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1.
Figure 1

Figure 1. Sulforaphane inhibited proliferation and induced apoptosis in breast cancer cells. From: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells.

(A) SUM159 and MCF7 cells growing in log phase were treated with increasing concentrations of sulforaphane for 48 hrs. The anti-proliferation effect of sulforaphane was measured by MTS assay. (B) Sulforaphane enhanced caspase-3 activity in SUM159 cells. Data are presented as mean ± SD(n≥ 3). SF = sulforaphane.

Yanyan Li, et al. Clin Cancer Res. ;16(9):2580-2590.
2.
Figure 3

Figure 3. Inhibitory effect of sulforaphane on ALDH-positive cell population. From: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells.

SUM159 cells were treated with sulforaphane (1 and 5 μM) or DMSO for 4 days, and subject to Aldefluor assay and flow cytometry analysis. (A) Sulforaphane decreased the percentage of ALDH-positive cells. Data are presented as mean ± SD(n =3). (B) A set of representative flow cytometry dot plots. R2 covers the region of ALDH-positive cells. SF = sulforaphane; ALDH = aldehyde dehydrogenase.

Yanyan Li, et al. Clin Cancer Res. ;16(9):2580-2590.
3.
Figure 2

Figure 2. Inhibitory effect of sulforaphane on mammosphere formation. From: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells.

MCF7 and SUM159 cells were cultured in mammosphere forming conditions. (A) Primary mammospheres were incubated with sulforaphane (0.5, 1, and 5 μM) or DMSO for 7 days. Sulforaphane treatment reduced the number of primary mammospheres. (B) Sulforaphane reduced the size of primary mammospheres(magnification × 100). The size of mammospheres was estimated using V = (4/3)π R3. (C) In the absence of drug, the 2nd and 3rd passages that were derived from sulforaphane-treated primary mammospheres yielded smaller numbers of spheres in comparison with control. Data are presented as mean ± SD(n = 3). *, P < 0.05; **, P < 0.01. SF = sulforaphane.

Yanyan Li, et al. Clin Cancer Res. ;16(9):2580-2590.
4.
Figure 5

Figure 5. Sulforaphane eradicated breast cancer stem cells in vivo asassessed by re-implantation in secondary mice. From: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells.

Each secondary NOD/SCID mouse received 50,000 cells from control tumorsin one side of mammary fat pad and another 50,000 cells from sulforaphane-treated tumorsin the contralateral fat pad. (A) Tumor growth curves of the recipient NOD/SCID mice. Data for group 1 are presented as mean ± SD(n = 4), and for group 2 are presented as mean ± SD(n = 3). Sulforaphane abrogated the tumorigenicity of breast cancer stem cells. (B) Percentage of tumor-free mice by the day of euthanization for each group. Four mice were euthanized at day 20 and three at day 33 due to the mass tumor burden on control side.

Yanyan Li, et al. Clin Cancer Res. ;16(9):2580-2590.
5.
Figure 4

Figure 4. Sulforaphane decreased tumor size and ALDH-positive cell population in primary breast cancer xenografts. From: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells.

NOD/SCID mice bearing SUM159 cells in fat pads as xenografts were treated with daily i.p. injection of control or 50 mg/kg sulforaphane for 2 weeks. Tumor volumes (A) and mouse body weights (B) were determined as described in “Materials and Methods”. Tumors in sulforaphane-treated mice were 50% the size of control animals at the end of drug treatment. (C) Sulforaphane decreased the percentage of ALDH-positive cells in xenograft breast tumors. (D) A set of representative flow cytometry plots. Data are presented as mean ± SD(n = 6). ALDH = aldehyde dehydrogenase.

Yanyan Li, et al. Clin Cancer Res. ;16(9):2580-2590.
6.

Figure 6. Sulforaphane down-regulated Wnt/β-catenin self-renewal pathway. From: Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells.

(A) Sulforaphane decreased protein levels of β-catenin and cyclin D1 in both SUM159 and MCF7 cell lines. (B) TOP-dGFP reporter lentivirus-infected MCF7 mammospheres were treated with indicated compounds (0.5 μM BIO and 5 μM sulforaphane) either alone or in combination for 2 days. Sulforaphane decreased the percentage of dGFP-positive cells by 30% ~40%. BIO increased this population, while sulforaphane decreased it by over 60% in the presence of BIO. Representative flow cytometry results of TOP-dGFP mammospheres and their pictures under fluorescence microscope(magnification × 100)are shown on the right. (C) Sulforaphane promoted β-catenin phosphorylation at Ser33/37/Thr41, while LiCl suppressed the phosphorylation by inactivating GSK3β (upper panel). Sulforaphane decreased phospho-GSK3β (Ser9)level, whereas total GSK3β remained unchanged(middle panel). LiCl increased protein level of β-catenin by phosphorylating/inactivating GSK3β at Ser9, while sulforaphane attenuated LiCl-induced GSK3β phosphorylation and β-catenin accumulation(bottom panel). SF = sulforaphane; dGFP = destabilized green fluorescent protein.

Yanyan Li, et al. Clin Cancer Res. ;16(9):2580-2590.

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