Effects Of AMD3100 On Growth And Invasion Of Pancreatic Cancer Cells

Objective: Pancreatic cancer is a common malignancy of the digestive tract with poor prognosis.Five-year survival of pancreatic cancer is less than 5%,which is largely attributed to its rapid growth and high tendency of metastasis.Recent studies have revealed an important role of the CXCL12-CXCR4 axis in regulation of tumor cell proliferation and metastasis,and AMD3100,a specific blocker of this axis,attracts much attention in the field of anti-tumor therapy.Some scholars have made breakthroughs in treating lung cancer,gastric cancer,and colon cancer with AMD3100,confirming its efficacy to inhibit proliferation and metastasis of several types of tumor cells.However,effect of AMD3100 on pancreatic cancer has rarely been studied,this study aims at this area,to provide basis for application of AMD3100 in the treatment of pancreatic cancer.Methods:Firstly,the culture of panc-1 of pancreatic cancer cells was carried out,and the experimental concentration was determined according to previous literature and preliminary experiments.After the above experiment,we made a macroscopic observation on the performance of Panc-1 cell biology behavior with AMD3100 from four parts:(1)Cell proliferation: To detect the cell proliferation of Panc-1 cells in logarithmic phase which had been intervened by different concentration gradient of AMD3100 in 24 h and 48 h with CCK-8.Each absorbance is measured by ELIASA to indirectly represents the number of cells.Each inhibition rate was calculated.(2)Apoptosis: To detect apoptosis of Panc-1 cell which had been intervened by AMD3100 in 24 h with Annexin V-FITC/PI,and the control group was established.To test and classify cells into normal cells,early apoptosis cells,cellular debris,and late apoptosis and death cell by Flow Cytometry and draw the scatterplot in order to compare the early apoptosis cells rate and the total apoptotic cells rate in control group and experimental group.(3)Cell migration: Wound scratch assay is that to contrast the migration distance over the artificial scratches of the Panc-1 cell cultivated in AMD3100 at 0 h,12 h and 24 h by the photograph,and the control group was established.Then,to calculate the migration rate of the groups in each time-point.The Transwell Chambers migration model was used to verify that the number of the cells which were passed through the semi-permeable membranes in the upper chamber after the Panc-1 cells were affected by the different concentration gradients of AMD3100 in 16 h.By the crystal violet staining,they were photographed and counted.(4)Cell invasion: The Transwell Chambers invasion model was used to verify that the number of the cell through the Matrix and the semi-permeable membrane after the Panc-1 cell under the influence of different concentration gradient of AMD3100 in 24 h,By the crystal violet staining they were photographed and counted.Finally,from the Angle of molecular proteins,using western blot technology test to detect the expression of CXCL12,CXCR4,MMP-9,VEGF-C by the Panc-1 cells under the influence of different concentration gradient of AMD3100 in 24 h,in order to make a preliminary validation about the mechanism of the effect of Panc-1 cells` biology behavior through AMD3100`s intervention.Results: 1.Cell proliferation: The average inhibition rates of 1,10,100 μg/ml AMD3100 for 24 h are 13.45%,15.53% and 25.97%,respectively.The average inhibition rates of 1,10,100 μg/ml AMD3100 for 48 h are 16.55%,28.06% and 47.43%,respectively.P < 0.05 in each group.The effect of inhibition on the proliferation of Panc-1 cells is proportional to the concentration and duration of AMD3100.2.Apoptosis: the early apoptotic cells in the control group was 5.41%,while the early apoptotic cells in the AMD3100 group was 19.31%,P < 0.05;The total apoptotic cells in the control group were 10.87% while the total apoptotic cells in the AMD3100 group was 36.03%,P < 0.05.The apoptotic cells of Panc-1 cells were significantly increased by AMD3100,and the apoptosis was induced by AMD3100.3.Cell migration: the migration rate of the blank control group at 12 h and 24 h was 45.27% and 77.18%,respectively.The migration rate of the AMD3100 group at 12 h and 24 h was 19.09% and 43.92%.P < 0.05 at the same time-point.AMD3100 can significantly reduce the migration of Panc-1 cells.Transwell Chambers migration model:the average numbers of the cells through the semi-permeable membrane in 0(control group)、 1 、 10 、 100μg/ml AMD3100 groups are 361,264,238,185,respectively(P < 0.05 in each group).The AMD3100 can significantly inhibit the migration ability of Panc-1 cells.Following the increase of AMD3100`s concentration,the number of the cells through the semi-permeable membrane is decreased and the inhibition effect was enhanced.4.Cell invasion: the numbers of the cells through the semi-permeable membrane in 0(control group)、1、10、100μg/ml AMD3100 groups in the Transwell Chambers invasion model are 359,265,197 and 122.P < 0.05 in each group.The AMD3100 can inhibit the invasive ability of Panc-1 cells.Following the increase of AMD3100`s concentration,the number of the cells through the semi-permeable membrane is decreased and the inhibition effect was enhanced.5.AMD3100 can significantly reduce the expression levels of MMP-9 and VEGF-C in Panc-1 cells,and the inhibiting effect is more significant following the increase of AMD3100`s concentration.The expression of CXCL2 and CXCR4 was not affected by AMD3100.CXCL12 and CXCR4 were expressed as usual when the concentration of AMD3100 was increased,.Conclusions: AMD3100 could inhibit Panc-1 cell`s proliferation,migration and invasion and induce cell apoptosis via down-regulation the expression of MMP-9 and VEGF-C by affecting the binding of CXCL12 and CXCR4.