Sulforaphane (SF) increases drug-mediated effects on cell viability, clonogenicity and induction of apoptosis in pancreatic cancer stem cells (CSCs). (a) CSC^high pancreatic cancer cells were left untreated (CO), or were treated with SF (5 µmol/l), cisplatin (CIS), gemcitabine (GEM), doxorubicin (DOX) or 5-flurouracil (5-FU) in concentrations indicated alone or in combination with SF as indicated. Seventy-two hours later viability was determined by MTT assay (upper panel). Data are presented as mean ± SD (*P < 0.05 compared with treatment in the absence of SF). Representative pictures of cells were taken at ×100 magnification (lower panel). (b) To examine clonogenic cell division, CSC^high pancreatic cancer cells were seeded in 6-well tissue culture plates and treated with SF (5 µmol/l) and GEM (5 nmol/l) alone or in combination (SF+GEM). Seventy-two hours later cells were trypsinized and re-plated at low density in 6-well plates. Ten days later colonies containing >50 cells were counted under a dissecting Zeiss Stemi DV4 microscope. Data are presented as mean ± SD. Photographs of the fixed and stained colonies are presented on the left panel. (c) MIA-PaCa2 cells were seeded in 6-well tissue culture plates and treated similar to the previous MTT assay. Induction of apoptosis was evaluated by annexin V staining of the cells and flow cytometry. Induction of apoptosis is presented as percentage of annexin-positive cells. Data are presented as mean ± SD (*P < 0.05).

Sulforaphane (SF) does not significantly increase cytotoxic drug effects to normal cells. Primary human fibroblasts, primary human umbilical vein endothelial cells (HUVECs) or human immortalized embryonic kidney (293) cells were treated with SF (5 µmol/l), gemcitabine (GEM) (5 nmol/l) or both together (SF+GEM). Viability was measured 72 hours later by MTT assay as described above.

Sulforaphane (SF) enhances gemcitabine (GEM)-mediated cytotoxic effects by prevention of tumor-initiating potential in mice. (a) Pancreatic CSC^high cells [4 ×10⁶ cells in 200 µl phosphate-buffered saline (PBS)] were injected subcutaneously into nude mice. After the tumors had reached a mean diameter of 8–10 mm, SF, GEM or both agents together (SF+GEM) were administered at days 5, 6, and 7 after tumor cell implantation (indicated by red arrows). Control animals (CO) received PBS injections only. The tumor volume was measured daily (upper left panel). Body weight of each individual mouse was set to 100% before treatment. Mice were weighted daily and relative changes in body weight are shown (lower left panel). Data are presented as mean of 6 animals ± SEM (n = 6) (*P < 0.05 compared with control and single treatment). A representative H&E staining of liver tissue after three consecutive treatments with SF and GEM is shown (scale bar = 200 µm) (left panel). (b) Pancreatic CSC^high cells were pretreated with SF (5 µmol/l), GEM (25 nmol/l) or both together in vitro. Control cells (CO) were left untreated. Seventy-two hours later an equal amount (5.7 × 10³) of live cells were transplanted subcutaneously with 50% Matrigel into the right anterior flank of 5–6 weeks old NMRI-nu (nu/nu) female mice, 6 mice per treatment group. External tumor size was measured using a caliper at time points indicated. The number of tumors, which start to re-grow in each group is indicated (tumor take). Data are presented as mean of six animals in the control group and as mean of two growing tumors in the GEM-treated group.

Sulforaphane (SF) increases chemotherapeutic drug effects in prostate cancer cells. (a) Prostate cancer cells DU145 were left untreated (CO) or were treated with SF (5 µmol/l) alone or in combination with taxol (TAX) or cisplatin (CIS) at doses indicated. Viability was determined 72 hours later as described above (*P < 0.05). Images of cells treated for 72 hours are shown in the lower panel. (b) DU145 cells were seeded in 6-well tissue culture plates and treated with SF (5 µmol/l) and TAX (5 nmol/l) alone or in combination. Seventy-two hours later, cells were trypsinized and re-plated at low density in 6-well plates. Ten days later colonies were stained with Coomassie blue and images of colonies were taken (left panel). Colonies containing >50 cells were counted under a dissecting Zeiss Stemi DV4 microscope and the amounts of the survival fractions are presented (right panel). (c) Prostate cancer cells were treated with SF, TAX, CUS or SF combined with a cytotoxic drug for 72 hours. Apoptosis induction was evaluated by annexin V staining and flow cytometry and is shown as percentage of annexin-positive cells. Data are presented as mean ± SD (*P < 0.05).

Sulforaphane (SF) enhances cytotoxic drug effects to pancreatic cancer stem cell (CSC) properties. (a) Cells were treated for 48 hours with SF (5 µmol/l) and gemcitabine (GEM) (25 nmol/l) or both agents together (SF+GEM). Expression of Notch-1 and c-Rel was evaluated by western blot analysis. (b) Pancreatic CSC^high cells were seeded at clonal density in low adhesion plates for spheroid formation. Twenty-four hours later cells were treated with SF (5 µmol/l), GEM (25 nmol/l), or both agents together (SF+GEM). Spheroids were photographed at day 7 under ×100 magnification or quantified (1st generation). Thereafter, 1st generation spheroids were dissociated to single cells and equal numbers of live cells pretreatment group were re-plated. Upon spheroid formation cells were treated as described above and 3 days later spheroid formation was quantified (2nd generation). (c) Pancreatic CSC^high were treated as described above. Three or 21 days later aldehyde dehydrogenase 1 (ALDH1) activity was analyzed by flow cytometry and the percentage of ALDH1-positive cells is presented. Data are presented as mean ± SD. (d) Likewise, proteins were harvested and expression of ALDH1 protein was analyzed by western blot. Expression of β-actin served as loading control. Lower panel: Twenty-one days after treatment cells were subjected to immunofluorescence analysis for ALDH1. Randomly chosen fields were examined under ×400 magnification using a Nikon Eclipse TS100 microscope and photographs were taken.