Combined impacts of microplastics and cadmium on the liver function, immune response, and intestinal microbiota of crucian carp (Carassius carassius).

Microplastics (MPs) and the heavy metal cadmium (Cd) have attracted global attention for their toxicological interactions in aquatic organisms. The purpose of this investigation was evaluating the effect of MPs (1 mg L-1) and Cd (5 mg L-1) on the liver function, immune response of crucian carp (Carassius carassius) after 96 h exposure, and intestinal microbiota after 21 days, respectively. Co-exposure to MPs and Cd significantly enhanced MP accumulation in the liver of the crucian carp compared to the accumulation with exposure to MPs alone. Co-exposure to MPs and Cd triggered notable histopathological alterations accompanied by increased hepatic cell necrosis and inflammation, and was associated with higher aspartate aminotransferase and alanine aminotransferase levels, lower superoxide dismutase and catalase activity levels, but higher malondialdehyde content and total antioxidant capacity in the liver. Moreover, the combined treatment of MPs and Cd led to the up-regulated transcription of genes related to immune response, such as interleukin 8 (il-8), il-10, il-1ß, tumor necrosis factor-α, and heat shock protein 70, both in the liver and spleen. Co-exposure to MPs and Cd reduced the variety and abundance of the intestinal microbiota in the crucian carp. Our research indicates that the combined exposure to MPs and Cd may exert synergistic toxic effects on crucian carp, which could impede the sustainable growth of the aquaculture industry and pose potential risks to food safety.

Ningmitai capsule promotes calculi expulsion after RIRS for 10-20-mm upper urinary stones: a multicenter, prospective, randomized controlled trial.

To evaluate the efficacy and safety of the use of Ningmitai capsule as an adjunctive stone expulsion therapy after RIRS. All patients were diagnosed with upper urinary tract calculi measuring 10-20 mm. The patients who successfully underwent RIRS were randomly assigned to the NMT capsule group (Ningmitai capsule, 1.52 g, three times daily) or the control group for 4 weeks based on the random number table method. The primary endpoints were the stone expulsion rate (SER) and stone-free rate (SFR). The average stone expulsion time (SET), average stone-free time (SFT) and complications were recorded. Between July 2, 2019, and December 17, 2020, 220 participants successfully underwent RIRS across 6 centers; 123 of them were randomized according to the exclusion criteria, and 102 (83%) were included in the primary analysis. The SERs on the 3rd, 7th, 14th and 28th days were significantly increased in the NMT capsule group compared with the control group (78.95% vs. 31.11%, 92.98% vs. 55.56%, 94.74% vs. 64.44%, 100% vs. 82.22%, respectively, p < 0.05). The SFRs on the 3rd and 7th days were not different (p > 0.05), while those on the 14th and 28th days were higher in the NMT capsule group (63.16% vs. 24.44% and 92.98% vs. 68.89%, p < 0.05). The average SET and average SFT of the NMT capsule group were remarkably shorter than those of the control group (p < 0.001). During the follow-up period, there were no significant differences in urine RBC counts between the two groups (p > 0.05). The urine WBC counts of the NMT capsule group were significantly lower than those of the control group on the 14th day (p = 0.011), but there was no difference on the 3rd, 7th or 28th day (p > 0.05). The analgesic aggregate of the NMT capsule group was also much lower (p = 0.037). There were no significant differences in adverse events (p > 0.05), and they improved significantly without sequelae. This study indicated that NMT capsules can significantly promote stone clearance and are more effective and safer for upper urinary calculi after RIRS.Trial registration Chinese Clinical Trial Registration No. ChiCTR1900024151.Date of registration June 28, 2019.

Identification of evodiamine as a suppressor of prostate cancer progression by reducing AR transcriptional activity via targeting Src.

