The Molecule Mechanism Research About P-gp On The Invasion And Metastasis Of The Drug-resistance Breast Cancer Cell

ObjectivesSeveral recent studies suggested that the acquisition of the multidrug resistance (MDR) phenotype is associated with an elevated invasion and metastasis of tumor cells. Our previous study showed that small interference RNA-mediated gene suppression demonstrated that Anxa2was required for enhanced cell invasion of the MCF-7/ADR cells. It plays an essential role in MDR-induced tumor invasion. It is widely except that the occurrence of MDR is predominantly contributed by enhanced drug extrusion from cells due to over-expression of ATP-binding cassette (ABC) drug transporters, including P-glycoprotein (P-gp). Our recent experiments revealed that in MCF-7cells, the expression of P-gp and Anxa2was concomitantly increased in a time-dependent manner following exposure to low concentrations of chemotherapeutic drugs for～4weeks, suggesting the existence of a possible functional linkage between Anxa2and P-gp. Here, we used Westren blottingting, co-immunoprecipitation and immunofluorescence to investigate the effect of Anxa2interacting with P-gp in migration and invasion on MDR MCF-7/ADR cells.Methods1. We used RNA interference technology to knockdown the expression of P-gp in MCF-7/ADR, and the expression of P-gp was detected by Westren blotting.2. The MTT assay was used to detect the effects of P-glycoprotein down-regulation on proliferation of MCF-7/ADR cells and determine cell sensitivity to adriamycin.3. Flow cytometry (FCM) was used to detect if P-gp modulators have the efficiency in inhibiting the activity of P-gp.4. Invasion and migration assays were performed to the influence of P-glycoprotein on MCF-7/ADR cells migration and invasion ability in vitro.5. Co-immunoprecipitation and immunofluorescence assays were used to test the interaction and co-localization of P-glycoprotein and Anxa2.6. Westren blotting was used to examine the expression of ERK1/2to explore the mechanism of P-glycoprotein interacted with Anxa2regulating the migration and invasion of MCF-7/ADR cells.Results1. We found that downregulation of P-gp decreased the invasion and migration rate of MCF-7/ADR breast cancer cells.2. Inhibition of P-gp activity, using selective P-gp modulators, significantly affected the invasion and migration abilities of MCF-7/ADR cells.3. Both suppression of P-gp pump activity and down-regulation of P-gp expression disrupted the Adriamycin-induced ERK1/2phosphorylation in MCF-7/ADR.4. P-gp and Anxa2proteins are colocalized and P-gp can regulate the tyrosine phosphorylation of Anxa2, a key player involved in the activation of ERK1/2.Conclusions1. Our data indicate that P-gp may promote the invasion and migration of MDR breast cancer cells through regulating tyrosine phosphorylation of Anxa2.2. The interaction between Anxa2and P-gp may be one of the potential molecular mechanisms behind the association between MDR and invasive/migration potentials in breast cancer cells.