| Objective: Human colon cancer cell lines were used as the major object, the main parts of this study is to investigate potential effect of p53 gene in regulating the expression of cancer stem cell surface marker CD133 and the related mechanism.In addition, the the radiosensitization efficiency of DNA-dependent protein kinase catalytic subunit inhibitor NU7026 was also evaluated on colon cancer cells HCT116 in which the subpopulation of CSC marker CD133 positive cells takes the most predominant ratio.Method: Polyacrylamide gel electrophoresis and immunoblotting hybridization(Western blot) was used to detect the proteins expression;In combined with the cancer stem cell(CSC) surface marker of CD133/CD44 flow cytometry was used to monitor and measure the ratio of the CSC subpopulation in wild-type HCT116p53+/+ and mutant HCT116p53-/- cells; CD133 and p53 mRNA relative expression was determinded by RT-PCR(quantitative real time PCR) method; Thep53-depressed cells or p53-overexpressed cells were estabolished by transfecting the p53-specific shRNA expression vectors or exogenous WT-p53 expression vectors separately.; The recombinant plasmid of CD133 promoter driving luciferase gene expression was constructed by Taihe Tec-biology company, and the dual chemical luminescence detection was performed for evaluating the trancscriptive activity of CD133 promoter-driving luciferase geneexpression. In order to investigate the radiosensitizing effect of DNA-PKcs inhibitor NU7026,The HCT116 cells with an high ratio of CSC subpopulation were divided into four groups: experimental control group, only 20?mol/L NU7026 group, 2 Gy ?-ray irradiation group, 20 ?mol/L NU7026 combined with 2Gy irradiation group, NU7026 treated the cells 2 hours before ?-ray irradiation. Cell clone formation and MTT methods were performed to measure cells proliferation; flow cytometry assay was used for the analyses of cell cycle, apoptosis, and cancer stem cell subpopulation.Immunofluorescence laser confocal microscopy was employed to observe and count the foci number of the phosphorylated histone H2 AX foci,which indicated the DNA double-strand breaks-induced by irradiation.Results: Differental p53 protein expression status was confirmed by Western blot analysis in p53 wild-type(WT) colon cancer cells HCT116p53+/+ and the mutant cells HCT116p53-/-. It has been found that the CD133 positive subpopulation was predominant in p53 wild typeHCT116 cells(84.84±0.05%), while it only took a less ratio in the p53 mutant HCT116 cells(4.13 ± 0.02%). The relative transcription level of CD133 mRNA was much higher in p53-WT cells(0.16 ± 0.041) than that in mutant cells(0.0081 ± 0.0039)(t=6.29, P < 0.01), the difference was statistical significant. shRNA-mediated depression of p53 resulted in decreased CD133 transcription from 85.78 ± 1.17% to 4.70 ± 0.17%,t=119.79, P < 0.0001, as well as decreased transcriptive activity of CD133 promoter in HCT116p53+/+ cell. On the other hand, expression of the exogenous p53 protein in HCT116p53-/- cell led to increased expression of CD133 mRNA and transcriptive activity of CD133 promoter. It was also observed a weak but statistical significant increase of CD133 positive subpopulation from 4.52 ± 0.35% to 5.83 ± 0.30%,t=4.92,P < 0.01. Moreover, shRNA-mediated depression of p53 in 293 T cells was also shown to increase the CD133 promoter activity. In the radiation treatment experiments, it was found that DNA-PKcs inhibitor NU7026 significantly sensitized p53 wild type HCT116 cells to 2 Gy of?-ray irradiation, an siginicant radiosensitization effect was observed in terms of decreased survival rate(t= 7.22, P < 0.01). The ratio of CSC marker CD133 positive subpopupation increased in survived cells post irradiation, while NU7026 largerly attenuated this enhancement of CD133 positive subpopupation. NU7026 treatment also significantly increased the occurrence of irradiation-induced irreversible G2/M phaseblock at 24 hours post-irradiation(t=7.67, P < 0.01), as well as increased the induction of early apoptosis within 48 hours. Obviously, NU7026 treatment significantly inhibited the cellular capacity of repairing DNA double-strand breaks induced by ?-ray irradiation.Conclusion: Our results demonstrated that p53 positively regulates the transcriptional expression of the CSC marker CD133 in human colon cancer HCT116 cells, and this effect is mechanistically related to the activation of CD133 gene promoter. DNA-PKcs inhibitor NU7026 can significantly sensitize the colon cancer HCT116 cells of CSCs as the superiority subsets to ionizing radiation. NU7026 attenuates the increasing of CSCs subpopulation after radiation treatment. There are multiple mechanisms for the radiosensitization effect of NU7026,including inhibition of DNA repair, inccrease of reversible G2/M phase arrest and apoptosis induction. |
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