Ginger extract (GE) affects cell-cycle progression kinetics by causing the S and G2/M arrest followed by an increase in sub-G1 cell population, suggesting apoptosis. Cell-cycle progression over (A) dose (0–1000 μg/ml) and (B) time (0–72 h) are depicted in a three-dimensional format. Cell populations in G0/G1 appear as 2N (unduplicated) DNA content and G2/M populations are indicated by 4N (duplicated) DNA content. (C) Immunoblots of cell lysates treated in the absence or presence of 250 μg/ml of GE for cyclin D1, cdk4, cyclin E, p21 and p-Rb. Uniform loading was confirmed by β-actin. (Di) Flow cytometric histogram profiles showing percentage of cells with cytosolic monomeric JC-1-associated green fluorescence (indicating collapse of mitochondrial membrane potential) in PC-3 cultures treated with dimethyl sulfoxide (DMSO; control, pink profile; ) or GE (blue profile; ) for 24 h. Representative data from a single experiment are shown. (Dii) Quantification of the increase in mean fluorescence intensity (MFI, i.e. the percentage of green JC-1-stained cells) in PC-3 cultures treated with DMSO (control) or GE for 24 h. (Ei) Histogram profiles showing a spectral shift and loss of red fluorescence, consistent with the loss of transmembrane potential on GE treatment (control, green; GE, red). (Eii) Quantification of the decrease in mean fluorescence intensity (i.e. the percentage of red JC-1-stained cells) in PC-3 cultures treated with DMSO (control) or GE for 24 h. Values are means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars (P < 0·05). (A colour version of this figure can be found online at www.journals.cambridge.org/bjn).

Ginger extract (GE) has potent antiproliferative activity. Human prostate cancer LNCaP ( ), DU145 ( ), PC-3 ( ), C4-2 ( ), C4-2B ( ) cells, as well as normal prostate epithelial cells (PrEC, ) and human dermal primary fibroblasts (HDF, ) were treated with gradient concentrations of GE for 72 h. The percentage of cell proliferation at indicated concentrations, compared with untreated control cells, was measured by the in vitro cell proliferation assay, as described in Materials and methods. (A) Plot of percentage of cell survival vs. GE concentrations used for the determination of half-maximal concentration of growth inhibition (IC₅₀) values. Values are means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars (P<0·05). (B) Bar graph representation of the IC₅₀ of the indicated cell lines. (C) Bar graph representation and photograph of crystal violet-stained surviving colonies from the control and GE-treated (250 and 1000 μg/ml) groups. (D) Fluorescence micrographs of control and GE-treated PC-3 cells stained for (Di) Ki67 (green) or (Dii) 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. (Diii) Quantification of Ki67-postive or DAPI-stained cells in control ( ) and 250 μg/ml of GE-treated PC-3 cells ( ) from random image fields totalling 200 cells. Values are means, with standard deviations represented by vertical bars. Mean values were significantly different from the controls (P<0·05). (A colour version of this figure can be found online at www.journals.cambridge.org/bjn).

Ginger extract (GE) induces mitochondrially mediated intrinsic apoptosis. (A) Immunoblot analyses for BAX, Bcl2, cytoplasmic cytochrome c (Cyt c), cleaved caspase-3 and poly(ADP-ribose)polymerase (PARP). β-Actin was used as a loading control. (B) Quantification of the time-dependent increase in caspase-3 (Casp-3) activity on GE treatment. Cells were treated with GE for 0, 12, 24 and 48 h, and caspase-3 activity was analysed using the fluorogenic substrate Ac-DEVD-7-amino-4-trifluoromethyl-coumarin. Values are means of three independent experiments performed in triplicate, with standard deviations represented by vertical bars (P<0·05). Immunofluoresence micrographs of control and 250 μg/ml of GE-treated cells stained for cleaved (Ci) caspase-3 and (Di) PARP. (Cii, Dii) Quantification of activated caspase-3-positive and cleaved PARP-positive cells. (A colour version of this figure can be found online at www.journals.cambridge.org/bjn).

Ginger extract (GE) caused in vivo inhibition of tumour growth in human PC-3 xenografts on dietary feeding of GE. (A) Progression profile of tumour growth in control vehicle-treated ( ) and GE-treated ( ) mice at the time of treatment. (B) GE treatment was well tolerated, and the body weights of the control ( ) and GE-treated ( ) groups were comparable. Values are means, with standard deviations represented by vertical bars (n = 6, P < 0·05). (C) Tumour micrographs from control and GE-treated mice, respectively, at 100× and 200× magnification. GE-treated tumour microsections reveal large areas of tumour cell death, consistent with the therapeutic effects of GE. Microsections from control tumour tissue show sheets of tumour cells with high-grade pleomorphic nuclei with minimal cell death. (D) Western blot analysis of tumour tissue lysates from control and GE-treated mice for cyclin B, cyclin D1, cyclin E, p21 and cleaved caspase-3. (A colour version of this figure can be found online at www.journals.cambridge.org/bjn).

(A) Immunohistochemical staining of paraffin-embedded tumour tissue sections from the control and ginger extract (GE)-treated groups for proliferation marker (Ki67) and apoptotic markers (cleaved caspase-3 (casp-3), cleaved poly(ADP-ribose)polymerase (PARP) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL)). (B) Quantification of Ki67, cleaved casp-3, cleaved PARP and TUNEL-positive cells counted from several randomly selected fields for a total of 200 cells. Values are means, with standard deviations represented by vertical bars (P < 0·05). Control, ; GE, . (A colour version of this figure can be found online at www.journals.cambridge.org/bjn).