PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (1179672)

Clipboard (0)
None

Related Articles

1.  Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis 
PLoS Medicine  2008;5(3):e54.
Background
MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.
Methods and Findings
By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1β, TNF-α, and IFN-γ was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-γ, TNF-α, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.
Conclusions
Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
Michael McGuckin and colleagues identify two mutations that cause aberrant mucin oligomerization in mice. The resulting phenotype, including endoplasmic reticulum stress, resembles clinical and pathologic features of human ulcerative colitis.
Editors' Summary
Background.
Inflammatory bowel diseases (IBD) are common disorders in which parts of the digestive tract become inflamed. The two main types of IBD are Crohn's disease, which mainly affects the small bowel, and ulcerative colitis (UC), which mainly affects the large bowel (colon). Both types tend to run in families and usually develop between 15 and 35 years old. Their symptoms include diarrhea, abdominal cramps, and unintentional weight loss. These symptoms can vary in severity, can be chronic (persistent) or intermittent, and may start gradually or suddenly. There is no cure for IBD (except removal of the affected part of the digestive tract), but drugs that modulate the immune system (for example, corticosteroids) or that inhibit “proinflammatory cytokines” (proteins made by the immune system that stimulate inflammation) can sometimes help.
Why Was This Study Done?
Although the clinical and pathological (disease-associated) features of Crohn's disease and UC are somewhat different, both disorders are probably caused by an immune system imbalance. Normally, the immune system protects the body from potentially harmful microbes in the gut but does not react to the many harmless bacteria that live there or to the food that passes along the digestive tract. In IBD, the immune system becomes overactive for unknown reasons, and lymphocytes (immune system cells) accumulate in the lining of the bowel and cause inflammation. In this study, the researchers use a technique called random mutagenesis (the random introduction of small changes, called mutations, into the genes of an organism using a chemical that damages DNA) to develop two mouse models that resemble human UC and that throw new light on to how this disorder develops.
What Did the Researchers Do and Find?
The researchers establish two mutant mouse strains—Winnie and Eeyore mice—that develop mild spontaneous inflammation of the colon and chronic diarrhea and that have more proinflammatory cytokines and more lymphocytes in their colons than normal mice. 25% and 40% of the Winnie and Eeyore mice, respectively, have severe clinical signs of colitis by 1 year of age. Both strains have a mutation in the Muc2 gene, which codes for MUC2 mucin, the main protein in mucus. This viscous substance (which coats the inside of the intestine) is produced by and stored in intestinal “goblet” cells. Mucus helps to maintain the intestine's immunological balance but is depleted in UC. The researchers show that the manufacture and assembly of Muc2 molecules is abnormal in Winnie and Eeyore mice, that less mucin is stored in their goblet cells than in normal mice, and that their intestinal mucus barrier is reduced. In addition, an incompletely assembled version of the molecule, called Muc2 precursor, accumulates in the endoplasmic reticulum (ER; the cellular apparatus that prepares newly manufactured proteins for release) of goblet cells, leading to overload with abnormal protein and causing a state of cellular distress known as the “ER stress response.” Finally, the researchers report that MUC2 precursor also accumulates in the goblet cells of people with UC and that even the noninflamed intestinal tissue of these patients shows signs of ER stress.
What Do These Findings Mean?
These findings indicate that mucin abnormalities and ER stress can initiate colitis in mice. Results from animal studies do not always reflect what happens in people, but these findings, together with those from the small study in humans, suggest that ER stress-related mucin depletion could be a component in the development of human colitis. The results do not identify the genetic changes and/or environmental factors that might trigger ER stress in human colitis, but suggest that once initiated, ER stress might interfere with MUC2 production, which would lead to a diminished mucus barrier, expose the lining of the intestine to more toxins and foreign substances, and trigger local mucosal inflammation. The release of inflammatory cytokines would then damage the intestine's lining and exacerbate ER stress, thus setting up a cycle of intestinal damage and inflammation. Clinical studies to look for genetic changes and environmental factors capable of triggering ER stress and for ER-stress related changes in human UC should now be undertaken to test this hypothesis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050054.
The MedlinePlus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
Information and support for patients with inflammatory bowel disease and their caregivers is provided by the Crohn's and Colitis Foundation of America and by the UK National Association for Colitis and Crohn's Disease
Wikipedia has pages on mucins and on mucus (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050054
PMCID: PMC2270292  PMID: 18318598
2.  Polymeric micelles for the solubilization and delivery of STAT3 inhibitor cucurbitacins in solid tumors 
International journal of pharmaceutics  2007;347(1-2):118-127.
Poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) and newly developed poly(ethylene oxide)-block-poly(α-benzyl carboxylate ε-caprolactone) (PEO-b-PBCL) micelles were evaluated for the solubilization and delivery of cucurbitacin I and B, poorly water soluble inhibitors of signal transducer and activator of transcription 3 (STAT3). Encapsulation of cucurbitacins in PEO-b-PCL and PEO-b-PBCL by co-solvent evaporation technique resulted in polymeric micelles <90 nm in diameter. The aqueous solubility of both derivatives increased from less than 0.05 mg/mL in the absence of the copolymer to around 0.30–0.44 and 0.65–0.68 mg/mL in the presence of 5000–5000 and 5000–24,000 PEO-b-PCL micelles, respectively. Maximum cucurbitacin solubilization was achieved with PEO-b-PBCL micelles for both derivatives. PEO-b-PCL micelles having longer PCL block were found to be more efficient in sustaining the rate of release for cucurbitacins. The anti-cancer and STAT3 inhibitory activity of polymeric micellar cucurbitacins were comparable with free drugs in B16.F10 melanoma cell line in vitro. Intratumoral injection of 1 mg/kg/day cucurbitacin I resulted in the regression of established B16.F10 mouse melanoma tumors in vivo. In comparison to free cucurbitacin I, PEO-b-PBCL micellar cucurbitacin I was found to provide comparable anti-cancer effects against B16.F10 tumors and limit drug levels in animal serum while maintaining high drug levels in tumor following intratumoral administration. The results indicate the potential of polymeric micelles as suitable vehicles for the delivery of cucurbitacin- I and B.
doi:10.1016/j.ijpharm.2007.06.032
PMCID: PMC2663961  PMID: 17681440
Polymeric micelles; Cucurbitacin; Solubilization; Drug delivery; STAT3
3.  Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models 
BMC Medicine  2012;10:80.
Background
HER2 is an oncogene, expression of which leads to poor prognosis in 30% of breast cancer patients. Although trastuzumab is apparently an effective therapy against HER2-positive tumors, its systemic toxicity and resistance in the majority of patients restricts its applicability. In this study we evaluated the effects of phenethyl isothiocyanate (PEITC) in HER2-positive breast cancer cells.
Methods
MDA-MB-231 and MCF-7 breast cancer cells stably transfected with HER2 (high HER2 (HH)) were used in this study. The effect of PEITC was evaluated using cytotoxicity and apoptosis assay in these syngeneic cells. Western blotting was used to delineate HER2 signaling. SCID/NOD mice were implanted with MDA-MB-231 (HH) xenografts.
