Fig 2: Correlation of APC with DOX sensitivity and the expression levels of MDR1 and MRP1 in human breast cancer. (A) Using patient tumor samples, the expression of APC was correlated with MDR1 expression levels. Protein levels were normalized to the normal epithelial cell lines. Lower levels of APC protein trended with higher levels of MDR1 protein (Pearson’s r = -0.3176) but this trend was not significant (p = 0.4049). (B) In the patients’ tumor samples, the protein level of APC was not significantly correlated with MRP1 protein levels. Higher APC protein levels trended with higher MRP1 protein levels, but this was not significant (Pearson’s r = 0.4370; p = 0.2066). (C) In a panel of human breast cancer cell lines, the expression levels of APC were correlated with the protein expression levels of MDR1 and MRP1. Protein levels were normalized to the average of the normal breast epithelial cell lines. Decreased APC levels trended with increased MDR1 levels (Pearson’s r = -0.3086), but the trend was not significant (p = 0.2448). (D) Increased APC protein levels correlated with increased MRP1 protein levels (Pearson’s r = 0.4950; p = 0.05124).

Fig 3: Inhibition of the ABC transporter restores DOX-induced apoptosis in MMTV-PyMT;ApcMin/+ cells. (A) DOX-treated MMTV-PyMT;ApcMin/+ cells have less CC3 compared with DOX-treated MMTV-PyMT;Apc+/+ cells. MDR1 inhibition sensitized MMTV-PyMT;ApcMin/+ cells to DOX-induced apoptosis. Densitometry was performed, and CC3 was normalized to Actin. (B) Quantification and representative Western blots showed that silencing MRP1 increased DOX-induced CC3 in MMTV-PyMT;ApcMin/+ cells as compared with the DOX-only treatment, similar to the CC3 levels in DOX-treated MMTV-PyMT;Apc+/+ cells. Densitometry was performed, and CC3 was normalized to Actin. (C) Treatment with a MDR1/MRP1 inhibitor plus DOX increased the CC3 in MMTV-PyMT;ApcMin/+ cells compared with DOX-only treatment. The combination treatment also increased DOX-induced apoptosis in MMTV-PyMT;Apc+/+ cells compared with the DOX-only treatment. Densitometry was performed, and CC3 was normalized to Actin. * p < 0.05, comparing treated cells to control-treated cells; ** p < 0.05 as compared with DOX-treated MMTV-PyMT;Apc+/+ cells; *** p < 0.05, comparing combination treatment in MMTV-PyMT;ApcMin/+ cells and MMTV-PyMT;Apc+/+ cells.

Fig 4: Increased MDR1 and MRP1 activity effluxes DOX extracellularly in APC-deficient cells. (A) MDR1 inhibition restores the accumulation of DOX in MMTV-PyMT;ApcMin/+ cells comparable to the accumulation of DOX in MMTV-PyMT;Apc+/+ cells. The mean fluorescence intensity (MFI) was normalized to that of the genotype control. (B) The accumulation of DOX increased in MMTV-PyMT;ApcMin/+ and MMTV-PyMT;Apc+/+ cells following MRP1 silencing. MFI was normalized to the genotype control. (C) Dual MDR1 and MRP1 inhibition increased intracellular DOX fluorescence in MMTV-PyMT;ApcMin/+ cells and MMTV-PyMT;Apc+/+ cells. MFI was normalized to the genotype control. * p < 0.05, comparing treated cells with control-treated cells; ** p < 0.05, comparing MMTV-PyMT;ApcMin/+ cells with MMTV-PyMT;Apc+/+ cells within the same treatment/transfection; *** p < 0.05, comparing the combination treatment with the DOX-only treatment.