Inhibition Effect of Mangiferin on Human Thyroid Papillary Carcinoma TPC-1 Cells and Its Anti-tumor Mechanism

Author:Zhang Lei

Supervisor:fu rong zhan


Degree Year:2019





BackgroundThyroid cancer is a common malignant tumor in the head and neck surgery.Statistics in the past 30 years show that the incidence of thyroid cancer is gradually increasing in the world.For the treatment of thyroid cancer,both domestic and foreign diagnostic and therapeutic guidelines recommend surgery as the main treatment,followed by radiotherapy and thyroid hormone endocrine therapy.Differentiated thyroid carcinoma(DTC)has a low degree of malignancy and a good prognosis.However,some of the poorly differentiated thyroid cancer also showed rapid tumor growth,high incidence of lymphatic metastasis,insensitivity to radiotherapy and chemotherapy,and recurrence and metastasis after surgery.Therefore,it is an important topic to explore the molecular mechanism of tumor pathogenesis and develop new and efficient treatment methods for thyroid malignant tumors.Obtaining small molecular compounds for treating diseases from natural plants has always been an important way to develop anti-tumor drugs.Mangiferin is a natural flavonoid,which is widely found in higher plants.Mangiferin has a wide range of pharmacological effects,and a large number of experiments have confirmed that mangiferin has the effects of inhibiting the central nervous system,anti-virus,anti-oxidation and immune regulation.Mangiferin also exhibits obvious anti-carcinogenic effects and potential value through the intervention and regulation of multiple molecular targets and signaling pathways.According to previous reports,mangiferin has obvious anti-tumor effect on various malignant tumors including brain,cervix,prostate,lung and breast in vivo and in vitro pharmacological studies.Through literature review,we find there are no studies on the application of mangiferin in the treatment of thyroid cancer,and the exact mechanism of the anti-carcinogenic effect of mangiferin is still unclear.This topic takes TPC-1 cells of human papillary thyroid carcinoma as the research object.In the experimental study of cell biological behavior.we found mangiferin can promote cell apoptosis,inhibit cell proliferation and migration.In order to analyze the molecular mechanism of mangiferin’s anti-tumor effect,we rely on the network pharmacological research platform,use reverse screening method to preliminarily predict the potential molecular targets of mangiferin’s pharmacological effect and further optimize and confirm the relevant targets through protein interaction analysis,pathway enrichment and forward molecular docking tools.In the following experiments,we selected molecular targets and signal pathways with high research value for preliminary verification,and elaborated the molecular mechanism of mangiferin-induced apoptosis of thyroid papillary cancer cells.Part One Anti-tumor Activity of Mangiferin on Thyroid Papillary Cancer Cells PurposeMangiferin treatment was used to interfere the growth of TPC-1 cells of thyroid cancer.The effects of mangiferin on promoting TPC-1 cell apoptosis,inhibiting cell proliferation and migration were verified by morphological observation and molecular biological detection.Methods1.Human papillary thyroid cancer cell lines were cultured in vitro.2.The effect of mangiferin on the proliferation of TPC-1 and BCPAP cells in human papillary thyroid carcinoma was detected by MTT assay.3.Transwell cell migration test was used to observe the changes of TPC-1 cell migration function after mangiferin intervention.4.DAPI staining was performed on the nucleus to observe the changes of TPC-1 nucleus morphology after mangiferin treatment,and the effect of mangiferin on cell apoptosis was evaluated from the perspective of cell morphology.5.DNA fragmentation was used to detect the effect of mangiferin treatment on the formation of DNA fragments in TPC-1 cells,and to evaluate the characteristic changes of cell DNA after apoptosis treatment.6.AO/EB double fluorescence staining was used to observe the survival status of TPC-1 cells treated with mangiferin and evaluate the changes of mangiferin-induced apoptosis cells at different stages.7.Western blot was used to detect the expression level of apoptosis-related proteins(Bid,Bax,Bad,Bcl-2,cytochrome C.caspase-3,caspase-9)in TPC-1 cells after mangiferin treatment,and analyze the effect of mangiferin intervention on apoptosis signal pathway.8.Immunofluorescence combined with DAPI counterstaining was used to observe the expression of proliferating cell nuclear antigen(PCNA)in