Echinacoside Induces Apoptotic Cancer Cell Tuag Los Ntawm Inhibiting Lub Nucleotide Pool Sanitizing Enzyme MTH1

Tshooj 1 Yuav ua li cas echinacoside inhibit cell apoptosis thiab txhim kho kev nco

Yog xav paub ntxiv thov hu rau: Ali.ma@wecistanche.com

Abstract:Inhibition ntawm lub pas dej nucleotide sanitizing enzyme MTH1 ua rau nws kim heev oxidative DNA puas thiab apoptosis nyob rau hauv cov qog nqaij hlav cancer thiab li no yuav siv tau raws li ib tug anticancer lub tswv yim. Raws li cov khoom ntuj tsim muaj cov khoom siv tshuaj lom neeg nplua nuj, hauv txoj kev tshawb fawb tam sim no, peb tau siv MTH1-catalyzed enzymatic cov tshuaj tiv thaiv raws li kev ua haujlwm siab hauv kev tshuaj ntsuam vitro txhawm rau tshawb nrhiav cov tshuaj ntuj muaj peev xwm inhibiting MTH1.Echinacoside, ib qho chaw muab tau los ntawm cov nroj tsuag tshuaj Cistanche thiab Echinacea, zoo inhibited cov catalytic kev ua si ntawm MTH1 nyob rau hauv ib tug in vitro assay. Kev kho mob ntawm ntau yam tib neeg cov kab mob qog noj ntshav nrogEchinacosideua rau muaj kev nce ntxiv hauv cellular theem ntawm oxidized guanine (8-oxoguanine), thaum cellular reactive oxygen hom tseem tsis hloov, qhia tiasEchinacosidekuj inhibited kev ua ntawm cellular MTH1. Yog li ntawd,EchinacosideKev kho mob ua rau muaj qhov nce sai thiab ua rau DNA puas cov cim thiab kev txhim kho ntawm G1 / S-CDK inhibitor p21, uas tau ua raws li cov cim apoptotic cell tuag thiab cell voj voog raug ntes hauv cov qog nqaij hlav tab sis tsis nyob hauv cov hlwb uas tsis yog mob cancer. Ua ke, cov kev tshawb fawb no tau txheeb xyuas cov khoom siv ntuj tsim los ua MTH1 inhibitor thiab qhia tias cov khoom ntuj tuaj yeem yog qhov tseem ceeb ntawm cov tshuaj tiv thaiv kab mob.

Ntsiab lus:Echinacoside, MTH1, 8-oxoG, DNA puas,apoptosis, cell voj voog ntes.

Nyem rau Cistanche siv rau kev nco

Taw qhia

Lub cellular thiab mitochondrial nucleoside triphosphate (NTP) thiab deoxynucleoside triphosphate (dNTP) pas dej ua ke yog lub hom phiaj tseem ceeb ntawm ntau hom reactive oxygen (ROS).1-3 Vim tias qhov tshwj xeeb ntawm DNA polymerases tsawg dua zoo meej, 4,5 oxidized dNTPs tuaj yeem ua tau. 6 Piv txwv li, lub hauv paus oxidized loj hauv lub pas dej nucleotide, 8-oxoguanine (8-oxoG), tuaj yeem ua khub nrog cytosine thiab adenine. Thaum muab tso rau hauv qhov sib txawv adenine thaum lub sij hawm DNA replication, nws muaj peev xwm ua rau T: A=G: C transversion,7 uas yog ib qho ntawm feem ntau mutations pom nyob rau hauv tib neeg mob qog noj ntshav.8,9 Txawm hais tias feem ntau ntawm cov oxidized dawb dNTPs. raug tshem tawm los ntawm nucleotide pas dej huv huv enzymes, kev tshawb fawb yav dhau los tau qhia tias lub pas dej nucleotide tseem yog ib qho tseem ceeb ntawm oxidized bases hauv DNA molecules thiab ib qho tseem ceeb contributor rau oxidative DNA puas.3,10 Mammalian hlwb yog armed nrog sophisticated tiv thaiv mechanisms. txo ROS-induced genomic lesions. Nucleotide pool sanitizing enzymes hydrolyze oxidized dNTPs thiab NTPs, yog li tiv thaiv lawv kev koom ua ke rau hauv DNA thiab RNA molecules.11,12 MTH1, ib tug tswv cuab ntawm Nudix hydrolase superfamily (li no kuj hu ua NUDT1), 13 yog triphosphatase lub luag hauj lwm rau tshem tawm NTPs thiab oxidized. dNTPs (8-oxodGTP, 2-OH-dATP, 8-OH-GTP, thiab 2-OH-ATP) los ntawm nucleotide pools.14 Txij thaum 8-oxoguanine (8-oxoG) yog lub hauv paus oxidized loj hauv cov hlwb, MTH1 yog qhov tseem ceeb tshaj plaws enzyme rau kev ua kom huv ntawm nucleotide pools.15 Txawm li cas los xij, txawm tias nyob rau hauv kev soj ntsuam los ntawm nucleotide pas dej huv huv enzymes, ib feem tseem ceeb ntawm oxidized dNTPs yog koom ua ke rau hauv DNA, thiab cov hauv paus hauv DNA molecules kuj tuaj yeem raug oxidized ncaj qha los ntawm ROS.6 Cells tom qab ntawd tso siab rau ntau cov tswv yim uas muaj feem xyuam nrog ntau yam DNA glycosylases, xws li OGG1 thiab MUTYH, thiab DNA kho cov txheej txheem, suav nrog kev kho qhov hauv paus excision (BER) thiab mismatch. kho (MMR), kho cov teeb meem hauv paus hauv DNA molecules.10, 16,17 BER thiab MMR ib txwm kho ntau lub hauv paus oxidized hauv DNA ib hnub thiab yog li ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kev ncaj ncees thiab kev ruaj ntseg ntawm genome.

