[Effect of Notch1 on cell cycle, apoptosis and migration of laryngeal squamous cell carninoma cell line Hep-2].
OBJECTIVE
To investigate the effect of Notch1 on cell cycle, apoptosis and migration of laryngeal squamous cell carcinoma cell line Hep-2 and explore its possible molecular mechanism.


METHODS
Hep-2 cells were divided into three groups: untreated group, empty vector group and Notch1 group. The effects of upregulation of Notch1 expression on cell proliferation, cell cycle and apoptosis were assessed by CCK-8 staining and flow cytometry, respectively, and effect of upregulation of Notch1 expression on cell migration of Hep-2 cells was studied using Boyden chamber assay, and further expression changes of genes related to cell proliferation, cell cycle, apoptosis and migration were detected by semi-quantitative RT-PCR and Western blotting.


RESULTS
Compared with the untreated group and empty vector group, cell proliferation of Hep-2 in the Notch1 group was significantly inhibited (P < 0.05). The results of flow cytometry revealed that the percentage of cells at G0/G1 phase in the Notch1 group was (70.43 +/- 1.36)%, significantly higher than the (46.39 +/- 1.19)% in the untreated group or (48.41 +/- 1.18)% in the empty vector group, and there was a significant difference among the three groups (P = 0.000). In addition, the percentage of apoptotic cells in the Notch1 group was (22.46 +/- 0.90)%, significantly higher than that in the untreated group [(5.77 +/- 0.37)%] or empty vector group [(6.09 +/- 0.20)%], with a significant difference among the three groups (P = 0.000). The results of Boyden chamber assay demonstrated that the number of cells migrated through membrane in the Notch1 group was evidently lower than that in the untreated group and empty vector group. Moreover, the results of semi-quantitative RT-PCR and Western blotting showed that cell proliferation inhibition and cell cycle arrest were closely associated with downregulation of cyclin D1, cyclin E and CDK2 expressions and upregulation of p53 expression. In addition, upregulation of Notch1 expression mediated cell apoptosis was tightly related to upregulation of caspase 3/9 expressions and downregulation of Bcl-2, while the decrease of Hep-2 cell migration ability was obviously correlated with downregulation of MMP-2/-9 expressions.


CONCLUSIONS
Notch1 signaling pathway may play a pivotal role in the occurrence and development of laryngeal squamous cell carcinoma. Further study may elucidate that Notchl signaling pathway may become a molecular target for therapy of laryngeal squamous cell carcinoma.
Department of Stomatology, Zhengzhou Hospital, Zhengzhou 450003, China.
Jing
J
Jiao
Sha
S
Li
D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D006801:Humans; D007822:Laryngeal Neoplasms / Q000378:metabolism / Q000473:pathology*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051881:Receptor, Notch1 / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C530104:CCND1 protein, human; C475820:CCNE1 protein, human; D019927:Cyclin E; C497761:NOTCH1 protein, human; D015513:Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051881:Receptor, Notch1; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9