TPC-1 cells treated with mangiferin and evaluate the inhibitory effect of mangiferin on TPC-1 cell proliferation.Results1.Mangiferin treatment significantly inhibited the proliferation of the two cells in a dose-dependent manner.Even low concentrations of mangiferin showed inhibitory effect on cell growth.The half inhibitory concentrations of mangiferin on the two cells were 3.69 μM(TPC-1 cells)and 9.90 μM(BCPAP cells),respectively.TPC-1 cells were more sensitive to mangiferin treatment.2.Transwell cell migration test showed that the migration function of TPC-1 cells was inhibited to different degrees after mangiferin treatment with different concentration gradients,and the inhibition of cell migration function was more obvious at the treatment concentration of 4μM.3.DAPI nuclear staining showed that after mangiferin treatment with different concentration gradient,the nuclear morphology changed to different degrees.Among them,4μM treatment caused typical apoptosis changes of nuclear lysis.At the same time,it can be observed that the number of nuclei in 4μM treatment is significantly less than that in the control group.4.The electrophoresis results of DNA fragments showed that TPC-1 cells showed characteristic DNA gradient formation after treatment with mangiferin at different concentrations,while control cells showed complete DNA bands.5.AO/EB staining showed that TPC-1 cells in both the experimental group and the control group had cell death.However,unlike the staining of necrotic cells,in mangiferin treated group,the staining of apoptotic TPC-1 cells showed bright red or orange.6.Western blot results showed that mangiferin treatment increased the expression level of pro-apoptotic proteins Bid,Bad and Bax in TPC-1 cells and decreased the expression level of anti-apoptotic protein Bcl-2.Meanwhile,mangiferin treatment significantly increased the expression levels of cytochrome C,caspase-9 and caspase-3.7.Immunofluorescence combined with DAPI counterstain showed that mangiferin treatment induced apoptosis and reduced the expression of proliferating cell nuclear antigen(PCNA)in cells.ConclusionMangiferin intervention inhibited the proliferation and migration of TPC-1 cells.Through morphological observation and analysis of TPC-1 cells,mangiferin treatment can induce apoptosis of TPC-1 cells.Molecular biological detection of apoptosis signal pathway shows that mangiferin treatment triggers both exogenous and endogenous apoptosis pathways.In addition,mangiferin also reduces the proliferation level of TPC-1 cells by inhibiting PCNA.The experimental results verify that mangiferin can induce cell apoptosis and inhibit proliferation and migration of thyroid cancer cells,suggesting the anti-carcinogenic potential of mangiferin.Part Two Screening of Anti-tumor Targets of Mangiferin Based on Molecular DockingPurposeThe potential molecular targets of mangiferin pharmacological action were preliminarily predicted by reverse screening method,and the related targets were further optimized and confirmed by protein interaction analysis,pathway enrichment and forward molecular docking tools.Further clarify the molecular mechanism of mangiferin’s anti-tumor effect,and provide reference for drug research and development with mangiferin as the lead compound.Method1.PharmMapper,the pharmacophore screening online server,is used to preliminarily predict the potential targets of mangiferin pharmacological effects,and the scores are sorted from high to low according to the fit score.2.Input mangiferin potential target information into the protein interaction network(PPI)online analysis database String to analyze the interaction of various drug targets and construct a "drug-target-disease”network.3.The key protein targets obtained by PPI analysis are mapped to the disease catalogue treated in KEGG and the main signal pathways involved,and a"component-target-pathway-disease" network is constructed.The enrichment degree of disease pathways is measured by rich factor and P value analysis.4.Download the three-dimensional structure of important target proteins from PDB database,use molecular docking software AutoDock to carry out forward molecular docking between mangiferin and these targets,and calculate the binding free energy of receptor and ligand.Result1.According to the screening results of PharmMapper database,the information of 600 targets of mangiferin was obtained,and 90 potential molecular targets were finally determined according to the Fit score ranking.2.The analysis of protein interaction network showed that 70 target proteins interacted with each other.In the core