The CUG-translated WT1, not AUG-WT1, is an oncogene.

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

Herbacetin suppresses cutaneous squamous cell carcinoma and melanoma cell growth by targeting AKT and ODC.

Herbacetin is a flavonol compound that is found in plants such as flaxseed and ramose scouring rush herb, it possesses a strong antioxidant capacity, and exerts anticancer effects on colon and breast cancer. However, the effect of herbacetin on skin cancer has not been investigated. Herein, we identified herbacetin as a dual V-akt murine thymoma viral oncogene homolog (AKT) and ornithine decarboxylase (ODC) inhibitor, and illustrated its anticancer effects in vitro and in vivo against cutaneous squamous cell carcinoma (SCC) and melanoma cell growth. To identify the direct target(s) of herbacetin, we screened several skin cancer-related protein kinases, and results indicated that herbacetin strongly suppresses both AKT and ODC activity. Results of cell-based assays showed that herbacetin binds to both AKT and ODC, inhibits TPA-induced neoplastic transformation of JB6 mouse epidermal cells, and suppresses anchorage-independent growth of cutaneous SCC and melanoma cells. The inhibitory activity of herbacetin was associated with markedly reduced NF-κB and AP1 reporter activity. Interestingly, herbacetin effectively attenuated TPA-induced skin cancer development and also exhibited therapeutic effects against solar-UV-induced skin cancer and melanoma growth in vivo. Our findings indicate that herbacetin is a potent AKT and ODC inhibitor that should be useful for preventing skin cancers.

Benzyl Isothiocyanate Inhibits Prostate Cancer Development in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) Model, Which Is Associated with the Induction of Cell Cycle G1 Arrest.

Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker), cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC.

Genetic ablation of caspase-7 promotes solar-simulated light-induced mouse skin carcinogenesis: the involvement of keratin-17.

Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17.

3,6,2',4',5'-Pentahydroxyflavone, an orally bioavailable multiple protein kinase inhibitor, overcomes gefitinib resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most lethal cancer, causing more than 150,000 deaths in the United States in 2013. The receptor tyrosine kinase inhibitors such as gefitinib are not perfect clinical therapeutic agents for NSCLC treatment due to primary or acquired tyrosine kinase inhibitor resistance. Herein, 3,6,2',4',5'-pentahydroxyflavone (36245-PHF) was identified as a multiple kinase inhibitor for NSCLC treatment based on the computational screening of a natural products database. 36245-PHF was shown to inhibit PI3K and Aurora A and B kinases and overcome gefitinib-resistant NSCLC growth. Our data clearly showed that 36245-PHF markedly inhibited anchorage-independent growth of gefitinib-resistant NSCLC cell lines and exerted a substantial chemotherapeutic effect following oral administration in a gefitinib-resistant NSCLC xenograft model. The evidence from three different subsequent methodological approaches, in vitro, ex vivo, and in vivo, all confirmed that 36245-PHF as a multiple protein kinase inhibitor. Overall, we identified 36245-PHF as a multiple protein kinase inhibitor and as a novel therapeutic agent to overcome gefitinib-resistant NSCLC growth, which could provide a new option for clinical NSCLC oral treatment.

Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR.

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.

Src is a novel potential off-target of RXR agonists, 9-cis-UAB30 and Targretin, in human breast cancer cells.

9-cis-UAB30 (UAB30) and Targretin are well-known retinoid X receptor (RXR) agonists. They were highly effective in decreasing the incidence of methylnitrosourea (MNU)-induced mammary cancers. However, whether the anti-mammary cancer effects of UAB30 or Targretin originate from the activation of RXR is unclear. In the present study, we hypothesized that UAB30 and Targretin not only affect RXR, but likely influence one or more off-target proteins. Virtual screening results suggest that Src is a potential target for UAB30 and Targretin that regulates extracellular matrix (ECM) molecules and cell motility and invasiveness. In vitro kinase assay data revealed that UAB30 or Targretin interacted with Src and attenuated its kinase activity. We found that UAB30 or Targretin substantially inhibited invasiveness and migration of MCF-7 and SK-BR-3 human breast cancer cells. We examined the effects of UAB30 and Targretin on the expression of matrix metalloproteinases (MMP)-9, which are known to play an essential role in tumor invasion. We show that activity and expression of MMP-9 were decreased by UAB30 or Targretin. Western blot data showed that UAB30 or Targretin decreased AKT and its substrate molecule p70(s6k), which are downstream of Src in MCF-7 and SK-BR-3 cells. Moreover, knocking down the expression of Src effectively reduced the sensitivity of SK-BR-3 cells to the inhibitory effects of UAB30 and Targretin on invasiveness. Taken together, our results demonstrate that UAB30 and Targretin each inhibit invasion and migration by targeting Src in human breast cancer cells.

