A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells

Comprehensive suppression of all apoptosis-induced proliferation pathways as a proposed approach to colorectal cancer prevention and therapy.

Mutations in the WNT/beta-catenin pathway are present in the majority of all sporadic colorectal cancers (CRCs), and histone deacetylase inhibitors induce apoptosis in CRC cells with such mutations. This apoptosis is counteracted by (1) the signaling heterogeneity of CRC cell populations, and (2) the survival pathways induced by mitogens secreted from apoptotic cells. The phenomena of signaling heterogeneity and apoptosis-induced survival constitute the immediate mechanisms of resistance to histone deacetylase inhibitors, and probably other chemotherapeutic agents. We explored the strategy of augmenting CRC cell death by inhibiting all survival pathways induced by the pro-apoptotic agent LBH589, a histone deacetylase inhibitor: AKT, JAK/STAT, and ERK signaling. The apoptosis-enhancing ability of a cocktail of synthetic inhibitors of proliferation was compared to the effects of the natural product propolis. We utilized colorectal adenoma, drug-sensitive and drug-resistant colorectal carcinoma cells to evaluate the apoptotic potential of the combination treatments. The results suggest that an effective approach to CRC combination therapy is to combine apoptosis-inducing drugs (e.g., histone deacetylase inhibitors, such as LBH589) with agents that suppress all compensatory survival pathways induced during apoptosis (such as the cocktail of inhibitors of apoptosis-associated proliferation). The same paradigm can be applied to a CRC prevention approach, as the apoptotic effect of butyrate, a diet-derived histone deacetylase inhibitor, is augmented by other dietary agents that modulate survival pathways (e.g., propolis and coffee extract). Thus, dietary supplements composed by fermentable fiber, propolis, and coffee extract may effectively counteract neoplastic growth in the colon

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

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

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

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