Nyob rau hauv cov hlwb uas nyob rau hauv siab oxidative kev nyuaj siab, muaj zog incorporation ntawm oxidized nucleotides rau hauv DNA yuav overwhelm lub cellular kho muaj peev xwm thiab ua rau DNA puas teb (DDR). Teem ib qho kev thaiv rau cov qog nqaij hlav.19 Txawm li cas los xij, txawm tias cov qog nqaij hlav qog nqaij hlav tuaj yeem ua rau muaj zog oxidative nruj heev thiab ua rau muaj peev xwm ua rau lub nra hnyav ntawm oxidized nucleotides, 20 lawv tuaj yeem muaj sia nyob ntawm DDR-koom nrog senescence lossis apoptosis.21 Ntau qhov kev tshawb fawb tau pom tias MTH1, los ntawm kev tshem tawm cov khoom txaus ntshai heev 8-oxodGTP thiab 2-OH-dATP hauv cov qog hlwb, ua lub luag haujlwm tseem ceeb hauv kev loj hlob ntawm cov qog nqaij hlav cancer.22 MTH1 tau pom tias muaj ntau yam mob qog noj ntshav, 23– 25 thiab 8-oxoG qib, thiab qhov ntau ntawm oxidative DNA lesions tau qis dua hauv cov qog ntau dua li cov ntaub so ntswg ib puag ncig.26–28 Ua haujlwm tau zoo, qhov overexpression ntawm MTH1 txo qis cov loog. er ntawm DNA kev hloov pauv pom nyob rau hauv MMR-tsis muaj nas embryonic fibroblasts10 thiab tso cai rau cov hlwb kom kov yeej oncogenic Ras-induced ntxov cellular senescence thiab apoptosis; 29 whereas kev tsuj ntawm MTH1 tiv thaiv kev loj hlob ntawm mob qog noj ntshav hauv OGG1- tsis muaj nas30 thiab txhawb ROS -induced DDR thiab cellular senescence nyob rau hauv Ras-transformed hlwb.3,29,31 Yog li, txawm hais tias MTH1 plays lub luag hauj lwm tiv thaiv los ntawm kev tiv thaiv ROS-induced mutations nyob rau hauv lub hlwb noj qab haus huv, cov kev tshawb fawb pom tias MTH1 yog tshwj xeeb yog yuav tsum tau rau kev tshwm sim thiab ciaj sia taus ntawm mob qog noj ntshav. hlwb. Cov kev tshawb fawb tsis ntev los no tau muab pov thawj ntxiv los txhawb lub luag haujlwm ntawm MTH1 hauv kev txhim kho mob qog noj ntshav.31–34 Kev Tiv Thaiv MTH1 los ntawm RNAi lossis cov tshuaj tiv thaiv me me hauv ntau cov kab mob qog noj ntshav ua rau muaj kev sib koom ua ke ntawm 8-oxodG rau hauv genomic DNA, ua rau kom dav DNA puas, apoptotic cell tuag, thiab txo cov qog nqaij hlav cancer. 32,33 Hauv cov kev tshawb fawb nas xenograft, inhibition ntawm MTH1 zoo suppressed kev loj hlob ntawm cov qog nqaij hlav cancer. Qhov tseem ceeb, kev txwv tsis pub MTH1 tsis cuam tshuam rau kev loj hlob thiab kev ciaj sia ntawm cov hlwb ib txwm, tej zaum vim cov hlwb ib txwm muaj qis ROS thiab yog li tsis tshua muaj nyob ntawm MTH1 rau kev ciaj sia.32,33 Cov kev tshawb pom no qhia tias inhibition ntawm MTH1 yog ib qho kev cog lus tshiab rau kev tawm tsam mob qog noj ntshav. .

Cov khoom siv ntuj tsim tau muaj cov khoom siv tshuaj lom neeg nplua nuj.35 Ntau yam kev kho mob niaj hnub no, suav nrog cov tshuaj tiv thaiv kab mob cancer, tau muab los ntawm cov tshuaj ntsuab lossis tshuaj ntsuab npaj. yuav tsum yog tus cwj pwm, feem ntau vim muaj teeb meem hauv kev tswj hwm lawv cov khoom sib xyaw kom tsis meej pem txhais lawv cov kev ua tau zoo thiab cov txheej txheem ntawm kev ua haujlwm.40 Cov txheej txheem tshiab uas tso cai rau kev tshawb nrhiav cov tshuaj nquag thiab kev tshuaj xyuas ntawm cov tshuab molecular yog xav tau. Nyob rau hauv no hais txog, lub hom phiaj-guided high-throughput nyob rau hauv vitro kev tshuaj ntsuam yuav ua tau instrumental nyob rau hauv kev nrhiav mechanical txhais cov tshuaj tebchaw los ntawm natural resources. Hauv txoj kev tshawb fawb tam sim no, peb tau siv MTH1-catalyzed enzymatic cov tshuaj tiv thaiv raws li kev ntsuas siab hauv vitro los tshawb nrhiav cov tshuaj ntuj uas tuaj yeem cuam tshuam MTH1. Kuj ceeb tias, qhov kev tshuaj ntsuam tau qhia tiasEchinacoside, ib yam khoom ntuj uas paub zoo tshaj plaws rau nws lub zog tiv thaiv kab mob, muaj peev xwm inhibiting MTH1.

