| Chemotherapy remains the main treatment option for advanced cancers,therefore it is critical to fully understand the antitumor mechanisms and mechanisms of resistance of chemotherapy drugs.This dissertation study includes two parts,Microarray technology and bioinformatics were used to find targets of methylselenic acid(MSA)in prostate cancer and Mass spectrometry combined with chemical proteomics was used to find novel methotrexate(MTX)-resistance targets in acute lymphoblastic leukemia(ALL).This study aimed at further elucidation of the mechanisms of action and resistance of MSA and MTX to further provide theoretical and experimental basis for their clinical application.1.The Role of GDF15 in the Anti-cancer Effect of MSA on Prostate CancerSelenium compounds play an important role in the prevention and treatment of cancer(including prostate cancer),and the metabolite methylselenol is the key to exert its anticancer activity.MSA is a newly synthesized selenium compound,which can be metabolized into active methylselenol after only one non-enzymatic reaction in cells.To date,the growth inhibitory efficacy of MSA in prostate cancer cells has been documented extensively.However,our understanding of the immediate targets that are key to the growth inhibitory effects of MSA remains limited.In this part of the dissertation,we first analyzed gene transcript changes in LNCa P and PC-3 prostate cancer cell lines induced by MSA.According to the microarray data,growth differentiation factor 15(GDF15)is the most significantly upregulated gene by MSA.Subsequently,using multiple preclinical prostate cancer models,we further demonstrated in vitro and in vivo that GDF15 was highly induced by MSA.We further showed that knockdown of GDF15 mitigated MSA inhibition on cell proliferation and induction of apoptosis.Further,pathways that are negatively correlated with GDF15 transcript levels in clinical samples are also negatively correlated with MSA treatment in cultured cells.Since most,if not all,of these pathways have been implicated in prostate cancer progression,suppression of their activities by inducing GDF15 is consistent with the anticancer effects of MSA in prostate cancer.These results demonstrate that GDF15 is a direct target of MSA.Furthermore,we explored the molecular mechanism by which MSA regulates the expression of GDF15.We found that the up-regulation of GDF15 expression induced by MSA was not related to p53,AR or ER stress.Finally,the clinical significance of up-regulation of GDF15 expression was investigated.Analysis of gene expression data from over 1000 primary and 200 metastatic prostate cancer samples revealed that GDF15 expression is decreased in metastatic prostate cancers compared to primary tumors and that lower GDF15 levels in primary tumors are associated with higher Gleason scores and shorter survival of the patients,suggesting that GDF15 is a potential prognostic marker in prostate cancer.Overall,this part of the disseration study indicates that GDF15 is a direct target of MSA,and GDF15 mediates cell proliferation inhibition and apoptosis induced by MSA.In addition,low levels of GDF15 expression predict higher pathologic progression and poorer clinical outcome in prostate cancer.These results provide a basis for fully elucidating the antitumor mechanism of MSA and support the further development of MSA for the treatment of prostate cancer.2.Regulation of Phosphoglycerate Kinase 1(PGK1)is also a Potential Mechanism of MTX Against Acute Lymphoblastic LeukemiaMTX has been widely used in the clinical treatment of many diseases,especially cancer,because of its inhibitory effect on cancer cell proliferation,and its anti-inflammatory and immune suppression function.MTX is also an effective drug for the treatment of ALL.However,relapse occurs due to ALL cell resistance to MTX.Thus,uncover the mechanism of resistance to MTX is critical for its more effective and safer use in the clinic.In the second part of this dissertation,we used quantitative proteomic analyses to deteact differentially expressed proteins before and after MTX treatment in CCRF-CEM cells and between the parental CEM and MTX-resistant CCRF-CEM(MRC)cells.The results showed that Phosphoglycerate kinase 1(PGK1)was the only protein that was upregulated upon MTX treatment in the CEM cells and down-regulated in the MRC cells compared to the parental CEM cells.We verified the up-regulation effect of MTX on PGK1 in ALL cells by real-time quantitative PCR and Western blots,consistent with up-regulation dihydrofolatereductase(DHFR),a known target of MTX.On the other hand,the expression levels of DHFR and PGK1 in the MRC cells were significantly lower than that in parental CEM cells.Removal of MTX resulted in up-regulation of PGK1 in the MRC cells.In addition,the expression levels of DHFR and PGK1 in the MRC cells and the ability of the MRC cells to restore proliferation positively correlated with the number of MTX exposures.These results suggest that transient MTX exposure results in compensatory up-regulation of PGK1 and DHFR,while long term MTX exposure results in adaptively down-regulation of PGK1 and DHFR.Subsequently,we demonstrated that MTX can specifically bind to PGK1 via MTX agarose bead enrichment assays and protein thermal stability analyses.Further,MTX treatment resulted in reduced levels of pyruvate,the downstream substrate of PGK1.These results indicate that PGK1 is a direct target of MTX.Through protein-protein interaction Network(PPI)analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis,we proposed that the dysfunction of intracellular glycolysis and amino acid synthesis is one of the important molecular mechanisms of MTX resistance in ALL cells.To summerize,the results of this part of the dissertation indicate that suppression of glycolysis and other metabolic pathways by directly acting on PGK1 is a potential molecular mechanism underlying the anti-ALL activity of MTX against ALL cells.This study provides a new clue to further elucidate the mechanism of action of MTX. |
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