Preliminary Study To Negative-regulative Mechanism Of Radiative-sensitivity By P-glycoprotein In MCF-7/ADR Cells

OBJECTIVE: Acquisition of a multidrug resistance (MDR) phenotype in tumour cells is a major obstacle to successful therpy. One of the mechanisms involved in the development of MDR in human cells is the overexpression of P-glycoprotein(P-gp). And tumour cells often display cross resistance due to the same regulative mechanism probably. P-gp maybe present resistance of tumour cells to X-ray, but its specific machanism was remain unclear. In the study, we investigated the radiative-sensitivity of P-gp positive cells, and discussed the possible mechanism preliminarily after irradiated. Our study maybe privide a clue to clarify the mechanism of P-gp to radiative-resistance, and further more find a possible pathway to reverse MDR and radiative-resistance .METHODS: The drug resistance of MCF-7 and MCF-7/Adr cells were tested by the MTT assay. Flow cytometry was performed to examine expression of P-gp for sensitive cells and drug resistant cells. In order to inhibit P-gp function, MCF-7/Adr cells were incubated with verapamil after appropriate X-ray-irradiation. Dynamic changes of apoptosis ratio, and mitochondrial membrane potenial (m), and expression of Cytochrome c in cytoplasm, and activity of Caspase-3 in cytoplasm were analyzed by FCM for MCF-7/Adr cells line after the irradiation of x-ray. All the data was analyzed by SPSS10.0 statistic computer program.RESULTS: The 50 percent inhibition concentration (IC50, μ/ml) of the MCF-7 cells to ADR and VCR was 0.52±0.25,0.75±0.61, respectively; that of MCF-7/Adr cells was 25.75±1.57,10.44±3.21 respectively. IC50 of theMCF-7/Adr to ADR and VCR was 49.5 and 13.9 times than that of the MCF-7 cells,respectively. The P-gp positive rate and mean fluorescent intensity (MFI) of the MCF-7/Adr cells was 87.50%±6.08% and 44.14± 4.91,respectively, and MCF-7 cells was 7.67%±1.46% and 4.52±0.69,respectively, The P-gp expression in MCF-7/Adr cells is higher than that in MCF-7 cells significantly (P<0.001). Apoptosis ratio of P-gp-blocked group cells was higher than control group cells at 6h, 12h, 24h after the irradiation of x-ray, respectively (p<0.01) , it is highest of all at 24h (F=47.870,p=0.000). There is a significant difference between P-gp-blocked group cells and control group cells (F=89.465,p=0.000). The m MFI of P-gp-blocked group cells was higher than control group cells at 6h, 12h, 24h after the irradiation of x-ray, respectively(p<0.01), it is highest of all at 12h(F=74.485,p=0.000). There is a significant difference between P-gp-blocked group cells and control group cells (F=141.907,p=0.000). The expression of cytochrome c in P-gp-blocked group cells was higher than control group cells at 6h, 12h, 24h after the irradiation of x-ray, respectively (p<0.05), it is highest of all at 6h (F=18.844,p=0.001). There is a significant difference between P-gp-blocked group cells and control group cells (F=43.951,p=0.000). The activity of Caspase-3 in P-gp-blocked group cells was higher than control group cells at 6h, 12h, 24h after the irradiation of x-ray,respectively, (p<0.01), it is highest of all at 24h(F=7.929,p=0.006). There is a significant difference between P-gp-blocked group cells and control group cells (F=126.623,p=0.000).CONCLUTION: 1. The apoptosis of MCF-7/ADR cells induced by X-ray was related with the function of P-gp. 2. P-gp down-regulated the radiative-sensitivity of MCF-7/ADR cells to X-ray. 3. P-gp inhibited the depolarization of mitochondrial membrane potential(m) in MCF-7/ADR cells. 4. The study suggests that radiative-sensitivity of MCF-7/ADR cells might be related with trans-membrane transportation or distribution of apoptosis factor such as Cyt-c,Csapase-3 etc.which transfered by P-gp.