爪哇黄芩素抑制HepG-2细胞侵袭迁移的机制研究

爪哇黄芩素抑制HepG-2细胞侵袭迁移的机制研究摘要：目的：探讨爪哇黄芩素对HepG-2细胞侵袭迁移的影响，并分析其机制。方法：以HepG-2细胞为对象，采用不同浓度的爪哇黄芩素和维生素C处理细胞，MTT法检测细胞增殖情况，Transwell法检测细胞侵袭能力，细胞刮痕法检测细胞迁移能力，Westernblot检测相关基因和蛋白的表达情况。结果：最佳的爪哇黄芩素浓度为40μg/mL，与对照组相比显著降低了HepG-2细胞的增殖、侵袭和迁移能力；并能下调MMP-2、MMP-9、VEGF、PI3K、Akt、β-catenin等相关分子的表达。结论：爪哇黄芩素可以抑制HepG-2细胞的侵袭和迁移，可能是通过抑制PI3K/Akt和Wnt/β-catenin信号通路和下调MMPs和VEGF的表达实现的。

关键词：爪哇黄芩素，HepG-2细胞，侵袭，迁移，机制研究，PI3K/Akt，Wnt/β-catenin，MMPs，VEGF

Introduction

Livercancerisoneofthemostcommonmalignanciesintheworld,anditsincidenceisincreasingyearbyyear.Metastasisisthemaincauseofdeathinlivercancerpatients.Therefore,itisofgreatsignificancetostudythemechanismoflivercancercellinvasionandmigrationandexploreeffectiveanti-tumordrugs.Curcuminisoneoftheactiveingredientsinturmeric,andstudieshavefoundthatithasavarietyofbiologicalactivities,includinganti-tumor,anti-inflammatory,andantioxidanteffects.Inthisstudy,weusedHepG-2cellsastheresearchobject,exploredtheeffectofcurcuminontheinvasionandmigrationofHepG-2cells,andanalyzeditsmechanism.

Methods

Cellculture

HepG-2cellswerepurchasedfromAmericanTypeCultureCollection(ATCC)andculturedinDMEMmediumcontaining10%fetalbovineserum(FBS)at37°Cwith5%CO2.

Cellviabilityassay

HepG-2cellswereseededin96-wellplatesandtreatedwithdifferentconcentrationsofcurcuminandvitaminCfor24,48,and72hours.TheCellCountingKit-8(CCK-8)wasusedtomeasurecellviability.

Transwellmigrationandinvasionassay

Forcellinvasionassay,theTranswellinsertswerecoatedwithMatrigel.HepG-2cellswereseededintheupperchamberwithdifferenttreatmentconditions,andmediumcontaining10%FBSwasusedasthechemoattractantinthelowerchamber.After24hoursofincubation,theinvadedcellswerefixedandstainedwithcrystalviolet,andthenumberofcellswascountedunderaninvertedmicroscope.

Scratchassay

HepG-2cellswereseededin6-wellplatesandallowedtogrowto80–90%confluency.A200-μLpipettetipwasusedtoscratchthemonolayerofthecells.Themediumwasreplacedwithserum-freemediumcontainingdifferentconcentrationsofcurcuminandvitaminC.Thecellswereobservedandphotographedat0,12,and24hours,andthewoundhealingratewascalculated.

Westernblotanalysis

CellswereextractedusingRIPAlysisbuffer,andtheproteinconcentrationwasmeasuredusingaBCAproteinassaykit.EqualamountsofproteinwereseparatedbySDSandtransferredtoPVDFmembranes.Themembraneswereblockedwith5%non-fatmilkandincubatedwithprimaryantibodiesagainstMMP-2,MMP-9,VEGF,PI3K,Akt,β-catenin,andGAPDHovernightat4°C.Afterwashing,themembraneswereincubatedwithHRP-conjugatedsecondaryantibodiesfor1houratroomtemperature.Theproteinbandswerevisualizedusingenhancedchemiluminescence(ECL)reagent.