Cov ntaub ntawv thiab cov txheej txheem

Khoom siv

Ntuj tshuaj ntsuab tebchaw tau yuav los ntawm Yuanye Biological Technology Co., Shanghai, Tib neeg lub koom pheej ntawm Tuam Tshoj. Lub npe, tus lej catalog, Chemical Abstracts Service (CAS) tus lej sau npe, thiab purity ntawm txhua qhov sib xyaw tau teev tseg hauv Table S1. Cov kev daws teeb meem ntawm cov tebchaw tau npaj nyob rau hauv 100 feem pua ​​​​dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO, USA), thiab cov kev daws teeb meem tau npaj rau hauv kev ntsuam xyuas tsis zoo lossis ua tiav cov kab lis kev cai ntawm tes. Tib cov tshuaj tsis muaj qhov sib xyaw ua ke tab sis muaj tib qhov nyiaj ntawm DMSO tau siv los tswj lub tsheb. (S)-crizotinib tau yuav los ntawm Selleck Chemicals, Houston, TX, USA.

Hauv vitro kev tshuaj ntsuam

Kev ntsuam xyuas hauv vitro enzymatic siv los tshuaj xyuas cov tshuaj ntsuab tshuaj ntsuab tau ua raws li cov txheej txheem piav qhia los ntawm Gad li al.32 Luv luv, serial dilutions ntawm ib tus neeg tebchaw tau npaj rau hauv qhov kev ntsuam xyuas tsis muaj 100 mM Tris- acetate (pH 8.{11}}), 40 mM NaCl, 10 mM Mg acetate, 0.005 feem pua ​​Tween 20, thiab 1 mM DTT. Tom ntej no, 0.5 nM recombinant tib neeg MTH1 (Abcam, Cambridge, UK), 100 μM dGTP (Thermo Fisher Scientific, Waltham, MA, USA), thiab 0.2 U / mL inorganic pyrophosphatases (Thermo Fisher Scientific) tau ntxiv, thiab cov phaj tau muab tso rau hauv. ntawm lub phaj shaker rau 1 teev ntawm chav tsev kub. Qhov kawg cov tshuaj tiv thaiv ntim yog 100 μL ntawm 96- phaj zoo. Thaum kawg ntawm cov tshuaj tiv thaiv, malachite ntsuab (J&K Scientific, Beijing, Tib neeg lub koom pheej ntawm Tuam Tshoj) tau ntxiv, 41, thiab cov phaj tau incubated ntawm chav tsev kub rau lwm 15 feeb. Absorbance ntawm 630 nm yog ntsuas los ntawm BioRad 680 microplate nyeem ntawv (Bio-Rad Laboratories Inc., Hercules, CA, USA). Inhibitory concentration (IC50) qhov tseem ceeb tau txiav txim los ntawm nonlinear regression tsom xam siv GraphPad Prism software.

Cell kab lis kev cai

Tib neeg MG-63 osteosarcoma, SK-HEP-1 hepatocarcinoma, MCF7 lub mis mob cancer, SW480 mob qog noj ntshav, HEK293 embryonic raum, thiab nas NIH/3T3 fibroblast cell kab yog los ntawm American Type Culture Collection, thiab tib neeg ib txwm daim siab cell kab L-O2 tau yuav los ntawm KenGen Biotech, Nanjing, Tib neeg koom pheej ntawm Tuam Tshoj. Cov hlwb no tau khaws cia ntawm 37 degree nyob rau hauv ib qho chaw humidified nrog 5 feem pua ​​​​CO2, ua raws li cov lus qhia los ntawm cov kws kho mob. Tsis muaj cov lus qhia txog kev coj ncaj ncees tau xav tau los ntawm pawg saib xyuas lub koom haum rau kev siv cov kab ntawm tes.

Kev ntsuas ntawm intracellular 8-oxoG

Intracellular {{0}} oxoG qib tau ntsuas los ntawm staining nrog Cy3-conjugated avidin.42 Tag nrho ntawm 1 × 104 hlwb tau cog rau ntawm ib puag ncig npog hauv 12- cov phiaj zoo. Hnub tom qab, cov hlwb raug kho nrog 0 μM, 15 μM, 30 μM, 60 μM, lossis 80 μMEchinacosiderau 5 teev, 12 teev, lossis 24 teev thiab tom qab ntawd kho nrog dej txias methanol rau 20 feeb, tom qab ntawd los ntawm incubation hauv Tris-buffered saline (TBS) nrog 0.1 feem pua ​​Triton X -100 (Sigma-Aldrich) rau 15 feeb. Cov qauv raug thaiv hauv TBS nrog 0.1 feem pua ​​​​Triton X-100 thiab 15 feem pua ​​​​ntawm fetal bovine serum rau 1 teev nyob rau hauv chav tsev kub thiab ces stained nrog Cy3-conjugated avidin (0.5 ug/ mL) (Rockland Immunochemicals, Limerick, PA, USA) hauv kev thaiv kev daws rau 1 teev ntawm 37 degree. Tom qab ntxuav hauv phosphate-buffered saline (PBS) 3 zaug rau 5 feeb, cov ntawv npog tau muab ntim rau ntawm iav slides hauv VECTASHIELD Mounting Medium nrog 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA , USA). Cov duab tau muab coj los soj ntsuam los ntawm Zeiss LCM 510 confocal microscope.

Kev ntsuas ntawm intracellular ROS

Intracellular ROS theem tau ntsuas los ntawm kev ntws cytometry siv cov khoom siv ntawm tes-based ROS (Beyotime Biotechnology, Haimen, Tib Neeg Lub Tebchaws ntawm Tuam Tshoj). Cells loj hlob nyob rau hauv 6-zoo daim hlau raug kho nrog 0 μM, 15 μM, 30 μM, 60 μM, los yog 80 μMEchinacosiderau 5 teev, 12 teev, lossis 24 teev, ntxuav ob zaug nrog PBS, thiab incubated nrog 10 μM dichlorofluorescein diacetate rau 30 feeb ntawm 37 degree. Cov hlwb tom qab ntawd tau sim sim thiab tshuaj xyuas los ntawm FACSCaliber flow cytometer (BD Biosciences, San Jose, CA, USA). Intracel-cellular ROS theem tau qhia raws li qhov nruab nrab dichloro di-hydro fluorescein fluorescence siv ntawm cov hlwb. Cov lej tau pom yog qhov nruab nrab ntawm peb qhov kev sim ywj pheej.

