Fabrication and characterization of dihydromyricetin-loaded microcapsules stabilized by glyceryl monostearate and whey protein-xanthan gum.

Dihydromyricetin (DMY) is a lipophilic nutrient with various potential health benefits; however, its poor storage stability and low solubility and bioavailability limit its applications. This study aims to encapsulate DMY in microcapsules by membrane emulsification and freeze-drying methods to overcome these issues. Glyceryl monostearate (GMS, solid lipid) and octyl and decyl glycerate (ODO, liquid lipid) were applied as the inner cores. Whey protein and xanthan gum (XG) were used as wall materials. The prepared microcapsules had an irregular blocky aggregated structure with rough surfaces. All the microcapsules had a DMY loading of 0.85 %-1.1 % and encapsulation efficiency (EE) >85 %. GMS and XG increased the DMY loading and EE. The addition of GMS and an increased XG concentration led to a decrease in the rehydration rate. The in vitro release and digestion studies revealed that GMS and XG controlled the release and digestion of DMY. The chemical stability results indicated that GMS and XG protected DMY against oxidation. An antioxidant capacity study showed that GMS and XG helped DMY in the microcapsules exert antioxidant effects. This research study provides a platform for designing microcapsules with good stability and high bioavailability to deliver lipophilic bioactive compounds.

Coenzyme Q10-loaded microcapsules stabilized by glyceryl monostearate and soy protein isolates-flaxseed gum: Characterization, in vitro release and digestive behavior.

This study aimed to stabilize microcapsules with core materials of glyceryl monostearate (GMS) and octyl and decyl glycerate, and wall materials of soy protein isolates (SPI) and flaxseed gum (FG) by complex coacervation method to overcome the drawbacks of coenzyme Q10 (CoQ10). It was demonstrated by the study that the obtained microcapsules were irregular aggregates. Differential scanning calorimetry and x-ray diffraction patterns indicated that CoQ10 was entrapped inside the disordered semisolid cores of microcapsules. The CoQ10 loading and encapsulation efficiency analysis revealed that GMS and FG helped CoQ10 better encapsulated inside the microcapsules. The in vitro release curve showed a "burst" release of CoQ10 absorbed on the surface of microcapsules for the first 180 min, followed by a sustained release of the encapsulated CoQ10. GMS and FG contributed to the sustained release and the release mechanism of the microcapsules was Fickian diffusion. The in vitro simulated digestion demonstrated that the constructed microcapsules improved the bio-accessibility of CoQ10. Finally, due to the protection of GMS and FG, microcapsules had good storage stability. In conclusion, this study emphasized the potential of using new microcapsules to deliver and protect lipophilic ingredients, providing valuable information for developing functional foods with higher bioavailability.

miR-27a-3p regulates intestinal cell proliferation and differentiation through Wnt/ß-catenin signalling.

Intestinal stem cells differentiate into absorptive enterocytes, characterised by increased brush border enzymes such as intestinal alkaline phosphatase (IAP), making up the majority (95%) of the terminally differentiated cells in the villus. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here, we show that the intestinal microRNA (miR)-27a-3p is an important regulator of intestinal epithelial cell proliferation and enterocyte differentiation. Repression of endogenous miR-27a-3p leads to increased enterocyte differentiation and decreased intestinal epithelial cell proliferation in mouse and human small intestinal organoids. Mechanistically, miR-27a-3p regulates intestinal cell differentiation and proliferation at least in part through the regulation of retinoic acid receptor α (RXRα), a modulator of Wnt/ß-catenin signalling. Repression of miR-27a-3p increases the expression of RXRα and concomitantly, decreases the expression of active ß-catenin and cyclin D1. In contrast, overexpression of miR-27a-3p mimic decreases the expression of RXRα and increases the expression of active ß-catenin and cyclin D1. Moreover, overexpression of the miR-27a-3p mimic results in impaired enterocyte differentiation and increases intestinal epithelial cell proliferation. These alterations were attenuated or blocked by Wnt inhibition. Our study demonstrates an miR-27a-3p/RXRα/Wnt/ß-catenin pathway that is important for the maintenance of enterocyte homeostasis in the small intestine.

