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Effects Of WKD-106 Down-regulation On ATM Protein In Human Colon Cancer SW620 Cells And Its Mechanism

Posted on:2020-09-19Degree:MasterType:Thesis
Country:ChinaCandidate:Y ZhuFull Text:PDF
GTID:2404330572984332Subject:Biology
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Objective:The effect of 13 pyrazole-containing monocarbonyl curcumin analogs synthesized in our laboratory on the activity of human colon cancer SW620 cells was screened,and the stronger curcumin analogue WKD-106 was selected for further study on its effect in SW620 cells and its mechanism.Methods:1.The effect of pyrazole-containing monocarbonyl curcumin analogs on the activity of SW620 cells:Human colon cancer SW620 cells were cultured and treated with 13 new compounds of pyrazole-containing monocarbonyl curcumin analogs WKD-101 to WKD-113?20?M?and curcumin?20?M?for 24 h,48 h and 72 h?Equal volume DMSO was added to the negative control group?,respectively.The effects of pyrazole-containing monocarbonyl curcumin analogs on the activity of colon cancer SW620 cells were determined by MTT assay.SW620 cells were treated with the selected new compounds WKD-101,WKD-106,WKD-113?20?M,10?M,5?M,2.5?M,1.25?M,0?M?for 24 h,48 h and 72 h.The OD value was determined by MTT assay,and the IC50 value was calculated.2.Effect of WKD-106 on apoptosis,cell cycle and related proteins in SW620 cells:Human colon cancer SW620 cells were seeded in 6-well plates,and compounds WKD-106?5?M,2.5?M,1.25?M,0?M?and curcumin?5?M?were used 24 h later.The cells were treated for 48 h,and cells were digested and counted for clone formation,migration and invasion experiments,and apoptosis and cycle were detected by flow cytometry.Human colon cancer SW620 cells were seeded in 6-well plates,after 24 h,the cells were treated with compounds WKD-106?5?M?and curcumin?5?M?for 48 h?Equal volume DMSO was added to the negative control group?.The expression of apoptosis and cell cycle related proteins Bcl-2,cleaved-caspase 3,PARP and cyclin B1and cyclin D1 were detected by Western Blot.3.The effect of WKD-106 on apoptosis and cell cycle of SW620 cells was related to its inhibition of ATM protein expression:Human colon cancer SW620 cells were seeded in a 6-well plate and treated with WKD-106?5?M?and curcumin?5?M?for 48 h?Equal volume DMSO was added to the negative control group?,and the expression of ATM protein was detected by Western Blot.On the other hand,after 24 h transfection of ATM overexpressed plasmid,the ATM protein expression was detected by 5?M WKD-106 for48 h to observe whether WKD-106 could regulate the protein expression of ATM.Human colon cancer SW620 cells were seeded in 6-well plates,after 24 h,the cells were treated according to the following four groups:negative control group,WKD-106?5?M,48 h?,ATM?overexpressing ATM plasmid?,WKD-106?5?M,48 h?+ATM,and the treated cells were digested and counted for clone formation,migration and invasion experiments,and apoptosis and cycle were detected by flow cytometry.The expression of apoptosis and cell cycle related proteins Bcl-2,cleaved-caspase 3,PARP and cyclin B1 and cyclin D1 were detected by Western Blot,and to clarify the effect of WKD-106on apoptosis and cell cycle of SW620 cells was related to its inhibition of ATM protein expression.Results:1.WKD-101,WKD-106,WKD-113 had better anti-tumor effect,and the effect was better than the positive drug curcumin,showing time-dose dependence,and the IC50value of WKD-106 is the smallest.2.Compound WKD-106 could down-regulate the ability of SW620 cells to clone formation,migration,invasion,and promoted cell apoptosis,leading to cell cycle arrest in S phase and G2/M phase.At the same time,WKD-106 could significantly down-regulate the protein expression of Bcl-2 and cyclin D1,and up-regulated the protein expression of cleaved-caspase 3,PARP and cyclin B1.3.WKD-106 was able to down-regulate ATM protein expression in SW620 cells.Overexpression of ATM could reduce the down-regulation of WKD-106 on the clone formation,migration and invasion of SW620 cells,partially offsetted the effect of WKD-106 on SW620 cells apoptosis and its cell cycle arrest.At the same time,overexpression of ATM could partially reverse WKD-106 induced the down-regulation of protein expression on Bcl-2 and cyclin D1,as well as the up-regulation of cleaved-caspase 3,PARP and cyclin B1.Conclusion:This study mainly observed the preliminary inhibitory effect of 13pyrazole-containing monocarbonyl curcumin analogs in SW620 tumor cells,among which WKD-101,WKD-106 and WKD-113 had good anti-SW620 tumor cell survival effect,and WKD-106 had the strongest effect.The mechanism may be achieved by down-regulating the ATM protein in SW620 cells to promote apoptosis and cycle arrest.
Keywords/Search Tags:WKD-106, SW620 cells, ATM protein, apoptosis, cycle arrest
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