plays an important regulatory role in tumor growth,such as in colorectal cancer,liver cancer,and breast cancer [37].The lipid phosphatase activity of PTEN is associated with the down-regulated activities of multiple downstream components of the phosphatydilinositol-3-kinase (PI3K)pathway,including the serine/threonine kinase Akt.The Akt/PTEN pathway has been substantially explored for its potential in developing novel anti-tumor drugs due to its key role in vital cellular processes[38,39].Our study indicated that Mv-3-gal increased the levels of PTEN and Akt,and suppressed the phosphorylation of Akt in a dose-dependent manner,thereby inhibiting the proliferation of HCC cells.Our conclusion is inconsistence with the previous report that red raspberry phytochemicals inhibited the proliferation of HCC cells by regulating Akt/PTEN signaling pathway[40].

Matrix metalloproteinase(MMPs)play an important role in cancer invasion,metastasis and angiogenesis.For example,MMP-2 and MMP-9 play important roles in the development of several tumors,such as breast and prostate cancer[41].Vascular endothelial growth factor (VEGF) is a highly specific vascular endothelial cell mitogen that can induce angiogenesis.The first step in the angiogenesis step is to degrade the basement membrane.MMP-2 and MMP-9 can degrade the basement membrane and specifically bind to the substrate to participate in various physiological and pathological changes in the body,including tumorigenesis and development [42].The genes of VEGF and MMPs may promote tumor metastasis by promoting the growth of malignant tumors and the formation of blood vessels.Our results showed that Mv-3-gal could inhibit the ability of migration and invasion of Huh-7 cells,and inhibit the expressions of MMP-2 and MMP-9 at mRNA level.

In conclusion,the results from this study demonstrated that Mv-3-gal suppressed hepatocellular carcinoma by inhibiting the proliferation,migration and invasion of Huh-7 cell,which was associated with its regulation on Akt/PTEN,MAPK and MMP pathways.These findings provide a theoretical basis for finding a potential auxiliary component or nutritional supplement for liver cancer prevention.

Declaration of Competing Interest

There are no conflicts to declare.

Acknowledgements

The work is thanks to supported by the national natural science foundation of China (31671863),the national key research and development plan(2016YFD0400200),innovative talent support program for institution of higher learning of Liaoning province(LR2017038),young and middle-aged technological innovation talent support for program of Shenyang science and technology bureau(RC170247),and thanks to the technically supported by the General Hospital of the Shenyang Military Region of the Chinese People’s Liberation Army.