Preparation and characterization of astaxanthin-loaded microcapsules stabilized by lecithin-chitosan-alginate interfaces with layer-by-layer assembly method.

Astaxanthin is a kind of keto-carotenes with various health benefits. However, its solubility and chemical stability are poor, which leads to low bio-availability. Microcapsules have been reported to improve the solubility, chemical stability, and bio-availability of lipophilic bioactives. Freeze-dried astaxanthin-loaded microcapsules were prepared by layer-by-layer assembly of tertiary emulsions with maltodextrin as the filling matrix. Tertiary emulsions were fabricated by performing chitosan and sodium alginate electrostatic deposition onto soybean lecithin stabilized emulsions. 0.9 wt% of chitosan solution, 0.3 wt% of sodium alginate solution and 20 wt% of maltodextrin were optimized as the suitable concentrations. The prepared microcapsules were powders with irregular blocky structures. The astaxanthin loading was 0.56 ± 0.05 % and the encapsulation efficiency was >90 %. A slow release of astaxanthin could be observed in microcapsules promoted by the modulating of chitosan, alginate and maltodextrin. In vitro simulated digestion displayed that the microcapsules increased the bio-accessibility of astaxanthin to 69 ± 1 %. Chitosan, alginate and maltodextrin can control the digestion of microcapsules. The coating of chitosan and sodium alginate, and the filling of maltodextrin in microcapsules improved the chemical stability of astaxanthin. The constructed microcapsules were valuable to enrich scientific knowledge about improving the application of functional ingredients.

A Novel Protozoa Parasite-Derived Protein Adjuvant Is Effective in Immunization with Cancer Cells to Activate the Cancer-Specific Protective Immunity and Inhibit the Cancer Growth in a Murine Model of Colorectal Cancer.

Cancer-specific CD8+ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8+ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable (either treated with mitomycin C or irradiated by X-ray) MC38 CRC cells in combination with rGRA6Nt adjuvant and received a challenge implantation of replication-capable MC38 tumor cells, those mice markedly inhibited the growth of the implanted tumors in association with a two-fold increase in CD8+ T cell density within the tumors. In addition, CD8+ T cells of the immunized mice secreted significantly increased amounts of granzyme B, a key mediator of the cytotoxic activity of CD8+ T cells, and IFN-γ in response to MC38 CRC cells in vitro when compared to the T cells from unimmunized mice. Notably, the protective effects of the immunization were specific to MC38 CRC cells, as the immunized mice did not exhibit a significantly inhibited growth of EL4 lymphoma tumors. These results indicate that rGRA6Nt is a novel and effective protein adjuvant when used in immunizations with non-replicable cancer cells to potently activate the protective immunity specifically against the cancer cells employed in the immunization.

Perfluorooctanesulfonic acid exposure leads to downregulation of 3-hydroxy-3-methylglutaryl-CoA synthase 2 expression and upregulation of markers associated with intestinal carcinogenesis in mouse intestinal tissues.

Perfluorooctanesulfonic acid (PFOS) is a widely recognized environment pollutant known for its high bioaccumulation potential and a long elimination half-life. Several studies have shown that PFOS can alter multiple biological pathways and negatively affect human health. Considering the direct exposure to the gastrointestinal (GI) tract to environmental pollutants, PFOS can potentially disrupt intestinal homeostasis. However, there is limited knowledge about the effect of PFOS exposure on normal intestinal tissues, and its contribution to GI-associated diseases remains to be determined. In this study, we examined the effect of PFOS exposure on the gene expression profile of intestinal tissues of C57BL/6 mice using RNAseq analysis. We found that PFOS exposure in drinking water significantly downregulates mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme, in intestinal tissues of mice. We found that diets containing the soluble fibers inulin and pectin, which are known to be protective against PFOS exposure, were ineffective in reversing the downregulation of HMGCS2 expression in vivo. Analysis of intestinal tissues also demonstrated that PFOS exposure leads to upregulation of proteins implicated in colorectal carcinogenesis, including ß-catenin, c-MYC, mTOR and FASN. Consistent with the in vivo results, PFOS exposure leads to downregulation of HMGCS2 in mouse and human normal intestinal organoids in vitro. Furthermore, we show that shRNA-mediated knockdown of HMGCS2 in a human normal intestinal cell line resulted in increased cell proliferation and upregulation of key proliferation-associated proteins such as cyclin D, survivin, ERK1/2 and AKT, along with an increase in lipid accumulation. In summary, our results suggest that PFOS exposure may contribute to pathological changes in normal intestinal cells via downregulation of HMGCS2 expression and upregulation of pro-carcinogenic signaling pathways that may increase the risk of colorectal cancer development.

