Co-treatment with manuka and taxol results in additive effect.

<p>B16.F1 cells were seeded at 1×10³ cells per well in a 96-well plate and incubated with the indicated concentrations of manuka, alone or in combination with taxol (10 ng/ml), for 72 hrs. Cell viability was determined using CellTiter-Glo luminescent assay. Results are expressed as percentage viability in treated cell cultures compared to untreated cells and are representative of 3 independent experiments. Asterisks denote statistically significant differences between corresponding cell cultures treated with each manuka concentration in absence or presence of taxol (*, <i>p</i><0.05).</p

Systemic administration of manuka honey is not associated with any alterations in hematological values.

<p>Mice were injected with saline or manuka (50% w/v) 2 times per week for a total of 3 weeks, following which blood was collected and analyzed for the indicated parameters. In each graph, the values for individual mice in a group are shown, together with the mean ± SEM. The shaded box in each graph represents the normal range for that particular parameter. The results are representative of three independent experiments.</p

Clinical chemistry parameters are unaltered in mice following intravenous injection with manuka honey.

<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055993#pone-0055993-g004" target="_blank">Figure 4</a> legend, following which blood was collected and analyzed for the indicated parameters. In each graph, the values for individual mice in a group are shown, together with the mean ± SEM. The shaded box in each graph represents the normal range for that particular parameter. The results are representative of three independent experiments.</p

Extent of tumor necrosis in experimental groups following various treatments.

<p>Tumors were excised from animals at day 20–24 post treatment with saline (panels <b><i>A–B</i></b>), manuka honey (panels <b><i>C–D</i></b>), taxol (panels <b><i>E–F</i></b>) or manuka+taxol (panels <b><i>G–H</i></b>). Tissue sections were stained with H&E, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055993#s2" target="_blank">Materials and Methods</a>. For each treatment, representative images at low (panels <b><i>A</i></b>, <b><i>C</i></b>, <b><i>E</i></b>, <b><i>G</i></b>; bar = 200 µm) and high magnifications (panels <b><i>B</i></b>, <b><i>D</i></b>, <b><i>F</i></b>, <b><i>H</i></b>; bar = 50 µm) are shown. Necrotic regions are indicated (n). The results are representative of two independent experiments.</p

Immunohistochemical staining for intratumor caspase-3⁺ apoptotic cells.

<p>Tumor tissue sections were prepared after treatment with saline (panel <b><i>A</i></b>), manuka honey (panel <b><i>B</i></b>), taxol (panel <b><i>C</i></b>) or manuka+taxol (panel <b><i>D</i></b>) and stained using caspase 3-specific antibody, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055993#s2" target="_blank">Materials and Methods</a>. Representative images at high magnification (bar = 50 µm) are shown. Arrows indicate representative, brown-staining, apoptotic cells. Necrotic regions are also indicated (n). The results are representative of two independent experiments. (<b><i>E</i></b>) Quantitative estimation of the number of caspase-3 positive cells in tumor sections of different treatment groups. The data is shown as the mean ± SEM of the number of positive cells per high power field. Tumors were obtained from 2–3 mice per treatment group and multiple sections were made from each tumor tissue. The number of positive cells was determined by counting the number of cells in 20 high power fields per section. Asterisks denote statistically significant differences between each experimental group and the saline control group; also shown is a comparison between manuka alone and manuka+taxol groups (*, <i>p</i><0.05).</p

Effect of systemic administration of manuka on tumor growth and host survival.

<p>(<b><i>A</i></b>) Animals with established tumors were treated i.v. with either manuka honey (50% w/v), taxol (10 mg/Kg), manuka+taxol, or saline as control. All treatments were given twice per week until the end of observation period. Each data point represents the mean ± SEM of 19–20 mice per group, pooled from 2 individual experiments. Asterisks denote statistically significant differences between each experimental group and the saline control group; also shown is a comparison between manuka alone and manuka+taxol groups (**, <i>p</i><0.01; ***, <i>p</i><0.001). (<b><i>B</i></b>) Co-treatment with taxol and manuka leads to a significant enhancement in host survival. Experimental animals were followed for survival for up to day 25 post treatment. Each data point represents the mean ± SEM of 19–20 mice per group, pooled from 2 individual experiments. Asterisks denote statistically significant differences between experimental and saline control groups; also shown is a comparison between taxol alone and manuka+taxol groups (**, <i>p</i><0.01; *, <i>p</i><0.05).</p

Manuka induces caspase-mediated apoptosis in cancer cells.

<p>B16.F1 melanoma cells were treated with manuka (5% w/v), taxol (10 or 50 ng/ml) or medium as control. After 24 hrs of culture, enzymatic activity of caspase 3/7 (graph <b><i>A</i></b>) and caspase 8 (graph <b><i>B</i></b>) were determined using specific kits and following manufacturer's recommendation. The data is presented as fold increase in caspase activity after normalization to the number of viable cells per culture. <b><i>C</i></b>. Western blot analysis of caspase-9 activation B16.F1 cells treated with manuka or taxol. Whole cell extracts were prepared after a 24-hr treatment with manuka (5% w/v) or taxol (10 ng/ml). Protein extracts were resolved on 10% SDS-PAGE and immunoblotted with caspase-9-specific ployclonal antibody capable of detecting both full length and cleaved forms of caspase-9. The cell extracts were also probed with an antibody against β-actin as a control for protein loading.</p

Evidence for late apoptotic events induced by manuka honey in cancer cells.

<p><b><i>A</i></b>. B16.F1 cells were incubated for 24 hrs or 72 hrs in the absence or presence of Manuka (<i>M</i>; 5%) or taxol (<i>T</i>; 50 ng/ml). Whole cell extracts (100 µg/lane) were resolved on 10% SDS-PAGE followed by Western blotting with an antibody specific to Bcl-2. <b><i>B</i></b>. Cells were treated for 24 hrs or 72 hrs with the indicated concentrations of manuka (0.6%–5.0%) or taxol (50 ng/ml). Whole cell extracts (100 µg/lane) were resolved on 10% SDS-PAGE followed by Western blotting with a PARP-specific antibody. The full-length (116 kD) and cleaved (89 kD) forms of PARP are indicated. The cell extracts were also probed with an antibody against β-actin as a control for loading. <b><i>C</i></b>. Following treatment for 72 hrs, cells were lysed and DNA extracted, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055993#s2" target="_blank">Materials and Methods</a>. Extracted DNA was resolved on 1.5% agarose gel and stained with ethidium bromide to visualize the oligonucleosomal fragments. The results are representati<b>v</b>e of two independent experiments.</p