Evodiamine (EVO) is a bioactive alkaloid that exerts antitumor activity in various cancers, including prostate cancer (PCa). In this paper, we further investigated the molecular mechanisms underlying the anti-PCa effect of evodiamine. In the present study, cell proliferation, colony formation, migration, and invasion were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and transwell assays, respectively. Animal studies were used to evaluate the effect of evodiamine on the tumorigenicity of LNCaP cells in vivo. The expression levels of steroid receptor coactivator (Src), androgene receptor (AR), and prostate-specific antigen (PSA) were detected by western blot, quantitative real-time PCR (qRT-PCR) or ELISA assay. Association between Src and AR was examined by Co-Immunoprecipitation (CoIP). The impact of evodiamine on AR-mediated transcriptional activity was confirmed by dual-luciferase reporter assay. The results showed that evodiamine reduced LNCaP and 22Rv1 cell proliferation, colony formation, migration, and invasion induced by dihydrotestosterone (DHT) in vitro, as well as diminished tumor growth in vivo. Mechanistically, evodiamine directly targeted Src and reduced DHT-induced Src activation. Moreover, the restoration of Src activation abolished evodiamine-mediated suppression of proliferation, migration, and invasion of DHT-treated LNCaP and 22Rv1 cells. Furthermore, evodiamine inhibited DHT-induced AR transcriptional activity through targeting Src. As a conclusion, our findings demonstrate the antitumor property of evodiamine in PCa by blocking AR transcriptional activity through targeting Src and provide a rationale for developing evodiamine as a promising antitumor agent against PCa.

Accurate Detection and Evaluation of the Gene-Editing Frequency in Plants Using Droplet Digital PCR.

Gene-editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been reported that detect mutations at targeted loci induced by the CRISPR/Cas system in different organisms, they are semiquantitative and have difficulty in the detection of mutants in processed food samples containing low initial concentrations of DNA and may not accurately quantify editing frequency, especially at very low frequencies in a complex polyploid plant genome. In this study, we developed a duplexed dPCR-based method for the detection and evaluation of gene-editing frequencies in plants. We described the design, performance, accurate quantification, and comparison with other detection systems. The results show that the dPCR-based method is sensitive to different kinds of gene-editing mutations induced by gene-editing. Moreover, the method is applicable to polyploid plants and processed food samples containing low initial concentrations of DNA. Compared with qPCR and NGS-based methods, the dPCR method has a lower limit of detection (LOD) of the editing frequency and a better relationship with the expected editing frequency in detecting the edited region of gene-edited rice samples. Taken together, the duplexed dPCR assay is accurate and precise, and it will be a powerful tool for the detection and evaluation of gene-editing frequencies in plants in gene-editing technology.

Burn-Induced Impairment of Ileal Muscle Contractility Is Associated with Increased Extracellular Matrix Components.

INTRODUCTION: Severe burns lead to marked impairment of gastrointestinal motility, such as delayed gastric emptying and small and large intestinal ileus. However, the cellular mechanism of these pathologic changes remains largely unknown. METHODS: Male Sprague Dawley rats approximately 3 months old and weighing 300-350 g were randomized to either a 60% total body surface area full-thickness scald burn or sham procedure and were sacrificed 24 h after the procedure. Gastric emptying, gastric antrum contractility ileal smooth muscle contractility, and colonic contractility were measured. Muscularis externa was isolated from the ileal segment to prepare smooth muscle protein extracts for Western blot analysis. RESULTS: Compared with sham controls, the baseline rhythmic contractile activities of the antral, ileal, and colonic smooth muscle strips were impaired in the burned rats. Simultaneously, our data showed that ileal muscularis ECM proteins fibronectin and laminin were significantly up-regulated in burned rats compared with sham rats. TGF-ß signaling is an important stimulating factor for ECM protein expression. Our results revealed that TGF-ß signaling was activated in the ileal muscle of burned rats evidenced by the activation of Smad2/3 expression and phosphorylation. In addition, the total and phosphorylated AKT, which is an important downstream factor of ECM signaling in smooth muscle cells, was also up-regulated in burned rats' ileal muscle. Notably, these changes were not seen in the colonic or gastric tissues. CONCLUSION: Deposition of fibrosis-related proteins after severe burn is contributors to decreased small intestinal motility.

Weighted gene co­expression network analysis for identifying hub genes in association with prognosis in Wilms tumor.

