The Research Of Notich1and Akt Singal On Tumor Growth In A Mouse Model Of Multiple Myeloma

BackgroundMultiple myeloma (multiple myeloma, MM) is a serious type of hematologicalmalignancies in patients, the occurrence and development of myeloma cells is closelyrelated to the abnormal proliferation of and micro-environment angiogenesis, the lowpercentage proliferation and the multi-the formation of drug resistance of myeloma cells,making the clinical treatment of MM is still very difficult. Notch signaling pathway hasclose ties the incidence of MM， myeloma tumor cells and bone marrow stromal cellsexpress a large number of Notch receptors and ligands，the activated Notch signalingpathway play an important role in the occurrence of drug-resistant myeloma. Akt is closelyrelate to tumor occurrence and development, it is reported that the Notch signaling andPI3k-Akt passway can actiate each other. γ-secretase inhibitors (gamma secretase inhibitor,GSI) can effectively inhibit the activation of Notch1, recent studies have reported that theγ-secretase inhibitors can induced apoptosis in majority of MM cell lines andaccompanied by reduced Notch1expression, GSI blocked Notch1can reduce Aktexpression and promote tumor apoptosis. Therefore, it is important to explore the role ofNotch1and Akt in MM using GSI reduced Notch Notch1expression in MM mouse model.ObjectiveTo investigate GSI inhibition Notch pathway and Akt on tumor growth, secretion ofcytokines and other biological behavior of tumor angiogenesis and changes and explore theeffect and mechanism of Notch1and Akt in MM mouse model, so as to reveal thepathogenesis of T-ALL and provide the basis for molecular targeted therapy.Materials and Methods 1. In vitro MM cell lines RPMI8226, the human myeloma cell line RPMI8226cellsby subcutaneous inoculation resistance in diabetic/severe combined immunodeficiency(NOD/SCID) mice to establish xenograft animal model.2. Continuous monitoring of myeloma cell growth in mice, observed changes intumor size.3. Application of GSI and saline inject intratumoral of a group of mice respectively,observed tumor growth in GSI group and saline group.4. Immunohistochemical stain: separation, removal of subcutaneous tumors in mice,with10%formalin-fixed, paraffin-embedded and the hematoxylin-eosin staining;immuneohistochemistry to detect Notch1, Akt expression and localization.5. Western blot detection of protein expression pathway: After grinding in liquidnitrogen, extraction of total cellular protein, Western blot detection of Notch1, Akt proteinexpression.Results1. Successfully established resistance in diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft animal model.2. The rate of tumor transplantation: vaccination myeloma cell lines RPMI8226cells5-14days (mean7days),18NOD/SCID mice were seen in the left or right groin masscan be measured, After three days of Tumor formation,the tumor average volume for5.26mm3, six days later the average volume of66.76mm3, nine days later the averagevolume of274.48mm3and twelve days later the average volume of510.26mm3, thetumor rate of100%.3. General condition of animals: saline injection group mice mental condition, activity,skin condition, the amount of drinking water showed no change; detect results show thatthe GSI of tumor volume changes inhibit multiple RPMI8226myeloma cells in tumor-bearing NOD/SCID mice transplanted tumor growth in vivo, and promote apoptosis oftumor cells. In a mouse model of myeloma, the average tumor volume of GSI group（683.5mm3） is smaller than in the saline injection group（1798.7mm3），p＜0.01.4. Immunohistochemical stain of tumors were detected Notch1, Akt expression andlocalization: Notch1was located in the cell cytoplasm or nucleus, Akt was mainly locatedin the cytoplasm, followed by positioning in the cell membrane or nucleus. Notch1positive rate of the experimental and control groups were11.1%and95.6%of Akt-positive rates were13.3%and93.3%，GSI Group expession of Notch1and Akt is lower than thesaline group.5. Western blot results showed that: GSI Group Notch1gene and protein expressionof Akt gene is lower than the saline group injection group, suggesting that GSI can inhibitNotch1, Akt protein expression.Conclusion1. GSI can down-regulate the expression of Akt molecules and Notch1of Notchsignaling pathway in multiple myelomaxenograft tumor animal.2. GSI can inhibit the growth of multiple myeloma RPMI-8226cells in tumor-bearingNOD/SCID mice transplantation tumor through the down of Notch1and Akt expression.3. Notch signaling is expected to become a new target for anticancer therapy ofmyeloma.