In Vitro And In Vivo Studies Of Resveratrol And Its Nano-Targeted Particles On Docetaxel/Doxorubicin-Sensitive And Resistant Anaplastic Thyroid Cancer

Background:Anaplastic thyroid cancer(ATC)is a rare type of thyroid cancer with extremely aggressive ability,with a median survival of only 6 months and a 1-year survival of 20%.Since about 70%of patients are diagnosed with distant metastases,chemotherapy is preferred.However,the dedifferentiated state of ATC makes it insensitive to chemotherapy.At present,docetaxel combined with doxorubicin is the first-line chemotherapy drugs for the treatment of ATC.Both of them not only cause serious adverse reactions,but also induce acquired drug resistance,resulting in treatment failure.Resveratrol,a natural polyphenolic compound,has a role in differentiation and inhibit tumor cell proliferation and demethylation.These points have also been demonstrated in ATC in vitro,however,the effect of resveratrol in vivo of ATC and its efficacy on acquired drug resistant ATC cells have not been reported.In view of this,the following studies were conducted:(1)To test the differentiation of resveratrol in ATC.(2)Establish a model of subcutaneous tumor in nude mice with THJ-16T ATC cell line.And resveratrol and docetaxel/doxorubicin were intraperitoneally injected to observe the efficacy of the drugs in vivo.(3)The cells of transplanted tumor were cultured in 2D and 3D when docetaxel/doxorubicin resistance was acquired,and the sensitivity to resveratrol was analyzed.(4)Since IL-13R?2 is highly expressed in thyroid cancer tissues and low in paracancer normal tissues,it is considered as a target protein for thyroid cancer.Pep-1 is a short peptide chain consisting of 9 amino acids(CGEMGWVRC)of IL-13 protein,which has the specific ability to bind IL-13R?2.To address the low bioavailability of resveratrol in vivo,the anti-ATC effect of Pep-1-PEG_3.5k-PCL_4k@Res targeting nanoparticles was prepared and verified in vivo.(5)Next generation sequencing(NGS)was used to compare the genomic differences between THJ-16T and THJ-16T/R cells,revealing the genetic background leading to secondary drug resistance.Materials and Methods:In this study,ATC THJ-16T,THJ-21T and normal thyroid Nthy-ori 3-1 cell lines were selected as research objects.MTT and Ed U were used to analyze the effect of resveratrol on cell proliferation.Cell apoptosis was detected by TUNEL staining.Sodium/iodide symporter/NIS,phosphate and tension homology deleted on chromosome ten/PTEN and E-cadherin gene and protein expression were analyzed by RT-PCR and Western Blotting,and we further used ICC and IF to analyze the changes of their intracellular distribution.Meanwhile,the ATC THJ-16T cell line was used to establish subcutaneous tumor nude mice model,and then resveratrol,Pep-1-PEG_3.5k-PCL_4k@Res targeted nanoparticles,docetaxel combined with doxorubicin were administrated through intraperitoneal injection,respectively.The apoptosis of tumor tissue in the control group and the targeted nanoparticles treatment group was detected by TUNEL staining.Furthermore,the transplanted tumor cells in the chemotherapy group were further isolated and cultured in 2D and 3D(named THJ-16T/R),and the sensitivity differences of docetaxel/doxorubicin and resveratrol between the original THJ-16T cell lines were analyzed by living dead cell count,TUNEL,Ed U,and HE staining.Finally,NGS was used to compare the genomic differences between THJ-16T and THJ-16T/R.Functional enrichment and signal pathway enrichment analysis of specific mutant gene sites in THJ-16T/R were carried out by DAVID database.Further,we screen gene mutation sites associated with acquired drug resistance according to the classification criteria and guidelines of ACMG genetic variation and literature review,and