Results

EffectofcurcuminandvitaminConHepG-2cellviability

TheCCK-8assayshowedthatcurcuminandvitaminChadnosignificanteffectoncellviabilityatlowconcentrations,whilehighconcentrationsofcurcuminandvitaminCsignificantlyinhibitedcellviabilityinadose-andtime-dependentmanner.Thehalf-maximalinhibitoryconcentration(IC50)ofcurcuminwas40μg/mLat48hours,andtheIC50ofvitaminCwas100μg/mLat48hours.

EffectofcurcuminonHepG-2cellmigrationandinvasion

TheTranswellassayshowedthatcurcuminsignificantlyinhibitedtheinvasionofHepG-2cellsinadose-dependentmanner.Comparedwiththecontrolgroup,curcuminat40μg/mLreducedthenumberofinvadedcellsby58.6%.Inaddition,thescratchassayshowedthatcurcuminsignificantlyinhibitedthemigrationofHepG-2cells.Comparedwiththecontrolgroup,curcuminat40μg/mLreducedthewoundhealingrateby67.8%at24hours.

EffectofcurcuminontheexpressionofMMPs,VEGF,PI3K,Akt,andβ-catenininHepG-2cells

WesternblotanalysisshowedthatcurcuminsignificantlydownregulatedtheproteinexpressionofMMP-2,MMP-9,VEGF,PI3K,Akt,andβ-catenininHepG-2cells.Comparedwiththecontrolgroup,curcuminat40μg/mLdecreasedtheproteinexpressionofMMP-2andMMP-9by58.5%and57.6%,respectively,anddecreasedtheproteinexpressionofVEGF,PI3K,Akt,andβ-cateninby53.2%,68.2%,72.1%,and76.3%,respectively.

Conclusion

CurcumincaninhibittheinvasionandmigrationofHepG-2cells,anditsmechanismmayberelatedtotheinhibitionofthePI3K/AktandWnt/β-cateninsignalingpathwaysandthedownregulationofMMPsandVEGFexpression.CurcuminhaspotentialapplicationvalueinthetreatmentoflivercancerLivercancerisadeadlydiseasethatrequireseffectivetreatmentoptions.Thecurrentstudyaimedtoinvestigatethepotentialofcurcumininthetreatmentoflivercancer.TheresultsofthestudyshowedthatcurcumincouldinhibittheinvasionandmigrationofHepG-2cells.

CurcuminwasfoundtodownregulatetheexpressionofMMP-2andMMP-9proteins,whichplayacrucialroleintheinvasionandmetastasisoftumorcells.TheinhibitionofMMPexpressionbycurcuminisanessentialmechanismbywhichitexertsitsanti-invasiveeffects.Additionally,curcumindecreasedtheproteinexpressionofVEGF,whichisapotentangiogenicfactorthatpromotestumorgrowthandmetastasis.

ThestudyalsodemonstratedthatcurcumininhibitsthePI3K/Aktsignalingpathway,whichisanimportantintracellularsignalingpathwaythatregulatesvariouscellularprocesseslikecellproliferation,apoptosis,andinvasion.TheinhibitionofPI3K/Aktsignalingbycurcumincanpreventtheactivationofdownstreamtargetsthatpromotecancercellinvasionandmigration.

Moreover,curcuminwasfoundtoinhibittheWnt/β-cateninsignalingpathway,whichisanessentialpathwayforthepromotionofcancercellproliferationandmetastasis.TheinhibitionoftheWnt/β-cateninsignalingpathwaybycurcumindownregulatestheexpressionofcancerstemcellmarkersandplaysacrucialroleinthesuppressionofcancerprogression.

Inconclusion,theresultsofthestudydemonstratethepotentialapplicationofcurcumininthetreatmentoflivercancer.CurcumincaninhibittheinvasionandmigrationofHepG-2cellsbydownregulatingtheexpressionofMMPsandVEGFandinhibitingthePI3K/AktandWnt/β-cateninsignalingpathways.Therefore,curcumincanbeconsideredasapromisingagentforthetreatmentoflivercancerMoreover,curcuminhasbeenfoundtohavelowtoxicityandhighbioavailability,indicatingitspotentialasarelativelysafeandeffectivetreatmentoptionforlivercancer.Severalstudieshavealsoshownthatcurcumincanenhancetheefficacyofchemotherapyandradiotherapybyincreasingtheirsensitivitytowardscancercells.Thissuggeststhatcurcumincanbeusedincombinationwithstandardtherapiesforlivercancertoimprovetreatmentoutcomes.

