Expetlmeiital Study Of Dll4/Notck Signaling Padiway In Non-small Cell Lung Cancer

Lung cancer ranks the first most common cancer and seriously affects the human lifeand health worldwide. About1.2million patients have been diagnosed lung cancer everyyear all over the world. In2008, people dead from lung cancer, accounted for18.4%of allcancer deaths and ranking the first cause of death in cancers. Non-small cell lung cancer(NSCLC) accounts for around84%of lung cancers. Despite the advance of surgicaltreatment, the5-year survival of NSCLC has remained at a dismal15%in the past threedecades. It is therefore imperative to deepen the understanding of the molecularmechanism of NSCLC and to find novel therapeutic strategies for this disease.Notch signaling pathway was discovered through observation of genetic mutations inDrosophila in1918. Mutations in Drosophila genes resulted in wings with a notched wingphenotype, hence the name. However, there was no successful cloning until the mid-1980sby scientists. Notch signaling pathway is highly-conserved and exists in both invertebratesand vertebrates. A complete Notch signaling pathway includes of Notch receptors, ligands,intracellular effectors CSL protein, the other effector, Notch regulation of molecules. Inmammals, there are four heterodimeric transmembrane receptors (Notch1-4), twoSerrate-like (Jagged-1and Jagged-2) and three Delta-like (Dll1, Dll3and Dll4) ligands.Notch signaling is one of the fundamental signaling pathways that enable cell-cellcommunication in multicellular organisms, regulating certain cellular response duringembryonic and postnatal development. It was reported that depletion of Notch interruptslung development in mice. Accumulating evidence has revealed the aberrant expression ofNotch components including Notch1, Notch3and HES1in NSCLC. However, the role ofNotch in lung cancer development remains elusive.As we all known, antiangiogenic therapy for cancer emerged in early1970s markedwith the publication of Folkman's hypothesis. Nowadays, this hypothesis has been widelyconfirmed by a large number of experiments and became a milestone in the field of cancerresearch. In the tumor angiogenesis study area, vascular endothelial growth factor (VEGF)has been recognized as the strongest, the most vascular-specific growth stimulating factor.Thereby, the researches of tumor antiangiogenesis treatment have been mainlyconcentrated on VEGF and its receptors. Although several medicines have effects on manypatients, a large mount of cancer patients were useless. As an endothelium-specific Notchligand, Dll4was the last successful cloning ligand in five Notch ligands and its expressionis restricted to endothelial cells during embryonic vascular development and angiogenesis of tumors. The vast majority of these studies focused on the effect of Dll4/Notchinteraction in tumor angiogenesis. However, it is largely unknown whether Dll4of vascularendothelial cells could regulate Notch signaling in lung cancer and its mechanism.We tried to design a serial of experiments to investigate the role of Dll4in vascularendothelial cell and whether endothelial Dll4could affect the lung cancer cells adjacentand and clarify its mechanisn. It would not only advance our understanding on thecross-talk between vascular endothelial cells and cancer cells, but also provide a novelstrategy for lung cancer therapy.Part I: Experiments study the role of Dll4in the biological behavior ofvascular endothelial cellsObjective: To explore whether Dll4could affect the biological behavior of vascularendothelial cells through cell experiments in vitro and its mechanism.Methods: The Dll4expression, shDll4and there control plasmid were transfected intoHUVECs and stable cell lines were built. Cell growth assay, wound healing assay and tubeformation assay were taken to investigate the difference among EC-Dll4, EC-shDll4andEC-EV cell lines in biological behaviors. We also tried to explore the mechanism byReal-time PCR and westernblot analysis.Results:1. The expression of Dll4was up-regulated in EC-Dll4cell line and Dll4wessignificantly decreased in EC-shDll4cell line compare with their control cells both inmRNA and protein levels.2. The results of CCK-8assay, wound healing assay and tubeformation assay were shown that Dll4over-expression could inhibit the proliferation ofECs, reduce the migration of ECs and decrease the tube-formation of ECs. It also shownthat when Dll4were silenced, the function of ECs in proliferation,magirtion andtube-formation were up-regulated compare with those of EC-EV cell line.3. In westernblotanalysis, we found that Dll4had no influence on Notch1, Notch2and Notch3in ECs.Conclusion: Endothelial-specfic Dll4could significantly inhibit