The Role Of Notch Signaling Pathway In Doxorubicin-Induced Epithelial-mesenchymal Transition Of Osteosarcoma Stem-like Cells

Osteosarcoma is a malignant tumor,which is originated from mesenchymal tissue.It is a most common of primary malignant bone tumor in children and adolescents.With the extensive development of neoadjuvant chemotherapy in clinic,the 5-year survival rate of patients with osteosarcoma has been greatly improved,however,the 5-year survival rate of osteosarcoma is maintained at about 70%,which is due to the reason of drug resistance,recurrence and early metastasis,and the distant metastasis is the most common cause of death for osteosarcoma.In recent years,with the development of the theory of cancer stem cell,it has become one of the most spots in cancer research,and it has been found in many tumor tissues,such as neoplasms,breast cancer,pancreatic cancer,prostate cancer,etc.The cancer stem cells and normal stem cells have many similar characteristics,which are self-renewing,multi-differentiation potential and strong in malignant.Therefore,the existence of cancer stem cells may be the basic cause in the failure of clinical chemotherapy.There is no consensus about the source of cancer stem cell.However,it is clear that the formation of cancer stem cells is the physiological balance was destroyed between the oncogene and tumor suppressor genes,in some cases also involves with the epithelial mesenchymal transition(EMT).The EMT is a physiological process of the biological characteristics of the differentiated mature epithelial cells acquire mesenchymal cells,the cancer stem cells in the process of tumor and cancer stem cell formation and tumor cell infiltration,metastasis is closely related.It has been found that osteosarcoma is regulated by many kinds of signal pathway and molecules in the process of metastasis,and the occurrence of EMT can promote the metastasis of osteosarcoma cells.EMT not only regulates the formation of multiple cancer stem cells and participates in the process of cancer drug resistance,but also is regulated by complex signaling pathways,such as Wnt,hedgehog,TGF-P,k-B and notch signaling pathways and so on.These signaling pathways play an important role in the induction of drug resistance in cancer cells.Therefore,through the study of the role of these signaling pathways in the formation of osteosarcoma stem cells,drug resistance and EMT process,the treatment of osteosarcoma stem cell resistance,metastasis and other characteristics of the regulatory pathway for targeted therapy specificity,to improve the treatment of osteosarcoma will have a significant.This topic by using low concentration of doxorubicin to enrich the osteosarcoma stem-like cells and induce the occurrence of the EMT,suggests that Notch signaling in maintaining osteosarcoma stem cell characteristics and regulation of EMT process have play an important role,and specific inhibition of Notch signaling pathway can effectively inhibit the characteristics expression of osteosarcoma stem cells and reduce the migration of osteosarcoma,tumorigenicity and provides a reliable experimental basis for the targeted therapy and combination therapy for osteosarcoma.Part-? The effect of low concentration of doxorubicin on the characteristics of osteosarcoma cancer stemness in vitroObjectives:The theory of Tumor stem cells has stimulated the correlation between osteosarcoma stem cells and chemotherapy drug resistance,but the specific mechanism is still equivocal.This study aims to explore the effects of low concentration of doxorubicin on the characteristics of osteosarcoma cancer sternness,enrich and expand the study of the characteristics of the tumor stem cells.Methods:The cck-8 assay was used to test the proliferation of osteosarcoma cells with different concentrations of doxorubicin,flow cytometry was used to examine the effect of low concentration of doxorubicin on the expression of the surface marker of stem cells;Quantitative RT-PCR was used to detect the mRNA levels of factor of transcription in osteosarcoma,Osteosphere formation assay was used to investigate the osteogenic ability of osteosarcoma cells.Results:1.Different concentrations of doxorubicin can lead to changes in the morphology of osteosarcoma cell lines 143B-TK and U20S cells,causing cell proliferation inhibited significantly;2.The expression of CD-117 and stro-1 double positive rate of stem cell markers was up-regulated under the condition of low concentration of doxorubicin;3.The ability of the