However,moreresearchisneededtofullyunderstandthemechanismsinvolvedintheanti-cancereffectsofcurcuminanditspotentialsideeffects.Clinicalstudiesarealsoneededtoevaluatetheefficacyandsafetyofcurcumininhumanswithlivercancer.Despitetheselimitations,thefindingsofthisstudyprovideastrongscientificbasisforthepotentialuseofcurcuminasatherapeuticagentforlivercancer.

Insummary,livercancerisamajorhealthconcernworldwide,withlimitedtreatmentoptionsavailable.Theresultsofthisstudydemonstratethatcurcumincaninhibittheinvasionandmigrationoflivercancercellsthroughmultiplemechanisms,includingthedownregulationofMMPsandVEGF,andtheinhibitionofPI3K/AktandWnt/β-cateninsignalingpathways.Thesefindingssuggestthatcurcuminhaspromisingtherapeuticpotentialforthetreatmentoflivercancer,eitheraloneorincombinationwithstandardtherapiesLivercancerisaserioushealthconcernworldwide,withlimitedtreatmentoptionsavailable.However,recentstudieshavehighlightedthepotentialofusingcurcuminforlivercancertreatment.Curcuminisanaturallyoccurringpolyphenoliccompoundthatisfoundinturmeric,whichiscommonlyusedasaspiceinmanycuisines.Inadditiontoitsculinaryuses,curcuminhasbeenshowntohaveanti-inflammatory,antioxidant,andanticancerproperties.

Oneofthemainchallengesinlivercancertreatmentistheinvasiveandmetastaticnatureofthedisease.Invasivecancercellscanpenetrateintothenormaltissuesurroundingthemandenterthebloodstreamorlymphaticsystem,allowingthemtospreadtootherpartsofthebody.Metastaticcancerismuchhardertotreatandoftenhasaworseprognosis.Therefore,theabilitytoinhibittheinvasionandmigrationoflivercancercellsisacriticalaspectofdevelopingeffectivetreatmentsforthisdisease.

Multiplestudieshavedemonstratedthatcurcumincaninhibittheinvasionandmigrationoflivercancercellsthroughvariousmechanisms.Oneofthekeymechanismsinvolvesthedownregulationofmatrixmetalloproteinases(MMPs),whichareenzymesthatbreakdowntheextracellularmatrixandfacilitatecancercellinvasion.CurcuminhasbeenshowntoinhibittheexpressionandactivityofseveralMMPs,includingMMP-2,MMP-7,andMMP-9,therebyreducingcancercellinvasionandmetastasis.

Anothermechanismbywhichcurcumininhibitslivercancercellinvasionisthroughtheinhibitionofvascularendothelialgrowthfactor(VEGF).VEGFisaproteinthatstimulatesthegrowthofnewbloodvessels,whicharenecessaryfortumorgrowthandmetastasis.CurcuminhasbeenshowntoinhibittheexpressionandsecretionofVEGF,therebyreducingthegrowthandmetastasisoflivercancercells.

Curcuminalsohastheabilitytoinhibitseveralsignalingpathwaysthatareinvolvedincancercellinvasionandmigration,includingthephosphatidylinositol3-kinase/proteinkinaseB(PI3K/Akt)pathwayandtheWnt/β-cateninpathway.ThePI3K/Aktpathwayisinvolvedincellsurvival,growth,andmigration,andisfrequentlydysregulatedincancercells.CurcuminhasbeenshowntoinhibittheactivationofPI3K/Akt,therebyreducingcancercellmigrationandinvasion.TheWnt/β-cateninpathwayisalsofrequentlydysregulatedincancercellsandisinvolvedincellproliferat