the function of growth,migration and tube-formation of vascular endothelial cells. The biological behavior of ECsinfluenecd by Dll4might not through Notch1, Notch2and Notch3receptor of endothelialcells.Part II: Experiments study the role of endothelial Dll4in the biological behavior of Non-small cell lung cancer cellObjective: to explore whether Dll4from endothelial cells could affect the function ofNSCLC cell in vitro and in vivo and the mechanism underlying.Methods: RhDll4was treated to A549cells to investigate its function on A549cell growth.We made use of a cell coculture model and flow cytometry to investigate the function ofDll4from endothelial cells on lung cancer cells neighboring in vitro. Nude mice weretaken to build tumor growth model of A549+EC-Dll4, A549+EC-shDll4and A549+EC-EVto explore the function of endothelial Dll4on tumor growth in vivo. We detected theexpression of Notch receptor and its target genes underlying in A549cells co-cultured withthree EC cell lines by real-time pcr, westernblot analysis. In further, the expression ofNotch receptor was changed by over-expression or knockdown technology to explore itsfunction on A549cells to result out the cross–talk between endothelial Dll4and Notchsignaling of lung cancer cells.Results:1. The proliferation of A549cells was significantly inhibited upon rhDll4treatment.2. In an in vitro assay, proliferation of NSCLC cells co-cultured with EC-Dll4was significantly inhibited compared with those co-cultured with EC-EV. In contrast,stably silencing Dll4in ECs by its specific shRNA remarkably increased the proliferationof NSCLC cells.3. Apoptotic A549cells were detected in this co-culture system, but nosignificant differences among the three groups was observed.4.A549cells mixed withEC-Dll4developed smaller tumors than those mixed with EC-EV, while tumors induced byA549cells mixed with EC-shDll4exhibited larger size.5.A549cells in xenograft ofEC-Dll4group showed limited growth, whereas the cells in EC-shDll4group proliferatedmore rapidly compared with the control group, indicating the suppressive effect ofendothelial Dll4on the proliferation of cancer cells in vivo.6.More microvasculars couldbeen seen in xenograft of A549+EC-shDll4group and less in xenograft of A549+EC-Dll4group than those in the xenograft of A549+EC-EV group by CD34staining.7. We foundthat expression of Notch1, HES1and HEY1were evidently increased in A549cellsco-cultured with EC-Dll4, and decreased in A549cells co-cultured with EC-shDll4 whatever in vitro or in vivo assays.8. The proliferation of A549cells was inhibited byN1ICD (Notch1intracellular domain) overexpression and enhanced by Notch1interference.9. Suppressive effect of rhDll4on cell proliferation was significantly impairedin A549cells stably expressing shNotch1as compared with control cells.Conclusion: We proved that Dll4-expressing ECs could significantly suppressedneighboring non-small cell lung cancer (NSCLC) cells and attenuated the growth ofNSCLC xenograft in nude mice. Furthermore, activation of Notch1, but not Notch2orNotch3, was enhanced in NSCLC cells cultured with EC-Dll4as well as in xenograftsinduced by a mixture of NSCLC cells and EC-Dll4. Interference of Notch1significantlyattenuated Dll4-mediated suppression of NSCLC cell proliferations, indicating thatDll4/Notch1signaling negatively modulates the NSCLC growth.Part III: Experiments study the corss-talk between Dll4/Notch1signaling and PTEN-PI3K/AKT signaling pathwayObjective: To explore whether there exist corss-talk between Dll4/Notch andPTEN-PI3K/AKT signaling pathway and its mechanismMethods: A549cells treated by rhDll4or co-cultured with EC-Dll4, EC-shDll4amdEC-EV were taken to detect the expression of PTEN or p-AKT by real-time pcr orwesternblot,etc. PTEN and p-PTEN were decteced in cells in which Notch1were overexpression or knockdown.Results:1. The expression of PTEN was much higher in A549cells co-cultured withEC-Dll4than A549cells co-cultured with EC-EV.2. PTEN expression was up-regulated intumors with EC-Dll4whatever in mRNA or protein levels, and it down-regulated in tumorswith EC-shDll4compared those in tumors with EC-EV.3. Both PTEN and PTENPhosphorylation expression was significantly increased in A549cells treated by rhDll4,and the expression of p-AKT was slightly down-regulated meantime.4. Over expression ofN1ICD in A549cells could up-regulated the PTEN protein.5. Both PTEN and PTENPhosphorylation expression was changed in cells in which exogenous Notch1over-expression or interference. Conclusion: Dll4/Notch1could positively regulate the the expression of PTEN andcross-talk with PI3K/AKT signaling pathway. Dll4/Notch1might influence the function oflung cancer by regulating tumor suppressor gene PTEN.