Glycolytic Regulation of Intestinal Stem Cell Self-Renewal and Differentiation.

BACKGROUND AND AIMS: The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. METHODS: Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase, the transcription factor atonal homolog 1, and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by Western blot, quantitative real-time reverse transcription polymerase chain reaction, or immunofluorescence, and immunohistochemistry staining. RESULTS: HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. CONCLUSIONS: Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells.

Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

Ketogenesis Attenuates KLF5-Dependent Production of CXCL12 to Overcome the Immunosuppressive Tumor Microenvironment in Colorectal Cancer.

The dynamic composition of the tumor microenvironment (TME) can markedly alter the response to targeted therapies for colorectal cancer. Cancer-associated fibroblasts (CAF) are major components of TMEs that can direct and induce infiltration of immunosuppressive cells through secreted cytokines such as CXCL12. Ketogenic diets (KD) can inhibit tumor growth and enhance the anticancer effects of immune checkpoint blockade. However, the role of ketogenesis on the immunosuppressive TME is not known. Here, we show that decreased ketogenesis is a signature of colorectal cancer and that an increase in ketogenesis using a KD decreases CXCL12 production in tumors, serum, liver, and lungs. Moreover, increasing ketogenesis by overexpression of the ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) or treatment with the ketone body ß-hydroxybutyrate markedly decreased expression of KLF5, which binds the CXCL12 promoter and induces CXCL12 expression in CAFs. KD decreased intratumoral accumulation of immunosuppressive cells, increased infiltration of natural killer and cytotoxic T cells, and enhanced the anticancer effects of PD-1 blockade in murine-derived colorectal cancer. Furthermore, increasing ketogenesis inhibited colorectal cancer migration, invasion, and metastasis in vitro and in vivo. Overall, ketogenesis is downregulated in the colorectal cancer TME, and increased ketogenesis represses KLF5-dependent CXCL12 expression to improve the immunosuppressive TME, which leads to the enhanced efficacy of immunotherapy and reduced metastasis. Importantly, this work demonstrates that downregulation of de novo ketogenesis in the TME is a critical step in colorectal cancer progression. SIGNIFICANCE: This study identifies ketogenesis as a critical regulator of the tumor microenvironment in colorectal cancer and suggests the potential for ketogenic diets as a metabolic strategy to overcome immunosuppression and prolong survival. See related commentary by Montrose and Galluzzi, p. 1464.

Regulation of SIRT2 by Wnt/ß-catenin signaling pathway in colorectal cancer cells.

