Differential expression of P-gp in bladder cancer cell lines

Objective The generation of drug resistance often leads to the failure of the bladder cancer chemotherapy.P-glycoprotein (P-gp) is an ATP-dependent drug efflux pump linked to development of multidrug resistance in cancer cells.Our laboratory has successfully established adriamycin-resistant human bladder cancer cell line (pumc-91/ADM) from its parental cell line (pumc-91). According to the drug resistant spectrum analysis, pumc-91/ADM cell line exhibited the characteristics of multi-drug resistance.However, the expression of P-gp in two cell lines was still unknown.In this paper, there was a comparison between pumc-91/ADM and pumc-91 about the differential expression of P-gp. Method To determine the expression and location of P-gp in pumc-91 and pumc-91/ADM, qRT-PCR, Western blot and immunocytochemistry were applied in the experiment.qRT-PCR was implemented to research the expression of P-gp mRNA in two cell lines (pumc-91/ADM and pumc-91). Western blot was adopted to investigate the expression of P-gp protein in pumc-91 and pumc-91/ADM cell lines.Immunocytochemistry technique was used to explore the cellular location of P-gp and affirm its expression in two cell lines visually.Student's t-test was employed for statistical analysis and P<0.05 was considered statistically significant. Results qRT-PCR analysis revealed that the expression of P-gp mRNA was upregulated in drug-resistant cell line pumc-91/ADM compared to parental cell line pumc-91.To normalize for differences in the amount of total RNA, GAPDH was selected as an endogenous RNA control.Compared with pumc-91, the expression of P-gp mRNA was upregulated 7.74 fold in pumc-91/ADM (t=11.97, P<0.05). Consistent with the qRT-PCR result, Western blot confirmed the protein of P-gp expressed differentially in two cell lines.The expression of P-gp protein was significantly increased in pumc-91/ADM compared to pumc-91.According to the results, the differences between pumc-91 and pumc-91/ADM had statistical significance (t=4.35, P<0.05). Immunocytochemical analysis results demonstrated that P-gp was not only located in cell membrane but also in cytoplasm of the two cell lines.The expression of P-gp in pumc-91/ADM increased distinctly.The difference was statistically significant (t=11.41, P<0.05). Conclusion Compared with pumc-91, the expression of P-gp in pumc-91/ADM was significantly upregulated.(Chin J Lab Med, 2015, 38: 277-280) Key words: Urinary bladder neoplasms; Carcinoma, transitional cell; Cell line, tumor; R Glycoprotein