| Background;Glioblastoma is the most common and vital primary brain tumor,and the most aggressive glioma,a collection of tumors arising from glial cells.It is also termed glioblastoma multiforme(GBM)because of its complex phenotype.The life expectancy of patients with glioblastoma multiforme(GBM),the most malignant glioma(World Health Organization WHO grade IV),using the current standard treatment is on average 15 months,after diagnosis despite aggressive surgery,radiation,and chemotherapies.The specificity of the brain is another reason for the high recurrence rate of glioblastoma,because the surgery principle for glioblastoma is to remove the tumor as much as possible and without any aggravating neurological dysfunction.Equivalent to maximally removing the tumor under a safe condition.However,the result is that the high invasiveness of glioblastoma and the limitation of the surgical resection make it facility to leave a certain number of tumor cells after surgery,which is a hidden danger for the future recurrence.This situation shows a serious challenge to postoperative adjuvant therapy to prevent tumor recurrence.During these recent years,resveratrol,a polyphenolic compound which found in plants,including polygonum cuspidatum,mulberry and grape,has beneficial biological effects such as prevention of tumors,cardiovascular and cerebrovascular diseases,anti-inflammatory and anti-oxidation.It has been found that resveratrol can effectively inhibit the growth of various tumor cells including glioblastoma cells in vitro,which inducing differentiation and apoptosis.However,different glioblastoma cell lines have different drug sensitivity for some unknown reason.Exosomes are a kind of disc-shaped microvesicles with a diameter between 30-200 nm,which are actively secreted by living cells into the body,and it contains a variety of complex RNAs and proteins.It could mediate cell-to-cell communication and migration,which is closely linked to tumor cell growth.Previous research found that tumor cells secrete a large number of exosomes carrying malignant transformation-related proteins,which is presumed to be closely related to the drug sensitivity on tumor cells.In view of the collection of the above relevant data,this study suggests that exosomes may be a potential reason in determining the resveratrol sensitivity difference in glioblastoma.In the end,the target of this experiment is:(1)Exploring the sensitivity different between U251 and LN428 glioblastoma cells to resveratrol.(2)Electron microscopy and Western-blotting were used to identify exosomes obtained by ultracentrifugation.(3)Exosome secretion levels of U251 and LN428 cells before and after resveratrol treatment were detected.(4)To explore the changes in the resveratrol sensitivity after co-culture tumor exosomes with U251 or LN428 cells.(5)To detect the expression levels of exosome proteins in U251 and LN428 cells before and after resveratrol treatment;and to explore the protein regulatory factors that cause the resveratrol sensitivity difference in glioma cells.(6)Explore the drug-sensitive regulatory mechanisms through bioinformatics analysis of protein regulatory factors.Method:A pair of resveratrol-sensitive and resistant human glioblastoma cell lines,U251 and LN428 were used as the research object.The sensitivity of resveratrol to these two glioblastoma cell lines was studied by MTT.The drug.Morphological changes with apoptosis was observed by H&E staining after treatment with resveratrol.At the same time,the exosomes in U251 and LN428 cell supernatants were extracted by ultracentrifugation before and after resveratrol administration,and the exosomes were also identified by electron microscopy and Western-blotting.Quantitative analysis of exosomes in each group was performed using nanoparticle tracking analysis to detect the exocrine of exosome.LN428 cells were co-incubated with U251/N or U251/Res exosomes for 48 h,then treated with 100?mol/L resveratrol for more 48 h;Vise versa,U251 cells were co-incubated with LN428/N or LN428/Res exosomes for 48 h,then treated with 100?mol/L resveratrol for more 48 h,and the cell viability in each group was detected by MTT assay.These two groups are used to explore the effects of exosomes on tumor drug sensitivity.In order to further explore the difference in sensitivity of different human glioblastoma to resveratrol,the total protein of exosomes was quantified by BCA,and then the exosomes proteins were qualitatively and quantitatively determined by liquid chromatography-tandem mass spectrometry.The changes of various indexes before and after drug treatment were analyzed,helping to find the factors causing drug sensitivity or tolerance.The function and molecular mechanism were predicted by GO enrichment analysis and KEGG bioinformatics analysis,and their correlation and influence were summarized latter.Results:1.After treating with resveratrol,the U251 cell shrinks and sheds.The growth and proliferation of U251 cells were significantly inhibited,and the number of cells was