Targeting breast stem cells with the cancer preventive compounds curcumin and piperine

Abstract

The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols, curcumin, and piperine are able to modulate the self-renewal of normal and malignant breast stem cells, we examined the effects of these compounds on mammosphere formation, expression of the breast stem cell marker aldehyde dehydrogenase (ALDH), and Wnt signaling. Mammosphere formation assays were performed after curcumin, piperine, and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells, selected by ALDH positivity. Wnt signaling was examined using a Topflash assay. Both curcumin and piperine inhibited mammosphere formation, serial passaging, and percent of ALDH+ cells by 50% at 5 microM and completely at 10 microM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 microM and completely at 10 microM. Curcumin and piperine separately, and in combination, inhibit breast stem cell self-renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment can be a mechanistic biomarker for use in human clinical trials.

Figure 1

Figure 1A. Primary mammospheres formed from unsorted normal human breast epithelial cells in suspension culture for 10 days. Left picture incubation with curcumin 5 μM, right picture piperine 5 μM treament. Figure 1B Primary mammosphere number (±SEM) formed from unsorted normal human breast epithelial cells in suspension culture with curcumin (C5= curcumin 5 μM, C10= curcumin 10 μM); 1C piperine treatment (P5= piperine 5 μM, and P10= piperine 10μM), 1D curcumin plus piperine treatment

Figure 1

Figure 1A. Primary mammospheres formed from unsorted normal human breast epithelial cells in suspension culture for 10 days. Left picture incubation with curcumin 5 μM, right picture piperine 5 μM treament. Figure 1B Primary mammosphere number (±SEM) formed from unsorted normal human breast epithelial cells in suspension culture with curcumin (C5= curcumin 5 μM, C10= curcumin 10 μM); 1C piperine treatment (P5= piperine 5 μM, and P10= piperine 10μM), 1D curcumin plus piperine treatment

Figure 2

Figure 2A. Secondary and Tertiary sphere formation from human normal single epithelial cells in suspension with curcumin and piperine Figure 2B. Number of differentiated colonies formed from secondary sphere forming human breast stem cells with curcumin and piperine from time of plating versus treatment 48 hrs. after plating. Figure 2C. Immunostaining for CK14 myoepithelial marker in blue and CK18 luminal marker in red and mixed differentiated colonies formed in the presence of curcumin and piperine

Figure 2

Figure 2A. Secondary and Tertiary sphere formation from human normal single epithelial cells in suspension with curcumin and piperine Figure 2B. Number of differentiated colonies formed from secondary sphere forming human breast stem cells with curcumin and piperine from time of plating versus treatment 48 hrs. after plating. Figure 2C. Immunostaining for CK14 myoepithelial marker in blue and CK18 luminal marker in red and mixed differentiated colonies formed in the presence of curcumin and piperine

Figure 2

Figure 2A. Secondary and Tertiary sphere formation from human normal single epithelial cells in suspension with curcumin and piperine Figure 2B. Number of differentiated colonies formed from secondary sphere forming human breast stem cells with curcumin and piperine from time of plating versus treatment 48 hrs. after plating. Figure 2C. Immunostaining for CK14 myoepithelial marker in blue and CK18 luminal marker in red and mixed differentiated colonies formed in the presence of curcumin and piperine

Figure 3

Figure 3A. Primary mammosphere number formed from ALDH+ presorted normal human breast stem/early progenitor cells in suspension culture with curcumin and piperine treatment (C5=curcumin 5 μM, c10=curcumin 10 μM, P5= piperine 5 μM, and P10=piperine 10 μM) compared to DMSO vehicle and no treatment (NT controls) Figure 3B Percent of ALDH1A1 positive cells after treatment of unsorted normal human breast epithelial cells with curcumin and piperine treatment

Figure 3

Figure 3A. Primary mammosphere number formed from ALDH+ presorted normal human breast stem/early progenitor cells in suspension culture with curcumin and piperine treatment (C5=curcumin 5 μM, c10=curcumin 10 μM, P5= piperine 5 μM, and P10=piperine 10 μM) compared to DMSO vehicle and no treatment (NT controls) Figure 3B Percent of ALDH1A1 positive cells after treatment of unsorted normal human breast epithelial cells with curcumin and piperine treatment

Figure 4

Figure 4A. Number of spheres formed from MCF7 or SUM 159 cells in suspension culture with 12 hrs. of curcumin and piperine treatment (C5=curcumin 5 μM;, C10=curcumin 10 μM; P5=piperine 5 μM; P10=piperine 10 μM; C5+P10=curcumin 5 μM and piperine 10 μM) Figure 4B Green fluorescence positivity in MCF7 Topflash assay with curcumin and piperine treatment

Figure 4

Figure 4A. Number of spheres formed from MCF7 or SUM 159 cells in suspension culture with 12 hrs. of curcumin and piperine treatment (C5=curcumin 5 μM;, C10=curcumin 10 μM; P5=piperine 5 μM; P10=piperine 10 μM; C5+P10=curcumin 5 μM and piperine 10 μM) Figure 4B Green fluorescence positivity in MCF7 Topflash assay with curcumin and piperine treatment