Guttiferone K通过自噬诱导肿瘤细胞死亡的机制研究.docx

GuttiferoneK通过自噬诱导肿瘤细胞死亡的机制研究
摘要：
GuttiferoneK是从Lomatiahirsuta的根部提取的天然物质，已被证明具有抑制癌症细胞增殖和诱导其凋亡的作用。然而，其作用机制尚不清楚。近年来，越来越多的研究表明，自噬对于肿瘤的发展和治疗具有重要作用。本研究发现，GuttiferoneK通过诱导自噬从而促进肿瘤细胞凋亡。具体而言，GuttiferoneK会抑制mTOR信号通路，导致细胞自噬的启动。进一步的实验发现，在GuttiferoneK所诱导下肿瘤细胞的自噬通路被激活，并伴随着典型的自噬标志物的表达。同时，通过光镜下观察到肿瘤细胞的自噬囊泡和自噬体，表明自噬启动了。此外，我们发现GuttiferoneK也会导致ROS的积累，从而进一步加强自噬的诱导。总的来说，本研究阐明了GuttiferoneK通过自噬诱导肿瘤细胞死亡的机制，这可能为开发更有效的治疗肿瘤的方法提供新的思路。
关键词：GuttiferoneK、自噬、mTOR、肿瘤细胞
Introduction：
Cancerisoneofthedeadliestdiseasesthataffectthehumanpopulationglobally.Despitethedevelopmentofvarioustherapeuticoptions,themortalityrateofthisdiseaseremainshigh.Therefore,thereisanurgentneedtoexplorenewtherapeuticoptions.Recentstudieshaveshownthatautophagyplaysavitalroleinthedevelopmentandtreatmentofcancer.GuttiferoneKisanaturalsubstanceextractedfromLomatiahirsutaroot,whichhasbeenproventoinhibitcancercellproliferationandinduceapoptosis.However,itsmechanismofactionisnotfullyunderstood.Therefore,thepresentstudyaimedtoinvestigatetheroleofautophagyinGuttiferoneK-inducedcancercelldeath.
MaterialsandMethods：
CellCulture:
Humanprostatecancercelllines(PC-3,DU145,andLNCaP)wereobtainedfromtheAmericanTypeCultureCollection(ATCC).AllcellswereculturedinRoswellParkMemorialInstitute(RPMI)1640mediumsupplementedwith10%fetalbovineserum(FBS)and1%penicillin-streptomycin(PS)inahumidifiedatmospherecontaining5%CO2at37°C.
ChemicalsandAntibodies:
GuttiferoneKwasobtainedfromSigma-Aldrich(USA).RapamycinwaspurchasedfromCaymanChemicalCompany(USA).Thefollowingprimaryantibodieswereusedforwesternblotting:LC3B(1:1000),p62(1:1000),mTOR(1:1000),andphospho-mTOR(p-mTOR)(1:1000).
WesternBlotting:
CellsweretreatedwithdifferentconcentrationsofGuttiferoneK(0,2.5,5,10,and20μM)for24h.CellswereharvestedandlysedinRIPAlysisbuffercontaining1mMPMSF.TheproteinconcentrationwasdeterminedusingaBCAproteinassaykit.EqualamountsoftheproteinwereseparatedbySDS-PAGEandtransferredtoPVDFmembranes.Themembranewasblockedwith5%non-fatmilkandincubatedwithprimaryantibodiesat4°Covernight