Immunofluorescent staining

Cov cell loj hlob ntawm cov ntawv npog tau kho nrog 0 μM, 15 μM, 30 μM, 60 μM, lossis 80 μMEchinacosiderau 5 teev, 12 teev, lossis 24 teev thiab tom qab ntawd ntxuav ib zaug nrog PBS, kho nrog 4 feem pua ​​​​paraformaldehyde hauv PBS rau 20 feeb, thiab thaiv hauv TBS nrog 0.1 feem pua ​​Triton X-100 thiab 15 feem pua fetal bovine serum rau 1 teev ntawm chav tsev kub. Cov hlwb ruaj khov tau stained rau 2 teev nyob rau hauv chav tsev kub nrog thawj cov tshuaj tiv thaiv kab mob tiv thaiv 8-oxoG (nas monoclonal anti-8-oxoG, Abcam), 53BP1 (liab anti-53BP1; Bethyl Laboratories, Montgomery , TX, USA), los yog active caspase-3 (Lavbit anti-caspase-3, Bioss, Beijing, People's Republic of China), tom qab los ntawm staining nrog Alexa 488-conjugated luav antimouse ( Abcam) los yog Cy3-conjugated tshis tiv thaiv luav (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) cov tshuaj tiv thaiv thib ob rau 1 teev ntawm chav tsev kub. Tom qab ntxuav hauv PBS 3 zaug rau 5 feeb, cov ntawv npog tau muab ntim rau ntawm iav slides hauv VECTASHIELD Mounting Medium nrog DAPI (Vector Laboratories). Cov duab tau coj los ntawm Zeiss LCM 510 confocal microscope.

Kev ntsuam xyuas Colony

Hnub ua ntej kev kho mob, cov hlwb tau muab noob rau hauv ib lub phaj rau lub qhov dej ntawm qhov concentration ntawm 1 × 104 hlwb rau ib qhov dej. Tom qab ntawd lawv tau kho nrog 0 μM, 60 μM, lossis 80 μM Echinacoside rau 7 hnub. Tom qab ntxuav nrog PBS, cov hlwb raug kho nrog dej khov-txias methanol thiab stained nrog crystal violet tov (Sigma Aldrich) (0.5 feem pua ​​​​ntawm 25 feem pua ​​​​methanol). Cov duab tau yees duab tom qab ziab daim hlau ib hmos. Cov siv lead ua violet tau yaj los ntawm kev ntxiv 70 feem pua ​​​​ethanol, thiab absorbance ntawm 595 nm yog ntsuas los ntawm BioRad 680 microplate nyeem. Cov ntaub ntawv tau txheeb xyuas siv GraphPad Prism software.

MTT kev xeem

Ib hnub ua ntej kev kho mob, cov hlwb tau muab noob rau hauv 96- cov phiaj zoo ntawm qhov concentration ntawm 1 × 104 hlwb rau ib qhov dej, ua raws li kev kho mob nrog serial dilutions ntawm Echinacoside rau 24 teev lossis 48 teev, lossis nrog 60 μM Echinacoside rau 5 teev. , 12 teev, lossis 24 teev. Qhov nruab nrab raug tshem tawm, thiab cov hlwb raug ntxuav nrog PBS, thiab tom qab ntawd, 20 μL ntawm 3-(4,5-dimethylthiazol-2-yl)-2,

5-diphenyl tetrazolium bromide (MTT) tov (5 mg/mL hauv PBS, pH 7.2) (Thermo Fisher Scientific) tau ntxiv rau txhua qhov dej. Cov phiajcim tau muab tso rau ntawm 37 degree rau lwm 4 teev. Thaum kawg, cov tshuaj MTT raug tshem tawm thiab 150 μL ntawm DMSO tau ntxiv rau txhua qhov dej. Cov phiajcim tau muab tso rau ntawm lub phaj shaker rau 10 feeb, thiab absorbance ntawm 570 nm yog ntsuas los ntawm BioRad 680 microplate nyeem. Txhua qhov kev sim tau ua ob zaug hauv triplicate. Cov ntaub ntawv tau txheeb xyuas siv GraphPad Prism software. IC50 qhov tseem ceeb tau txiav txim siab los ntawm kev txheeb xyuas qhov tsis sib xws.

Kev txheeb xyuas lub voj voog ntawm tes

Cells nyob rau hauv 6-zoo daim hlau raug kho nrog 0 μM, 6{{10}} μM, los yog 80 μM Echinacoside rau 24 teev, sau nrog trypsin (Life Technologies, Carlsbad, CA, USA) , ntxuav ob zaug nrog PBS, thiab tom qab ntawd kho nrog dej khov-txias ethanol (70 feem pua) rau 2 teev ntawm −20 degree. Tsau hlwb raug ntxuav ob zaug hauv qhov txias PBS thiab rov ua dua tshiab hauv 300 μL ntawm PBS tshiab tshiab nrog 0.1 feem pua ​​Triton X-100, 0.2 mg/mL DNase-free RNase A (Sigma Aldrich), 10 ug/mL propidium iodide (PI (Hoffman-La Roche Ltd., Basel, Switzerland). Tom qab incubation ntawm 37 degree nyob rau hauv qhov tsaus ntuj rau 20 feeb, lub hlwb tau lim los ntawm ib tug Falcon nylon mesh (BD Biosciences), loaded mus rau lub FAC-Calibur flow cytometer, thiab soj ntsuam siv ModFit software (BD Biosciences).