Results
Our results show that treatment of MDA-MB-231 and MCF-7 cells with varying concentrations of PEITC for 24 h extensively reduced the survival of the cells with a 50% inhibitory concentration (IC50) of 8 μM in MDA-MB-231 and 14 μM in MCF-7 cells. PEITC treatment substantially decreased the expression of HER2, epidermal growth factor receptor (EGFR) and phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr-705. The expression of BCL-2-associated × (BAX) and BIM proteins were increased, whereas the levels of B cell lymphoma-extra large (BCL-XL) and X-linked inhibitor of apoptosis protein (XIAP) were significantly decreased in both the cell lines in response to PEITC treatment. Substantial cleavage of caspase 3 and poly-ADP ribose polymerase (PARP) were associated with PEITC-mediated apoptosis in MDA-MB-231 and MCF-7 cells. Notably, transient silencing of HER2 decreased and overexpressing HER2 increased the effects of PEITC. Furthermore, reactive oxygen species (ROS) generation, mitochondrial depolarization and apoptosis by PEITC treatment were much higher in breast cancer cells expressing higher levels of HER2 (HH) as compared to parent cell lines. The IC50 of PEITC following 24 h of treatment was reduced remarkably to 5 μM in MDA-MB-231 (HH) and 4 μM in MCF-7 (HH) cells, stably overexpressing HER2. Oral administration of 12 μM PEITC significantly suppressed the growth of breast tumor xenografts in SCID/NOD mice. In agreement with our in vitro results, tumors from PEITC-treated mice demonstrated reduced HER2, EGFR and STAT3 expression and increased apoptosis as revealed by cleavage of caspase 3 and PARP. In addition our results show that PEITC can enhance the efficacy of doxorubicin.
Conclusions
Our results show a unique specificity of PEITC in inducing apoptosis in HER2-expressing tumor cells in vitro and in vivo and enhancing the effects of doxorubicin. This unique specificity of PEITC offers promise to a subset of breast cancer patients overexpressing HER2.
doi:10.1186/1741-7015-10-80
PMCID: PMC3412708  PMID: 22824293
apoptosis; doxorubicin; EGFR; ERBB2/HER2; in vivo; mitochondria; STAT3
4.  HDAC4-regulated STAT1 activation mediates platinum resistance in ovarian cancer 
Cancer research  2011;71(13):4412-4422.
Ovarian cancer frequently acquires resistance to platinum chemotherapy, representing a major challenge for improving patient survival. Recent work suggests resistant clones exist within a larger drug sensitive cell-population prior to chemotherapy, implying that resistance is selected for rather than generated by treatment. We sought to compare clinically-derived, intra-patient paired models of initial platinum response and subsequent resistant relapse to define molecular determinants of evolved resistance. Transcriptional analysis of a matched cell-line series from three patients with high-grade serous ovarian cancer before and after development of clinical platinum resistance (PEO1/PEO4/PEO6, PEA1/PEA2, PEO14/PEO23) identified 91 up- and 126 down-regulated genes common to acquired resistance. Significantly enhanced apoptotic response to platinum treatment in resistant cells was observed following knockdown of HDAC4, FOLR2, PIK3R1 or STAT1 (p<0.05). Interestingly, HDAC4 and STAT1 were found to physically interact. Acetyl-STAT1 was detected in platinum sensitive but not HDAC4 over-expressing platinum resistant cells from the same patient. In resistant cells, STAT1 phosphorylation/nuclear translocation was seen following platinum exposure, whereas silencing of HDAC4 increased acetyl-STAT1 levels, prevented platinum induced STAT1 activation and restored cisplatin sensitivity. Conversely, matched sensitive cells were refractory to STAT1 phosphorylation on platinum treatment. Analysis of 16 paired tumor biopsies taken before and after development of clinical platinum resistance showed significantly increased HDAC4 expression in resistant tumors (n=7/16[44%]; p=0.04). Therefore, clinical selection of HDAC4 overexpressing tumor cells upon exposure to chemotherapy promotes STAT1 deacetylation and cancer cell survival. Together, our findings identify HDAC4 as a novel, therapeutically tractable target to counter platinum resistance in ovarian cancer.
doi:10.1158/0008-5472.CAN-10-4111
PMCID: PMC3130134  PMID: 21571862
5.  Cis3/Socs3/Ssi3 Plays a Negative Regulatory Role in Stat3 Activation and Intestinal Inflammation 
Immune and inflammatory systems are controlled by multiple cytokines, including interleukins (ILs) and interferons. These cytokines exert their biological functions through Janus tyrosine kinases and signal transducer and activator of transcription (STAT) transcription factors. We recently identified two intrinsic Janus kinase (JAK) inhibitors, JAK binding protein (JAB; also referred to as suppressor of cytokine signaling [SOCS1]/STAT-induced STAT inhibitor [SSI1]) and cytokine-inducible SH2 protein (CIS)3 (or SOCS3/SSI3), which play an essential role in the negative regulation of cytokine signaling. We have investigated the role of STATs and these JAK inhibitors in intestinal inflammation. Among STAT family members, STAT3 was most strongly tyrosine phosphorylated in human ulcerative colitis and Crohn's disease patients as well as in dextran sulfate sodium (DSS)-induced colitis in mice. Development of colitis as well as STAT3 activation was significantly reduced in IL-6–deficient mice treated with DSS, suggesting that STAT3 plays an important role in the perpetuation of colitis. CIS3, but not JAB, was highly expressed in the colon of DSS-treated mice as well as several T cell–dependent colitis models. To define the physiological role of CIS3 induction in colitis, we developed a JAB mutant (F59D-JAB) that overcame the inhibitory effect of both JAB and CIS3 and created transgenic mice. DSS induced stronger STAT3 activation and more severe colitis in F59D-JAB transgenic mice than in their wild-type littermates. These data suggest that hyperactivation of STAT3 results in severe colitis and that CIS3 plays a negative regulatory role in intestinal inflammation by downregulating STAT3 activity.
PMCID: PMC2195913  PMID: 11181699
Janus kinase; CIS/SOCS; interleukin 6; ulcerative colitis; negative regulation
6.  Differential in vivo mechanism of chemoprevention of tumor formation in azoxymethane/dextran sodium sulfate mice by PEITC and DBM 
Carcinogenesis  2009;31(5):880-885.
Previously, phenethyl isothiocyanate (PEITC) and dibenzoylmethane (DBM) had been shown to inhibit intestinal carcinogenesis in Apc(Min/+) mice. In this study, we investigated the chemopreventive efficacy of PEITC and DBM in the azoxymethane (AOM)-initiated and dextran sodium sulfate (DSS)-promoted colon cancer mouse model and to compare their potential in vivo mechanisms leading to chemoprevention. The mice were fed with diet supplemented with 0.05% PEITC or 1% DBM before or after AOM initiation. Our results showed that AOM/DSS mice fed with PEITC- or DBM-supplemented diet had lower tumor incidence, lower colon tumor multiplicities and smaller polyps as compared with mice fed with the standard AIN-76A diet. PEITC was effective even after AOM initiation, whereas DBM was not as effective when fed after AOM initiation. Hematoxylin and eosin staining showed that mice fed with PEITC or DBM had attenuated loss of crypt, a marker of inflammation. To examine potential in vivo mechanisms involved in chemoprevention, western blotting was performed and showed that inhibition of growth of adenomas by PEITC was associated with an increase of apoptosis (increased cleaved caspase-3 and-7) and cell cycle arrest (increased p21). In contrast DBM's effect on cell cycle arrest and apoptosis markers was not as substantial as PEITC. Instead, DBM showed increased induction of NF-E2-related factor-2 (Nrf2) transcription factor and phase II detoxifying enzymes, which appears to correlate with in vitro cell lines results that DBM is a more potent Nrf2 activator than PEITC. In summary, our present study shows that PEITC and DBM are potent natural dietary compounds for chemoprevention of colon cancer induced by AOM/DSS and appears to be associated with different in vivo mechanism of actions. PEITC's chemopreventive effect appears to be due to induction of apoptosis and cell cycle arrest, whereas DBM's effect is due to prevention of AOM initiation via induction of Nrf2 and phase II detoxifying enzymes.
doi:10.1093/carcin/bgp285
PMCID: PMC2864406  PMID: 19959557
7.  Leptin antagonist ameliorates chronic colitis in IL-10−/− mice 
Immunobiology  2013;218(12):1439-1451.
