Role Of Methylseleninic Acid And ER Selenoproteins In Apoptosis And ER Stress Signaling Of Multiple Myeloma Cells

During the past five years,there is increasing morbidity of multiple myeloma(MM)due to the aggravation of aging population worldwide,which makes MM becoming the second hematological malignancies only following non-Hodkin lymphoma(NHL).In spite of the improved outcome by the immunomodulator and proteasome inhibitor,nearly all patients with MM experience relapse and refractory disease.Up to now,MM remains uncurable.Hence,it has become the focus of researchers to find more effective drugs with low toxicity in treating multiple myeloma.As one backbone agent,bortezomib(BTZ)has triggered drug resistance in MM due to the extensive application of BTZ,therefore,high effective complementary drugs are definitely in need to enhance BTZ's effects.Methylseleninic acid(MSA),as one kind of organic selenium compounds,has a wide range of anti-cancer effect.MSA stands out for its characteristics of acting on target cells directly without being transformed by metabolic enzyme in vivo in advance and as an intermediate of dietary selenium nutrition metabolism,the effects of MSA for adjuvant therapy of tumor have been confirmed,so it is expected to be an auxiliary drug with promising application prospects.In this thesis we take an attempt to explore MSA affect MM cell apoptosis and its signal mechanism,meanwhile to observe the ER selenoprotein levels of MM cells and its possible role in the apoptosis induced by BTZ,thus providing theoretical and experimental basis for multiple myeloma treatment as well as for its further research.Major research findings are listed in the following part:1.The apoptosis effects in MM cells induced by BTZ and its signal mechanism are both promoted by MSA.Firstly,three specific multiple myeloma cell lines namely U266,RPMI-8226,OPM-2 are chosen as the research objects.Then,MTT experiment is adopted to test different concentration gradient of MSA and BTZ single drug or the combination of both anti-proliferative effects of multiple myeloma cells,and the result shows that MSA has enhanced the inhibitory effect of BTZ on the proliferation of these three myeloma cells.Secondly,with Annexin ? and PI double staining as research methods,the flow cytometry instrument is adopted to examine the apoptosis effects of MSA and BTZ respectively or in combination on cells and cell-cycle arrests when the cell proliferation rate reaches the IC50 concentration,and it turns out that MSA can significantly enhance apoptosis of MM cells induced by BTZ meanwhile cause cell arrests at G2/ M phase.And then with Western Blot experiment,we find that the combination of MSA and BTZ can obviously reduce the Bcl-2 signal levels,meanwhile increasing Bax levels to a large extent.And some experiments also indicate that Bcl-2 family is also involved in apoptosis induced by endoplasmic reticulum stress(ER stress),suggesting that ER stress may be the apoptosis pathways in this study.Given the fact that BTZ is a proteasome inhibitor which can generate a large amount of non-functional beta 2 MM cells-microglobulin,the role that the ER stress mediated by folded protein response(unfolding protein response,UPR)may play are thus inferred.In order to study the effects of the ER stress on apoptosis of MM cells induced by BTZ and MSA,the Western Blot is adopted to examine UPR and several protein levels in the ER stress and we find that MSA and BTZ be clearly activated ER Stress signal.And the above results are further verified by the enzyme digestion experiment of XBP-1.The results indicate that MSA enhances the apoptosis of the MM cells induced by BTZ through the way of ER Stress.2.How ER selenoprotein group of MM cells responds to BTZ.First,the fluorescent quantitative PCR method is employed to examine mRNA levels of three kinds of MM cells in the endoplasmic reticulum of 7 kinds of selenoprotein gene.Based on the research results,the focus is placed on SelS,and further analysis is carried out on its role in mediating MM cell apoptosis in gene and its protein levels respectively.In this study,the organic selenium compounds are applied to the MM for the very first time,and the study indicates that it can serve as an auxiliary drug for treating MM.Therefore,this study enriches the MM treatment,and also find MSA may be a nice auxiliary drug in MM.Second,this thesis paves the way for the further study of the endoplasmic reticulum selenoprotein in MM.