| Cross-resistance or multidrug resistance (MDR) remains a major obstacle in the treatment of colon carcinoma that displays a characteristic chemoresistance towards various anticancer drugs, which may have different structures and anticancer mechanisms. The investigation of the mechanisms underlying multidrug resistance might reveal possible avenues for therapy. Our aims of the study were: (1) To establish a MDR model in vitro by increasing concentrations of adriamycin and observing the expression level of multidrug resistance related genes in different time point during introduction process in order to elucidate possible mechanisms of MDR involved in colon carcinoma. (2) We studied the relationship between phenotype changes of MDR cells and a multidrug resistance. (3) To investigate the mechanism of PKC regulating MDR.Establishment of a MDR model A MDR subline, LoVo/Adr cell line was established by long-term exposure of the parental line LoVo cell to increasing concentrations of adriamycin, the concentration of which in the medium increasing from 0.04 to 1.0 μg/ml. over more than 8 months, 55 passages of culture. LoVo/Adr cell line was MDR subline by. proved by methyl tetrazolium assay (MTT) . The drug resistance index of LoVo/Adr to adriamycin, vincristine, mitomycin, cytoxan was 61, 14, 3, and 9-times stronger than the native line respectively, but remained unchanged tostronger than the native line respectively, but remained unchanged to 5-fluorouracil. To trace the acquired MDR gene, the reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the expression of MDR-related protein (MRP), mdrl, glutathione transferase(GST- π), and tropoisomerase II a mRNA during the process of resistance increasing to adriamycin. The results were as follows: The LoVo cells showed no mdrl expression and the level of mdrl mRNA expression was increased gradually along with the concentration increasing of ADR in resistant cell lines, and the level of GST- n mRNA was only increased significantly in the initial stage, MRP mRNA expression was detected neither in parental nor resistant cell lines. The level of Topo II a mRNA remained unchanged. The protein level of these genes, which had significantly changed, was detected by immunohistochemistry. P-gp, which is coded by mdrl, was negative in LoVo cells, while intensive positive in LoVo/Adr cells. The protein expression of GST- Jt in LoVo/Adr cells was significantly higher than that in LoVo cells. The activity of GST was further tested using the 1-C1-2, 4-dinitrobenzene method. The result showed the activity of GST was not significantly different in LoVo/Adr and LoVo cells. The above result suggested that LoVo/Adr cell line offers a model with a typical MDR phenotype, in which, the resistant mechanisms were mediated by mdrl and GST- π not MRP and Topo II α .The relationship between phenotype changes of MDR cells and a multidrug resistance We used light microscopy, scanning electron microscopy to observe the morphologic changes in parental and resistant cells. The result showed that compared with LoVo cells, LoVo/Adr cells were larger and mixed with giant cells of different sizes. Growth curve and doubling time of cells were counted by continuous cell count. When compared with LoVo cells, the resistant subline had a lower growth rate. The doubling time of LoVo and LoVo/Adr cells were 20.9 and 33.58 hours respectively. The doubling time of LoVo/Adr cells was therefore 1.7-times longer than LoVo cells. Cell cycles were analyzed by flow cytometry. Flow cytometry analyses showed that in the resistant cell pool there were a greaterpercentage of cells in the GI phase and a lesser percentage of cells in the S phase. We made use of adriamycin intrinsic fluorescence to examine the uptake of ADR in cells by flow cytometry. The results were as follows: The uptake of ADR was 2.04±0.70 in LoVo/Adr cells, and 3.99±1.87 in LoVo cellsCP<0.01). The uptake ability for ADR increased to 2.67±1.13 (P<0.01) in LoVo/Adr cells when pulsing VP(verapamil). The...
|
PDF Full Text Request