Background
Although the etiology of two major forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) are unknown and evidence suggests that chronic intestinal inflammation is caused by an excessive immune response to mucosal antigens. Previous studies support the role for TGF-β1 through 3 in the initiation and maintenance of tolerance via the induction of regulatory T cells (Tregs) to control intestinal inflammation. Leptin, a satiety hormone produced primarily by adipose tissue, has been shown to increase during colitis progression and is believed to contribute to disease genesis and/or progression.
Aim
We investigated the ability of a pegylated leptin antagonist (PG-MLA) to ameliorate the development of chronic experimental colitis.
Results
Compared to vehicle control animals, PG-MLA treatment of mice resulted in an (1) attenuated clinical score; (2) reversed colitis-associated pathogenesis including a decrease in body weight; (3) reduced systemic and mucosal inflammatory cytokine expression; (4) increased insulin levels and (5) enhanced systemic and mucosal Tregs and CD39+ Tregs in mice with chronic colitis. The percentage of systemic and mucosal TGF-β1, -β2 and -β3 expressing CD4+ T cells were augmented after PG-MLA treatment. The activation of STAT1 and STAT3 and the expression of Smad7 were also reduced after PG-MLA treatment in the colitic mice. These findings clearly suggest that PG-MLA treatment reduces intestinal Smad7 expression, restores TGF-β1-3 signaling and reduces STAT1/STAT3 activation that may increase the number of Tregs to ameliorate chronic colitis.
Conclusion
This study clearly links inflammation with the metabolic hormone leptin suggesting that nutritional status influences immune tolerance through the induction of functional Tregs. Inhibiting leptin activity through PG-MLA might provide a new and novel therapeutic strategy for the treatment of IBD.
doi:10.1016/j.imbio.2013.04.020
PMCID: PMC3778116  PMID: 23726523
Inflammation; Inflammatory bowel disease (IBD); Leptin; Antagonist; Pegylated; Ulcerative Colitis (UC); Crohn’s Disease (CD)
8.  Phenethyl Isothiocyanate inhibits STAT3 activation in prostate cancer cells 
This study was undertaken to investigate the mechanism by which phenethyl isothiocyanate (PEITC), a natural compound from cruciferous vegetables, exhibits antitumor effect on prostate cancer cells. Cell proliferation, cell cycle, western blot, gene transfer and reporter assays were used to test the effects of PEITC on the growth and IL6/JAK/STAT3 pathway in prostate cancer. The result showed that PEITC significantly inhibited DU145 cell proliferation in a dose-dependent manner and induced the cell arrest at G2-M phase. PEITC inhibited both constitutive and interleukin 6 (IL-6)-induced STAT3 activity in DU145 cells. IL-6-stimulated phosphorylation of JAK2, an STAT3 upstream kinase, was also attenuated by PEITC. Moreover, an antioxidant reagent, N-acetyl-l-cysteine (NAC) which suppresses reactive oxygen species (ROS) generation, reversed the early inhibitory effects of PEITC on cell proliferation, constitutive or IL-6-mediated JAK-STAT3 phosphorylation in PCa cells. Taken together, our data demonstrated that PEITC can inhibit the activation of the JAK-STAT3 signal-cascade in prostate cancer cells and the underlying mechanism may be partially involved with blocking cellular ROS production during the early stage of the signaling activation by IL-6.
doi:10.1002/mnfr.200800253
PMCID: PMC3964815  PMID: 19437484
Phenethyl isothiocyanate; IL-6; STAT3; Androgen independent growth
9.  Analysis of Germline GLI1 Variation Implicates Hedgehog Signalling in the Regulation of Intestinal Inflammatory Pathways 
PLoS Medicine  2008;5(12):e239.
Background
Ulcerative colitis (UC) and Crohn's disease (CD) are polygenic chronic inflammatory bowel diseases (IBD) of high prevalence that are associated with considerable morbidity. The hedgehog (HH) signalling pathway, which includes the transcription factor glioma-associated oncogene homolog 1 (GLI1), plays vital roles in gastrointestinal tract development, homeostasis, and malignancy. We identified a germline variation in GLI1 (within the IBD2 linkage region, 12q13) in patients with IBD. Since this IBD-associated variant encodes a GLI1 protein with reduced function and our expression studies demonstrated down-regulation of the HH response in IBD, we tested whether mice with reduced Gli1 activity demonstrate increased susceptibility to chemically induced colitis.
Methods and Findings
Using a gene-wide haplotype-tagging approach, germline GLI1 variation was examined in three independent populations of IBD patients and healthy controls from Northern Europe (Scotland, England, and Sweden) totalling over 5,000 individuals. On log-likelihood analysis, GLI1 was associated with IBD, predominantly UC, in Scotland and England (p < 0.0001). A nonsynonymous SNP (rs2228226C→G), in exon 12 of GLI1 (Q1100E) was strongly implicated, with pooled odds ratio of 1.194 (confidence interval = 1.09–1.31, p = 0.0002). GLI1 variants were tested in vitro for transcriptional activity in luciferase assays. Q1100E falls within a conserved motif near the C terminus of GLI1; the variant GLI protein exhibited reduced transactivation function in vitro. In complementary expression studies, we noted the colonic HH response, including GLI1, patched (PTCH), and hedgehog-interacting protein (HHIP), to be down-regulated in patients with UC. Finally, Gli1+/lacZ mice were tested for susceptibility to dextran sodium sulphate (DSS)-induced colitis. Clinical response, histology, and expression of inflammatory cytokines and chemokines were recorded. Gli1+/lacZ mice rapidly developed severe intestinal inflammation, with considerable morbidity and mortality compared with wild type. Local myeloid cells were shown to be direct targets of HH signals and cytokine expression studies revealed robust up-regulation of IL-12, IL-17, and IL-23 in this model.
Conclusions
HH signalling through GLI1 is required for appropriate modulation of the intestinal response to acute inflammatory challenge. Reduced GLI1 function predisposes to a heightened myeloid response to inflammatory stimuli, potentially leading to IBD.
Charlie Lees and colleagues identify a reduced-function variant of the hedgehog signaling pathway protein GLI1 that associates with inflammatory bowel disease, and investigate its role in a mouse model of colitis.
Editors' Summary
Background.
