Differences In Perifosine Resistance Between Euploid And Aneuploid Colon Cancer Cells And Relative Mechanisms

Aims:Aneuploidy refers to an abnormal number of chromosomes in a cell,which might be induced by aberrancies such as spindle assembly checkpoint(SAC)dysfunction during mitosis.Aneuploidy has been found in most tumors and highly aneuploid tumors show poor prognosis in clinic.Previous studies demonstrate that aneuploid tumor cells are resistant to conventional chemotherapeutic drugs such as cisplatin,oxaliplatin and hydroxyurea.Therefore,exploring the effects of more categories of chemotherapeutic drugs on aneuploid tumor cells is of great significance for clinical tumor therapy.Perifosine is a serine/threonine kinase 1/Protein kinase B(AKT also known as PKB,)inhibitor in clinical trials.It can increase the expression of the tumor suppressor p53 by inhibiting AKT activity,and thus induce apoptosis in tumor cells.Whether perifosine affects the proliferation of euploid and aneuploid tumor cells differentially and relative mechanisms remain elusive.In this study,by establishing a model of aneuploidy on the basis of euploid colon cancer cells,we examined the differences between euploid and aneuploid colon cancer cells in sensitivity to perifosine treatment and explore relative mechanisms.Methods:First,the HCT116-MAD2 cell line was generated by the lentiviral system containing the pT3G-MAD2 lentiviral plasmid.Upon doxycycline(Dox)treatment,the cells could temporally express MAD2 shRNA to knock down the SAC component MAD2 by activating the tet-on system and induce the formation of aneuploidy(Dox(+)cells)by disturbing SAC function.On the other hand,HCT116-MAD2 cells without Dox treatment were set as euploid control(Dox(-)cells).Fluorescence in the cells after Dox treatment was observed under the microscope to confirm the expression of MAD2 shRNA.Protein expression of MAD2 was examined by Western blot.Cell karyotypes of euploid Dox(-)and aneuploid Dox(+)cells were determined by chromosome spread analysis.Second,the differences in proliferation between euploid Dox(-)and aneuploid Dox(+)cells treated with perifosine at different concentrations for 24 hours(h),48 h and 72 h were examined by cell number count and CCK-8 analysis.Apoptosis in the cells was determined by the expression levels of the apoptotic marker protein cleaved-PARP and cleaved-Caspase 3 through Western blot analyses after 48 h of perifosine treatment,in order to compare the different perifosine sensitivity between euploid and aneuploid HCT116-MAD2 cells.Third,Western blot was applied to detect the expression of total AKT,phosphorylated AKT at Ser473(p-AKT)and p53 in euploid Dox(-)and aneuploid Dox(+)cells after 48 h of perifosine treatment,in order to compare the activation of the AKT-p53 pathway in the cells.Fourth,quantitative real time reverse transcript PCR(qRT-PCR)was applied to detect the mRNA expression of p53 downstream apoptosis-related genes BAX,PUMA and NOXA.The protein levels of PUMA and BAX were also examined by Western blot analyses to determine the activation of p53 downstream pathway in euploid and aneuploid colon cancer cells.Fifth,AKT was stably knocked down in HCT116-MAD2 cells by lentiviral system with two different shRNAs to generate the HCT116-MAD2-AKT-1,HCT116-MAD2-AKT-2 and control HCT116-MAD2-Con cell lines.Under Dox treatment,aneuploidy can be induced in the two cell lines.Cell karyotypes of the resultant Control shRNA Dox(-),Control shRNA Dox(+),shRNA AKT1 Dox(-),shRNA AKT1 Dox(+),shRNA AKT2 Dox(-)and shRNA AKT2 Dox(+)cells were analyzed by metaphase chromosome spread.Cell counting was applied to compare cell growth in the six groups of cells.qRT-PCR and Western blot were used to examine apoptosis and the activation of the AKT-p53 pathway.Results:First,HCT116-MAD2 cells were treated with Dox for 12 h(Day 0)to induce MAD2 shRNA expression.Red fluorescence was observed in aneuploid Dox(+)cells three days after Dox removal and the fluorescence decreased on Day 6,suggesting transient expression of MAD2 shRNA.No fluorescence was detected in euploid Dox(-)cells.Compared with euploid Dox(-)cells,MAD2 expression in aneuploid Dox(+)cells was significantly decreased on Day 3 and recovered to normal level on Day 6.Chromosome spread analyses showed that 90%of Dox(+)cells were aneuploid,while the aneuploidy ratio in Dox(-)cells was only 14%.Second,under the treatment of perifosine at different concentrations for different time spans,the cell viability of aneuploid Dox(+)cells was significantly higher than that of euploid Dox(-)cells.Although the expression of cleaved-PARP and cleaved-Caspase 3 increased in both euploid and aneuploid cells after perifosine treatment,the increment of cleaved-PARP and cleaved-Caspase 3 was significantly lower in the aneuploid cells.Third,after perifosine treatment,the expression of p-AKT decreased and the levels of p53 increased in both Dox(-)and Dox(+)cells.Notably,less increase in p53 was observed in Dox(+)cells compared with Dox(-)cells.Fourth,both the mRNA levels of p53 downstream targets BAX,PUMA and NOXA and the protein levels of BAX and PUMA increased to a lower extent in aneuploid Dox(+)cells in comparison with euploid Dox(-)cells.Fifth,MAD2 shRNA was transiently expressed by treating HCT116-MAD2-Con,HCT116-MAD2-AKT-1 and HCT116-MAD2-AKT-2 cells with Dox for 12 h on Day 0 to induce aneuploidy.Chromosome spread analysis showed that the aneuploid ratio in Control shRNA Dox(-),Control shRNA Dox(+),shRNA AKT1 Dox(-),shRNA AKT1 Dox(+),shRNA AKT2 Dox(-)and shRNA AKT2 Dox(+)cells was 13%,90%,10%,87%,13%and 87%respectively.In comparison with euploid colon cancer cells,aneuploid colon cancer cells showed less decrease in proliferation,lower expression of the apoptotic marker cleaved-PARP,less expression of p53 and its downstream genes BAX and PUMA mRNA after knockdown of AKT.Conclusion:First,an aneuploid model with aneuploidy as the main abnormality was established by transiently disturbing SAC function through the tet-on-system-controlled expression of MAD2 shRNA in euploid colon cancer cells.Second,compared with euploid Dox(-)cells,aneuploid Dox(+)cells are more resistant to perifosine treatment.Third,the differences in perifosine sensitivity between euploid Dox(-)and aneuploid Dox(+)cells is related to differential activation of the p53 pathway in the cells,and AKT expression showed less influence on the viability of aneuploid colon cancer cells than euploid colon cancer cells.