In Vitro And In Vivo Reversal Of P-gp-mediated Multidrug Resistance Of Breast Cancer By Fulvestrant (ICI182,780)

Breast cancer is one of the most common malignancies and a leading cause of cancer-related mortality in women. Doxorubicin and paclitaxel are widely used as the first-line scheme of chemotherapy. However, multidrug resistance (MDR) remains a major obstacle to the successful cancer chemotherapy and is associated with poor clinical response. P-glycoprotein (P-gp) which belongs to the ATP-binding cassette (ABC) transporter superfamily is a well-known transmembrane glycoprotein and functions as an ATP-dependent efflux pump with a variety of substrates. Thus, current studies have focused on blocking specific efflux pathways using various MDR modulators, especially for P-gp.Fulvestrant (ICI182,780, Faslodex) is a new type of pure ER antagonist without known agonist effects. In our previous study, we found that the combination of fulvestrant could dramatically reverse the ER-mediated resistance and sensitize ER-positive BCap37cells which derived from stable transfection of an ER-α expression vector into ER-negative BCap37cells to antimicrotubule agents such as paclitaxel and vinca alkaloids. Recently, we established two independent novel paclitaxel-resistant cell lines Bats-72and Bads-200from the breast cell line BCap37by two-stage screening strategy (TSSS), both of them were ER-negative and showed cross-resistance to other anticancer drugs including doxorubicin and vinorelbine.In this thesis research, we investigated whether fulvestrant could reverse MDR and its potential mechanisms using the novel ER-negative MDR cell lines and tumor xenograft models. By a series of cytotoxicity assays in vitro, we found that fulvestrant significantly sensitized paclitaxel and doxorubicin-induced cytotoxicity in a dose dependent manner in ER-negative MDR cell lines including Bats-72, Bads-200and KBv200. Comparing with other modulators, the reversal potency of fulvestrant was similar to that of verapamil and more effective than that of tamoxifen when in combination with doxorubicin at the same doses. Fulvestrant potentiated doxorubicin-induced apoptosis, G2/M arrest and cyclin B1expression in Bats-72and Bads-200cell lines. Fulvestrant increased intracellular accumulation and retention of doxorubicin in Bats-72and Bads-200cells, which implied that the reversal of drug resistance by fulvestrant may be attributable to the inhibition of P-gp-mediated drug transport. Fulvestrant not only restored doxorubicin accumulation but also tried to relocalize them to the nuclei in Bats-72and Bads-200cells. These results implied that fulvestrant might increase the amount of doxorubicin to access to nuclear targets in MDR cells, which may be another potential reason related to its reversal potency to P-gp mediated doxorubicin resistance. Furthermore, fulvestrant was able to stimulate the ATPase activity of P-gp, indicating that fulvestrant acted as a substrate of P-gp to inhibit its function of drug-efflux. Finally, in animal studies, we found that coadministration of fulvestrant potentiated the antitumor activity of paclitaxel against Bats-72xenografts.In summary, the obtained results indicate that fulvestrant is a selective and effective modulator of P-gp mediated MDR. It can significantly reverse P-gp-mediated multidrug resistance by inhibiting P-gp activity independent of estrogen receptor signal pathway. Fulvestrant not only take part in ER-mediated pathway in breast cancer therapy, the combination of fulvestrant with chemotherapy agents plays an important role in combating P-gp-mediated multidrug resistance. This study may provide useful clues for understanding the novel anticancer mechanism of fulvestrant and supporting the clinical application of fulvestrant for the treatment of metastatic and progressive breast cancer.