Role for ERK signaling in the apoptosis of CRC cells exposed to dietary agents and clonal growth ability of neoplastic and normal cells exposed to such agents.

<p>A. HCT116 cells were exposed for 24 h to mock (M), 1 mg/ml roasted coffee extract (R), 5 mM butyrate and 100 µg/ml propolis (BP), or butyrate/propolis and increasing concentrations of coffee extract: 0.25, 0.5 or 1.0 mg/ml. Apoptosis was measured by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#s2" target="_blank">Materials and Methods</a>. B. HCT-116 cells were treated as in (A) for 17 hours and total cell lysates were analyzed by western blotting. C. HCT-116 and LT 97 cells were exposed for 20 h, and HCT-R cells were exposed for 42 h to mock (M), 0.5 µM AZD6244 (A), the combination; 5 mM butyrate, 100 µg/ml propolis, 1 mg/ml roasted coffee extract (BPR), or BPR and 0.5 µM AZD6244 (BPR-A). Apoptosis was measured by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#s2" target="_blank">Materials and Methods</a>. Statistically significant differences in apoptotic levels are noted by asterisks (P<0.05). D. Clonal growth assays. Percent clonal growth was calculated by dividing the number of cell colonies by the number of plated cells, and multiplying by 100. The ratios of clonal growth were calculated by dividing the percent clonal growth in mock-treated samples by the percent clonal growth in agent-treated samples. Experiments were repeated four to six times with triplicate samples per experiment. Statistically significant differences among cell lines in their mean ratios were determined by one-way ANOVA. For cells exposed to butyrate, F(6,31) = 10.26, P<0.0001; for cells exposed to propolis and coffee extract, F(6,28) = 5.493, P = 0.0007, and for cells exposed to butyrate, propolis and coffee extract, F(6,26) = 10.56, P<0.0001. The post-test calculations used the Bonferroni correction to adjust for multiple comparisons with 95% confidence. Among the cells exposed to butyrate, statistically significant differences (P<0.05, CI 95%) were detected in HCT116 vs. CCL92, HCT116 vs. HEK293, HCT116 vs. SW13, HCT116 vs. LA1-5s, HCT116 vs. HCT-R, CCD841CoN vs. HEK293, and CCD841CoN vs. HCT-R cells. Among the cell lines exposed to propolis and coffee extract, statistically significant differences (P<0.05, CI 95%) were detected in HCT116 vs. SW13, HCT-R vs. CCL92, CCD841CoN vs. HCT-R cells, HEK293 vs. HCT-R, and SW13 vs. HCT-R cells. Among cell lines exposed to butyrate, propolis, and coffee extract statistically significant differences (P<0.05, CI 95%) were detected in HEK293 vs. SW13 cells, and HCT116 vs. all other cell lines except for HCT-R cell line.</p

The combination treatment of butyrate, propolis, and coffee extract induces highest levels of apoptosis in colon adenoma and carcinoma cells.

<p>A–C. Cells were exposed to mock (M), 5 mM butyrate (B), 1 mg/ml roasted coffee extract (R), 100 µg/ml propolis (P), butyrate and coffee extract (BR), butyrate and propolis (BP), propolis and coffee extract (PR), or butyrate, propolis and coffee extract (BPR). Apoptosis was measured by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#s2" target="_blank">Materials and Methods</a>. D–F. Cells were exposed to treatments as described above for 17 h, and total cell lysates were analyzed by western blotting.</p

Ability of ICAP and propolis to augment chemotherapeutics-induced apoptosis.

<p>A. HCT-R or HCT-116 cells were exposed for 48 h or 24 h respectively, to mock (M), 50 nM LBH589 (L), LBH589 and 100 µg/ml propolis (LP), LBH589 and the ICAP (LC), 100 µg/ml propolis (P), or the ICAP alone (C). B. HCT-R cells were exposed to the treatments described in A for 17 h. and total cell lysates were analyzed by western blotting. C. HCT-R and HCT-116 cells were exposed for 48 h or 24 h respectively, to mock (M), 10 µM 5-fluorouracil (F), 5-fluorouracil and 100 µg/ml propolis (FP), 5-fluorouracil and the ICAP (FC), propolis (P), or the ICAP alone (C).</p

LBH589-induced apoptosis is augmented by suppressing three survival pathways.