Inflammatory bowel diseases (IBDs) are common disorders in which parts of the digestive tract become repeatedly or continuously inflamed. The immune system normally protects the body from entities it identifies as foreign, but in IBD it mistakenly recognizes gut tissue, and immune system cells accumulate in the lining of the bowel, which causes inflammation. There are two main types of IBD—Crohn's disease (CD), which mainly affects the small bowel, and ulcerative colitis (UC), which affects only the large bowel (colon). Both types tend to run in families and usually develop between the ages of 15 and 35 years. Symptoms—including diarrhea, abdominal cramps, and unexplained weight loss—can be mild or severe and the disease can develop slowly or suddenly. There is no cure for IBD except surgical removal of the affected part of the digestive tract. However, drugs that modulate the immune system (for example, corticosteroids) or that specifically inhibit “proinflammatory cytokines” (proteins made by the immune system that stimulate inflammation) are often helpful in reducing symptoms.
Why Was This Study Done?
Why the immune system becomes unbalanced in people with IBD is not clear but it is known that IBD is “polygenic,” that is, a disease caused by the combined actions of two or more inherited gene variants. Although UC and CD are clinically different diseases, they share several “susceptibility loci” (regions of the genome that harbor disease-associated gene variants), including the IBD2 locus. The identification of the actual gene within the IBD2 locus that is altered in people who are susceptible to IBD might provide new insights into what causes the immune imbalance in IBD and into how to treat the disease. In this study, the researchers test the hypothesis that a variant of a gene called GLI1, which lies in the IBD2 locus, is associated with IBD susceptibility. GLI1 encodes a transcription factor (a protein that regulates the production of proteins) that is a central component in the signaling pathway named for a protein called “hedgehog.” This pathway is involved in the development of many organs, including the digestive tract.
What Did the Researchers Do and Find?
The researchers used a technique called gene-wide haplotype tagging to look for inherited GLl1 variants in patients with IBD and in healthy people living in Scotland, England, and Sweden. A specific variant of the GLI1 gene, resulting in alteration of a single amino acid component of the GLI1 protein, was associated with IBD (particularly with UC) in both Scotland and England; the same variant was weakly associated with IBD in the Swedish population. The variant GLI1 protein was only half as active as the normal protein in a laboratory assay, and, consistent with this result, the expression of several components of the hedgehog signaling pathway was lower in colon samples taken from patients with UC than in samples taken from healthy individuals. Finally, Gli1+/lacZ mice (which express half the normal amount of Gli1 protein) developed severe intestinal inflammation more rapidly than wild-type mice when they were treated with dextran sodium sulfate (DSS), a chemical that induces acute (sudden) colitis. Cellular analysis revealed that myeloid cells (cells that sense and modify the inflammatory response) are direct targets of the hedgehog signaling pathway. Furthermore, the expression of several pro-inflammatory cytokines (in particular, one called IL-23) increased more markedly in the Gli1+/lacZ mice than in the wild-type mice after DSS treatment.
What Do These Findings Mean?
These findings suggest that the normal response of the mammalian gut to challenge with inflammatory substances involves hedgehog signaling through GLI1 and that reduced GLI1 function might be one trigger for IBD. More specifically, the human genetic studies identify a GLI1 variant that is associated with IBD (at least in certain north European populations), the laboratory experiments indicate that this GLI1 variant encodes a protein with reduced activity, and the animal studies show that a similar reduction in Gli1 activity is sufficient to heighten intestinal inflammatory responses. Although this last result needs to be confirmed in animal models of chronic colitis that more closely resemble human IBD, these findings suggest that drugs that modulate hedgehog signaling might be useful in the treatment of IBD.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050239.
The MedlinePlus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
MedlinePlus provides links to other information Crohn's disease and ulcerative colitis (in English and Spanish)
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
The UK National Health Service Direct Encyclopedia also provides information on Crohn's disease and on ulcerative colitis
Wikipedia has a page on the hedgehog signaling pathway (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050239
PMCID: PMC2596854  PMID: 19071955
10.  An Antibiotic-Responsive Mouse Model of Fulminant Ulcerative Colitis  
PLoS Medicine  2008;5(3):e41.
Background
The constellation of human inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease, which both display a wide spectrum in the severity of pathology. One theory is that multiple genetic hits to the host immune system may contribute to the susceptibility and severity of IBD. However, experimental proof of this concept is still lacking. Several genetic mouse models that each recapitulate some aspects of human IBD have utilized a single gene defect to induce colitis. However, none have produced pathology clearly distinguishable as either ulcerative colitis or Crohn's disease, in part because none of them reproduce the most severe forms of disease that are observed in human patients. This lack of severe IBD models has posed a challenge for research into pathogenic mechanisms and development of new treatments. We hypothesized that multiple genetic hits to the regulatory machinery that normally inhibits immune activation in the intestine would generate more severe, reproducible pathology that would mimic either ulcerative colitis or Crohn's disease.
Methods and Findings
We generated a novel mouse line (dnKO) that possessed defects in both TGFβRII and IL-10R2 signaling. These mice rapidly and reproducibly developed a disease resembling fulminant human ulcerative colitis that was quite distinct from the much longer and more variable course of pathology observed previously in mice possessing only single defects. Pathogenesis was driven by uncontrolled production of proinflammatory cytokines resulting in large part from T cell activation. The disease process could be significantly ameliorated by administration of antibodies against IFNγ and TNFα and was completely inhibited by a combination of broad-spectrum antibiotics.
Conclusions
Here, we develop to our knowledge the first mouse model of fulminant ulcerative colitis by combining multiple genetic hits in immune regulation and demonstrate that the resulting disease is sensitive to both anticytokine therapy and broad-spectrum antibiotics. These findings indicated the IL-10 and TGFβ pathways synergize to inhibit microbially induced production of proinflammatory cytokines, including IFNγ and TNFα, which are known to play a role in the pathogenesis of human ulcerative colitis. Our findings also provide evidence that broad-spectrum antibiotics may have an application in the treatment of patients with ulcerative colitis. This model system will be useful in the future to explore the microbial factors that induce immune activation and characterize how these interactions produce disease.
Paul Allen and colleagues describe the development of a mouse model of fulminant ulcerative colitis with multiple genetic hits in immune regulation which can be moderated by anti-cytokine therapy and broad-spectrum antibiotics.
Editors' Summary
Background.
Inflammatory bowel disease (IBD), a group of disorders characterized by inflammation (swelling) of the digestive tract (the tube that runs from the mouth to the anus), affects about 1.4 million people in the US. There are two main types of IBD. In Crohn's disease, which can affect any area of the digestive tract but most commonly involves the lower part of the small intestine (small bowel), all the layers of the intestine become inflamed. In ulcerative colitis, which primarily affects the colon (large bowel) and the rectum (the part of the bowel closest to the anus), only the lining of the bowel becomes inflamed, the cells in this lining die, and sores or ulcers form. Both types of IBD most commonly develop between the ages of 15 and 35 years, often run in families, and carry an increased risk of cancer. Symptoms—usually diarrhea and abdominal cramps—can be mild or severe and the disorder can develop slowly or suddenly. There is no medical cure for IBD, but drugs that modulate the immune system (for example, corticosteroids) can help some people. Some people benefit from treatment with drugs that specifically inhibit “proinflammatory cytokines,” proteins made by the immune system that stimulate inflammation (for example, TNFα and INFγ). When medical therapy fails, surgery to remove the affected part of the bowel may be necessary.
Why Was This Study Done?
