*2.2. The E*ff*ect of ABCC Small Molecule Inhibitors on Breast Cancer Cell Proliferation*

Having established that both cell lines expressed the ABCC proteins, we investigated the effect of small molecule inhibitors of these proteins on the ability of the cells to proliferate. The small molecule inhibitors used in the study are detailed in Table 1.


**Table 1.** ABCC inhibitors used in this study.

The first parameter investigated was the clonogenic capacity of the cells, i.e., the ability to reproduce from a single cell. Examples of the assay are shown in Figure 2a,b, where it can be seen that, following the treatment of MDA-MB-231 cells with MK571 or Reversan, at increasing concentrations, both the number of colonies formed over seven days and the size of those colonies are reduced. The average data shown in Figure 2c–e show that Reversan and MK571 affect the colony formation for both MDA-MB-231 and MCF-7 cells, but the effect is more pronounced for the MDA-MB-231 cells, perhaps correlating with the higher level of ABCC protein expression in these cells. In contrast, Ceefourin 1 and 2 and Indomethacin had no effect at all on the colony formation.

–

**Figure 2.** MK571 and Reversan affect the clonogenic capacity of breast cancer cells. 100 cells/well were seeded in 6-well plates and cultured at 37 ◦C for 24 h, after which cells were treated with the indicated concentrations of inhibitors. Control wells were untreated cells. After 7 days of culture, the colonies formed were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, the colonies counted and measured. Example results for MDA-MB-231 cells treated with (**a**) MK571 or (**b**) Reversan. Average results for the number (**c**,**d**) and size (**e**,**f**) of colonies for MCF-7 cells (**c**,**e**) and MDA-MB-231 (**d**,**f**). Data are mean ± sem, n ≥ 6. Data were analyzed using a one-way ANOVA with a Dunnett's post hoc test; \* *p* < 0.05, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001 significantly lower than the untreated sample.

The effect of these inhibitors on cellular proliferation was also investigated using an MTT assay. As can be seen in Figure 3, the presence of the inhibitors did not affect the proliferation of either cell line for the first 24 h. However, after this time, MK571 and Reversan had a significant impact on the proliferation of both MCF-7 and MDA-MB-231 cells, whereas Ceefourin 1 and 2 and Indomethacin did not. To confirm the results obtained with the MTT assay, and to make sure it was not due to an

a Dunnett's post hoc test

indirect effect on the enzyme required to reduce MTT, proliferation was also measured by a trypan blue exclusion and cell counting approach (Figure S2). Although the errors are larger using this approach, the key findings replicate those of the MTT assay.

≥

≥ way ANOVA with a Dunnett's post **Figure 3.** MK571 and Reversan affect the proliferation of breast cancer cells. Fifteen thousand MCF-7 cells (**a**,**b**) or 6000 MDA-MB-231 cells (**c**,**d**) were seeded in 24-well plates. After 4 h of culture, cells were treated with inhibitors, as detailed. Cell viability was assessed at 6, 12, 24, 48, and 72 h after treatment using an MTT assay and absorbance measured at 570 nm. Data are mean ± SD, n ≥ 6. Data were analyzed using a two-way ANOVA with a Dunnett's post hoc test. \*\*\* *p* < 0.001 and \*\*\*\* *p* < 0.0001 significantly lower than the untreated sample.