Activation of the Wnt/ß-catenin pathway is one of the hallmarks of colorectal cancer (CRC). Sirtuin 2 (SIRT2) protein has been shown to inhibit CRC proliferation. Previously, we reported that SIRT2 plays an important role in the maintenance of normal intestinal cell homeostasis. Here, we show that SIRT2 is a direct target gene of Wnt/ß-catenin signaling in CRC cells. Inhibition or knockdown of Wnt/ß-catenin increased SIRT2 promoter activity and mRNA and protein expression, whereas activation of Wnt/ß-catenin decreased SIRT2 promoter activity and expression. ß-Catenin was recruited to the promoter of SIRT2 and transcriptionally regulated SIRT2 expression. Wnt/ß-catenin inhibition increased mitochondrial oxidative phosphorylation (OXPHOS) and CRC cell differentiation. Moreover, inhibition of OXPHOS attenuated the differentiation of CRC cells induced by Wnt/ß-catenin inhibition. In contrast, inhibition or knockdown of SIRT2 decreased, while overexpression of SIRT2 increased, OXPHOS activity and differentiation in CRC cells. Consistently, inhibition or knockdown or SIRT2 attenuated the differentiation induced by Wnt/ß-catenin inhibition. These results demonstrate that SIRT2 is a novel target gene of the Wnt/ß-catenin signaling and contributes to the differentiation of CRC cells.

Ketogenesis alleviates TNFα-induced apoptosis and inflammatory responses in intestinal cells.

The disturbance of strictly regulated self-regeneration in mammalian intestinal epithelium is associated with various intestinal disorders, particularly inflammatory bowel diseases (IBDs). TNFα, which plays a critical role in the pathogenesis of IBDs, has been reported to inhibit production of ketone bodies such as ß-hydroxybutyrate (ßHB). However, the role of ketogenesis in the TNFα-mediated pathological process is not entirely known. Here, we showed the regulation and role of HMGCS2, the rate-limiting enzyme of ketogenesis, in TNFα-induced apoptotic and inflammatory responses in intestinal epithelial cells. Treatment with TNFα dose-dependently decreased protein and mRNA expression of HMGCS2 and its product, ßHB production in human colon cancer cell lines HT29 and Caco2 cells and mouse small intestinal organoids. Moreover, the repressed level of HMGCS2 protein was found in intestinal epithelium of IBD patients with Crohn's disease and ulcerative colitis as compared with normal tissues. Furthermore, knockdown of HMGCS2 enhanced and in contrast, HMGCS2 overexpression attenuated, the TNFα-induced apoptosis and expression of pro-inflammatory chemokines (CXCL1-3) in HT29, Caco2 cells and DLD1 cells, respectively. Treatment with ßHB or rosiglitazone, an agonist of PPARγ, which increases ketogenesis, attenuated TNFα-induced apoptosis in the intestinal epithelial cells. Finally, HMGCS2 knockdown enhanced TNFα-induced reactive oxygen species (ROS) generation. In addition, hydrogen peroxide, the major ROS contributing to intestine injury, decreased HMGCS2 expression and ßHB production in the intestinal cells and mouse organoids. Our findings demonstrate that increased ketogenesis attenuates TNFα-induced apoptosis and inflammation in intestinal cells, suggesting a protective role for ketogenesis in TNFα-induced intestinal pathologies.

SIRT2 Contributes to the Regulation of Intestinal Cell Proliferation and Differentiation.

BACKGROUND AND AIMS: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis. METHODS: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated. RESULTS: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/ß-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression. CONCLUSION: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-ß-catenin signaling and is important for maintenance of IEC proliferation and differentiation.

Regulation of PTEN expression in intestinal epithelial cells by c-Jun NH2-terminal kinase activation and nuclear factor-kappaB inhibition.

The tumor suppressor protein phosphatase and tensin homologue deleted on chromosome ten (PTEN) plays an important role in intestinal cell proliferation and differentiation and tumor suppression by antagonizing phosphatidylinositol 3-kinase. Despite its importance, the molecular mechanisms regulating PTEN expression are largely undefined. Here, we show that treatment of the colon cancer cell line HT29 with the differentiating agent sodium butyrate (NaBT) increased PTEN protein and mRNA expression and induced c-Jun NH2-terminal kinase (JNK) activation. Inhibition of JNK by chemical or genetic methods attenuated NaBT-induced PTEN expression. In addition, our findings showed a cross-talk between nuclear factor kappaB (NF-kappaB) and JNK with respect to PTEN regulation. Overexpression of the NF-kappaB superrepressor increased PTEN expression and JNK activity, whereas overexpression of the p65 NF-kappaB subunit reduced both basal and NaBT-mediated JNK activation and PTEN expression. Moreover, we showed that overexpression of PTEN or treatment with NaBT increased expression of the cyclin-dependent kinase inhibitor p27(kip1) in HT29 cells; this induction was attenuated by inhibition of PTEN or JNK expression or overexpression of p65. Finally, we show a role for PTEN in NaBT-mediated cell death and differentiation. Our findings suggest that the JNK/PTEN and NF-kappaB/PTEN pathways play a critical role in normal intestinal homeostasis and colon carcinogenesis.

