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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway.

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.

Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells.

The nuclear factor of activated T-cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation. We identified REDD1, a negative regulator of mammalian target of rapamycin (mTOR) through the tuberous sclerosis complex (TSC1/2 complex), as a new molecular target of NFATc3. We show that treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus ionophore A23187 (Io), which induces NFAT activation, increased REDD1 mRNA and protein expression and inhibited mTOR signaling; pretreatment with the calcineurin inhibitor cyclosporin A (CsA), an antagonist of NFAT signaling, decreased REDD1 induction and mTOR inhibition. Knockdown of NFATc3, not NFATc1, NFATc2, or NFATc4, attenuated PMA/Io-induced REDD1 expression. Treatment with PMA/Io increased REDD1 promoter activity and increased NFATc3 binding to the REDD1 promoter. Overexpression of NFATc3 increased REDD1 mRNA and protein expression and increased PMA/Io-mediated REDD1 promoter activity. Treatment with PMA/Io increased expression of the goblet cell differentiation marker MUC2; these changes were attenuated by pretreatment with CsA or knockdown of REDD1 or NFATc3. Overexpression of NFATc3 increased, while knockdown of TSC2 decreased, MUC2 expression. We provide evidence showing NFATc3 inhibits mTOR via induction of REDD1. Our results suggest a role for the NFATc3/REDD1/TSC2 axis in the regulation of intestinal cell differentiation.

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.

NFATc1 regulation of TRAIL expression in human intestinal cells.

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

Nuclear factor of activated T cells (NFAT) signaling regulates PTEN expression and intestinal cell differentiation.

The nuclear factor of activated T cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation and adaptation. Previously we demonstrated that inhibition of phosphatidylinositol 3-kinase or overexpression of PTEN enhanced intestinal cell differentiation. Here we show that treatment of intestinal-derived cells with the differentiating agent sodium butyrate (NaBT) increased PTEN expression, NFAT binding activity, and NFAT mRNA expression, whereas pretreatment with the NFAT signaling inhibitor cyclosporine A (CsA) blocked NaBT-mediated PTEN induction. Moreover, knockdown of NFATc1 or NFATc4, but not NFATc2 or NFATc3, attenuated NaBT-induced PTEN expression. Knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt and downstream targets Foxo1 and GSK-3α/ß. Furthermore, overexpression of NFATc1 or the NFATc4 active mutant increased PTEN and p27(kip1) expression and decreased Akt phosphorylation. In addition, pretreatment with CsA blocked NaBT-mediated induction of intestinal alkaline phosphatase (IAP) activity and villin and p27(kip1) expression; knockdown of either NFATc1 or NFATc4 attenuated NaBT-induced IAP activity. We provide evidence showing that NFATc1 and NFATc4 are regulators of PTEN expression. Importantly, our results suggest that NFATc1 and NFATc4 regulation of intestinal cell differentiation may be through PTEN regulation.

Suppression of neurotensin receptor type 1 expression and function by histone deacetylase inhibitors in human colorectal cancers.

Neurotensin, a gut peptide, stimulates the growth of colorectal cancers that possess the high-affinity neurotensin receptor (NTR1). Sodium butyrate (NaBT) is a potent histone deacetylase inhibitor (HDACi) that induces growth arrest, differentiation, and apoptosis of colorectal cancers. Previously, we had shown that NaBT increases nuclear GSK-3beta expression and kinase activity; GSK-3beta functions as a negative regulator of extracellular signal-regulated kinase (ERK) signaling. The purpose of our current study was to determine: (a) whether HDACi alters NTR1 expression and function, and (b) the role of GSK-3beta/ERK in NTR1 regulation. Human colorectal cancers with NTR1 were treated with various HDACi, and NTR1 expression and function were assessed. Treatment with HDACi dramatically decreased endogenous NTR1 mRNA, protein, and promoter activity. Overexpression of GSK-3beta decreased NTR1 promoter activity (> 30%); inhibition of GSK-3beta increased NTR1 expression in colorectal cancer cells, indicating that GSK-3beta is a negative regulator of ERK and NTR1. Consistent with our previous findings, HDACi significantly decreased phosphorylated ERK while increasing GSK-3beta. Selective MAP/ERK kinase/ERK inhibitors suppressed NTR1 mRNA expression in a time- and dose-dependent fashion, and reduced NTR1 promoter activity by approximately 70%. Finally, pretreatment with NaBT prevented neurotensin-mediated cyclooxygenase-2 and c-myc expression and attenuated neurotensin-induced interleukin-8 expression. HDACi suppresses endogenous NTR1 expression and function in colorectal cancer cell lines; this effect is mediated, at least in part, through the GSK-3beta/ERK pathway. The downregulation of NTR1 in colorectal cancers may represent an important mechanism for the anticancer effects of HDACi.

PTEN loss induces epithelial--mesenchymal transition in human colon cancer cells.

BACKGROUND: The epithelial-mesenchymal transition is a critical early event in the invasion and metastasis of many types of cancer, including colorectal cancer (CRC). Chronic inflammation is an inducer of several cancer types and inflammatory cytokines have been implicated in tumor invasion. MATERIALS AND METHODS: Human colon cancer cell lines HCT116 and SW480 were transfected with phosphatase and tensin homolog deleted on chromosome 10 (PTEN) siRNA or non-targeting control (NTC). Invasiveness was measured using a modified Boyden chamber assay and migration was assessed using a scratch assay. RESULTS: PTEN knockdown increased the invasion and migration of CRC cells and the addition of medium containing tumor necrosis factor-alpha (TNF-alpha) further enhanced the migration and invasion. PTEN knockdown resulted in nuclear beta-catenin accumulation and increased expression of downstream proteins c-Myc and cyclin D1. CONCLUSION: Our study supports the findings of clinical studies identifying an association of PTEN loss with late stage cancer. Cellular factors secreted from the surrounding tumor milieu likely act in concert with genetic changes in the tumor cells and contribute to enhanced tumor invasion.

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.