Exactly what causes IBD is not clear, but people with IBD seem to have an overactive immune system. The immune system normally protects the body from harmful substances but in IBD it mistakenly recognizes the food substances and “good” bacteria that are normally present in the human gut as foreign and hence reacts against them. As a result, immune system cells accumulate in the lining of the bowel and cause inflammation. Several different pathways usually prevent inappropriate immune activation, so could IBD be caused by alterations in one or several of these immune regulatory pathways? In previous studies, mice with a defect in just one pathway have developed mild intestinal abnormalities but not the problems seen in the most severe forms of IBD. In this study, therefore, the researchers have generated and characterized a new mouse line with defects in two immune regulatory pathways to see whether this might be a better animal model of human IBD.
What Did the Researchers Do and Find?
To make their new mouse line, the researchers mated mice that had a defective TGFβ signaling pathway in their T lymphocytes with mice that had a defective IL-10 signaling pathway. Both these pathways are anti-inflammatory, and mice with defects in either pathway develop mild and variable inflammation of the colon (colitis) by age 3–4 months. By contrast, the doubly defective mice (dnKO mice) failed to thrive, lost weight, and died by 4–6 weeks of age. The colons of 4- to 5-week old dnKO mice were inflamed and ulcerated (some changes were visible in 3-week-old mice) and contained many immune system cells. Mice with a single defective signaling pathway had no gut abnormalities at this age. The dnKO mice, just like people with IBD, had higher than normal blood levels of IFNγ, TNFα, and other proinflammatory cytokines; these raised levels were the result of abnormal lymphocyte activation. Treatment of the dnKO mice with a combination of agents that neutralize IFNγ and TNFα (anti-cytokine therapy) greatly reduced the colitis seen in these mice; neutralization of IFNγ alone had some beneficial effects, but neutralization of TNFα alone had no effect. Finally, early treatment of the dnKO mice with broad-spectrum antibiotics completely inhibited colitis.
What Do These Findings Mean?
These findings suggest that dnKO mice are a good model for fulminant (severe and rapidly progressing) ulcerative colitis and support the idea that IBD involves multiple genetic defects in immune regulation. They also indicate that the IL-10 and the TGFβ signaling pathways normally cooperate to inhibit the inappropriate immune responses to intestinal bacteria seen in IBD. This new mouse model should help researchers unravel what goes wrong in IBD and should also help them develop new treatments for ulcerative colitis. More immediately, these findings suggest that combined anti-cytokine therapy may be a better treatment for ulcerative colitis than single therapy. In addition, they suggest that clinical studies should be started to test whether broad-spectrum antibiotics can ameliorate ulcerative colitis in people.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050041.
The Medline Plus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
Information is available from the UK National Health Service Direct Health Encyclopedia about Crohn's disease and ulcerative colitis
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
Information and support for patients with inflammatory bowel disease and their caregivers is provided by the Crohn's and Colitis Foundation of America and by the UK National Association for Colitis and Crohn's Disease
doi:10.1371/journal.pmed.0050041
PMCID: PMC2270287  PMID: 18318596
11.  Notch Activation by Phenethyl Isothiocyanate Attenuates Its Inhibitory Effect on Prostate Cancer Cell Migration 
PLoS ONE  2011;6(10):e26615.
Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive component of edible cruciferous vegetables with in vivo efficacy against prostate cancer in experimental rodents. Cancer chemopreventive response to PEITC is characterized by its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-κB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch signaling in malignant as well as normal human prostate cells. Exposure of human prostate cancer cells (LNCaP, PC-3, and DU145) and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, and increased transcriptional activity of Notch. In PC-3 and LNCaP cells, PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2 (PC-3), overexpression of γ-secretase complex components Presenilin1 and Nicastrin (PC-3), nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by knockdown of Notch2 protein as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving a Notch inhibitor.
doi:10.1371/journal.pone.0026615
PMCID: PMC3200337  PMID: 22039516
12.  Phenethyl Isothiocyanate Suppresses Inhibitor of Apoptosis Family Protein Expression in Prostate Cancer Cells in Culture and In Vivo 
The Prostate  2011;72(10):1104-1116.
BACKGROUND
Cruciferous vegetable constituent phenethyl isothiocyanate (PEITC) causes apoptosis in prostate cancer cells through mechanisms not fully understood. The present study was designed to determine the role of inhibitor of apoptosis (IAP) family proteins in PEITC-induced apoptosis induction.
METHODS
Effect of PEITC treatment on protein and mRNA expression of IAP in cells was determined by western blotting and reverse transcription PCR, respectively. Immunohistochemistry was performed to determine the in vivo effect of PEITC administration on X-linked inhibitor of apoptosis (XIAP) and Survivin protein expression. Overexpression of desired protein was achieved by transient transfection. Cell viability was determined by trypan blue dye exclusion assay, whereas apoptosis was quantified by measurement of histone-associated DNA fragment release into the cytosol. Transwell chamber assay was used to determine cell migration.
RESULTS
Exposure of PC-3 and LNCaP human prostate cancer cells to PEITC resulted in downregulation of XIAP and Survivin proteins and Survivin mRNA. PEITC administration to Transgenic Adenocarcinoma of Mouse Prostate mice caused modest but significant downregulation of XIAP and Survivin proteins in the dorsolateral prostate. Proapoptotic response to PEITC was significantly attenuated by ectopic expression of XIAP and Survivin proteins. Survivin overexpression also conferred modest but significant protection against PEITC-mediated inhibition of PC-3 cell migration.
CONCLUSIONS
The present study demonstrates that cellular responses to PEITC, including apoptosis induction and inhibition of cell migration, in prostate cancer cells are mediated by downregulation of XIAP and/or Survivin, which may serve as valid biomarkers of PEITC response in future clinical investigations.
doi:10.1002/pros.22457
PMCID: PMC3310272  PMID: 22161756
Phenethyl Isothiocyanate; XIAP; Survivin; Chemoprevention
13.  Relationships between the physicochemical properties of an amphiphilic triblock copolymers/DNA complexes and their intramuscular transfection efficiency 
Nucleic Acids Research  2006;35(3):728-739.
Poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer (PEO-PPO-PEO) based plasmid delivery systems are increasingly drawing attention in the field of nonviral gene transfer because of their proven in vivo transfection capability. They result from the simple association of DNA molecules with uncharged polymers. We examined the physicochemical properties of PEO-PPO-PEO/DNA mixtures, in which the PEO-PPO-PEO is Lutrol® (PEO75-PPO30-PEO75), formulated under various conditions. We found that interactions between PEO-PPO-PEO and DNA are mediated by the central hydrophobic block within the block copolymer. Dynamic light scattering and cryo-electron microscopy showed that the mean diameter of transfecting particles as well as their stability depended on the PEO-PPO-PEO/DNA ratio and on the ionic composition of the formulating medium. The most active formulation promoting a good tissue-distribution and an optimal transfection was characterized by a reduced electrophoretic mobility, a mean hydrodynamic diameter of ∼250–300 nm and by a conserved B-DNA form as shown by circular dichroism studies. Our study also revealed that the stability of these formulations strongly depended on a concentration balance between the DNA and the PEO-PPO-PEO, over which the DNA conformation was modified, micron-sized particles were generated, and the transgene expression was declined. We showed that the physicochemical properties of PEO-PPO-PEO/DNA formulations directly impact the level of gene expression in transfected muscles.
doi:10.1093/nar/gkl860
PMCID: PMC1807968  PMID: 17182627
14.  Inflammation-associated gene expression is altered between normal human ovarian surface epithelial cells and cell lines derived from ovarian adenocarcinomas 
British Journal of Cancer  2005;92(10):1927-1933.