DNA fragmentation tsom xam

Cells loj hlob nyob rau hauv rau-zoo daim hlau tau kho nrog 0 μM, 60 μM, los yog 80 μM Echinacoside rau 24 teev thiab tsau nyob rau hauv cov dej khov-txias 4 feem pua ​​paraformaldehyde (Sigma-Aldrich) rau 20 feeb. Tom qab ntxuav nrog PBS, cov hlwb raug kaw hauv VECTASHIELD Mounting Medium nrog DAPI (Vector Laboratories). Nuclear morphology tau yees duab nrog Olympus fluorescent microscope. Txhawm rau txheeb xyuas DNA tawg ntawm agarose gels, DNA tau muab rho tawm siv QIAamp DNA micro cov khoom siv (Qiagen NV, Venlo, Netherlands), thiab electrophoresis tau ua ntawm 2 feem pua ​​agarose gels uas muaj 0.1 ug / mL ethidium bromide.

Kev tsom xam ntawm apoptosis los ntawm kev ntws cytometry

Cov hlwb Apoptotic tau kuaj xyuas siv cov khoom siv Annexin V-FITC Apoptosis kuaj pom (Zoo tshaj, Shanghai, Tib Neeg Lub Tebchaws ntawm Tuam Tshoj), ua raws li cov chaw tsim khoom cov lus qhia. Cells loj hlob nyob rau hauv 96- cov phiaj zoo tau kho nrog 0 μM, 15 μM, 30 μM, 60 μM, 80 μM, lossis 160 μM Echinacoside rau 2 teev, 5 teev, 12 teev, lossis 24 teev, ntxuav ob zaug nrog PBS, thiab tom qab ntawd resuspended nyob rau hauv ib tug binding tsis. Tom ntej no, 5 μL ntawm Annexin V-FITC thiab 5 μL ntawm PI (Roche) tau ntxiv ua ntu zus, thiab cov hlwb raug tsim rau 15 feeb hauv qhov tsaus ntuj ntawm chav tsev kub. Cov hlwb raug thauj mus rau FACSCalibur flow cytometer (BD Biosciences), thiab cov ntaub ntawv raug tshuaj xyuas siv Cell Quest software (BD Biosciences).

Western blot tsom xam

Cells loj hlob nyob rau hauv 6-zoo daim hlau tau kho nrog 0 μM, 6{{10}} μM, los yog 80 μM Echinacoside rau 5 teev, 12 teev, los yog 24 teev, ntxuav ob zaug nrog PBS thiab muab tshem tawm ntawm lub phaj siv 100 μL ntawm radioimmunoprecipitation buffer (150 mM NaCl, 1.0 feem pua ​​​​IGEPAL CA-630, 0.5 feem pua ​​sodium deoxycholate, 0.1 feem pua ​​sodium dodecyl sulfate , thiab 50 mM Tris, pH 8.0) (Sigma-Aldrich) muaj 1 mM phenylmethane sulfonyl fluoride. Cov qauv raug centrifuged ntawm 12, 000 × g rau 20 feeb ntawm 4 degree . Protein concentrations nyob rau hauv lub supernatants tau txiav txim los ntawm lub Bradford reagent (Dingguo, Changchun, neeg lub koom pheej ntawm Tuam Tshoj). Cov qauv raug denatured ntawm 95 degree rau 10 feeb thiab sib cais ntawm 12 feem pua ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Tom qab electrophoresis, cov proteins raug xa mus rau polyvinylidene fluoride daim nyias nyias (Millipore), tom qab ntawd thaiv hauv Tris-buffered saline nrog Tween 20 (10 mM Tris, pH 7.5; 100 mM NaCl; 0.1 feem pua ​​Tween 20) muaj 5 feem pua ​​(w/v) mis nyuj tsis muaj rog rau 1 teev ntawm chav tsev kub. Blots tau soj ntsuam nrog thawj cov tshuaj tiv thaiv p21 (Abcam), poly (ADP-ribose) polymerase (Bioss), lossis activated caspase-3 (Bioss), tom qab ntawd los ntawm horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Cov teeb liab tau tsim los siv cov khoom siv kho kom zoo chemiluminescence Western blotting nrhiav kom pom (Trans gen Biotech, Changchun, Tib Neeg Lub Tebchaws ntawm Tuam Tshoj). Cov kev sim tau rov ua dua peb zaug.

Kev ntsuas ntawm mitochondrial

membrane muaj peev xwm

Mitochondrial membrane muaj peev xwm ntsuas tau siv JC {{0}} dye (Biotechnology), ua raws li cov chaw tsim khoom cov lus qhia. Cells loj hlob nyob rau hauv 6-zoo daim hlau raug kho nrog 0 μM, 60 μM, los yog 80 μM Echinacoside rau 5 teev, 12 teev, los yog 24 teev, ntxuav ob zaug hauv PBS thiab ces incubated nrog JC -1 rau 20 feeb. Cov duab tau muab coj los siv Olympus fluorescent microscope.

Kev txheeb cais

Kev txheeb cais tau ua tiav siv GraphPad Prism Software. Qhov tseem ceeb tau suav nrog kev txheeb xyuas ib-txoj kev ntawm qhov sib txawv, thiab P0.05 tau suav tias yog qhov tseem ceeb. Cov txiaj ntsig tau qhia tias txhais tau tias ± tus qauv sib txawv.