Regulation of Ketogenic Enzyme HMGCS2 by Wnt/ß-catenin/PPARγ Pathway in Intestinal Cells.

The Wnt/ß-catenin pathway plays a crucial role in development and renewal of the intestinal epithelium. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting ketogenic enzyme in the synthesis of ketone body ß-hydroxybutyrate (ßHB), contributes to the regulation of intestinal cell differentiation. Here, we have shown that HMGCS2 is a novel target of Wnt/ß-catenin/PPARγ signaling in intestinal epithelial cancer cell lines and normal intestinal organoids. Inhibition of the Wnt/ß-catenin pathway resulted in increased protein and mRNA expression of HMGCS2 and ßHB production in human colon cancer cell lines LS174T and Caco2. In addition, Wnt inhibition increased expression of PPARγ and its target genes, FABP2 and PLIN2, in these cells. Conversely, activation of Wnt/ß-catenin signaling decreased protein and mRNA levels of HMGCS2, ßHB production, and expression of PPARγ and its target genes in LS174T and Caco2 cells and mouse intestinal organoids. Moreover, inhibition of PPARγ reduced HMGCS2 expression and ßHB production, while activation of PPARγ increased HMGCS2 expression and ßHB synthesis. Furthermore, PPARγ bound the promoter of HMGCS2 and this binding was enhanced by ß-catenin knockdown. Finally, we showed that HMGCS2 inhibited, while Wnt/ß-catenin stimulated, glycolysis, which contributed to regulation of intestinal cell differentiation. Our results identified HMGCS2 as a downstream target of Wnt/ß-catenin/PPARγ signaling in intestinal epithelial cells. Moreover, our findings suggest that Wnt/ß-catenin/PPARγ signaling regulates intestinal cell differentiation, at least in part, through regulation of ketogenesis.

Alternative medicine products as a novel treatment strategy for inflammatory bowel disease.

Inflammatory bowel disease (IBD) affects the mucosal lining of the gastrointestinal tract; the etiology is unknown and treatment is directed at systemic immunosuppression. Natural products, including medicinal herbs, have provided approximately half of the drugs developed for clinical use over the past 20 years. The purpose of our current study was to determine the effects of a novel combination of herbal extracts on intestinal inflammation using a murine model of IBD. Female Swiss-Webster mice were randomized to receive normal water or 5% dextran sulfate sodium (DSS) drinking water to induce colitis. Mice were treated with either a novel combination of herbal aqueous extracts or vehicle control per os (po) or per rectum (pr) every 24 hours for 7-8 days. Disease activity index score (DAI) was determined daily; mice were sacrificed and colons were analyzed by H & E staining, MPO assay, and cytokine (TNF-alpha, IL-6) ELISAs. Mice treated with the combination of herbal extracts, either po or pr, had significantly less rectal bleeding and lower DAI scores compared to the vehicle-treated group. Moreover, colonic ulceration, leukocytic infiltration, and cytokine levels (TNF-alpha and IL-6) were also decreased in the colons of herbal-treated mice, reflected by H & E staining, MPO assay, and cytokine ELISA. Treatment with the combination of medicinal herbs decreases leukocyte infiltration and mucosal ulceration, ameliorating the course of acute colonic inflammation. This herbal remedy may prove to be a novel and safe therapeutic alternative in the treatment of IBD.

Deptor Is a Novel Target of Wnt/ß-Catenin/c-Myc and Contributes to Colorectal Cancer Cell Growth.