Ovulation is believed to contribute to the development of ovarian cancers that derive from the ovarian surface epithelium (OSE). The process of ovulation is synonymous with inflammation and inflammatory cytokines such as interleukin-1α (IL-1α) have recently been shown to induce both inflammatory and anti-inflammatory responses in human OSE (HOSE) cells. In this study we directly compared levels of IL-1α-induced gene expression by analysing the levels of 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 (11βHSD-1) and 2 (11βHSD-2), cyclooxygenase-2 (COX-2), IL-1 receptor (IL-1R) and glucocorticoid receptor α (GRα) mRNA between normal HOSE cells and cell lines derived from poorly differentiated (SKOV-3, BG-1, PEO-4) and well-differentiated (PEO-14) ovarian adenocarcinoma. In HOSE cell cultures, and to a lesser extent PEO-14 cells, the basal mRNA levels of COX-2 and 11βHSD-1 were relatively high and further shown to be induced in response to IL-1α (for HOSE cells; >20-fold, P<0.05 and PEO-14 cells; >3fold, P<0.05). However, whereas HOSE cells expressed a low level of 11βHSD-2 mRNA that was only mildly responsive to IL-1α (1.3-fold, P<0.001), all cell lines exhibited a higher basal level of 11βHSD-2 mRNA that was in some cases further stimulated in PEO-4 cells (five-fold; P<0.05) or suppressed in SKOV-3 cells (two-fold; P<0.01) in response to IL-1α. All cells tested expressed IL-1R and, with the exception of BG-1, GRα. These results indicate that cell lines derived from ovarian cancers have lost the ability to respond normally to inflammatory cytokines such as IL-1α. The finding that normal OSE cells, in contrast to cell lines derived from patients with ovarian adenocarcinoma, abundantly express 11βHSD-1 mRNA but are essentially devoid of 11βHSD-2 mRNA supports the concept that the pattern of 11βHSD isoform gene expression is a defining feature of neoplastic cellular transformation, which might have particular relevance to the ovary.
doi:10.1038/sj.bjc.6602568
PMCID: PMC2361768  PMID: 15870720
ovarian cancer; ovarian surface epithelium; 11βHSD-1/2; inflammation
15.  T Cell–mediated Pathology in Two Models of Experimental Colitis Depends Predominantly on the Interleukin 12/Signal Transducer and Activator of Transcription (Stat)-4 Pathway, but Is Not Conditional on Interferon γ Expression by T Cells  
The Journal of Experimental Medicine  1998;187(8):1225-1234.
The requirements for interleukin (IL)-12/signal transducer and activator of transcription (Stat)-4 signaling and induction of T cell–specific interferon (IFN)-γ expression in the development of T helper cell (Th)1–type pathology were examined in two different models of experimental colitis. In each model, abnormal reconstitution of the T cell compartment in immunodeficient mice by adoptive cell transfer leads to a wasting syndrome and inflammation of the colon, induced by IFN-γ and tumor necrosis factor (TNF)-α–producing T cells. We show here that treatment with anti–IL-12 antibodies in one of the models, or reconstitution with T cells from Stat-4–deficient (Stat-4null) mice in both models resulted in a milder disease in the majority of recipient animals, compared with those that were left untreated or that had been reconstituted with wt cells. Protected mice in each group also harbored lower frequencies of IFN-γ–producing T cells than did diseased mice, suggesting that effects on wasting and colitis resulted from the attenuation of IFN-γ expression by T cells. To test whether the development of pathogenic T cells in the two colitis models was directly dependent on T cell–specific IFN-γ expression, IFN-γnull donors were used for T cell reconstitution in each system. Surprisingly, large numbers of IFN-γnull–reconstituted mice developed wasting and colitis, which in many cases was of comparable severity to that seen in animals reconstituted with wt cells. Furthermore, T cells from these animals expressed TNF-α, demonstrating that they had retained the ability to produce another proinflammatory cytokine. Taken together, these results demonstrate that in some forms of chronic experimental colitis the development of pathogenic T cells is influenced predominantly, though not exclusively, by IL-12 via the actions of Stat-4 proteins. Furthermore, our data suggest that in the models of colitis studied here the effects of IL-12/Stat-4 or other Th1 promoting pathways are not limited to the induction of IFN-γ gene expression in T lymphocytes.
PMCID: PMC2212228  PMID: 9547334
16.  STAT6 deficiency ameliorates severity of oxazolone colitis by decreasing expression of claudin-2 and Th2-inducing cytokines 
Patients suffering from ulcerative colitis (UC) exhibit chronic colonic inflammation caused by a dysregulated mucosal immune response and epithelial barrier disruption. Th2 cytokines, including IL-13, have been implicated in the pathogenesis of UC. IL-13 induces phosphorylation of STAT6, and we have previously demonstrated increased epithelial phosphorylated (p)STAT6 in children with UC. Here, we investigated the role of STAT6 in oxazolone colitis, a murine model of UC, by inducing colitis in STAT6-deficient (STAT6−/−) and wild type (WT) mice. We observed increased epithelial cell, T cell, macrophage, and NKT cell STAT6 phosphorylation, and increased pSTAT6+ IL-13-producing NKT cells, in colitic WT mice. Colitis was attenuated in STAT6−/− mice with improvements in weight, colon length, and histopathology. There was decreased induction of the pore-forming tight junction protein claudin-2 in STAT6−/− mice. Similarly, shRNA STAT6 knockdown reduced claudin-2 induction and transepithelial resistance decrease in IL-13-treated human T84 cells. Tissue expression of IL-13, IFN-γ, IL-17, and IL-10 mRNA was similarly induced in WT and STAT6−/− colitic mice; however, we observed increased mRNA expression for the Th2-inducing cytokines IL-33 and thymic stromal lymphopoietin (TSLP) in WT mice with colitis, which was abrogated in STAT6−/− mice. Mesenteric lymph node (MLN) cells from STAT6−/− mice with colitis exhibited reduced secretion of IL-4, IL-5, IL-13, and IFN-γ. IL-33 augmented MLN cell secretion of IL-5, IL-13, IL-6, and IFN-γ. These data implicate STAT6 in the pathogenesis of colitis in vivo with important roles in altering epithelial barrier function and regulating Th2-inducing cytokine production.
doi:10.4049/jimmunol.1201373
PMCID: PMC3563924  PMID: 23303670
17.  Twinkle mutations associated with autosomal dominant progressive external ophthalmoplegia lead to impaired helicase function and in vivo mtDNA replication stalling 
Human Molecular Genetics  2008;18(2):328-340.