Cov txiaj ntsig

Kev txheeb xyuas ntawm Echinacoside raws li MTH1 inhibitor

Txhawm rau tshawb nrhiav cov khoom xyaw ntuj uas muaj peev xwm inhibiting MTH1, peb tau siv MTH1-catalyzed enzymatic cov tshuaj tiv thaiv raws li kev ua haujlwm siab hauv Vitro tshuaj ntsuam xyuas. Dhau li ntawm oxidized purine nucleotides, MTH1 tseem tuaj yeem ua rau lub cev deoxyguanosine triphosphate los tsim deoxyguanosine monophosphate thiab pyrophosphate. Nyob rau hauv lub xub ntiag ntawm ntau inorganic pyrophosphatase, tag nrho cov pyrophosphates tau hloov mus rau hauv inorganic phosphates, uas muaj peev xwm yuav quantified siv ib tug malachite ntsuab-raws li absorbance assay, 41 yog li tso cai rau indirect ntsuas ntawm MTH1 kev ua ub no.32 Rau kev ntsuam xyuas qhov system, peb thawj zaug ntsuas qhov. Cov nyhuv ntawm (S)-crizotinib, ib qho kev sib xyaw nrog cov pov thawj muaj zog raws li MTH1 inhibitor.33 Cov txiaj ntsig tau pom tias (S)-crizotinib tiag tiag muaj peev xwm inhibited MTH1 (Daim duab 1A) nrog IC50 ntawm 500 nM, uas yog xya npaug siab tshaj qhov tau tshaj tawm. los ntawm Huber et al (72 nM).33 Qhov no yuav yog vim qhov sib txawv ntawm txoj kev tshawb nrhiav los yog qhov chaw ntawm cov tshuaj thiab enzymes. Peb tau txais yuav ib txoj hauv kev uas tsis tshua muaj chromogenic, thaum Huber li al siv txoj kev bioluminescent rhiab heev.33 Ua raws li qhov no hauv siab, peb tau tshuaj xyuas 12 cov khoom lag luam muaj nyob hauv ntuj (Table S1), txhua qhov yog cov khoom tseem ceeb ntawm cov tshuaj ntsuab uas muaj peev xwm antitumor. kev ua si. Echinacoside, ib qho tshuaj purified los ntawm cov kab mob parasitic medicinal cog Cistanche salsa (Daim duab 1C), cuam tshuam cov tshuaj tiv thaiv (Daim duab 1B), nrog rau IC50 ntawm 7.01 ± 2.13 μM (Daim duab 1D). Ntxiv 50 zaug ntau pyrophosphatase tsis muaj kev cuam tshuam rau qhov tshwm sim thaum ntxiv tsib zaug ntau dua MTH1 protein ua rau txo qis ntawm qhov inhibition, qhia tias Echinacoside tshwj xeeb inhibited qhov kev ua ntawm MTH1 nyob rau hauv vitro enzymatic assay.

Echinacoside inhibited cellular MTH1 kom nce intracellular 8-oxoG

Tom ntej no, peb nug yog tias Echinacoside tuaj yeem cuam tshuam kev ua haujlwm ntawm MTH1 intracellular. Inhibition ntawm cellular MTH1 yuav nce intracellular 8-oxoG. Avidin tau pom tias khi rau 8-oxoG nrog qhov tshwj xeeb; 42 yog li ntawd, peb siv cov tshuaj tiv thaiv kab mob nrog Cy3-conjugated avidin los sib piv intracellular 8-oxoG qib hauv ntau cov kab mob qog noj ntshav ua ntej thiab tom qab kho Echinacoside. Tib neeg MG-63 osteosarcoma, SK-HEP-1 hepatocarcinoma, MCF{{10}} mob qog noj ntshav mis, thiab SW480 cov qog nqaij hlav hauv plab tau kho nrog 0 μM, 15 μM, 30 μM, 60 μM, lossis 80 μM Echinacoside rau 5 teev, 12 teev, lossis 24 teev. Staining nrog Cy3-conjugated avidin qhia tias kev kho mob nrog 60 μM Echinacoside rau 24 teev kom meej meej thiab nce qib ntawm cellular 8-oxoG (Cy3-avidin reactive substance) hauv cov qog nqaij hlav cancer no ( Daim duab 2A thiab B). Cov concentration siab dua (80 μM) ntawm Echinacoside ua rau muaj zog cellular 8-oxoG staining (Daim duab 2B), qhia txog kev sib raug zoo ntawm koob tshuaj. Cov txiaj ntsig zoo sib xws tau txais los ntawm immunofluorescent staining nrog nas monoclonal anti-8-oxoG antibody (Daim duab S1A).43 Qhov siab ntawm Echinacoside (60 μM) xav tau rau kev nce ntxiv hauv cellular 8-oxoG ntau dua. IC50 (7.01 μM) los ntawm kev soj ntsuam hauv vitro. Qhov no yuav yog vim qhov sib txawv ntawm qhov rhiab heev ntawm ob qhov kev ntsuam xyuas. Immunofluorescent staining yog deb tsawg rhiab tshaj li qhov kev ntsuam xyuas hauv vitro enzymatic; Tsis tas li ntawd, tus naj npawb ntawm inhibitor molecules uas tuaj yeem ncav cuag thiab cuam tshuam nrog cellular MTH1 yog cuam tshuam los ntawm cov txheej txheem lom neeg; Tsis tas li ntawd, hauv kev ntsuam xyuas hauv vitro, qhov tsis nyiam dGTP yog siv los ua cov substrate, thiab yog li nws yuav yooj yim dua (siv cov inhibitors tsawg) los inhibit MTH1, uas ua rau qis dua IC50 hauv qhov kev ntsuam xyuas hauv vitro.