Wilms tumor (WT) is the most common type of renal malignancy in children. Survival rates are low and high­risk WT generally still carries a poor prognosis. To better elucidate the pathogenesis and tumorigenic pathways of high­risk WT, the present study presents an integrated analysis of RNA expression profiles of high­risk WT to identify predictive molecular biomarkers, for the improvement of therapeutic decision­making. mRNA sequence data from high­risk WT and adjacent normal samples were downloaded from The Cancer Genome Atlas to screen for differentially expressed genes (DEGs) using R software. From 132 Wilms tumor samples and six normal samples, 2,089 downregulated and 941 upregulated DEGs were identified. In order to identify hub DEGs that regulate target genes, weighted gene co­expression network analysis (WGCNA) was used to identify 11 free­scale gene co­expressed clusters. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were annotated using KEGG Orthology Based Annotation System annotation of different module genes. The Search Tool for the Retrieval of Interacting Genes was used to construct a protein­protein interaction network for the identified DEGs, and the hub genes of WGCNA modules were identified using the Cytohubb plugin with Cytoscape software. Survival analysis was subsequently performed to highlight hub genes with a clinical signature. The present results suggest that epidermal growth factor, cyclin dependent kinase 1, endothelin receptor type A, nerve growth factor receptor, opa­interacting protein 5, NDC80 kinetochore complex component and cell division cycle associated 8 are essential to high­risk WT pathogenesis, and they are closely associated with clinical prognosis.

Luteolin-Mediated Inhibition of Hepatic Stellate Cell Activation via Suppression of the STAT3 Pathway.

Hepatic stellate cell (HSC) activation is responsible for hepatic fibrogenesis and is associated with an overexpression of transcription 3 (STAT3). Luteolin, a common dietary flavonoid with potent anti-inflammatory properties, has previously demonstrated antifibrogenic properties in HSCs but the mechanism has not been fully elucidated. Activated human and rat hepatic stellate cell lines LX-2 and HSC-T6 were used to study the effects of luteolin on HSCs. Cellular proteins were determined by western blot and immunofluorescence. Cell proliferation was assessed with Alamar Blue assay. Luteolin significantly decreased LX-2 and HSC-T6 cell viability in a time-and-dose-dependent manner, as well as decreased HSC end-products α-smooth muscle actin (α-SMA), collagen I, and fibronectin. Luteolin decreased levels of total and phosphorylated STAT3, suppressed STAT3 nuclear translocation and transcriptional activity, and attenuated expression of STAT3-regulated proteins c-myc and cyclin D1. STAT3 specific inhibitors stattic and SH-4-54 demonstrated similar effects on HSC viability and α-SMA production. In LX-2 and HSC-T6 cells, luteolin demonstrates a potent ability to inhibit hepatic fibrogenesis via suppression of the STAT3 pathway. These results further elucidate the mechanism of luteolin as well as the effect of the STAT3 pathway on HSC activation.

Burn Trauma Acutely Increases the Respiratory Capacity and Function of Liver Mitochondria.

BACKGROUND: A complete understanding of the role of the liver in burn-induced hypermetabolism is lacking. We investigated the acute effect of severe burn trauma on liver mitochondrial respiratory capacity and coupling control as well as the signaling events underlying these alterations. METHODS: Male BALB/c mice (8-12 weeks) received full-thickness scald burns on ∼30% of the body surface. Liver tissue was harvested 24 h postinjury. Mitochondrial respiration was determined by high-resolution respirometry. Citrate synthase activity was determined as a proxy of mitochondrial density. Male Sprague-Dawley rats received full-thickness scald burns to ∼60% of the body surface. Serum was collected 24 h postinjury. HepG2 cells were cultured with serum-enriched media from either sham- or burn-treated rats. Protein levels were analyzed via western blot. RESULTS: Mass-specific (P = 0.01) and mitochondrial-specific (P = 0.01) respiration coupled to ATP production significantly increased in the liver after burn. The respiratory control ratio for ADP (P = 0.04) and the mitochondrial flux control ratio (P = 0.03) were elevated in the liver of burned animals. Complex III and Complex IV protein abundance in the liver increased after burn by 17% and 14%, respectively. Exposure of HepG2 cells to serum from burned rats increased the pAMPKα:AMPKα ratio (P < 0.001) and levels of SIRT1 (P = 0.01), Nrf2 (P < 0.001), and PGC1α (P = 0.02). CONCLUSIONS: Severe burn trauma augments respiratory capacity and function of liver mitochondria, adaptations that augment ATP production. This response may be mediated by systemic factors that activate signaling proteins responsible for regulating cellular energy metabolism and mitochondrial biogenesis.

