Cdc6 Contributes To Stemness Of Cancer Cells And Norcantharidin Induces Apoptosis Of Cancer Stem Cells

BackgroundCancer stem cell(CSC)refers to a group of cells with infinite self-renewal ability in cancer cell,which is the source of infinite growth of cancer.CSC can resist the killing of chemotherapy and radiotherapy,lead to cancer recurrence and metastasis.Therefore,how to effectively suppress or clear CSC has become a research hotspot in the field of cancer.It is the core problem of CSC to study the molecular mechanism of CSC's multi-potential "sternness".Cdc6(cell division cycle 6)is the key regulator of DNA replication.It is over expressed in cancer cells and is related to the malignant process of cancer.The high expression of Cdc6 is not only the result of strong cancer proliferation,but also Cdc6 itself has the ability to promote malignant transformation of cells.It has been reported that Cdc6 can inhibit transcription of INK4/ARF as a transcriptional inhibitor in cancer cells,and the transcriptional inhibition of this gene is an important feature of CSC's "sternness".However,whether Cdc6 exert same function in CSC is had not been reported.The cancer suppressor gene INK4/ARF can encode three proteins simultaneously:p16INK4A,p15INK4B and p14ARF,which can regulate the cell cycle through pRb and p53 pathway respectively.In human cancers,the INK4/ARF gene is frequently inactivated,and closely related to cancer occurrence,development and prognosis.Norcantharidin(NCTD)is China's independent developed anti-cancer drug,and is currently widely used in the treatment of a variety of cancers.But its specific anti-cancer mechanism has not yet been elaborated.Our prevous researches proved that NCTD can degrade Cdc6 protein in cancer cells.Objective1.Screening and identification of CSCs from the cancer cell lines by limited dilution method.2.To investigate the expression of Cdc6 protein in CSC and to study whether it is involved in the regulation of INK4/ARF gene.3.To investigate the effect of norcantharidin on CSC.Methods1.Screening of cancer stem cells:HepG2 or UMUC-3 cell lines were cultured in ultra-low attachment culture plates in serum-free medium(containing EGF 20 ng/mL,b-FGF 20 ng/mL,LIF 20ng/mL,B27 20?L/mL and BSA 4?g/mL in DMEM/F12 medium)for 2 months or more.2.Identification of cancer stem cells:The cancer stem cell markers CD133,CD44,NANOG,OCT4 and ABCG2 were detected by qPCR and flow cytometry.3.Comparison of CSC functional:In vitro cell proliferation;cell tolerance to chemotherapeutic drugs;EdU proliferation assay;flow cytometry to detect cell cycle and in vivo cell tumorigenesis experiments.4.The total amount of Cdc6 protein and the expression of chromatin neutral and non-chromatin were detected by Chromatin binding.5.The interaction between Cdc6 and INK4/ARF gene was detected by Chromatin Immunoprecipitation.6.The apoptosis was detected by flow cytometry.Results:1.Culture HepG2 and UMUC-3 by limited dilution five days later,can able to see monoclonal cell growth pelletization,monoclonal cell culture taken after 20 days group continue to expand to 60 days culture.2.Identification of cancer stem cells:Flow cytometry showed that the percentage of CD 133+ in HepG2 cells was 90.4±1.1%,which was significantly higher than that of parent cancer cells(0.5±0.2%),and the ratio of CD44 +(0.5±0.12%)also higher than parent cancer cells(0.1±0.04%).The mRNA levels of CD133,CD44,NANOG,OCT4 and ABCG2 were significantly higher than those of parent cancer cells.In vivo tumorigenesis experiments showed that the cancerr formation rate of HepG2 cells(1×105 cells/per)was 1/5,5×106 cells/per cancer formation rate was 3/5,the corresponding parental HepG2 None of the tumor.These results indicate that the cells selected by limited dilution method are CSC.3.CSC resistance to paclitaxel(PTX),the MTS results showed that:The IC50 of PTX for HepG2-CSC was much more than 500 nM/mL much higher than that of IC50 of 60.9±5.7 on parent HepG2.IC50 of UMUC-3-CSC is much more than 500 nM/mL much higher than IC50 of parent UMUC-3:63.0 ± 4.8.4.CSC for still dormant cells,Flow cytometry showed that the percentage of GO/G1 phase in CSC was significantly increased(HepG2-CSC:70.72 ± 5.2%,UMUC-3-CSC:70.24 ± 4.9),indicating that CSC was dormancy period.EdU incorporation experiments showed that CSC had almost no DNA replication.After the release of serum dormant CSC re-enter the cell cycle,and after four days the release of serum,CSC proliferation rate is significantly higher than parent cancer cells.5.In the CSC,Cdc6 was only bound to the RDINK4/ARF gene(-311--157)region and not in the RDINK4/ARF gene(-175--51)region by ChIP.6.The percentage of HepG2-CSC and UMUC-3-CSC apoptosis(70.54±0.26%,29.93±1.32%)increased significantly(35.00±0.89%,15.11±0.78%)after culture added NCTD.Conclusions1.Dormant CSC can be obtained from cancer cell lines by the limiting dilution method.2.In dormant CSC,Cdc6 is still expressed in chromatin,and was bined with RDINK4/ARF to regulate the function of INK4/ARF3.NCTD can make dormancy CSC apoptosis.