Patient demographics.

<p>*NA; not applicable.</p

Luminal bacteria penetrate colon tissue of Muc2^−/− mice and inflamed UC patients.

<p>qPCR was used to determine the ratio of 23SrRNA to 18SrRNA to assess bacterial presence in mouse colon tissue (<b>A</b>), mouse MLN (<b>B</b>) and biopsies from UC patients with active inflammation, from UC patients in clinical remission and from non-inflamed controls (<b>C</b>). Results were analyzed using the ΔΔCT method with the CT value of 18SrRNA as the endogenous reference gene. (<b>D</b>) Differential gene expression of Relmβ was analyzed using the ΔΔCT method with the CT value of HPRT as the endogenous reference gene. (<b>E</b>) Dot plots show intracellular iNOS expression by gated viable (7AAD⁻) CD11c⁺MHC-II^hiCD11b⁺ cells from the MLN of a representative inflamed Muc2^−/− mouse (left) and a WT mouse (right). (<b>F</b>) Scatter plot show the percent iNOS-expressing cells among total CD11c⁺MHC-II^hiCD11b⁺ cells from the MLN of WT mice (grey circles), Muc2^−/− mice (open circles) and inflamed Muc2^−/− mice (black circles). Mice in the “Muc2^−/− inflamed” group had a higher frequency of colon PMNs relative to other Muc2^−/− mice indicating they were colitic and were analyzed as a separate group; see text for details. Mouse data was obtained from 3 independent experiments with 8–11 mice in each group. Each symbol represents an individual. Mice were between 8–16 weeks of age except 2 WT mice that were 7 weeks old. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001).</p

A Unifying Mechanism for Cancer Cell Death through Ion Channel Activation by HAMLET

<div><p>Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl₂), preventing the changes in free cellular Na⁺ and K⁺ concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET’s broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET’s documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.</p> </div

Histology of colon tissue from Muc2^−/− mice and UC patients.

<p>(<b>A</b>) Carnoy fixed whole colon sections with preserved mucus from WT and Muc2^−/− mice were analyzed by FISH (red) for bacteria localization and counterstained with DAPI. The mucus separating bacteria from the epithelium in WT mice is indicated by a double arrow and bacteria in contact with the epithelium in Muc2^−/− mice are marked by arrows. Scale bars are 100 µm. (<b>B</b>) Sigmoid sections of human biopsies from a control patient and a patient with active UC (Mayo endoscopic score 2) were stained for MUC2 (green) and DAPI. Mucus separating bacteria and the epithelium is indicated by a double arrow and bacteria within the remaining mucus in the UC patient are marked by arrows. Scale bars are 20 µm. (<b>C–D</b>) Representative sections from the proximal, middle or distal colon from (<b>C</b>) Muc2^+/− (18 weeks of age) and (<b>D</b>) inflamed Muc2^−/− (18 weeks of age) are shown. The neutrophil frequency determined by flow cytometry in parallel samples of the displayed tissues is indicated in the inlays. Original magnification is 5x. (<b>E–F</b>) Human rectal tissue from representative sections of (<b>E</b>) an inflamed UC patient at the time of diagnosis and (<b>F</b>) a non-inflamed control are shown. Original magnification is 40x. All tissues were sliced in 5 µm sections and stained with H&E.</p

p38 inhibition rescues carcinoma and lymphoma cells from death in response to HAMLET.

<p>(A, B) p38 MAPK inhibition (SB202190, 20 µM, 30 minutes pre-incubation) rescued carcinoma (A549 and A498) and T-cell lymphoma (Jurkat) cells from death in response to HAMLET (7–49 µM, 3 h). Viability was quantified by Trypan blue exclusion (A) or as ATP levels (B). Data are means±SEMs for 3 independent experiments. The p38 inhibitor alone had no significant effect on cell death (leftmost bars in each graph). (C) Real-time images of cell morphology after HAMLET exposure, showing that p38 inhibition prevents morphological changes in carcinoma cells (nuclear condensation, rounding up and blebbing). Alexa-Fluor 568-labeled HAMLET is red and the nuclei are blue (Hoechst 33342). (D–E) A549 lung carcinoma cells were transfected using siRNA against p38α and/or p38β or non-targeting siRNA and compared to non-transfected controls (NTF). Relative p38α and p38βmRNA levels are shown (p38/GAPDH, in % of non-transfected cells) as means<u>+</u>SEMs for four independent experiments. Knockdown was also confirmed on the protein level by western blotting against total p38 MAPK (representative blot shown, *p<0.05, ***p<0.001.). GAPDH was used as a loading control. (F) The cytotoxic effect of HAMLET was quantified 48 hours after transfection as a reduction in ATP levels. Data are means+SEM for four independent experiments. *p<0.05, ***p<0.001. For effects of the p38 inhibitor Birb0796, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058578#pone.0058578.s006" target="_blank">Figure S6</a>.</p