*2.3. E*ff*ect of Inhibitors on Breast Cancer Cell Migration*

– In addition to rapid proliferation, enhanced migration is a hallmark of aggressive cancers. Therefore, the effects of ABCC inhibitors on breast cancer cell migration was measured using a scratch assay, as shown in Figure 4a. The MDA-MB-231 cells migrated faster than the MCF-7 cells (Figure 4b,c). Most of the inhibitor treatments had no significant effect on the migration. However, the treatment with MK571 did significantly decrease the migration of MDA-MB-231 cells (Figure 4c). This was not due to an effect on proliferation, since after 10–12 h when the migration was most affected, no effect on the proliferation was observed (Figure 3c).

MK571, which inhibits both ABCC1 and ABCC4, and Reversan, which inhibits ABCC1, were the only inhibitors to affect the proliferation of the breast cancer cells. MK571 was the only drug to affect the cell migration. Ceefourin 1 and 2 and Indomethacin, which inhibit ABCC4, had no effects. This might suggest that ABCC1 plays a role in the proliferation of breast cancer cells. Similarly, it might suggest that ABCC1, or maybe both ABCC1 and ABCC4 together, are involved in the migration. However, one of the challenges associated with using inhibitors of multidrug transporters is the lack of specificity. In addition to inhibiting ABCC1 and ABCC4, MK571 can inhibit other ABCC family members, and it is also a leukotriene antagonist, inhibiting the binding of leukotriene D<sup>4</sup> (LTD4) to cysteinyl leukotriene receptor 1 [50]. Reversan is an inhibitor of ABCC1 but can also inhibit the related protein ABCB1 [54]. Indomethacin inhibits ABCC4 but was developed as an inhibitor of cyclooxygenase. Therefore, the effect of the genetic knockdown of ABCC1 and ABCC4 was investigated.

**Figure 4.** MK571 decreases the rate of migration by MDA-MB-231 cells. Cells were seeded in 24-well plates to reach 100% confluency the day of the assay. A scratch across the monolayer of the cells was carefully made, and the medium was replaced with fresh prewarmed culture medium. Cells were treated with the inhibitors as described above. Three image positions were selected from each well, and images were taken at 1-h intervals using the Cell-IQ. Representative images of MDA-MB-231 scratch assay (**a**). Pink lines represent the scratch edges as defined by the Cell IQ software, and the blue lines are the distance measurement between the edges. Average results for MCF-7 (**b**) and MDA-MB-231 (**c**) cell migration in the presence of inhibitors. Data are mean ± sem, n ≥ 6. Data were analyzed using a two-way ANOVA with a Dunnett's post hoc test. \* *p* < 0.05 significantly lower than the untreated sample.