Cellular {{0}}oxoG yog tsim los ntawm ROS, uas yog antagonized los ntawm antioxidants. Yog li, kev nce hauv cellular ROS lossis kev tawm tsam ntawm kev ua haujlwm ntawm cov tshuaj antioxidants kuj yuav ua rau muaj kev nce hauv cellular 8-oxoG qib. Echinacoside nws tus kheej yog ib qho muaj zog antioxidant44,45 thiab yog li yuav tsum txhim kho es tsis txhob txwv cov kev ua ntawm antioxidants. Txhawm rau tshuaj xyuas seb qib cellular ROS tau hloov pauv los ntawm kev kho Echinacoside, tib lub qog qog noj ntshav tau kho nrog 0 μM, 15 μM, 30 μM, 60 μM, lossis 80 μM Echinacoside rau 5 teev, 12 teev, lossis 24 teev thiab tom qab ntawd txheeb xyuas los ntawm ntws. cytometry tom qab staining nrog fluorescent sojntsuam 2′,7′-dichlorofluorescein diacetate. Cov txiaj ntsig tau pom tias tsis muaj ib qho ntawm cov kev kho mob no hloov cellular ROS theem (Daim duab 2C thiab D). Yog li, qhov nce cellular 8- qib oxoG feem ntau yuav tshwm sim los ntawm inhibition ntawm MTH1 los ntawm Echinacoside.

Echinacoside ua rau muaj kev puas tsuaj ntau DNA tshwj xeeb hauv cov qog nqaij hlav cancer Kev koom ua ke ntawm {{0}}oxoG rau hauv DNA yuav txhawb nqa BER thiab MMR, uas tsim ib leeg-strand DNA so (SSB) thiab qhov khoob.16,17 Nquag, cov kev so no, thiab qhov khoob khoob tau raug kaw los ntawm kev kho cov txheej txheem uas muaj qhov sib txawv ntawm qhov sib txawv thiab ligation los ntawm DNA polymerases thiab ligases.46,47 Txawm li cas los xij, kev nce sai hauv cov cellular SSB tuaj yeem ua kom cov cellular kho muaj peev xwm, uas ua rau tsim ntau ntau ob-strand DNA so (DSB) 18 thiab DDR signaling. Txhawm rau tshuaj xyuas yog tias Echinacoside-induced nce hauv cellular 8-oxoG ua rau DNA puas, peb tau tshuaj xyuas ib qho ntawm cov cim thaum ntxov ntawm DNA puas, 53BP1, uas khi rau qhov chaw ntawm DSB raws li cov lus teb ntxov tshaj plaws ntawm tes rau DSB.48 Lub MG -63, SK-HEP-1, MCF-7, thiab SW480 kab mob qog noj ntshav thiab cov kab mob qog noj ntshav tsis yog tib neeg lub siab L-O2,49 tib neeg lub raum embryonic HEK 293, thiab nas fibroblast NIH /3T3 tau kho nrog 0 μM, 15 μM, 30 μM, 60 μM, lossis 80 μM Echinacoside rau 5 teev, 12 teev, lossis 24 teev. Fluorescent immunostaining qhia tau hais tias kev kho mob nrog 60 μM Echinacoside rau 24 teev tshwj xeeb nce cov cell nrog tsib los yog ntau dua stained nuclear 53BP1 foci nyob rau hauv tag nrho cov mob qog noj ntshav tab sis tsis nyob rau hauv ib qho ntawm cov kab mob qog noj ntshav (Daim duab 2E thiab F). Ib qho kev nce ntxiv ntawm 53BP1 ntxiv rau cov hlwb tau pom thaum ntxov li 5 teev tom qab pib kho Echinacoside (Daim duab 2F). Qhov siab dua (80 μM) ntawm Echinacoside lossis lub sijhawm kho mob ntev dua (12 teev thiab 24 teev) ua rau muaj kev nce ntxiv hauv ob qho tib si ntawm 53BP1 ntxiv rau cov hlwb (Daim duab 2F) thiab cov xov tooj ntawm tes 53BP1 foci. Ua ke, cov txiaj ntsig no qhia tias inhibition ntawm MTH1 los ntawm Echinacoside ua rau 8-oxo tsub zuj zuj thiab ntau DNA puas tsuaj hauv cov qog noj ntshav tab sis tsis nyob hauv cov hlwb uas tsis yog mob cancer.

Echinacoside suppressed mob qog noj ntshav cell proliferation

Nyob rau hauv cycling hlwb, unrepaired DNA strand so yuav ua rau lub cev qhuav dej ntawm DNA replication diav rawg, uas yuav ua rau blockage ntawm cell proliferation los ntawm induction ntawm cell voj voog ntes thiab apoptosis.50 Txhawm rau kuaj xyuas cov teebmeem ntawm Echinacoside ntawm kev loj hlob ntawm tes thiab kev loj hlob, peb ua ntej. txheeb xyuas qhov kev loj hlob ntawm Echinacoside-kho MG-63, SK-HEP-1, MCF-7, thiab SW480 cov qog nqaij hlav qog noj ntshav los ntawm kev tshuaj ntsuam xyuas colony. Cov txiaj ntsig tau pom tias cov kab mob qog noj ntshav tau kho nrog 60 μM lossis 80 μM Echinacoside tsim muaj tsawg dua, thiab cov cheeb tsam uas tsim muaj tsawg dua (Daim duab 3A thiab B), qhia tau hais tias muaj kev cuam tshuam loj heev. Tom ntej no, peb txheeb xyuas qhov kev loj hlob ntawm cov qog nqaij hlav cancer no, ua ke nrog L-O2, HEK 293, thiab NIH/3T3 cov hlwb uas tsis yog qog noj ntshav los ntawm kev soj ntsuam MTT. Cov txiaj ntsig tau pom tias Echinacoside koob tshuaj-raws li inhibited kev loj hlob ntawm cov qog nqaij hlav tab sis tsis yog cov qog nqaij hlav cancer (Daim duab 3C). Kev tshawb fawb txog lub sij hawm ntawm cov qog nqaij hlav cancer kho nrog 60 μM Echinacoside pom tias tom qab 5 teev, tsis muaj qhov sib txawv tseem ceeb ntawm cov pab pawg tsis kho thiab kho, tab sis tom qab 12 teev, kev loj hlob ntawm cov qog nqaij hlav cancer tau cuam tshuam loj heev los ntawm 60 μM Echinacoside (Daim duab 3D).