Butein, a novel dual inhibitor of MET and EGFR, overcomes gefitinib-resistant lung cancer growth.

Lung cancer is a leading cause of death worldwide and MET amplification is a major therapeutic limitation in acquired-resistance lung cancer. We hypothesized that butein, a phytochemical, can overcome gefitinib-induced resistance by targeting both EGFR and MET in non-small cell lung cancer (NSCLC). To investigate the ability of butein to target EGFR and MET, we used in silico docking, a library of natural compounds and kinase assays. The effects of butein on growth, induction of apoptosis and expression of EGFR/MET signaling targets were examined in HCC827 (gefitinib-sensitive) and HCC827GR (gefitinib-resistant) NSCLC cells. Results were confirmed in vivo by a HCC827 or HCC827GR cell xenograft mouse model, each treated with vehicle, butein or gefitinib. Butein inhibited phosphorylation and kinase activity of EGFR and MET as well as soft agar colony formation and decreased viability of HCC827 and HCC827GR cells. Butein increased apoptosis-related protein expression in these cells. Results were confirmed by co-treatment with inhibitors of EGFR/MET or double knock-down. Finally, xenograft study results showed that butein strongly suppressed HCC827 and HCC827GR tumor growth. Immunohistochemical data suggest that butein inhibited Ki-67 expression. These results indicate that butein has potent anticancer activity and targets both EGFR and MET in acquired-resistance NSCLC.

CInQ-03, a novel allosteric MEK inhibitor, suppresses cancer growth in vitro and in vivo.

The mitogen-activated protein kinase kinase 1 and 2 signaling pathway is a major component of the RAS (Rat sarcoma)/RAF (Radpidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERKs (Extracellular signal-regulated kinases) signaling axis that regulates tumorigenesis and cancer cell growth. MEK is frequently activated in various cancers that have mutations in the KRAS and BRAF oncogenes. Therefore, MEK has been suggested as a therapeutic target for inhibitor development against tumors that are dependent on the activating mutations in mitogen-activated protein kinase signaling. Herein, we report the discovery of three novel MEK inhibitors, herein referred to as CInQ-01, CInQ-03 and CInQ-06. All three inhibitors were highly effective in suppressing MEK1 and MEK2 in vitro kinase activity as well as anchorage-dependent and anchorage-independent cell growth. The inhibitory activity was associated with markedly reduced phosphorylation of ERKs and ribosomal S6 kinases. Furthermore, administration of CInQ-03 inhibited colon cancer cell growth in an in vivo xenograft mouse model and showed no skin toxicity. Overall, these results suggest that these novel MEK inhibitors might be used for chemotherapy or prevention.

Mechanisms by which licochalcone e exhibits potent anti-inflammatory properties: studies with phorbol ester-treated mouse skin and lipopolysaccharide-stimulated murine macrophages.

In this study we found that licochalcone E (LicE), a recently isolated retrochalcone from Glycyrrhiza inflata, exhibits potent anti-inflammatory effects in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema and lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage models. Topical application of LicE (0.5-2 mg) effectively inhibited TPA-induced (1) ear edema formation; (2) phosphorylation of stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK), c-Jun, and extracellular signal regulated kinase 1/2; and (3) expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins in mouse skin. The treatment of RAW 264.7 cells with LicE (2.5-7.5 µmol/L) induced a profound reduction in LPS-induced (1) release of NO and prostaglandin E2; (2) mRNA expression and secretion of interleukin (IL)-6, IL-1ß and tumor necrosis factor-α; (3) promoter activity of iNOS and COX-2 and expression of their corresponding mRNAs and proteins; (4) activation of AKT, p38 mitogen activated protein kinase (MAPK), SAPK/JNK and c-Jun; (5) phosphorylation of inhibitor of κB (IκB) kinase-αß and IκBα, degradation of IκBα, translocation of p65 (RelA) to the nucleus and transcriptional activity of nuclear factor (NF)-κB; and (6) transcriptional activity of activator protein (AP)-1. These results indicate that the LicE inhibition of NF-κB and AP-1 transcriptional activity through the inhibition of AKT and MAPK activation contributes to decreases in the expression of pro-inflammatory cytokines and the inducible enzymes iNOS and COX-2.

