Multi‐drug resistance (MDR) occurs when cancer cells become resistant to a diverse array of chemotherapeutics and xenobiotics. Among the many mechanisms of MDR, one of the most prominent is the overexpression of ATP‐binding cassette (ABC) transporters. ABC transporters harness the energy from ATP hydrolysis to transport xenobiotics, drugs, and other toxic compounds out of the cell. When ABC transporters are overexpressed in cancer cells, their efflux activity can lower the intracellular concentration of medicinal compounds to sub‐therapeutic levels. One member of the ABC transporter family, the Breast Cancer Resistance Protein (BCRP, or ABCG2), confers MDR to a variety of cancers. Transient inhibition of BCRP should therefore restore sensitivity of resistant cells to chemotherapeutics. In previous work by our lab, several potential BCRP inhibitors were identified via computational methods. Using cell viability assays, we tested the potential BCRP inhibitors using a BCRP overexpressing cell line. Here we compare the effectiveness of the inhibitors at re‐sensitizing the cells to Mitoxantrone relative to the parental cell line which does not overexpress BCRP. The potential toxicities of the experimental compounds were also assessed using the non‐cancerous HFL1 cell line.

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to multiple chemotherapeutic drugs that are functionally and structurally different. MDR usually leads to failure of chemotherapy treatment and subsequently to poor patient prognosis. Cancers can become resistant to antineoplastic drugs by over‐expressing one or more ATP‐binding cassette (ABC) transporters that act as drug efflux pumps and expel xenobiotics from cells. One such protein is the Breast Cancer Resistance Protein (BCRP, or ABCG2), which is believed to confer MDR to several types of cancer. Inhibition of BCRP cellular activity is therefore thought as a way to restore sensitivity to chemotherapeutics. In previous work, we identified several potential inhibitors of BCRP using high‐throughput in silico screens. Using cell viability and cellular accumulation assays of BCRP substrates, we assessed these compounds for toxicity to cells and the ability to reverse MDR in the BCRP‐overexpressing breast cancer cell line, MCF7‐M100. Here we expand our studies of the potential BCRP inhibitors to two other BCRP‐overexpressing cell lines, the BCRP overexpressing colon cancer and non‐small cell lung cancer cell lines, S1M1‐80 and MX20, respectively. For each cell line, the level of BCRP overexpression was assessed at RNA and protein level and the MDR phenotype was confirmed using cell viability assays. The effects of our newly discovered inhibitors on reversing MDR in these two cell lines is reported. Support or Funding Information This work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.

ATP‐binding cassette (ABC) transporters are transmembrane proteins that act as drug efflux pumps and are associated with multi‐drug resistance (MDR) in cancers. Two specific proteins that belong to this protein superfamily are P‐glycoprotein (P‐gp) and breast cancer resistant protein (BCRP). These two proteins are known to pump chemotherapeutics out of cells, rendering treatments of some cancers less efficient. Certain cancer cell lines overexpress these proteins, leading to MDR. If a compound can be identified that inhibits these efflux pumps, then sensitivity to chemotherapeutics can be restored. The levels of expression of P‐gp and BCRP were quantitatively measured in four cancer cell lines that are used by us to screen for inhibitors of P‐gp or BCRP. The expression of P‐gp, BCRP, and MRP1 (multidrug resistance‐associated protein 1) was measured using quantitative One‐Step qPCR in a prostate cancer cell line, DU145, and compared to an MDR derivative line that overexpresses P‐gp, DU145 TXR. We found that DU145 TXR expresses P‐gp, BCRP, and MRP1 by about 14,000‐fold, 4‐fold, and 0.75‐fold, respectively, when compared to the parental cell line, DU145. A breast cancer cell line, MCF‐7, and its mitoxantrone‐resistant derivative, MCF‐7 M100, were also analyzed. The MCF‐7 M100 cell line was found to express BCRP, P‐gp, and MRP1 by about 600‐fold, 0.3‐fold, and 1.6‐fold, respectively, compared to the parental cell line, MCF‐7. The results were qualitatively confirmed at the protein level using Western Blot analyses. In other experiments, we demonstrated that P‐gp and BCRP inhibitors identified by us that reversed multidrug resistances in the MDR prostate and breast cancer cell lines, DU145 TXR and MCF‐7 M100, did not down‐regulate expression levels of P‐gp or BCRP, suggesting that reversal of MDR was accomplished by inhibiting transport processes and not transporter expression levels.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM09477102] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas, as well as the Hamilton Undergraduate Research Scholars Program and the SMU Summer Research Assistantship and Undergraduate Research Assistantship Programs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP‐binding cassette (ABC) transporters, P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are two well‐studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P‐gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain. Using computational methods, we previously identified a novel class of P‐gp inhibitors that were not transport substrates of the pump. Here we describe the effects of chemical variants of the previously identified P‐gp inhibitor, SMU‐29. The variants were generated using computational approaches or by structure‐based design to improve protein binding interactions. All variants showed improved efficacy in reversing paclitaxel resistance in the P‐gp over‐expressing DU145 TXR prostate cancer cell line and their ability of increasing P‐gp substrate calcein in these cells. Five of these variants were not transported by P‐gp and three of them were substrates of the pump. Four variants only affected P‐gp, but not BCRP function, making them more selective than the parental compound, SMU 29. The three variants that affected BCRP reversed MDR in a BCRP over‐expressing breast cancer cell line, MCF‐7 M100, and increased accumulation of BCRP substrates such as Hoechst 33342, mitoxantrone and daunorubicin in these cells, indicating the potential to be used as BCRP inhibitors. The variants that affected both P‐gp and BCRP also showed higher accumulation of P‐gp and BCRP substrates in a blood brain barrier model cell line, hCMEC/D3, showing the potential to open the blood brain barrier for increased drug uptake into the brain.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.