Construction Of T24 - Resistant Strain Of Bladder Cancer And Analysis Of Its Biological Characteristics

Background: Bladder cancer is one of the most common urogenital cancers inwestern society, and the incidence in men ranks4th, women ranks9th. In our countryit,s the first malignant tumor in urogenital system and there is a growing trend.Surgical resection combined with intravesical therapy is the major method for clinicaltreatment of superficial bladder cancer, but still have the recurrence rate of40%-80%.Chemotherapy resistant is one of the main reasons of recurrence, but the mechanismis not clear. Therefore, we establish a human drug-resistant bladder cancer cell lineT24/MMC, and to investigate its characteristics, in order to study the mechanism ofits formation.Experimental Design：Human bladder cancer cell line T24was established usinggradually increased concentration of mitomycin (MMC) in culture. Basiccharacteristics of both the parental cell line T24and the drug-resistant cell lineT24/MMC were analyzed and compared. The shapes of the cells were observed by ahigh-powered microscope. The cell growth curve and proliferation activity weredetected by MTT assay. The changes of single cell proliferation were detected bycolony formation assay. The migration and invasion ability were detected bymigration and invasion assay. The sphere-forming ability and enrichment of cancerstem cells were detected by serum-free culture medium method.Results:1. Using gradually increased concentration of mitomycin(MMC) in culture,which lasted4months, until the cells can growth well and stablly, and can sustainedgrow, forming T24bladder cancer drug-resistant cell line.2. Compared with T24, the T24/MMC cells grew more rapidly after the withdrawal,and the growth capacity increased in the test of MTT. Through the colony formation,transwell migration/invasion experiment found T24/MMC cells colony-forming，migration and invasion ability increased. That means the T24/MMC have a high degree of malignancy.3. In serum-free suspension culture, T24/MMC cells began to form sphere on thethird day, and the sphere foming with shorter time, high rate, more tightness, andbigger volume. It indicates that T24/MMC cells were enriched with cancer stem cells.Conclusion: Gradually increased concentration of MMC can induce drug-resistantbladder cancer cell line, and it was enriched of cancer stem cells.