Osteosphere formation was enhanced by the low concentration of doxorubicin;4.Low concentration of doxorubicin can promote the expression of cancer stem cell related genes in osteosarcomaConclusions:Our study has provided evidences that low concentration of doxorubicin can enhance tumor stem cell properties and metastasis.The low concentration of doxorubicin in osteosarcoma cells stimulated the expression of the stem cell characteristics,and promote the proliferation and metastasis of osteosarcoma cells,although it can kill the tumor cells.And it brought a negative effect in the clinical treatment.Part-? The critical role of Notch signaling pathway in human osteosarcoma stem-like cellsObjectives:The mechanism of Notch signaling pathway in regulated the development of tumor is very complicated,but in the field of osteosarcoma stem cells,it has not been reported.In this chapter,we will enrich the osteosarcoma stem-like cells by low concentration of doxorubicin,to study the effect of Notch signaling pathway on the characteristics and metastasis of osteosarcoma cells,and to provide experimental evidence for the role of Notch signaling pathway in osteosarcoma stem cells.Methods:Notch signal reported virus was constructed and the stable transfection of osteosarcoma cell lines were screened,the effect of low concentration of doxorubicin on the expression of Notch gene was detected by flow cytometry.Quantitative RT-PCR and Western blotting were used to detect the mRNA and protein levels of Notch-1 and its downstream target gene HES-1 between resistant group and control group.Plate clone formation assay and Osteosphere formation assay were used to evaluate the effect of Notch signaling pathway inhibitor on the osteogenic ability of human osteosarcoma stem-like cells in vitro.Results:1.The signal of intracellular fluorescence was significantly enhanced among the cells,which stable transfected with Notch signal reported virus,that under low concentration of doxorubicin.2.The Notch-1 and its downstream target gene HES-1 were significantly higher expressed in the low concentration doxorubicin group compare with those in the control group.3.The DAPT,a kinds of inhibitor for Notch signaling pathway,can significantly reduce the number of tumor cells in the low concentration of doxorubicin and the ability of osteosphere.Conclusions:Low concentration of doxorabicin can promote the activation of Notch signaling pathway,and inhibit the Notch signaling pathway could produce a significant inhibitory effect on human osteosarcoma sternness,proved that Notch signaling pathway plays an essential role in regulating the formation and drug resistance of osteosarcoma stem-like cells.Part-? The effect of Notch signaling pathway on EMT of human osteosarcoma stem-like cellsObjectives:Activation of the Epithelial-Mesenchymal Transition(EMT)is the key mechanism for tumor cells to invasion and metastasis,and the Notch signal pathway is one of the signal pathways in regulated of EMT,it also plays an important role in the process of drug resistance,metastasis.However,there is no report about whether the EMT transformation and the Notch signaling pathway could be involved in the regulation of EMT of osteosarcoma stem cells.In this chapter,we will explore the expression of EMT in osteosarcoma stem-like cells,verify the regulatory role of Notch signaling pathway in the process of EMT of osteosarcoma stem cell,and provide new ideas and targets for targeted therapy for osteosarcoma.Methods:Western-Blotting was used to detect the transcription level of EMT markers in osteosarcoma stem-like cells;Transwell chamber model and Cell scratch repair were used to detect the migration of osteosarcoma stem-like cells induced by low concentration of doxorubicin.Notch signaling pathway inhibitor(DAPT)was used to detect the negativity effect of EMT on human osteosarcoma stem-like cells.Results:1.Low concentration of doxorubicin can promote the process of EMT in osteosarcoma cell lines;2.low concentration of doxorubicin could strengthen the migration and migration of osteosarcoma stem-like cells;3.Notch signaling pathway inhibitors can inhibit the EMT induced by the low concentration of doxorubicin on osteosarcoma stem-like cells.Conclusions:Low concentration of doxorubicin could promote the EMT of osteosarcoma stem-like cells,and the Notch signaling pathway was involved in the invasion and metastasis of osteosarcoma stem cells,that also play a synergistic role in the occurrence of EMT in osteosarcoma stem-like cells.