The Regulation And Its Mechanism Of The Notch Signaling Pathway In Small Cell Lung Cancer

The pulmonary neuroendocrine tumors(NET)are derived from the tumor stem cell with potential of multi-directional differentiation.They have several significant deviations with the ordinary carcinoma of lung such as the heterogeneity on tissue structure,the biological behaviour on oncobiology and drug-responsibility.NET in lung can generate many neuroendocrine markers and hormones such as chromogranin A(CgA),neurone specific enolase(NSE),ACTH,ADH which can lead to a lot of pathological changes in body organs or systems such as endocrine,nervous,digestive, blood,kidney,bone,joint,and skin.Those pathological changes with corresponding clinical manifestations are called "paraneoplastic syndromes".The small cell lung cancer(SCLC)which originates from Kulchitsky cell or stem cell in epithelium mucosae of bronchus is a kind of pulmonary neuroendocrine tumors.Among all kinds of lung cancer,SCLC has a higher incidence(20%-25%in primary lung cancer)that makes it a common type of lung cancer besides squamous carcinoma and adenocarcinoma.SCLC is a severe malignant tumor with rapid progression.Patient with SCLC is usually unsuitable for surgery due to the early metastasis and die in one year.So far there has no breakthrough progress in its therapy and prognosis.Abnormal Notch signaling pathways contribute to tumorigenesis,progression and phenotype of many tumors.Notch gene encodes a 300 kD transmembrane receptor which can release a active intracellular region(NIC)after the enzymolysis ofγ-secretase.NIC can be transferred into nuclei and regulate expression of downstream gene,In mammals,one of the direct targets of NIC is CBF-1 which inhibits expression of the corresponding downstream gene and can transform when binding to NIC.The other targets of NIC include HES family and Cell cycle-related proteins.HES encodes a group of DNA-bind protein with specialαHLH structure which can bind to the hASH1 promoter and inhibit the gene expression.Up to now,the molecular mechanism of regulation and maintenance of neuroendocrine differentiation are unclear yet.But study also reveals that the expression of hASH1 have close relationship to the differentiation of neuroendocrine tumor.So notch signal transduction pathway may have an effect to inhibition of HES-to-hASH1 and then have an indirectly regulation to neuroendocrine tumors.Existing research on the regulation of Notch signaling to SCLC is mainly focused on molecules within the pathway and morphological changes of the cells,for example:Notchl reduces the expression of hASH1 in SCLC cells,Notch signaling inhibits the growth and proliferation of SCLC cells,etc.Evidence has shown that Notch signaling pathway can inhibit the generation of neuroendocrine markers in pancreatic cancer cells,but similar effects of Notch signaling on SCLC remain unclear.Therefore we transfected recombinant plasmid expressing Notch1 into SCLC cells and later conducted a series of tests,in order to further investigate the function of the Notch signaling in SCLC,especially to examine the expression of neuroendocrine markers,so as to provide some clues to gene therapy for SCLC patients lacking effective treatments now.Materials and methodsCells of SCLC cell line NCI-H446 were cultured in RPMI 1640 medium with 10%FBS.Recombinant plasmid pEFBOS-NIC-MYC expressing Notch gene and sham vector pEFBOS-neo(without insert)were provided by the Department of Medical Genetics and Developmental Biology,the Fourth Military Medical University. PEFBOS-NIC-MYC plasmid were first transfected into competent E.coli and extracted after sequencing.Then pEFBOS-NIC-MYC and pEFBOS-neo plasmid were transfected into NCI-H446,and stable cell lines were selected by G418 antibiotics. After the cells were transfected with pEFBOS-NIC-MYC and pEFBOS-neo,MTT assay,RT-PCR analysis,western blot analysis and immunocytochemistry were carried out to test the cell growth rate and the expression of genes(Notch1,HES1 and hASH1) in the Notch signaling pathway and the change of neuroendocrine markers(CgA,NSE).Results1.RT-PCR analysis:The expression of Notchl and HES1 is found in the pEFBOS-NIC-MYC group,while absent in the blank control group and the pEFBOS-neo group.Compared to the blank control group and the pEFBOS-neo group,the expression of hASH in the pEFBOS-NIC-MYC group is significantly decreased.2.Growth assays:Cells of the three groups were seeded in 96-well plates(2 X 103 cells per well)and the cell growth rate was tested once a day for a succession of 6 days(each experiment was repeated for at least three times).The statistical analysis suggests that transfected cells with pEFBOS-NIC-MYC plasmid showed a significantly slower growth rate compared with the two control groups(P=0.037 and 0.043,Student's t-test).3.Immunocytochemistry:The cultured cells were smeared onto slides and immunocytochemistry staining.Gray scale analysis was conducted to compare the stain results of the three groups.It is found that the pEFBOS-NIC-MYC transfected cells express a significantly lower level of NSE and CgA,compared to the two control groups(P＜0.001,Student's t-test).4.Western blot analysis:The NIC-MYC protein is detected in the pEFBOS-NIC-MYC group cells rather than in the two control group cells.The blank control group and the pEFBOS-neo group cells both express a higher level of neuroendocrine markers(CgA and NSE)than the pEFBOS-NIC-MYC group cells by density analysis of strap used Quantity one system(P＜0.001,Student's t-test).Conclusion1.Our data suggest for the first that Notch signaling pathway may have an inhibitory effect on the expression of SCLC neuroendocrine markers,which provides very valuable clues to gene therapy for SCLC patients.2.The Notch signaling pathway regulates SCLC cells through its inhibitory effect on hASH1 transcription via HES1. 3.The Notch signaling pathway can inhibit the proliferation of SCLC cells.