Activation of the Wnt/ß-catenin signaling pathway drives colorectal cancer growth by deregulating expression of downstream target genes, including the c-myc proto-oncogene. The critical targets that mediate the functions of oncogenic c-Myc in colorectal cancer have yet to be fully elucidated. Previously, we showed that activation of PI3K/Akt/mTOR contributes to colorectal cancer growth and metastasis. Here, we show that Deptor, a suppressor of mTOR, is a direct target of Wnt/ß-catenin/c-Myc signaling in colorectal cancer cells. Inhibition of Wnt/ß-catenin or knockdown of c-Myc decreased, while activation of Wnt/ß-catenin or overexpression of c-Myc increased the expression of Deptor. c-Myc bound the promoter of Deptor and transcriptionally regulated Deptor expression. Inhibition of Wnt/ß-catenin/c-Myc signaling increased mTOR activation, and the combination of Wnt and Akt/mTOR inhibitors enhanced inhibition of colorectal cancer cell growth in vitro and in vivo Deptor expression was increased in colorectal cancer cells; knockdown of Deptor induced differentiation, decreased expression of B lymphoma Mo-MLV insertion region 1 (Bmi1), and decreased proliferation in colorectal cancer cell lines and primary human colorectal cancer cells. Importantly, our work identifies Deptor as a downstream target of the Wnt/ß-catenin/c-Myc signaling pathway, acting as a tumor promoter in colorectal cancer cells. Moreover, we provide a molecular basis for the synergistic combination of Wnt and mTOR inhibitors in treating colorectal cancer with elevated c-Myc.Significance: The mTOR inhibitor DEPTOR acts as a tumor promoter and could be a potential therapeutic target in colorectal cancer. Cancer Res; 78(12); 3163-75. ©2018 AACR.

Curcumin inhibits neurotensin-mediated interleukin-8 production and migration of HCT116 human colon cancer cells.

PURPOSE: Neurotensin, a gut tridecapeptide, acts as a potent cellular mitogen for various colorectal and pancreatic cancers that possess high-affinity neurotensin receptors. Cytokine/chemokine proteins are increasingly recognized as important local factors that play a role in the metastasis and invasion of multiple cancers. The purpose of this study was to (a) determine the effect of neurotensin on cytokine/chemokine gene expression and cell migration in human cancer cells and (b) assess the effect of curcumin, a natural dietary product, on neurotensin-mediated processes. EXPERIMENTAL DESIGN: The human colorectal cancer, HCT116, was treated with neurotensin, with or without curcumin, and interleukin (IL)-8 expression and protein secretion was measured. Signaling pathways, which contribute to the effects of neurotensin, were assessed. Finally, the effect of curcumin on neurotensin-mediated HCT116 cell migration was analyzed. RESULTS: We show that neurotensin, acting through the native high-affinity neurotensin receptor, induced IL-8 expression in human colorectal cancer cells in a time- and dose-dependent fashion. This stimulation involves Ca2+-dependent protein kinase C, extracellular signal-regulated kinase-dependent activator protein-1, and extracellular signal-regulated kinase-independent nuclear factor-kappaB pathways. Curcumin inhibited neurotensin-mediated activator protein-1 and nuclear factor-kappaB activation and Ca2+ mobilization. Moreover, curcumin blocked neurotensin-stimulated IL-8 gene induction and protein secretion and, at a low concentration (i.e., 10 micromol/L), blocked neurotensin-stimulated colon cancer cell migration. CONCLUSIONS: Neurotensin-mediated induction of tumor cell IL-8 expression and secretion may contribute to the procarcinogenic effects of neurotensin on gastrointestinal cancers. Furthermore, a potential mechanism for the chemopreventive and chemotherapeutic effects of curcumin on colon cancers may be through the inhibition of gastrointestinal hormone (e.g., neurotensin)-induced chemokine expression and cell migration.

Ketogenesis contributes to intestinal cell differentiation.