An integrated analysis for long noncoding RNAs and microRNAs with the mediated competing endogenous RNA network in papillary renal cell carcinoma.

Papillary renal cell carcinoma (PRCC) is the second most common subtype of renal cell carcinoma, and it lacks effective therapeutic targets and prognostic molecular biomarkers. Attention has been increasingly focused on long noncoding RNAs (lncRNAs), which can act as competing endogenous RNA (ceRNA) to compete for shared microRNAs (miRNAs) in the tumorigenesis of human tumors. Therefore, to clarify the functional roles of lncRNAs with respect to the mediated ceRNA network in PRCC, we comprehensively integrated expression profiles, including data on mRNAs, lncRNAs and miRNAs obtained from 289 PRCC tissues and 32 normal tissues in The Cancer Genome Atlas. As a result, we identified 2,197 differentially expressed mRNAs (DEmRNAs) and 84 differentially expressed miRNAs (DEmiRNAs) using a threshold of |log2 (fold change)| >2.0 and an adjusted P-value <0.05. To determine the hub DEmRNAs that could be key target genes, a weighted gene co-expression network analysis was performed. A total of 28 hub DEmRNAs were identified as potential target genes. Seven dysregulated DEmiRNAs were identified that were significantly associated with the 28 hub potential target genes. In addition, we found that 16 differentially expressed lncRNAs were able to interact with the DEmiRNAs. Finally, we used Cytoscape software to visualize the ceRNA network with these differently expressed molecules. From these results, we believe that the identified ceRNA network plays a crucial role in the process of PRCC deterioration, and some of the identified genes are strongly related to clinical prognosis.

Involvement of erbB4 and tumor marker genes in renal carcinoma regulatory network.

BACKGROUND: Renal carcinoma is a common urologic tumor, and there is no ideal tumor marker for clinical diagnosis except for imaging diagnosis. This study aims to screen the serum tumor markers closely related with the benign and malignant of renal carcinoma out and chart out the regulatory network that involves renal carcinoma-related genes. METHODS: Based on 96 pathologically diagnosed renal cancer patients, factors strongly linked to renal carcinoma character were selected using Fisher discriminant analysis. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were utilized to manipulate function annotation of erbB4 and the selected genes and pathway analysis. RESULTS: Four essential tumor markers CYFRA21-1, CA125, VHL and HIF-1ß were successfully screened out. Using GO and KEGG databases, the regulatory network of renal cancer cell escaping apoptosis was charted out on the basis of erbB4 signaling pathway. CONCLUSION: Serum tumor marker genes play a certain part in the genesis and development of renal carcinoma. We preliminarily illustrated the molecular mechanism of these markers to predict tumor, laying a foundation for further exploration in renal carcinoma.

Transformation of primary human hepatocytes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. Currently, there is limited knowledge of neoplastic transformation of hepatocytes in HCC. In clinical practice, the high rate of HCC local recurrence suggests the presence of different hepatocyte populations within the liver and particularly in the tumor proximity. The present study investigated primary human hepatocyte cultures obtained from liver specimens of patients affected by cirrhosis and HCC, their proliferation and transformation. Liver samples were obtained from seven HCC cirrhotic patients and from three patients with normal liver (NL). Immediately after surgery, cell outgrowth and primary cultures were obtained from the HCC lesion, the cirrhotic tissue proximal (CP, 1-3 cm) and distal (CD, >5 cm) to the margin of the neoplastic lesion, or from NL. Cells were kept in culture for 16 weeks. Morphologic analyses were performed and proliferation rate of the different cell populations compared over time. Glypican-3, Heppar1, Arginase1 and CD-44 positivity were tested. The degree of invasiveness of cells acquiring neoplastic characteristics was studied with a transwell migration assay. We observed that HCC cells maintained their morphology and unmodified neoplastic characteristics when cultured. Cells isolated from CP, showed a progressive morphologic transformation in HCC-like cells accompanied by modification of markers expression with signs of invasiveness. Absence of HCC contamination in the CP isolates was confirmed. In CD samples some of these characteristics were present and at significantly lower levels. With the present study, we are the first to have identified and describe the existence of human hepatocytes near the cancerous lesion that can transform in HCC in vitro.