<p>A. Western blot analyses of HCT-R CRC cells exposed to mock (C) treatment, 50 nM LBH589 (L), 1 µM MK2206 (M), 0.5 µM AZD6244 (A), or 1 µM pyridone 6 (P) for 20 hours. Equal number of cells were lysed directly in Laemmli buffer and analyzed for expression levels of phosphorylated and total levels of AKT, ERK1/2, and STAT3. B. Apoptotic analyses of HCT-R cells exposed for 48 h to the treatments described in (A). Asterisk indicates statistically significant differences (P<0.05) between the apoptotic levels. Additional statistically significant differences are indicated in the text.</p

Hypothetical mechanisms of the immediate resistance (IMR) that facilitate survival of CRC cells in the presence of HDACis.

<p>Stochastic differences in signaling levels are always present in cell populations, and these differences are augmented by lateral inhibition-type interactions, microenvironment, and exposure to apoptotic agents <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#pone.0115068-Lazarova1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#pone.0115068-Brabletz1" target="_blank">[12]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#pone.0115068-Huang1" target="_blank">[15]</a>. Due to their different signaling levels (in particular, variability in WNT/beta-catenin activity), not all CRC cells commit to apoptosis within 24 hours of exposure to HDACis. Apoptosis-induced mitogens <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#pone.0115068-Bordonaro2" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#pone.0115068-Bordonaro3" target="_blank">[22]</a> may allow for the survival of a limited number of cells; in <i>in vivo</i> conditions, such cells could accumulate resistance-conferring mutations in time.</p

CAPE-containing propolis augments the apoptotic effect of butyrate on CC cells.

<p>(A). Representative western blot analysis of HCT-R cells exposed to mock (M), 5 mM butyrate (B), 1.2 µg/ml CAPE, butyrate and CAPE (BC), 100 µg/ml propolis (P), or butyrate and propolis (BP) for 19 h. (B, C) HCT-R (B) and HCT-116 (C) cells were exposed to mock (M), 5 mM butyrate (B), 1.2 µg/ml CAPE (C), butyrate and CAPE (BC), 100 µg/ml propolis (P), or butyrate and propolis (BP) for 50 h. Propolis preparation contains 0.3 µg CAPE per 100 µg powder. A minimum three independent experiments were carried out with triplicate samples each, values are mean ± SD.</p

Propolis Augments Apoptosis Induced by Butyrate via Targeting Cell Survival Pathways

<div><p>Diet is one of the major lifestyle factors affecting incidence of colorectal cancer (CC), and despite accumulating evidence that numerous diet-derived compounds modulate CC incidence, definitive dietary recommendations are not available. We propose a strategy that could facilitate the design of dietary supplements with CC-preventive properties. Thus, nutrient combinations that are a source of apoptosis-inducers and inhibitors of compensatory cell proliferation pathways (e.g., AKT signaling) may produce high levels of programmed death in CC cells. Here we report the combined effect of butyrate, an apoptosis inducer that is produced through fermentation of fiber in the colon, and propolis, a honeybee product, on CC cells. We established that propolis increases the apoptosis of CC cells exposed to butyrate through suppression of cell survival pathways such as the AKT signaling. The programmed death of CC cells by combined exposure to butyrate and propolis is further augmented by inhibition of the JNK signaling pathway. Analyses on the contribution of the downstream targets of JNK signaling, c-JUN and JAK/STAT, to the apoptosis of butyrate/propolis-treated CC cells ascertained that JAK/STAT signaling has an anti-apoptotic role; whereas, the role of cJUN might be dependent upon regulatory cell factors. Thus, our studies ascertained that propolis augments apoptosis of butyrate-sensitive CC cells and re-sensitizes butyrate-resistant CC cells to apoptosis by suppressing AKT signaling and downregulating the JAK/STAT pathway. Future <i>in vivo</i> studies should evaluate the CC-preventive potential of a dietary supplement that produces high levels of colonic butyrate, propolis, and diet-derived JAK/STAT inhibitors.</p></div

Role of JAK/STAT signaling in the apoptosis of butyrate/propolis-treated HCT-R cells.