Identification of mammalian target of rapamycin as a direct target of fenretinide both in vitro and in vivo.

N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic retinoid that has been tested in clinical trials as a cancer therapeutic and chemopreventive agent. Although 4HPR has been shown to be cytotoxic to many kinds of cancer cells, the underlying molecular mechanisms are only partially understood. Until now, no direct cancer-related molecular target has been reported to be involved in the antitumor activities of 4HPR. Herein, we found that 4HPR inhibited mammalian target of rapamycin (mTOR) kinase activity by directly binding with mTOR, which suppressed the activities of both the mTORC1 and the mTORC2 complexes. The predicted binding mode of 4HPR with mTOR was based on a homology computer model, which showed that 4HPR could bind in the ATP-binding pocket of the mTOR protein through hydrogen bonds and hydrophobic interactions. In vitro studies also showed that 4HPR attenuated mTOR downstream signaling in a panel of non-small-cell lung cancer cells, resulting in growth inhibition. Moreover, knockdown of mTOR in cancer cells decreased their sensitivity to 4HPR. Results of an in vivo study demonstrated that i.p. injection of 4HPR in A549 lung tumor-bearing mice effectively suppressed cancer growth. The expression of mTOR downstream signaling molecules in tumor tissues was also decreased after 4HPR treatment. Taken together, our results are the first to identify mTOR as a direct antitumor target of 4HPR both in vitro and in vivo, providing a valuable rationale for guiding the clinical uses of 4HPR.

Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model.

INTRODUCTION: Tumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors. METHODS: 4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted. RESULTS: The co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone. CONCLUSION: The crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.

A high-fat diet increases angiogenesis, solid tumor growth, and lung metastasis of CT26 colon cancer cells in obesity-resistant BALB/c mice.

We evaluated whether high-fat diet (HFD), in the absence of increased calorie intake, increases colon cancer growth and metastasis. Four-week-old male BALB/c mice were fed on an HFD (60 kcal% fat) or control diet (10 kcal% fat) for 16 wk, after which CT26 colon cancer cells were subcutaneously injected into the right flank. Solid tumor growth and the number and volume of tumor nodules in the lung were increased markedly in the HFD group with only a slight increase in body weight (5.9%). HFD feeding increased tumor tissue levels of Ki67, cyclin A, cyclin D1, CDK2, Bcl-xL, and Bcl-2; reduced p53 levels and TUNEL-positive apoptotic cells; increased the levels of CD45, CD68, CD31, VEGF, P-VEGF receptor-2, iNOS, and COX-2 as well as hemoglobin content; and increased the levels of HIF-1α, P-STAT3-Y705, P-STAT3-S727, P-IκB-α, P-p65, p65, P-c-Jun, P-Akt, P-ERK1/2, P-p38, and P-SAPK/JNK. HFD feeding increased the serum levels of EGF, insulin, IGF-I, IFN-γ, leptin, RANTES, MCP-1, IL-1ra, and SDF-1α and media conditioned by epididymal fat tissue explants from HFD-fed mice caused an increase in microvessel outgrowth from the mouse aorta and tube formation of human umbilical vein endothelial cells. These results indicate that the chronic consumption of an HFD increases colon cancer cell proliferation, tumor angiogenesis, and lung metastasis in mice in the absence of discernible weight gain. HFD feeding increases the levels of growth factors which activate transcription factors, thereby inducing the expression of many genes involved in the stimulation of inflammation, angiogenesis, and cellular proliferation.

