ABSTRACT
OBJECTIVE：To stud y the effects of sinapine thiocyanate （ST） on the proliferation ，epithelial mesenchymal transformation（EMT）and metastasis of human cutaneous squamous cell carcinoma SCL- 1 cells，and to investigate its possible mechanism. METHODS ：Human cutaneous squamous cell carcinoma SCL- 1 cells were divided into blank control group （0.1％ DMSO） and ST different concentration groups （5，10，20 μmol/L）. CCK- 8 assay，5-ethynyl-2′-deoxyuridine（EDU）test， scratch test and Transwell chamber invasion test were adopted to test the proliferation ，migration and invasion ability. The expression of N-cadherin and E-cadherin were detected by Western blot and immunofluorescence assay . Other SCL- 1 cells were collected and divided into blank control group （0.1％ DMSO），ST group （20 μmol/L），ST+NSC228155 group [ 20 μmol/L ST+100 μmol/L NSC228155（EGFR agonist ）] and ST+SC 79 group [ 20 μmol/L ST+20 μmol/L SC79（PI3K/Akt agonist ）]. The proliferation ，migration and invasion ability of SCL- 1 cells in each group were detected by CCK- 8 assay，scratch test and Transwell chamber invasion assay. The expression of epidermal growth factor receptor （EGFR），phosphatidylinositol 3 kinase（PI3K），phosphorylated phosphatidylinositol 3 kinase（p-PI3k），protein kinase B （Akt）and phosphorylated protein Akt （p-Akt）protein of cells in blank control group and ST different concentration groups（5，10，20 μmol/L）were determined by Western blot assay so as to validate the relationship between ST effect and EGFR/ PI3K/Akt signaling pathway. SCL- 1 cells and human normal skin fibroblasts cell WS 1 were divided into blank control group （0.1％ DMSO），ST group （20 μmol//L），ZD1839 group（positive control ，20 μmol//L，EGFR inhibitor ）and LY 294002 group（positive control，20 μmol//L，PI3K/Akt inhibitor ）. CCK- 8 assay was used to detect the cell proliferation in order to evaluate the cells cytotoxicity of ST. RESULTS ：Compared with blank control group ，the proliferation ，migration and invasion ability of SCL- 1 cells were significantly decreased in 5，10，20 μmol/L ST groups（P＜0.05）. Western blot and immunofluorescence assay showed that the expression of N-cadherin in SCL- 1 cells were decreased significantly in 5，10，20 μmol/L ST groups（P＜0.05），while the protein expression of E-cadherin was increased significantly （P＜0.05）；the protein expressions of EGFR ，p-PI3K and p-Akt were significantly decreased （P＜0.05）. Compared with ST group ，the proliferation ，migration and invasion ability of SCL- 1 cells were increased significantly in ST + NSC 228155 group and ST + SC 79 group （P＜0.05）. Compared with blank control group ，the proliferation ability of WS 1 cells had no significant change in ST group ，while the proliferation ability of SCL- 1 cells was decreased significantly （P＜0.05）；the proliferation ability of the two kinds of cells were decreased significantly in ZD 1839 group and LY 294002 group（P＜0.05）. Compared with ST group ，the proliferation ability of WS 1 cells was decreased significantly in ZD1839 group and LY 294002 group（P＜0.05），but there was no significant difference in the proliferation ability of SCL- 1 cells （P＞0.05）. CONCLUSIONS ：ST may inhibit the proliferation ，EMT and metastasis of SCL- 1 cells through inhibiting the activation of EGFR/PI 3K/Akt signaling pathway ，and its side effects are few.