Mutations in the mitochondrial helicase Twinkle underlie autosomal dominant progressive external ophthalmoplegia (PEO), as well as recessively inherited infantile-onset spinocerebellar ataxia and rare forms of mitochondrial DNA (mtDNA) depletion syndrome. Familial PEO is typically associated with the occurrence of multiple mtDNA deletions, but the mechanism by which Twinkle dysfunction induces deletion formation has been under debate. Here we looked at the effects of Twinkle adPEO mutations in human cell culture and studied the mtDNA replication in the Deletor mouse model, which expresses a dominant PEO mutation in Twinkle and accumulates multiple mtDNA deletions during life. We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. This indicated severe replication pausing or stalling and caused mtDNA depletion. A strongly enhanced accumulation of replication intermediates was evident also in six-week-old Deletor mice compared with wild-type littermates, even though mtDNA deletions accumulate in a late-onset fashion in this model. In addition, our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype and might be of relevance in adPEO pathophysiology. Finally, in vitro assays showed functional defects in the various Twinkle mutants and broadly agreed with the cell culture phenotypes such as the level of mtDNA depletion and the level of accumulation of replication intermediates. On the basis of our results we suggest that mtDNA replication pausing or stalling is the common consequence of Twinkle PEO mutations that predisposes to multiple deletion formation.
doi:10.1093/hmg/ddn359
PMCID: PMC2638771  PMID: 18971204
18.  Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo 
Angiogenesis is a hallmark of tumor development and metastatic progression, and antiangiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti-proliferative and anti-angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO-1 and cisplatin resistant, BRCA2 proficient PEO-4 ovarian cancer cells. Both PEO-1 and PEO-4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF-A and Glut-1 in PEO-1 and PEO-4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR-5 cells. Plumbagin challenge also restricts the VEGF induced pro-angiogenenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR-5 tumor-bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti-cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.
doi:10.1002/ijc.27724
PMCID: PMC3496826  PMID: 22806981
Plumbagin; Ovarian cancer; Cytotoxicity; Angiogenesis
19.  Kaempferol nanoparticles achieve strong and selective inhibition of ovarian cancer cell viability 
Ovarian cancer is one of the leading causes of cancer death for women throughout the Western world. Kaempferol, a natural flavonoid, has shown promise in the chemoprevention of ovarian cancer. A common concern about using dietary supplements for chemoprevention is their bioavailability. Nanoparticles have shown promise in increasing the bioavailability of some chemicals. Here we developed five different types of nanoparticles incorporating kaempferol and tested their efficacy in the inhibition of viability of cancerous and normal ovarian cells. We found that positively charged nanoparticle formulations did not lead to a significant reduction in cancer cell viability, whereas nonionic polymeric nanoparticles resulted in enhanced reduction of cancer cell viability. Among the nonionic polymeric nanoparticles, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nanoparticles incorporating kaempferol led to significant reduction in cell viability of both cancerous and normal cells. Poly(DL-lactic acid-co-glycolic acid) (PLGA) nanoparticles incorporating kaempferol resulted in enhanced reduction of cancer cell viability together with no significant reduction in cell viability of normal cells compared with kaempferol alone. Therefore, both PEO-PPO-PEO and PLGA nanoparticle formulations were effective in reducing cancer cell viability, while PLGA nanoparticles incorporating kaempferol had selective toxicity against cancer cells and normal cells. A PLGA nanoparticle formulation could be advantageous in the prevention and treatment of ovarian cancers. On the other hand, PEO-PPO-PEO nanoparticles incorporating kaempferol were more effective inhibitors of cancer cells, but they also significantly reduced the viability of normal cells. PEO-PPO-PEO nanoparticles incorporating kaempferol may be suitable as a cancer-targeting strategy, which could limit the effects of the nanoparticles on normal cells while retaining their potency against cancer cells. We have identified two nanoparticle formulations incorporating kaempferol that may lead to breakthroughs in cancer treatment. Both PEO-PPO-PEO and PLGA nanoparticle formulations had superior effects compared with kaempferol alone in reducing cancer cell viability.
doi:10.2147/IJN.S33670
PMCID: PMC3410694  PMID: 22866004
nanochemoprevention; kaempferol; ovarian cancer; nanoparticles; viability; natural compound
20.  A Molecular Link Between Interleukin 22 and Intestinal Mucosal Wound Healing 
Advances in Wound Care  2012;1(6):231-237.
Background
Interleukin 22 (IL-22) and signal transducer and activator of transcription 3 (STAT3) are two important regulators of inflammation. Crohn's disease and ulcerative colitis are considered inflammatory bowel diseases (IBDs), due to the belief that these diseases result from dysregulated responses of the intestinal immune system to bacteria present in the commensal flora.
The Problem
It is debated whether a breakdown of immune tolerance is the primary cause of these diseases or occurs downstream of an initial defect of the intestinal barrier and intestinal epithelial cells (IECs).
Basic/Clinical Science Advances
Recent reports suggest a crucial role for IL-22 in the regulation of gut inflammation as well as epithelial barrier integrity. Local IL-22 gene delivery enhances expression of its downstream effector, STAT3, within colonic epithelial cells and induces both STAT3-dependent expression of mucus-associated molecules and restitution of mucus-producing goblet cells. IEC-specific deletion of STAT3 results in significant susceptibility to experimental colitis with a striking defect in epithelial restitution. STAT3 activation, thus, may regulate immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.
Clinical Care Relevance
The importance of IL-22/STAT3 signaling in IEC wound healing suggests a critical role for epithelial homeostasis in IBDs.
Conclusion
Effective healing of the IECs could be considered a primary target in the development of treatments for IBDs. IL-22/STAT3 signaling exerts a protective role in the process of intestinal mucosal wound healing and may thereby provide a promising therapeutic approach to the treatment of IBDs.
doi:10.1089/wound.2011.0334
PMCID: PMC3623586  PMID: 24527311
21.  Attenuation of murine antigen-induced arthritis by treatment with a decoy oligodeoxynucleotide inhibiting signal transducer and activator of transcription-1 (STAT-1) 
The transcription factor STAT-1 (signal transducer and activator of transcription-1) plays a pivotal role in the expression of inflammatory gene products involved in the pathogenesis of arthritis such as various cytokines and the CD40/CD40 ligand (CD40/CD40L) receptor-ligand dyad. The therapeutic efficacy of a synthetic decoy oligodeoxynucleotide (ODN) binding and neutralizing STAT-1 was tested in murine antigen-induced arthritis (AIA) as a model for human rheumatoid arthritis (RA). The STAT-1 decoy ODN was injected intra-articularly in methylated bovine serum albumin (mBSA)-immunized mice 4 h before arthritis induction. Arthritis was evaluated by joint swelling measurement and histological evaluation and compared to treatment with mutant control ODN. Serum levels of pro-inflammatory cytokines, mBSA-specific antibodies and auto-antibodies against matrix constituents were assessed by enzyme-linked immunosorbent assay (ELISA). The transcription factor neutralizing efficacy of the STAT-1 decoy ODN was verified in vitro in cultured synoviocytes and macrophages. Single administration of STAT-1 decoy ODN dose-dependently suppressed joint swelling and histological signs of acute and chronic arthritis. Delayed-type hypersensitivity (DTH) reaction, serum levels of interleukin-6 (IL-6) and anti-proteoglycan IgG titres were significantly reduced in STAT-1 decoy ODN-treated mice, whereas mBSA, collagen type I and type II specific immunoglobulins were not significantly affected. Intra-articular administration of an anti-CD40L (anti-CD154) antibody was similarly effective. Electrophoretic mobility shift analysis (EMSA) of nuclear extracts from synoviocytes incubated with the STAT-1 decoy ODN in vitro revealed an inhibitory effect on STAT-1. Furthermore, the STAT-1 decoy ODN inhibited the expression of CD40 mRNA in stimulated macrophages. The beneficial effects of the STAT-1 decoy ODN in experimental arthritis presumably mediated in part by affecting CD40 signalling in macrophages may provide the basis for a novel treatment of human RA.
doi:10.1186/ar1869
PMCID: PMC1526583  PMID: 16507120
22.  Mechanism of Action of Phenethylisothiocyanate and Other Reactive Oxygen Species-Inducing Anticancer Agents 
Molecular and Cellular Biology  2014;34(13):2382-2395.