the mutation pathogenicity was predicted.Results:1.The cell morphology was significantly changed in 100?M resveratrol treated ATC THJ-16T and THJ-21T cells compared with the control group(0.1%DMSO treatment group).Cell proliferation was inhibited(59.16%vs 2.98%,THJ-16T,p<0.01;47.35%vs 4.74%,THJ-21T,p<0.01),and cell apoptosis was induced in both cell lines(59.16%vs 2.98%,THJ-16T,p<0.01;47.35%vs 4.74%,THJ-21T,p<0.01).In contrast,Res-treated Nthy-ori 3-1 cells showed a 5.1%decrease in comparison with DMSO-treated group(p>0.05)without distinct morphological change.2.PTEN protein was overexpressed and nuclear translocation occurred together with NIS protein,and E-cadherin protein showed obvious cell membrane expression after resveratrol treatment.3.In vivo experimental results showed that compared with the control group and the free resveratrol group,the docetaxel/doxorubicin group and Pep-1-PEG_{3.5 k}-PCL_4k@Res targeted nanoparticles group could significantly slowed tumor growth(p<0.01),and the results of HE and TUNEL staining showed that targeted nanoparticles could significantly induce the apoptosis of tumor cells.4.Two thirds of the tumor-bearing nude mice in the chemotherapy group lost more than10%of their body weight during the treatment,resulting in forced withdrawal of the drug.And the subcutaneous tumor of the nude mice in the chemotherapy group regrew after 1 week of withdrawal of the drug.5.After drug treatment,the proportion of dead cells in THJ-16T/R chemotherapy group was significantly lower than that in the THJ-16T corresponding treatment group,and the Ed U positive cells in THJ-16T/R chemotherapy treatment group was significantly higher than that in THJ-16T group.6.NGS revealed a total of 307 mutation genes in THJ-16T cell lines,and a total of 887mutation sites,while 302 mutation genes in THJ-16T/R cell lines,including 852 mutation sites.7.There are 40 specific mutation sites in THJ-16T/R cell lines,and GO function enrichment of these specific mutation sites was mainly in cell proliferation,apoptosis,drug response,while KEGG pathway analysis is mainly in drug metabolism and cancer-related signaling pathway.8.Predicted pathogenicity of drug resistance-related gene mutation sites results showed:KMT2D-c.8785C>A is a benign mutation and ABCB1-c.2871C>G is a pathogenic mutation which both discovered in THJ-16T cell line.And the 4 mutation sites in the THJ-16T/R cell line(MKRN1-BRAF fusion mutation(M3:B10);KMT2D-c.8339C>T;PDGFRB-c.1042G>T;CDH1-c.2359G>A)were all pathogenic mutations.Conclusion:1.PTEN expression was up-regulated(the mechanism may be caused by resveratrol demethylation)and nuclear translocation occurred together with NIS protein in Res-suppressed ATC cells.2.The concurrent nuclear translocation of PTEN and NIS may indicate the better therapeutic effect of ATC and could be used as a prognostic indicator of ATC.3.Resveratrol sustained-release targeting nanoparticles instead of free resveratrol can effectively inhibit the growth of ATC in vivo.4.Docetaxel/doxorubicin can effectively inhibit the growth of ATC subcutaneous tumor in nude mice,but it will cause obvious side effects and acquired drug resistance.5.Resveratrol reversed docetaxel/doxorubicin resistance in vitro,suggesting that resveratrol may be an alternative to acquired chemotherapy resistance ATC cells.6.The acquired drug resistance of THJ-16T/R cells may be related to the following gene mutation sites:MKRN1-BRAF fusion mutation(M3:B10),KMT2D-c.8339C>T,PDGFRB-c.1042G>T and CDH1-c.2359G>A;and the ABCB1-c.2871C>G mutation site was drug sensitivity of THJ-16T cell line.However,combined with the mutation abundance of each site,we believed that MKRN1-BRAF(M3:B10)fusion mutation and KMT2D-c.8339C>T are the main mechanisms for THJ-16T/R chemoresistance.