Luteolin decreases IGF-II production and downregulates insulin-like growth factor-I receptor signaling in HT-29 human colon cancer cells.

BACKGROUND: Luteolin is a 3',4',5,7-tetrahydroxyflavone found in various fruits and vegetables. We have shown previously that luteolin reduces HT-29 cell growth by inducing apoptosis and cell cycle arrest. The objective of this study was to examine whether luteolin downregulates the insulin-like growth factor-I receptor (IGF-IR) signaling pathway in HT-29 cells. METHODS: In order to assess the effects of luteolin and/or IGF-I on the IGF-IR signaling pathway, cells were cultured with or without 60 µmol/L luteolin and/or 10 nmol/L IGF-I. Cell proliferation, DNA synthesis, and IGF-IR mRNA levels were evaluated by a cell viability assay, [3H]thymidine incorporation assays, and real-time polymerase chain reaction, respectively. Western blot analyses, immunoprecipitation, and in vitro kinase assays were conducted to evaluate the secretion of IGF-II, the protein expression and activation of IGF-IR, and the association of the p85 subunit of phophatidylinositol-3 kinase (PI3K) with IGF-IR, the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)1/2, and cell division cycle 25c (CDC25c), and PI3K activity. RESULTS: Luteolin (0 - 60 µmol/L) dose-dependently reduced the IGF-II secretion of HT-29 cells. IGF-I stimulated HT-29 cell growth but did not abrogate luteolin-induced growth inhibition. Luteolin reduced the levels of the IGF-IR precursor protein and IGF-IR transcripts. Luteolin reduced the IGF-I-induced tyrosine phosphorylation of IGF-IR and the association of p85 with IGF-IR. Additionally, luteolin inhibited the activity of PI3K activity as well as the phosphorylation of Akt, ERK1/2, and CDC25c in the presence and absence of IGF-I stimulation. CONCLUSIONS: The present results demonstrate that luteolin downregulates the activation of the PI3K/Akt and ERK1/2 pathways via a reduction in IGF-IR signaling in HT-29 cells; this may be one of the mechanisms responsible for the observed luteolin-induced apoptosis and cell cycle arrest.

Multifragmentary patellar fracture has a distinct fracture pattern which makes coronal split, inferior pole, or satellite fragments.

The present study aimed to map the location and frequency of fracture lines on the coronal articular and sagittal planes in multifragmentary patellar fractures. 66 multifragmentary patellar fractures were digitally reconstructed using the 3D CT mapping technique. The coronal articular surface and midsagittal fracture maps were produced by superimposing each case over a single template. Each fracture line was classified based on the initial displacement and orientation. We evaluated the frequency and direction of the fracture line, coronal split fragment area, and satellite and inferior pole fragment presence. Coronal articular surface fracture mapping identified primary horizontal fracture lines between the middle and inferior one-third of the articular surface in 63 patients (95.4%). Secondary horizontal fracture lines running on the inferior border of the articular facet were confirmed (83.3%). Secondary vertical fracture lines creating satellite fragments were mostly located on the periphery of the bilateral facet. Midsagittal fracture mapping of primary and secondary horizontal fracture lines with the main coronal fracture line revealed a predominantly X-shaped fracture map. The consequent coronal split fragment and inferior pole fracture were combined in most cases. In conclusion, the multifragmentary patellar fracture has a distinct pattern which makes coronal split, inferior pole, or satellite fragments.

p21 produces a bioactive secretome that places stressed cells under immunosurveillance.

Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates retinoblastoma protein (Rb)­dependent transcription at select gene promoters to generate a complex bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21 within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently induces proliferative arrest and immunosurveillance of cells under duress.

Characteristics of veterinary antibiotics in intensive livestock farming watersheds with different liquid manure application programs using UHPLC-q-orbitrap HRMS combined with on-line SPE.