ABSTRACT
OBJECTIVE：To study the effects and potential mechani sm of deoxyschizandrin on the proliferation ，migration and invasion of nasopharyngeal carcinoma cell HONE- 1. METHODS ：HONE-1 cell was set as cell model ，while CCK- 8 test，wound healing assay and Transwell chamber test were used to detect the proliferation ，migration and invasion ability changes of HONE- 1 cells after treatment with different concentrations [ 0（blank control ），10，20，40 μmol/L] of deoxyschizandrin. Computer molecular docking was performed to analyze the binding ability between deoxyschizandrin and Met protein. Western blotting assay was used to detect the relative protein expressions of p-Met ，p-PI3K，p-Akt，Bcl-2 and N-cadherin in cells. RESULTS ：Compared with blank control ，the proliferation ，migration and invasion ability of cells after treated with 10，20，40 μmol/L deoxyschizandrin were all decreased significantly （P＜0.05）. Results of molecular docking revealed that deoxyschizandrin could stably bind with the activity pocket of Met protein. Results of Western blotting assay demonstrated that compared with blank control ，10，20，40 μmol/L deoxyschizandrin all decreased the relative protein expressions of p-Met ，p-PI3K，p-Akt，Bcl-2 and N-cadherin in cells significantly（P＜0.05）. CONCLUSIONS ：Deoxyschizandrin can inhibit the proliferation ，migration and invasion of HONE- 1 cell via inhibiting the activation of Met/PI 3K/Akt signaling pathway.
ABSTRACT
Objective: To investigate the expressions of Calpain 1 and Calpain 2 proteins, as well as the effects of regulating Calpain 2 expression on the migration and invasion of pancreatic cancer PANC-1 cells in hypoxia microenvironment. Methods: The expressions of Calpain 1 and Calpain 2 mRNAs in pancreatic cancer tissues and their survival prognosis value were analyzed using GEPIA (Gene Expression Profiling Interactive Analysis) database online tool. The pancreatic cancer PANC-1 cells were cultured in normoxia (21% O2) and hypoxic (1% O2) environment, then the expressions of Calpain 1 and Calpain 2 mRNAs and proteins were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The siRNA targeting CAPN2gene(encoding Calpain 2) was transfected into pancreatic cancer PANC-1 cells, then the silencing effect of CAPN2 gene was detected by real-time fluorescent quantitative PCR and Western blotting. The PANC-1 cells with Calpain 2 low expression were cultured under the normoxia and hypoxic conditions, then the migration and invasion abilities of these cells were detected by wound healing test and Transwell chamber assay, the expressions of E-cadherin and Vimentin were detected by Western blotting, and the cell morphology was observed by inverted microscopy. Results: The expressions of Calpain 1 and Calpain 2 mRNAs in human pancreatic cancer tissues were higher than those in normal pancreatic tissues (both P < 0.01), and the high expression of Calpain 2 was associated with the poor prognosis of pancreatic cancer patients (P = 0.013). Compared with the normoxia, the expression levels of Calpain 2 mRNA and protein were increased in hypoxia microenvironment (both P< 0.01), but the expressions of Calpain 1 mRNA and protein were not changed. The Calpain 2 low-expressed PANC-1 cells were established successfully. Under normoxia condition, the down-regulation of Calpain 2 expression inhibited the migration and invasion of PANC-1 cells (both P < 0.05); hypoxia promoted the migration and invasion of PANC-1 cells (both P < 0.05); but the down-regulation of Calpain 2 expression could reverse the promotion effects of hypoxia on the migration and invasion of PANC-1 cells (both P < 0.05). Furthermore, the expression of E-cadherin increased, the expression of Vimentin decreased in PANC-1 cells after the down-regualtion of Calpain 2 expression under normoxia condition (both P < 0.05), while the size of irregular cells increased, and the part of cells had apoptotic change; but the expression of E-cadherin decreased, the expression of Vimentin increased in PANC-1 cells cultured under hypoxia condition (both P < 0.05), while the most of cells had mesenchymal-like changes, showing long shuttle type; but the down-regulation of Calpain 2 expression could reverse the effect of hypoxia on the epithelial-mesenchymal transition of PANC-1 cells (both P< 0.05). Conclusion: Stimulated by hypoxia microenvironment, the expression of Calpain 2 was increased, so as to promote the migration and invasion of PANC-1 cells via regulating E-cadherin and Vimentin expressions.