Hydroxyurea Resistance Of Aneuploid Colon Cancer Cells And Relevant Mechanism

Objective:To study the chemoresistance of aneuploid colon cancer cells to hydroxyurea and investigate possible mechanisms.Methods:1.HCT116 cell line expressing inducible MAD2L1 shRNA was generated(HCT116-MAD2L1-shRNA),in which MAD2L1 shRNA can be induced by doxycycline(Dox).After been treated with Dox for 16 h,MAD2L1 gene was knocked down in P53+/+ HCT116-MAD2L1-shRNA cells,which were referred to as Dox(+)cells;While the HCT116-MAD2L1?shRNA cells treated with the same amount of dimethyl sulfoxide(DMSO)were served as control group,namely Dox(-);2.On the third and sixth days after drug removal,the expression of MAD2L1 protein was detected by Western blot;3.On the sixth day after drug removal,chromosomal spread method was used to detect the aneuploidy ratio of the two groups of cells.The drug resistance of two groups of cells to different concentrations of hydroxyurea(HU)was detected by CCK8 analysis.4.On the sixth day after drug removal,5 mM HU was applied to the two groups of cells for 48 h,and relative control groups(Dox(+)and Dox(-)cells without HU treatment)were established.RT-PCR was used to detect the expression of PUMA,BAX,NOXA and p21 genes.Western blot was used to detect the protein levels of Caspase-3,PUMA,BAX,p21 and y-H2AX.5.p53 knockout HCT116-MAD2L1-shRNA cells were treated the same as above described to detect cell growth and apoptosis.6.The p53+/+ and p53-/-cells were cultured in serum-free medium for 24 h,and the control group was cultured under normal conditions.The cells were treated with different concentrations of HU for 48 h and cell viability was detected by CCK8 method;After treating the cells with 5 mM HU for 48 h,the expression of PUMA,BAX,NOXA,and p21 genes were detected.7.Hydroxyurea resistance was determined in four other kinds of colon cancer cells of different karyotypes and p53 status by CCK8 analysis.Results:1.On the third day after Dox removal,compared with euploid Dox(-)cells,the expression of MAD2L1 protein in the aneuploid Dox(+)cells decreased significantly;on the sixth day after drug removal,the expression of MAD2L1 protein in the aneuploid Dox(+)cells recovered to its original level;2.On the sixth day after drug removal,the aneuploidy ratio of p53+/+ Dox(+)cells was 92%,while the aneuploidy ratio of DMSO-treated cells was 6%;3.After treatment with different concentrations of HU for 48 h,the viability of Dox(+)cells was significantly higher than that of Dox(-)cells,indicating that aneuploid Dox(+)cells were resistant to HU;4.After treatment with 5 mM HU,the expression of apoptotic genes increased in both groups of cells,but the increasing levels of PUMA,BAX,NOXA,p21 mRNA in Dox(+)cells were less than Dox(-)cells;With the increase of HU drug concentration,the increasing levels of y-H2AX,p21,PUMA,BAX,Caspase-3 proteins in Dox(+)cells were less than Dox(-)cells.5.In p53 knockout cells,after treatment with different HU concentrations for 48 h,Dox(+)cells had no resistance to HU.6.In p53 knockout cells,as HU concentration increased,Dox(+)cells showed comparable increase in the expression of PUMA,BAX,NOXA,p21 mRNA and Caspase-3?PUMA?BAX?y-H2AX protein with Dox(-)cells.7.Compared with serum-cultured HCT116 cells,serum-free HCT116 cells showed resistance to HU,and the increasing levels of apoptotic genes PUMA,BAX,NOXA?p21 mRNA were significantly lower.8.In p53 wild-type colon cancer cells,aneuploid Caco-2 cells were more resistant to HU treatment compared with euploid HCT116 cells.,whereas in p53 mutant colon cancer cells,no HU resistance was detected when comparing aneuploid SW480 cells with euploid HCT15 cells.Conclusion:1.Compared to euploid cells,p53 wild-type aneuploid colon cancer cells were resistant to HU;2.In p53-deficient cells,HU resistance of aneuploid cells disappeared;3.Aneuploidy activated p53 and led to growth inhibition,resulting in drug resistance to HU,which specifically affected proliferating cells.