Composted livestock manures, in both solid and liquid form, are used as fertilizers in cropland. However, excess solid and liquid manures in agricultural watersheds are considered as nonpoint pollution sources because of their high nutrient and heavy metal contents of, as well as their antibiotic contents, especially veterinary antibiotics (VAs). In this study, 21 VAs under nine classes (i.e., cephems, ionophores, lincosamides, penicillins, pleuromutilins, quinolones, streptogramins, sulfonamides, and tetracyclines) found in agricultural watersheds were simultaneously analyzed via UHPLC-q-orbitrap high-resolution mass spectrometry using an on-line solid-phase extraction system. The residues of VAs in the surface water of two intensive livestock rearing watersheds (Cheongmi and Gwangcheon streams) in Korea were successfully quantified, and the values were found to range from 1.84 ± 0.42 ng L-1 to 835.6 ± 31.9 ng L-1. Time lags of 2-3 months were observed between the periods of liquid manure application and the periods with the maximum concentrations of VAs. In both watersheds, samples from points close to areas with extensive application of liquid manure exhibited high concentrations of most of the 21 VAs. Between the watersheds, the one with heavier application of liquid manure showed higher concentrations of the target VAs. To the best of our knowledge, this study represents the first attempt at evaluating the correlation between liquid manure application and environmental occurrence of VAs in surface water. The findings reveal that liquid manure application plays an important role in introducing VAs into aquatic environments.

ARC Is a Critical Protector against Inflammatory Bowel Disease (IBD) and IBD-Associated Colorectal Tumorigenesis.

The key functional molecules involved in inflammatory bowel disease (IBD) and IBD-induced colorectal tumorigenesis remain unclear. In this study, we found that the apoptosis repressor with caspase recruitment domain (ARC) protein plays critical roles in IBD. ARC-deficient mice exhibited substantially higher susceptibility to dextran sulfate sodium (DSS)-induced IBD compared with wild-type mice. The inflammatory burden induced in ARC-deficient conditions was inversely correlated with CCL5 and CXCL5 levels in immune cells, especially CD4-positive T cells. Pathologically, ARC expression in immune cells was significantly decreased in clinical biopsy specimens from patients with IBD compared with normal subjects. In addition, ARC levels inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens. ARC interacted with TNF receptor associated factor (TRAF) 6, regulating ubiquitination of TRAF6, which was associated with NF-κB signaling. Importantly, we identified a novel ubiquitination site at lysine 461, which was critical in the function of ARC in IBD. ARC played a critical role in IBD and IBD-associated colon cancer in a bone marrow transplantation model and azoxymethane/DSS-induced colitis cancer mouse models. Overall, these findings reveal that ARC is critically involved in the maintenance of intestinal homeostasis and protection against IBD through its ubiquitination of TRAF6 and subsequent modulation of NF-κB activation in T cells. SIGNIFICANCE: This study uncovers a crucial role of ARC in the immune system and IBD, giving rise to a novel strategy for IBD and IBD-associated colon cancer therapy.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

BACKGROUND: 3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. METHODS: HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. RESULTS: The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. CONCLUSION: Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation.

Inflammation is a complex biological host reaction to tissue damage, infection and trauma. Extensive study of the inflammatory response has led to the identification of several protein kinases that are essential for signaling and could be potential therapeutic targets. The RSK family of kinases has multiple cellular functions. In our study, we found that RSK2 is a mediator for inflammation signaling and interacts with TRAF6. In vitro kinase assay results indicated that RSK2 strongly phosphorylates TRAF6 at serines 46, 47 and 48. Ectopic overexpression of TRAF6 or knocking down RSK2 expression confirmed that RSK2 is a positive regulator of TRAF6 K63 ubiquitination. TRAF6 is also required for RSK2 ubiquitination. TRAF6 bridges the TNF receptor superfamily and intracellular signaling for the induction of proinflammatory cytokines. We developed a colon inflammation model using RSK2 wild type (WT) and knockout (KO) mice. As expected, F4/80 and CD3 infiltration were significantly upregulated in WT mice compared to RSK2 KO mice. Furthermore, inflammation signaling, including Ikkα/ß, p38 and JNKs, was dramatically upregulated in WT mice. Colon tissue immunoprecipitation results further confirmed that TRAF6 K63 ubiquitination was lower in RSK2 KO mice. Overall, these results indicate that phosphorylation of TRAF6 (S46, 47, 48) by RSK2 is required for TRAF6 K63 ubiquitination and inflammation signaling.