Establishment And Characterization Of Novel Breast Cancer Drug Resistant Cells And Reversal Of Their Drug Resistance

Cancer has become the leading cause of human death.It is expected that the number of cancer patients will further increase in the next 20 years. Currently, chemotherapy is one of the most important treatments for cancer. Moreover, drug resistance often results in the failure of chemotherapy. Statistics shows that over 90% of the death of cancer patients is related to drug resistance. Therefore, drug resistance is one of the key obstacles in successful cancer treatment, and must be resolved urgently.Tumor cell lines are important tools for cancer research. In vitro tests should be conducted before applying in clinics. The studies based on drug-resistant cell lines play a key role in clarifying the potential mechanisms of tumor drug resistance (TDR), as well as finding means to reverse it. In this study, through different "two stages screening" strategies, we successfully established two novel drug resistant breast cancer cell lines Bats-72 and Bads-200 from the parental BCap37 cell line by using paclitaxel as a selective drug. Bats-72 was selected based on a high concentration of paclitaxel with time-stepwise increment exposure, while Bads-200 was selected based on dose-stepwise increment exposure to paclitaxel. The proliferation of Bats-72 and Bads-200 were slower than BCap37 in vitro, wheras their growth rates in vivo were faster than BCap37. Compared to parental BCap37 cell, both Bats-72 and Bads-200 were highly resistant to paclitaxel-induced cytotoxicity, cell cycle arrest and apoptosis. However, the drug resistance statuses of Bats-72 and Bads-200 were significantly different. First, Bats-72 was less resistant to MDR-related drugs than Bads-200. Second, the resistant spectrum of Bats-72 was larger thanthe one of Bads-200. Last but not least, in the absence of paclitaxel, the drug resistance of Bats-72 was stable, for over 300 days. While the drug resistance of Bads-200 decreased gradually, and it disappeared after 45 days.The new breast cancer resistant cell lines Bats-72 and Bads-200 which share the same origin were established using two different strategies by paclitaxel. Interestingly, Bats-72 and Bads-200 showed significantly different drug resistance and represented two different types of tumor drug resistance. Together with BCap37, Bats-72 and Bads-200 were valuable for studies on TDR. By using them as cell models, we carried out further studies to elucidate the mechanisms of TDR, screen new biomarkers for TDR and develop suitable strategies to overcome TDR.ATP-binding cassette (ABC) transporters can actively transport chemotherapy agents out of the cell. In this way, the intracellular drug concentration is decreased and TDR occurs. It has been reported that there are 15 ABC transporters associated with TDR. In particular, P-gp, BCRP, and MRP1 have been suggested to be the key elements for TDR. In this study, the ABC transporter modulators, verapmail and tetrandrine, were found to be able to efficiently reverse the drug resistance in Bats-72 and Bads-200. It indicated that ABC transporters might involve in the drug resistance of Bats-72 and Bads-200. Further, using cDNA microarray, RT-PCR, and Western Blot assays, we investigated the mechanisms of drug resistance in Bats-72 and Bads-200. Bats-72 and Bads-200 overexpressed ABCB1 and P-gp. The expression level of P-gp was lower in Bats-72 than in Bads-200. This could explain why Bats-72 was less resistant to MDR-related drugs than Bads-200. In addition, compared with Bads-200, Bats-72 overexpressed both ABCG2 and ABCC6 which mediate the resistance to methotrexate and gemcitabine leading to a broader resistance spectrum. The expression level of P-gp was very stable in Bads-72, but gradually descreased in Bads-200 when it was cultured in paclitaxel-free medium. This is consistent with the finding that the drug resistance of Bats-72 was long-term stable, wheras the drug resistance of Bads-200 decreased gradually in the absence of paclitaxel.TDR is associated with a variety of mechanisms, co-existing and interacting with each other. P-gp, BCRP, MRP1, topoisomerase?(Topo-?), glutathione-S-transferase (GST), and protein kinase C (PKC) are the most well known biomarkers for TDR. When applying P-gp monoclonal antibody, a un-identified protein was found to be expressed in drug resistant cell lines (i.e., Bats-72, Bads-200, and MCF7/ADR). Since the molecular weight of this protein was over 250 KDa, we named it M250. However, the same protein was not detectable in the drug sensitive cell line BCap37. Further analyses suggested that M250 shared the homology sequences with P-gp, and was associated with TDR. Followed, M250 had been isolated by immunoprecipitation. more studies will be carried out to identify M250 as a new biomarker for TDR.Fulvestrant (ICI 182,780) is a pure selective estrogen receptor antagonist. It can selectively down-regulate the expression of Estrogen receptor (ER). In our previous studies, the application of fulvestrant could reverse ER-mediated resistance to paclitaxel and vinca alkaloids. Interestingly, we found that fulvestrant could also reverse the drug resistance of Bats-72 and Bads-200, which are ER-negative, in a dose-dependent manner. Therefore, it indicates that fulvestrant can not only render chemotherapeutic sensitization by blocking ER signaling pathways, but also reverse TDR via a mechanism which is independent of ER signaling pathway.In summary, we successfully established two novel drug resistant breast cancer cell lines Bats-72 and Bads-200. Our experiments confirmed that Bats-72 and Bads-200 represented two different types of TDR. The expression of specific ABC transporters plays an important role in the drug resistance of Bats-72 and Bads-200. An unknown protein M250 was found and isolated from drug resistance cell lines. In addition, our work indicated that fulvestrant could not only sensitize tumor cells to chemotherapeutic drugs by blocking ER-oc signaling pathways, but also reverse TDR via a mechanism independent of ER-?signaling pathway. In a word, this study provided two novel cell models for understanding the molecular mechanism of TDR and developed a potential effective strategy to overcome the drug resistance.