Echinacoside induced cell voj voog ntes

Raws li kev soj ntsuam ntawm kev loj hlob ntawm tes thiab kev loj hlob, Western blot tsom xam pom tias 24 teev kev kho mob nrog 60 μM thiab 80 μM Echinacoside nce qib protein ntau ntawm G1 / S-CDK blocker thiab DNA synthesis inhibitor p21Cip / WAF1 hauv MG-63, SK-HEP-1, MCF-7, thiab SW480 qog nqaij hlav cancer (Daim duab 4A thiab B). Ntxiv mus, zoo ib yam li 53BP1, qhov nce ntxiv ntawm p21 protein ntau tau pom thaum ntxov li 5 teev tom qab pib kho Echinacoside (Daim duab 4B). Kev soj ntsuam ntawm cov xov tooj ntawm cov theem sib txawv ntawm lub voj voog ntawm tes los ntawm kev khiav cytometry qhia tias Echinacoside kho koob tshuaj-raws li txo cov feem pua ​​​​ntawm cov hlwb hauv ob theem S thiab G2 / M, thaum feem pua ​​​​ntawm cov hlwb hauv G1 theem nce (Daim duab 4C. thiab D). Tom qab 80 μM Echinacoside kev kho mob ntawm MG-63 cov hlwb rau 24 teev, cov hlwb hauv G2 / M theem tau txo qis los ntawm 15 feem pua ​​​​mus rau zero, thiab cov hlwb hauv S theem txo qis los ntawm 37 feem pua ​​​​rau 17 feem pua, thaum cov hlwb hauv G0 / 1 theem nce ntawm 43 feem pua ​​​​rau 80 feem pua ​​(Daim duab 4E). Cov txiaj ntsig no qhia tau hais tias kev kho mob nrog Echinacoside ua rau lub voj voog ntawm tes raug ntes thiab thaiv cov qog nqaij hlav cancer hauv theem G1.

Echinacoside induced apoptosis hauv cov qog nqaij hlav cancer

Raws li kev soj ntsuam ntawm cell proliferation, Western blot tsom xam pom tias kev kho mob nrog 60 μM thiab 80 μM Echinacoside rau 24 teev nce qib ntawm cov nquag ua haujlwm caspase-3 thiab cleaved poly (ADP-ribose) polymerase proteins hauv cov qog nqaij hlav cancer (Daim duab 5A thiab B), tawm tswv yim induction ntawm caspase-dependent apoptosis. Tom qab ntawd peb tshuaj xyuas ob peb lub xov tooj ntawm tes ntawm apoptosis. Ua ntej, nuclear morphology ntawm Echinacoside-kho cov qog nqaij hlav cancer tau tshwm sim los ntawm staining nrog DNA dye DAPI, uas pom tau tias 24 teev kev kho mob nrog 60 μM thiab 80 μM Echinacoside nce tus naj npawb ntawm cov cim ntawm apoptosis, suav nrog pyknosis thiab condensed chromatin (ci dua nuclei. ) (Daim duab 5C). Thib ob, DNA muab rho tawm los ntawm cov kab mob qog noj ntshav zoo sib xws tau txheeb xyuas los ntawm electrophoresis ntawm agarose gel, uas qhia txog tus qauv apoptotic ntaiv (Daim duab S1B). Thaum kawg, cellular active caspase{13}} tau kuaj pom los ntawm immunofluorescent staining, uas qhia tau hais tias muaj zog activated caspase{14}} cov cim hauv Echinacoside-kho cov qog nqaij hlav cancer (Daim duab S1C). Tom ntej no, qhov feem pua ​​​​ntawm cov hlwb apoptotic tau ntsuas los ntawm Annexin V-FITC thiab PI ob chav staining thiab ntws cytometry. Kev soj ntsuam ntawm cov hlwb kho nrog 0 μM, 15 μM, 30 μM, 60 μM, 80 μM, lossis 160 μM Echinacoside rau 2 teev, 5 teev, 12 teev, lossis 24 teev pom tias 60 μM lossis siab dua ntawm Echinacoside induced ib qho tseem ceeb. lub MG-63, SK-HEP-1, MCF-7, thiab SW480 cov qog nqaij hlav cancer (Daim duab 6A), tab sis tsis nyob hauv cov qog nqaij hlav L-O2, HEK 293, thiab NIH/3T3 hlwb (Daim duab S2). Tom qab kev kho mob ntawm MG-63 hlwb nrog 60 μM, 80 μM, lossis 160 μM Echinacoside rau 24 teev, feem pua ​​​​ntawm cov hlwb apoptotic tau nce los ntawm 8.89 feem pua ​​​​rau 33.72 feem pua, 39.01 feem pua ​​, thiab 48.12 feem pua ​​​​, raws li 6A (Fig) . Kev tshawb fawb lub sij hawm qhia tau hais tias tom qab 5 teev, tsis muaj qhov sib txawv ntawm cov pab pawg tsis kho thiab kho, tab sis qhov tseem ceeb apoptosis tau pom tom qab kho nrog 60 μM lossis siab dua ntawm Echinacoside rau 12 teev (Figures 6B thiab S3).

Thaum kawg, kev ntsuas ntawm mitochondrial membrane muaj peev xwm los ntawm JC -1 fluorescent dye kom meej meej pom qhov poob ntawm mitochondrial membrane muaj peev xwm tom qab kev kho mob nrog 60 μM Echinacoside rau 12 teev (Daim duab 6C), tab sis tsis yog tom qab 5 teev, qhia txog kev ua haujlwm ntawm txoj hauv kev apoptosis nyob rau hauv raws li DNA puas tsuaj los ntawm elevated 8-oxodG.