greatly reduced(85.7%).After treating with resveratrol,the LN428 cell line showed no change in cell morphology,and the cell numbers was not significantly reduced.MTT cell proliferation assay revealed that OD value(0.310 ± 0.020,cell viability = 50.1%)of resveratrol-treated U251 cells was significantly reduced in comparison with that(0.618 ± 0.103,p < 0.01)of the untreated counterpart;the mean OD values(0.743 ± 0.047)of resveratrol-treated LN428 cells and untreated cells(0.722 ± 0.185,p = 0.375)have no significant different.2.Transmission electron microscopy of exosomes showed the presence of a large number of oval or circular disc-shaped vesicle,which size is between 30-200 nm.Western blot analysis revealed that the exosomes typical protein CD63 was enriched in all exosome samples,while ?-actin is undetectable.These indicate that the exosome from the sample was successful purified without significant remaining cellular protein contamination.3.Nanoparticle tracking analysis of exosomes secreted by glioma cells U251 and LN428 before and after treating with resveratrol,NTA revealed the exosome size distribution is from 30 nm–200 nm,which can be scattered or aggregated.NTA-based exosome quantification showed that,resveratrol promoted exosome release especially in U251 cell in the extent of 415.9%,while exosome of LN428 cells increased by 12.1% respectively.4.In co-culture of exosomes and cells,the result of the MTT assay revealed a reduction of proliferation rates of U251/N/Exo-(OD = 0.624 ± 0.027)rather than U251/Res/Exo-(OD = 0.703 ± 0.047,#,p = 0.043)or phosphate buffered saline(PBS)-pre-incubated LN428(OD = 0.743 ± 0.040,*,p = 0.011)after being treated by resveratrol.For U251 cells,the resveratrol sensitive properties of U251(OD = 0.310 ± 0.020)remained unchanged,irrespective to LN428/N/Exo(0.0.295 ± 0.020,p = 0.145)or LN428/Res/Exo(0.334 ± 0.036,p = 0.173)pre-incubation.5.Using Label-free liquid chromatography-mass spectrometry(LC-MS/MS),it was found that total of 123 types of protein were identified in U251 exosomes,while 216 proteins in LN428 exosomes,respectively.U251/N/Exo have higher keratin(KRT including KRT18,KRT19),H2 A histone family member X levels(H2AX including H2 AFX,HIST1H2AD)and lower Ras-related protein 1(Rap1 includes Rap1 B,Rap1A),polymer filaments form actin levels(F-actin including ACTB,ACTA2)and guanine nucleotide binding proteins(G proteins including GNAS,GNAI).In contrast,the proteomic pattern of LN428/N/Exo is distinct to the proteome pattern of U251/N/Exo,which expressed lower KRT,H2 AX and higher Rap1 and F-actin.The proteomic patterns of U251 and LN428 cells are altered by resveratrol in terms of KRT,H2 AX,Rap1,F-actin and G protein being conversed.In contrast,the proteomic pattern of LN428/N/Exo is distinct to that of U251/N/Exo by showing lower KRT,H2 AX and higher Rap1 and F-actin.The proteomic patterns of U251 and LN428 cells are altered by resveratrol in terms of KRT,H2 AX,Rap1,F-actin and G protein being conversed.6.Based on gene ontology enrichment(GO)analysis,bioinformatics analysis was performed on all exosome-derived proteins.After further quantitative analysis and comparison,it was found that in biological processes,U251/N/Exo tend to have higher expression on chromatin silencing and epidermis development,while U251/R/Exo contains more proteins involved in oxygen transport,G-protein coupled receptor signaling pathway and cellular protein metabolic process.Molecular functional analysis showed that U251/N/Exo focus more on protein heterodimerization activity and DNA binding.U251/Res/Exo expression is higher than U251/N/Exo at certain precision molecular function,such as binding activity(G-protein complex binding and guanyl nucleotide binding)and GTPase activity.Both LN428/N/Exo and LN428/Res/Exo share a number of proteins involved in the key biological processes and molecular function.After screening,the top 5 filtered biological processes were ?nucleosome assembly‘,?microtubule-based process‘,?chromatin silencing‘,?cytoskeleton organization‘,and ?oxygen transport‘.In addition,the most abundant molecular functional activity are the structural components and enzymatic activities of the cytoskeleton(including structural molecular activity,protein heterodimerization activity,GTPase activity and oxygen transport activity).7.Using KEGG signaling pathway enrichment analysis,U251/N/Exo may enhance the drug sensitivity of drug-resistant LN428 cells to resveratrol by regulating Rap1 signaling pathway and necrotic apoptosis pathway.Conclusions:1.Resveratrol can increase the exosome secretion activity in sensitive U251 cells.2.U251/N/Exo can enhance the sensitivity of drug-resistant cell LN428 to resveratrol.3.Resveratrol can increase the level of exosome drug resistance protein in glioma cells regardless of tumor cells‘ drug sensitivity.4.Beyond exosomes,the activation on tumor development-related pathways is more critical,and they determines the fate of resveratrol-treated glioblastoma cells. |
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