Enhanced anti-fibrogenic effects of novel oridonin derivative CYD0692 in hepatic stellate cells.

Oridonin, isolated from Rabdosia rubescens, has been proven to possess various anti-neoplastic and anti-inflammatory properties. Previously, we reported the anti-fibrogenic effects of oridonin for liver in vitro. In the present study, we investigated the effects of a newly designed analog CYD0692 in vitro. Cell viability was measured by Alamar Blue assay. Cell apoptosis was assessed by Cell Death ELISA and Yo-Pro-1 staining. Western blots were performed for cellular proteins. Flow cytometry was used to measure cell cycle regulation. CYD0692 significantly inhibited LX-2 cells proliferation in a dose- and time-dependent manner with an IC50 value of ~0.7 µM for 48 h, ~tenfold greater potency than oridonin. Similar results were observed in HSC-T6 cells. In contrast, on the human hepatocyte cell line C3A, only 12 % of the cell growth was inhibited with 5 µM of CYD0692 treatment for 48 h, while 30 % inhibited at 10 µM. After CYD0692 treatment on LX-2 cells, apoptosis and S-phase cell cycle arrest were induced; cleaved-PARP, p21, and p53 were activated while cyclin-B1 levels declined. In addition, α-smooth muscle actin, type I Collagen, and fibronectin (FN) were markedly down regulated. Transforming growth factor ß1 (TGF ß1) has been identified as a dominant stimulator for ECM production in HSC. Our results indicated that pretreatment with CYD0692 blocked TGF ß1-induced FN expression, thereby decreasing the downstream factors of TGF ß1 signaling, such as Phospho-Smad2/3 and phospho-ERK. In comparison with oridonin, its novel derivative CYD0692 has demonstrated to be a more potent and potentially safer anti-fibrogenic agent for the treatment of hepatic fibrosis.

Oridonin inhibits hepatic stellate cell proliferation and fibrogenesis.

BACKGROUND: Liver fibrosis is a common response to liver injury and, in severe cases, leads to cirrhosis. The hepatic stellate cells (HSCs) become activated after liver injury and play a significant role in fibrogenesis. The activated HSC is characterized by increased proliferation, overexpression of α smooth muscle actin, and excessive production of extracellular matrix (ECM) proteins. Oridonin, a naturally occurring diterpenoid, has been shown to induce apoptosis in liver and gastric cancer cells. However, its effects on the HSC are unknown. METHODS: We tested the effects of oridonin on the activated human and rat HSC lines LX-2 and HSC-T6, and the human hepatocyte cell line C3A. Transforming growth factor ß1 (TGF-ß1) was used to stimulate LX-2 cells. RESULTS: Oridonin significantly inhibited LX-2 and HSC-T6 proliferation. In contrast, oridonin had no antiproliferative effect on C3A cells at our tested range. Oridonin induced apoptosis and S-phase arrest in LX-2 cells. These findings were associated with an increase in p53, p21, p16, and cleaved Poly (ADP-ribose) Polymerase (PARP), and with a decrease in Cyclin-dependent kinase 4 (Cdk4). Oridonin markedly decreased expression of α smooth muscle actin and ECM protein type I collagen and fibronectin, blocked TGF-ß1-induced Smad2/3 phosphorylation and type I collagen expression. CONCLUSIONS: Oridonin induces apoptosis and cell cycle arrest involving the p53-p21 pathway in HSC and appears to be nontoxic to hepatocytes. In addition, oridonin suppressed endogenous and TGF-ß1-induced ECM proteins. Thus, oridonin may act as a novel agent to prevent hepatic fibrosis.

