Preliminary Research On Chemotherapy Agent Sorting Bladder Cancer Stem Cells

Background Bladder transitional cell cancers(BTCC) is the most common urologic tumor in China and it has unfavorable prognosis. The current therapy effect is not so satisfatory. In1977, the cancer stem cell(CSC) hypothesis was initially proposed. CSCs hypothesis indicate that the reason for tumorigenesis, metastasis and recurrence of tumors is the existence of CSCs in tumors. CSCs is the targets for tumor targeting therapy.Cancer stem cells (CSCs) were verified experimentally in acute myeloid leukemia in the last decades. Though the existence of CSCs were subsequently verified in various tumors, CSCs of bladder transitional cell cancers(BTCC) had not been identified by the common methods reported. According to the phenomenon that CSCs were resistant to chemotherapy, we used mitomycin C for BTCC CSCs identification.Methods BTCC cell line T24were handled with mitomycin C for24hours. Cell inhibition was measured by WST-8Cell proliferation and cytotoxicity assay kit. Drug resistant cells were obtained to continue further experiments and the concentration gradient of mitomycin C was designed by the results of Cell inhibition assay:0.03mg/ml,0.5mg/ml,2mg/ml and they were divided into three groups (Group B, C and D). The same volume of PBS was used as controls(Group A). Drug resistant cells and control cells were used to investigate cell cycle distributions by flow cytometry. Real-time reverse transcription-PCR and Western blotting were used to assess expression levels of OCT-4and NANOG in drug resistant cells and control cells.Results Cell inhibition assay revealed that the inhabitation rate was increased with the concentrations gradient of mitomycin C. The maximum and minimum was92.5%±1.0versus64.1%±1.4(P<0.001). Drug resistant cells just comprised approximately7.5%of the total cells. Cell cycle results suggested more drug resistant cells than controls were in the G0G, phase (83.99%versus24.00%, P<0.05) and fewer were in the S phase (15.33%versus63.24%, P<0.05). Two important embryonic stem cell markers (OCT-4and NANOG) were highly expressed in protein and gene level in BTCC cell line T24after24-hour mitomycin C exposure and interestingly, the drug concentration gradient was in accord with OCT-4and NANOG expression. Conclusions After handled with mitomycin C for24hours, residual BTCCs (drug resistant cells) had stem cell properties. Associating with serum-free culture medium, our study may supply a complete new method for identifying BTCC CSCs.