<p>(A). A representative western blot analysis of HCT-R cells exposed to mock (M), 5 mM butyrate and 100 µg/ml propolis treatment (BP), 5 mM butyrate and 1.2 µg/ml CAPE (BC) for 19 h. (B). A representative immunoblot of HCT-R cells exposed for 19 h to mock (M), 1 µM JAK inhibitor pyridone 6 (Ji), 5 mM butyrate and 100 µg/ml propolis treatment (BP), the combination of JAK inhibitor, butyrate, and propolis (Ji/BP), 5 mM butyrate and 1.2 µg/ml CAPE (BC), and the combination of JAK inhibitor and butyrate/CAPE (Ji/BC). (C and D) Apoptotic analyses of HCT-R cells exposed for 35 hours to (M), 5 mM butyrate and 100 µg/ml propolis treatment (BP), 1 µM JAK inhibitor (Ji), 5 mM butyrate and 1.2 µg/ml CAPE (BC), JAK inhibitor and butyrate/propolis (Ji/BP), or JAK inhibitor and butyrate/CAPE (Ji/BC). A minimum of three independent experiments were carried out with triplicate samples each, values are mean ± SD.</p

Role of cJUN in the apoptosis of butyrate/propolis-treated HCT-R cells.

<p>(A) Silencing of c<i>JUN</i> expression via nucleofection of c<i>JUN</i> siRNA suppresses total and phosphorylated (p) cJUN levels in HCT-R cells, as ascertained by western blot analyses. (B) A representative apoptotic analysis of HCT-R cells that were nucleofected with control or c<i>JUN</i> siRNA. Cells were exposed to mock or butyrate/propolis treatment for 24 h, and analyzed at 48 h post-nucleofection. Each treatment was in triplicate, values are mean ± SD. (C) Detection of TAM67-GFP in HCT-116 (H) or HCT-R (R) cells transfected stably with control (c) or TAM67 (t) expression vector. (D) AP1 luciferase transcription assays of control (C) and TAM67 (T) cells exposed for 19 h to mock (m) or 5 mM butyrate and 100 µg/ml propolis (bp) treatment. (E) Apoptotic assays of control and TAM67-expressing HCT-116 cells exposed for 24 h to mock or 5 mM butyrate and 100 µg/ml propolis were carried out as described in Methods. Three independent experiments were carried out with triplicate samples each, values are mean ± SD. (F) Expression of TAM67-GFP, endogenous cJUN, and beta-catenin in nuclear lysates of HCT-116 cells transfected with GFP (control) or TAM67-GFP vector. Cells were exposed to butyrate/propolis as in Fig. 1A for 19 h. TAM67 and endogenous cJUN were visualized with an antibody to the DNA-binding domain of c-JUN (sc-44, Santa Cruz Biotechnology). (G) Immunoprecipitations with nuclear lysates described in (F) with a control (IgG) antibody or a cJUN antibody that recognizes the amino-terminal domain of the endogenous cJUN protein (sc-45, Santa Cruz Biotechnology). Beta-catenin that co-immunoprecipitates with endogenous cJUN was labeled with an antibody from Santa Cruz Biotechnology (sc-53483). (H,I) Luciferase transcription assays of control (C) and TAM67 (T) cells exposed for 19 h to mock (m) or 5 mM butyrate and 100 µg/ml propolis (bp) were carried out as described in Methods. Three independent experiments were carried out with duplicate samples each, values are mean ± SD.</p

Diet-derived pAKT inhibitors mimic the effects of MK2206 in augmenting HDACi-induced apoptosis in CC cells.

<p>(A) Diet-derived compounds suppress butyrate-induced pAKT levels in CC cells. Representative western blot of cells exposed for 14 hrs to mock treatment (M), 5 mM butyrate (B), 5 mM butyrate and 4 µg/ml CAPE (BC), 5 mM butyrate and 20 µM sulforaphane (BS), 5 mM butyrate and 40 µM DATS (BD). Detection of pAKT, total AKT (AKT), and ACTIN was carried out with total cells lysates. (B) Apoptotic levels in HCT-116 and HCT-R cells exposed for 28 hrs to mock treatment (M), 4 µg/ml CAPE (C), 5 mM butyrate (B), or butyrate and 4 µg/ml CAPE (BC). Apoptotic assays were performed by flow cytometry. Percent apoptosis and necrosis was calculated by dividing the number of apoptotic and necrotic cells by the total number of analyzed cells. Each experiment had triplicate samples per treatment; data represent the mean of three experiments. Statistically significant differences (P<0.05) are noted by a star. (C) CAPE suppresses the phospho-p55 PI3K levels in butyrate-treated HCT-R cells. Cells were treated as in (B) for 17 hrs, and total cell lysates were analyzed by western blotting. Phospho-p85 levels were not detected; however, phospho-p55 levels were detected and the bar graph next to the western blot represents the densitometry quantification of phospho-p55 PI3K in three independent experiments.</p