The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body ß-hydroxybutyrate (ßHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21Waf1). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/ßHB signaling in intestinal cells. In contrast, treatment of intestinal cells with ßHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/ßHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/ßHB to maintain intestinal homeostasis.

Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF-kappaB-dependent pathway.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are cytoplasmic adapter proteins that link a wide variety of cell surface receptors to the apoptotic signaling cascade. The purpose of this study was to delineate the signaling pathways and TRAF1 promoter elements responsible for phorbol ester-mediated TRAF1 induction in human colon cancers. Here, we found that the PKC activators, phorbol 12-myristate 13-acetate (PMA) and bryostatin I, induced TRAF1 mRNA expression; pretreatment with actinomycin D blocked PMA-mediated TRAF1 expression suggesting induction at the transcriptional level. In contrast, expression of other TRAFs (TRAF2, 3 and 4) was minimally altered by PMA. Various PKC isoform-selective inhibitors blocked PMA-mediated TRAF1 mRNA and promoter stimulation; rottlerin, a selective PKCdelta inhibitor, had no effect suggesting that Ca(2+)-dependent PKC isoforms (e.g., PKCalpha and betaI) play a role in TRAF1 regulation. In addition, the MEK/ERK inhibitors, PD98059 and UO126, suppressed PMA-stimulated TRAF1 promoter activity indicating a role for ERK in TRAF1 induction. Moreover, cotransfection of a dominant-negative Raf-1 (Raf-C4) significantly reduced PMA-stimulated TRAF1 promoter activity whereas transfection of dominant-negative Ras or treatment with Ras inhibitors had minimal to no effect on TRAF1 induction suggesting dependence on Raf, but not Ras, activation. Finally, site-specific mutagenesis of functional NF-kappaB sites (particularly the most proximal site) in the TRAF1 promoter significantly decreased PMA-mediated promoter activity. In conclusion, our results demonstrate selective induction of TRAF1 in human colon cancer cells through a Ca(2+)-dependent PKC/Raf-1/ERK/NF-kappaB-dependent pathway.

Augmentation of sodium butyrate-induced apoptosis by phosphatidylinositol 3'-kinase inhibition in the KM20 human colon cancer cell line.

PURPOSE: We have recently shown that inhibition of the phosphatidylinositol3'-kinase (PI3k) pathway enhances sodium butyrate (NaBT)-mediated differentiation of human colon cancer cells. The purpose of this study was to determine whether PI3k inhibition can enhance the inhibitory effect of NaBT on an aggressive human colon cancer cell line, KM20. EXPERIMENTAL DESIGN: The KM20 cell line, established from a metastatic colon cancer, was treated in vitro with NaBT, gemcitabine, or 5-fluorouracil either alone or in combination with the PI3k inhibitors wortmannin or LY294002; DNA fragmentation and cell viability were measured. As further indicators of apoptosis, protein was extracted to determine caspase-9 and caspase-3 activation and cleavage of poly(ADP-ribose) polymerase. In addition, the effect of NaBT and wortmannin on in vivo KM20 tumor growth was determined. RESULTS: We demonstrate that inhibition of PI3k enhanced NaBT-mediated apoptosis and decreased KM20 cell viability; the nonspecific caspase inhibitor zVAD-fmk blocked the induction of apoptosis by the combination treatment. Either wortmannin or LY294002, combined with NaBT, enhanced activation of caspase-9 and caspase-3 and the subsequent cleavage of poly(ADP-ribose) polymerase. Furthermore, inhibition of PI3k increased the sensitivity of KM20 cells to gemcitabine and 5-fluorouracil. Wortmannin alone inhibited KM20 xenograft growth in vivo; the combination of wortmannin and NaBT demonstrated an enhanced effect compared with either agent alone. CONCLUSIONS: Our results are the first to show that inhibition of PI3k enhances NaBT-mediated colon cancer cell apoptosis through the activation of caspase-9 and caspase-3. Moreover, these findings suggest that agents that selectively target the PI3k pathway may enhance the effects of standard chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.