Reactive oxygen species (ROS)-inducing anticancer agents such as phenethylisothiocyanate (PEITC) activate stress pathways for killing cancer cells. Here we demonstrate that PEITC-induced ROS decreased expression of microRNA 27a (miR-27a)/miR-20a:miR-17-5p and induced miR-regulated ZBTB10/ZBTB4 and ZBTB34 transcriptional repressors, which, in turn, downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells. Decreased expression of miR-27a/miR-20a:miR-17-5p by PEITC-induced ROS is a key step in triggering the miR-ZBTB Sp cascade leading to downregulation of Sp TFs, and this is due to ROS-dependent epigenetic effects associated with genome-wide shifts in repressor complexes, resulting in decreased expression of Myc and the Myc-regulated miRs. Knockdown of Sp1 alone by RNA interference also induced apoptosis and decreased pancreatic cancer cell growth and invasion, indicating that downregulation of Sp transcription factors is an important common mechanism of action for PEITC and other ROS-inducing anticancer agents.
doi:10.1128/MCB.01602-13
PMCID: PMC4054319  PMID: 24732804
23.  Anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity. 
British Journal of Cancer  1996;74(10):1545-1552.
The chemosensitising effects of poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide) (PEO-PPO-PEO) block copolymers (Pluronic) in multidrug-resistant cancer cells has been described recently (Alakhov VY, Moskaleva EY, Batrakova EV, Kabanov AV 1996, Biocon. Chem., 7, 209). This paper presents initial studies on in vivo evaluation of Pluronic copolymers in the treatment of cancer. The anti-tumour activity of epirubicin (EPI) and doxorubicin (DOX), solubilised in micelles of Pluronic L61, P85 and F108, was investigated using murine leukaemia P388 and daunorubicin-sensitive Sp2/0 and -resistant Sp2/0(DNR) myeloma cells grown subcutaneously (s.c.). The study revealed that the lifespan of the animals and inhibition of tumour growth were considerably increased in mice treated with drug/copolymer compositions compared with animals treated with the free drugs. The anti-tumour activity of the drug/copolymer compositions depends on the concentration of the copolymer and its hydrophobicity, as determined by the ratio of the lengths of hydrophilic PEO and hydrophobic PPO segments. The data suggest that higher activity is associated with more hydrophobic copolymers. In particular, a significant increase in lifespan (T/C> 150%) and tumour growth inhibition (> 90%) was observed in animals with Sp2/0 tumours with EPI/P85 and DOX/L61 compositions. The effective doses of these compositions caused inhibition of Sp2/0 tumour growth and complete disappearance of tumour in 33-50% of animals. Future studies will focus on the evaluation of the activity of Pluronic-based compositions against human drug-resistant tumours.
PMCID: PMC2074856  PMID: 8932333
24.  Chemopreventative Potential of the Cruciferous Vegetable Constituent Phenethyl Isothiocyanate in a Mouse Model of Prostate Cancer 
Background
This study was undertaken to determine the chemopreventative efficacy of phenethyl isothiocyanate (PEITC), a bioactive constituent of many edible cruciferous vegetables, in a mouse model of prostate cancer, and to identify potential biomarker(s) associated with PEITC response.
Methods
The chemopreventative activity of dietary PEITC was investigated in Transgenic Adenocarcinoma of Mouse Prostate mice that were fed a control diet or one containing 3 μmol PEITC/g (n = 21 mice per group) for 19 weeks. Dorsolateral prostate tissue sections were stained with hematoxylin and eosin for histopathologic evaluations and subjected to immunohistochemistry for analysis of cell proliferation (Ki-67 expression), autophagy (p62 and LC3 protein expression), and E-cadherin expression. Autophagosomes were visualized by transmission electron microscopy. Apoptotic bodies were detected by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling. Plasma proteomics was performed by two-dimensional gel electrophoresis followed by mass spectrometry to identify potential biomarkers of PEITC activity. All statistical tests were two-sided.
Results
Administration of PEITC (3 μmol/g diet) decreased incidence (PEITC diet vs control diet, mean = 21.65 vs 57.58%, difference = −35.93%, 95% confidence interval = −45.48% to −13.10%, P = .04) as well as burden (affected area) (PEITC diet vs control diet, mean = 18.53% vs 45.01%, difference = −26.48%, 95% confidence interval = −49.78% to −3.19%, P = .02) of poorly differentiated tumors in the dorsolateral prostate of transgenic mice compared with control mice, with no toxic effects. PEITC-mediated inhibition of prostate carcinogenesis was associated with induction of autophagy and overexpression of E-cadherin in the dorsolateral prostate. However, PEITC treatment was not associated with a decrease in cellular proliferation, apoptosis induction, or inhibition of neoangiogenesis. Plasma proteomics revealed distinct changes in the expression of several proteins (eg, suppression of clusterin protein) in the PEITC-treated mice compared with control mice.
Conclusions
In this transgenic model, dietary PEITC suppressed prostate cancer progression by induction of autophagic cell death. Potential biomarkers to assess the response to PEITC treatment in plasma were identified.
doi:10.1093/jnci/djr029
PMCID: PMC3071352  PMID: 21330634
25.  Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin 
Molecular nutrition & food research  2011;55(10):1572-1581.
Naturally-occurring chemopreventive agent phenethyl isothiocyanate (PEITC), derived primarily from watercress, has been shown to inhibit cell growth and induce apoptosis in cancer cells. In this study, we examined the potential of PEITC in enhancing cisplatin-induced apoptosis in cervical cancer cells. HeLa cells were exposed to PEITC, cisplatin or both. Pretreatment of cells with PEITC strongly enhanced cisplatin-induced cytotoxicity. PEITC activated the mitogen-activated protein kinases, including JNK, ERK, and p38. The synergistic induction of apoptosis was significantly attenuated by MEK1/2 inhibitor U0126, but not by JNK or p38 inhibitor, suggesting that ERK activation is responsible for the synergistic effect. We found that NF-κB signaling pathway is not involved in the synergistic effect. Sulforaphane and benzyl isothiocyanate, two other members of the isothiocyanate family, also sensitize HeLa cells to apoptosis induced by cisplatin. Furthermore, we found that the synergistic effect was not seen in normal cells. Finally, we demonstrated that Noxa induction was associated with apoptosis induced by PEITC plus cisplatin. Taken together, this study shows that PEITC can sensitize cancer cells to apoptosis induced by cisplatin and this effect is mediated through ERK activation, suggesting the potential of PEITC to be used as an adjuvant with cisplatin in combination therapeutic treatments.
doi:10.1002/mnfr.201000560
PMCID: PMC3561716  PMID: 21595016
Phenethyl isothiocyanate; cisplatin; apoptosis; ERK activation; sensitization

Results 1-25 (1179672)