region of protein interaction network,there are intensive interactions among multiple targets.Analysis of the interaction between mangiferin and potential targets shows that mangiferin pharmacophore can act on many target genes,suggesting the multi-target action characteristics of mangiferin.3.A total of 68 genes,involving 28 related diseases and signal pathways,were screened out in KEGG enrichment analysis.Target genes are highly enriched in mitogen-activated protein kinase pathways and multiple cancer pathogenesis pathways.TGFβ RII,CASPS3,JNK and EGFR are shown to be key nodal proteins in these signal pathways.4.Positive molecular docking results of mangiferin and TGFβ R11,CASPS3,JNK and EGFR show that all four protein targets have certain binding potential with mangiferin,among which JNK has higher binding potential.ConclusionMangiferin has the characteristics of multi-target pharmacological action,and has high binding potential with key node proteins in tumor-related signaling pathways.The calculated simulation angle predicts that mangiferin can reasonably act on these target proteins,suggesting that mangiferin has the structural basis of anti-tumor activity.Molecular docking shows that TGFβ RⅡ,CASPS3,JNK and EGFR may be key targets of mangiferin’s anti-tumor effect.Part Three Mangiferin Targeted JNK Inhibits Proliferation of Thyroid Cancer TPC-1 Cells and Induces ApoptosisPurposeTo further verify the targeted binding effect of mangiferin and JNK protein,and the molecular mechanism of mangiferin anti-tumor effect and the effects of targeted JNK on inhibiting TPC-1 cell proliferation and inducing apoptosis.Method1.The effects of mangiferin and JNK pathway inhibitor SP600125 on the proliferation of TPC-1 cells of human papillary thyroid carcinoma were detected by MTT assay.2.Flow cytometry(FCM)was used to detect the effects of mangiferin and JNK pathway inhibitor SP600125 on TPC-1 cell apoptosis in human papillary thyroid carcinoma.3.Western blot was used to detect the effects of mangiferin and JNK pathway inhibitor SP600125 on the expression of JNK,p-JNK,FasL,Bid,Bax,Bcl-2 and caspase-3 in TPC-1 cells.4.Real-time fluorescence quantitative PCR(qPCR)was used to detect JNK mRNA level in TPC-1 cells.Result1.Mangiferin inhibited TPC-1 cell proliferation in a time-dependent manner,but the degree of cell proliferation decline gradually slowed down after 24 hours.The half inhibitory concentration(IC50)measured at 72h time point was 3.92 μM.SP600125 can inhibit TPC-1 cell proliferation in a concentration and time dependent manner.IC50 value calculated at 72h time point is 117.89nM.2.Flow cytometry was used to detect the apoptosis rate of cells in each group.There was no significant difference in the apoptosis rate between the control group and SP600125 group.There was significant difference in the apoptosis rate between the control group and mangiferin group.Compared with the control group,the apoptosis rate was still significantly increased after combined administration.3.Mangiferin treatment inhibited the expression of JNK and p-JNK.With the increase of mangiferin concentration,the inhibition of JNK and p-JNK expression gradually increased,but the inhibition of p-JNK was more significant.The treatment of SP600125 significantly inhibited the expression of p-JNK,mangiferin and SP600125 had synergistic effect.The expression of apoptosis-related proteins FasL,Bid,Bcl-2 and Bax were down-regulated by SP600125 and mangiferin monotherapy.Mangiferin monotherapy inhibited Bcl-2 more obviously,and the ratio of Bcl-2/Bax was also significantly reduced.SP600125 single drug treatment significantly reduced the expression of apoptosis executive protein caspase-3,while mangiferin treatment increased the expression of caspase-3 compared with the control group.4.Real-time fluorescence quantitative PCR was used to detect the expression level of JNK mRNA in TPC-1 cells.Compared with the control group,there was no significant difference in JNK mRNA expression in TPC-1 cells after different concentrations of mangiferin treatment.ConclusionMangiferin affects JNK phosphorylation through targeted binding with JNK protein.Mangiferin inhibited the proliferation of TPC-1 cells by inhibiting the activity of JNK signaling pathway.Compared with JNK signaling pathway specific inhibitor SP600125,mangiferin induces apoptosis of TPC-1 cells by adjusting the ratio of pro-apoptotic protein to anti-apoptotic protein and up-regulating the expression of caspase-3.The effect of mangiferin on promoting apoptosis may also be related to the influence of other target molecules targeted for regulation.