Raf-1 kinase inhibitory protein (RKIP) mediates ethanol-induced sensitization of secretagogue signaling in pancreatic acinar cells.

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca(2+) homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca(2+) signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca(2+) signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca(2+) signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.

mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON.

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.

Characterization of promoter elements regulating the expression of the human neurotensin/neuromedin N gene.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels of constitutive expression of the NT/N gene. In this study, we demonstrated multiple cis-regulatory elements including a proximal region containing a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein (CREB)/ATF proteins (c-Jun, ATF-1, ATF-2, JunD, and CREB). Similar to the rat NT/N gene, this region is critical for constitutive hNT/N gene expression. Moreover, we identified a novel region that binds the orphan hormone receptor, NR2F2. We have demonstrated that the C terminus of NR2F2 strongly represses hNT/N transcription, whereas an N-terminal domain antagonizes this repressive effect. Regulation of NT/N expression by NR2F2 may have important consequences for lipid metabolism. We speculate that a complex interplay between the proximal CRE/AP-1-like motif and NR2F2 binding region exists to regulate hNT/N expression, which is critical for the high level of constitutive expression of NT/N in enteroendocrine cells. Finally, the BON cell line provides a unique model to characterize the factors regulating expression of the hNT/N gene and to better understand the mechanisms responsible for terminal differentiation of the N cell lineage in the gut.

Triptolide inhibits proliferation and migration of colon cancer cells by inhibition of cell cycle regulators and cytokine receptors.

BACKGROUND: Phytochemicals are an important source of emerging preventive and therapeutic agents for cancer. Triptolide/PG490, an extract of the Chinese herb Tripterygium wilfordii Hook F, is a potent anti-inflammatory agent that also possesses anticancer activity. While its antiproliferative effects are well-established, the potential antimigratory effects of triptolide have not been characterized. MATERIAL AND METHODS: Effects of triptolide on the proliferation and invasion of colon cancer cells and expression of cancer-related genes and proteins were assessed. RESULTS: Triptolide potently inhibited HT29 and HCT116 colon cancer cell growth and reduced basal and stimulated HCT116 migration through collagen by 65% to 80%. Triptolide inhibited mRNA expression of the positive cell cycle regulatory genes c-myc, and A, B, C, and D-type cyclins in multiple colon cancer cell lines. Additionally, we show that triptolide treatment decreased expression of VEGF and COX-2, which promote cancer progression and invasion, and inhibited the expression of multiple cytokine receptors potentially involved in cell migration and cancer metastasis, including the thrombin receptor, CXCR4, TNF receptors, and TGF-ß receptors. CONCLUSIONS: Triptolide is a potent inhibitor of colon cancer proliferation and migration in vitro. The down-regulation of multiple cytokine receptors, in combination with inhibition of COX-2 and VEGF and positive cell cycle regulators, may contribute to the antimetastatic action of this herbal extract.

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.

Curcumin inhibits proliferation of colorectal carcinoma by modulating Akt/mTOR signaling.

BACKGROUND: Curcumin, a natural polyphenol product of the plant Curcuma longa, has been shown to inhibit the growth and progression of colorectal cancer; however, the anticancer mechanism of curcumin remains to be elucidated. MATERIALS AND METHODS: Colorectal cancer cells were treated with curcumin and changes in proliferation, protein and mRNA levels were analyzed. RESULTS: Curcumin inhibited proliferation of colorectal cancer cells. This effect was mediated by inhibition of mammalian target of rapamycin (mTOR) signaling as evidenced by decreased phosphorylation of downstream effectors of mTOR complex 1 (mTORC1), p70S6K and 4E-BP1. Curcumin decreased total expression of mTOR, Raptor and Rictor protein and mRNA levels. Surprisingly, curcumin induced phosphorylation of Akt(Ser 473); this effect may be attributed to a decrease in levels of the PHLPP1 phosphatase, an inhibitor of Akt. CONCLUSION: Our data suggest that curcumin, a natural compound, may exert its antiproliferative effects by inhibition of mTOR signaling and thus may represent a novel class of mTOR inhibitor.

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.