Down-regulation Of Notch1Resulting In Breast Cancer Stem Cell Sensitizes To Paclitaxel In Vitro And Vivo

Stem cells is defined as undifferentiated populations that have the ability of self-renewal and asymmetric cell division. Recently increasing number of evidence confirmcancer cells do exit some sort of hierarchy, which means that there are differentdifferentiation stage of cancer cells. Some cancer cells sharing similar properties withnormal stem cells terms of their capacity for self-renewal and playing a decisive role intumor formation are named cancer stem cells or tumor-initiating cells. In1979, Dickand colleagues, who transfected AML from human patients to NOD/SCID mice,confirmed the hypothesis that AML originated from leukemia stem cell. Since then,putative cancer stem cells have been isolated from many other tumors such as breast,brain, colon, pancreas, prostate, lung and head and neck tumors.Notch signaling in the regulation of stem cell proliferation, differentiation,apoptosis, adhesion and tumor angiogenesis and other direction has a very importantrole. Solid tumors and blood malignancies, mostly detected wild-type Notch receptors,ligands and target gene expression disorders. At the same time, Notch signaling canfunction in precursor cells, to promote a special type of myoepithelial cell proliferation.Phillips et al reported that breast cancer stem cells can be phenotypic markers to identifyand controlled by the Notch signaling pathway in breast cancer Famie and other studieshave shown that the Notch signaling pathway in breast cancer, the development processplay an important role in the transfer expressed in breast disorders。In recent years, paclitaxel has been one of the best natural anti-cancer drug forclinical effective against a wide range of solid tumors such as lung cancer. Themechanism of action of paclitaxel was initially considered to be fundamentally differentfrom that of previous anti-mitotic compounds in that paclitaxel increased polymer massand stabilized the microtubules. Inherent or acquired multidrug resistance is one of themajor problems in clinical widespread use of paclitaxel. The breast cancer resistance protein (BCRP) gene, formally known as ATP-binding cassette transporter G2(ABCG2)gene, encodes an ABC half transporter that causes resistance to certain cancerchemotherapeutic drugs when transfected and expressed in drug sensitive cancer cells.The ABCG2transporter is an efficient efflux pump and this property is significance ofcancer recurrence and chemotherapy resistance. Breast cancer stem cells can escape thechemotherapy through highly expressing BCRP. And it would be higher expressingafter chemotherapy to adapt the new micro-environment which makes breast cancerbecome one of the most refractory cases. Therefore, breast cancer stem cells shoule beconsider as a significant research and therapy target.At the beginning, we isolate the breast cancer stem cells which surface phenotypeis CD44+/CD24-from breast cancer cell line named MCF-7. And detecting theexpressing of Notch1signaling pathway between cells with CD44+/CD24-andnon-CD44+/CD24-in order to know more effect of Notch1signaling pathway. What’smore, we using pGPU6/GFP/Neo to build a stably transfected MCF-7cells which cansupport the study of paclitaxel and MCF-7cell proliferation, cell cycle and apoptosis.Then to discussion the breast cancer stem cells is important with respect to multi-drugresistance in the development of breast cancer.PartⅠ：The mechanism of Notch-1signaling pathway inthe breast cancer stem cellsObjective: To evaluate the expression of Notch-1signaling pathway in the breastcancer stem cells.Methods: The suspension-cultured breast cancer cells were divided intoCD44+/CD24-and non-CD44+/CD24-cells by magnetic-activated cell sorting (MACS).Then to evaluate the expression of Notch-1signaling pathway in the breast cancer stemcells by RT-PCR and Western blot assays.Results: Almost7.38%of cells were CD44+/CD24-by magnetic-activated cellsorting which expressed the marker of cancer stem cells ALDH1A1at the same time.The expression of the Notch-1signaling pathway between CD44+/CD24-andnon-CD44+/CD24-cells were significantly different.Conclusion: The Notch-1signaling pathway highly expresses in CD44+/CD24-cells and it would maintain the cells shape and function when the signaling pathway is activated. PartⅡRNAi on Notch1Effect on Carcinogenesis of Breast CancerObjective: To down-regulation the effect of Notch1gene on biological behaviorof breast cancer cell.Methods: The shRNA Notch1probe was designed to interfere and reduce theNotch1gene expression in MCF-7cell.The RT-PCR and western blot were used todetect them in mRNA and protein of Notch1in breast cancer MCF-7cells, which weretransfected for48hours by the mediation of Sofast-kit.Results: It was verified by partial nucleotide sequencing that the constructedeukaryotic vectors expressing shRNA of Notch1were correct. In the Notch1shRNA-1group, Notch1shRNA-2group, Notch1shRNA-3group, Notch1shRNA-4group, blankgroup, negative control group and MOCK group of MCF-7cells, the expression ofNotch1mRNA relative to GAPDH was0.77±0.07,0.98±0.08,0.71±0.05,0.57±0.05,0.96±0.08,1.01±0.08,1.00±0.11, respectively. Contrast to MOCK group, the Notch1mRNA expression in Notch1shRNA-1group, Notch1shRNA-3group, Notch1shRNA-4group declined, moreover the Notch1mRNA expression in Notch1shRNA-4group obviously lower than other3groups.Conclusions: Notch1shRNA visibly inhibited the expression of Notch1 Part ⅢNotch1of siRNA combined with paclitaxel inhibition breast cancerstem cell growth and sensitizes to paclitaxel in vitroObjective: Down-regulate Notch1gene expression in breast cancer cell anddefine the drug resistance mechanisms in breast cancer stem cell.Methods: By stable down Notch1in MCF-7cells, colony formation experimentsand the CCK8experimental detection paclitaxel on the proliferation of breast cancer after disturbance, Hochest33342and the AV/PI double staining detection of breastcancer MCF-7cells apoptosis. The breast cancer stem cell formation assay in breastcancer stem cell proliferation. Flow cytometry to detect paclitaxel in the treatment ofbreast cancer after the proportion of stem cells. Western blot analysis protein expressionof ABCG2Results: lowered Notch1expression and paclitaxel in breast cancer MCF-7cellsproliferation inhibition significantly increased the proportion of apoptosis, the G2/Mphase cells increased. Breast cancer stem cell self-renewal capacity to reduce thedecline in the proportion of cancer stem cells in breast cancer cells, reduced expressionof ABCG2protein.Conclusion: The ABCG2may be downstream of Notch1signaling pathway, bydownregulating Notch1protein, increasing the sensitivity of breast cancer stem cells topaclitaxel. Part ⅣNotch1of siRNA combined with paclitaxel inhibition breast cancerstem proliferation and tumor growth in vivoObjective: Understanding of Notch1of siRNA combined with paclitaxelinhibition of nude mice in vivo breast cancer stem cell proliferation mechanismMethods: The normal group and Notch1stability down the cell group wereinoculated in the removal of fat pads of nude mice breast site of tumor growth bypaclitaxel treatment.Results: of Notch1reduced the inhibition of paclitaxel to the tumor significantlyincreased necrosis increased, smaller tumor volume, weight reduction. Reduce theproportion of stem cells in the tumor tissue. Expression of Notch1signaling pathway ofNotch1, of NICD, of Hes-1and ABCG2were reduced.Conclusion: Down-regulate the expression of Notch1can improve the sensitivityof breast cancer stem cells to paclitaxel.