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1.  An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes 
Journal of ethnopharmacology  2010;130(2):222-230.
Aim of the study
Ginseng has been used as a folk medicine for thousands of years in Asia, and has become a popular herbal medicine world-wide. Recent studies have revealed that ginseng, including American ginseng, exerts antioxidant effects in the cardiovascular system; however, the underlying mechanisms are not fully understood. Thus, we investigated role of Nrf2, a master transcription factor of endogenous anti-oxidative defense systems, in the regulation of American ginseng-mediated anti-oxidative actions in cardiomyocytes.
Materials and methods
A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. H9C2 cells, a rat cardiomyocyte cell line, were exposed to angiotensin II (Ang II) or tumor necrosis factor alpha (TNFα) to induce oxidative stress that was examined by measuring formation of reactive oxygen and nitrogen species. Oxidative stress-induced cell death was induced by exogenous addition of hydrogen peroxide (H2O2). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR. Nrf2-driven transcriptional activity was assessed by antioxidant response element (ARE)-luciferase reporter assay. Direct Nrf2 binding to its target gene promoters was determined by chromatin immunoprecipitation assay. Adenoviral overexpression of Nrf2 shRNA was utilized to knock down Nrf2 in H9C2 cells. Immunochemical staining was applied for Nrf2 expression in the heart.
Results
American ginseng induced dramatic increases in Nrf2 protein expression, Nrf2 nuclear translocation, Nrf2 transcriptional activity, direct Nrf2 binding to its target gene promoters, and expression of a group of anti-oxidative genes driven by Nrf2 in H9C2 cells. In addition, American ginseng inhibited Ang II- or TNFα-induced free radical formation and H2O2-induced cell death in H9C2 cells over-expressed with control shRNA but not in the cells over-expressed with Nrf2 shRNA. Finally, oral administration of American ginseng markedly increased Nrf2 activity in murine hearts.
Conclusion
These results demonstrate that American ginseng suppresses oxidative stress and oxidative stress-induced cell death in cardiomyocytes through activating the Nrf2 pathway, thereby providing cardioprotection against pathological cardiac remodeling.
doi:10.1016/j.jep.2010.03.040
PMCID: PMC3920583  PMID: 20447451
Panax quinquefolius; American ginseng; Nrf2; Oxidative stress; Cardiomyocytes; Cell death
2.  Taming the beast within: resveratrol suppresses colitis and prevents colon cancer 
Aging (Albany NY)  2010;2(4):183-184.
PMCID: PMC2881508  PMID: 20436227
Inflammation; complementary and alternative medicine; SIRT-1; NF-kappaB; inflammatory bowel disease
3.  Nitric Oxide Inactivates the Retinoblastoma Pathway in Chronic Inflammation 
Cancer research  2007;67(19):9286-9293.
Patients with chronic inflammatory bowel disease have a high risk of colon cancer. The molecules that initiate and promote colon cancer and the cancer pathways altered remain undefined. Here, using in vitro models and a mouse model of colitis, we show that nitric oxide (NO) species induce retinoblastoma protein (pRb) hyperphosphorylation and inactivation, resulting in increased proliferation through the pRb-E2F1 pathway. NO-driven pRb hyperphosphorylation occurs through soluble guanylyl cyclase/guanosine 3′,5′-cyclic monophosphate signaling and is dependent on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK/ERK and phosphatidylinositol 3-kinase/AKT pathways. Our results reveal a link between NO and pRb inactivation and provide insight into molecules that can be targeted in the prevention of the inflammation-to-cancer sequence.
doi:10.1158/0008-5472.CAN-07-2238
PMCID: PMC2752153  PMID: 17909036
4.  Nitric oxide as a target of complementary and alternative medicines to prevent and treat inflammation and cancer 
Cancer letters  2008;268(1):10-30.
Nitric oxide (NO) and associated reactive nitrogen species (RNS) are involved in many physiological functions. There has been an ongoing debate to whether RNS can inhibit or perpetuate chronic inflammation and associated carcinogenesis. Although the final outcome depends on the genetic make-up of its target, the surrounding microenvironment, the activity and localization of nitric oxide synthase (NOS) isoforms, and overall levels of NO/RNS, evidence is accumulating that in general, RNS drive inflammation and cancers associated with inflammation. To this end, many complementary and alternative medicines (CAMs) that work in chemoprevention associated with chronic inflammation, are inhibitors of excessive NO observed in inflammatory conditions. Here we review recent literature outlining a role of NO/RNS in chronic inflammation and cancer, and point toward NO as one of several targets for the success of CAMs in treating chronic inflammation and cancer associated with this inflammation.
doi:10.1016/j.canlet.2008.03.024
PMCID: PMC2680023  PMID: 18440130
5.  D-amino acid based protein arginine deiminase inhibitors: Synthesis, pharmacokinetics, and in cellulo efficacy 
ACS medicinal chemistry letters  2012;3(12):1081-1085.
The protein arginine deiminases (PADs) are known to play a crucial role in the onset and progression of multiple inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and cancer. However, it is not known how each of the five PAD isozymes contributes to disease pathogenesis. As such, potent, selective, and bioavailable PAD inhibitors will be useful chemical probes to elucidate the specific roles of each isozyme. Since D-amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of D-amino acid analogs of our pan-PAD inhibitor Cl-amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-amidine and d-o-F-amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-amidine were moderately improved over those of l-Cl-amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.
doi:10.1021/ml300288d
PMCID: PMC3572853  PMID: 23420624
PADs; d-Amino Acids; Cl-amidine; MDA-MB-231 Cells; Pharmacokinetics
6.  Seeing Citrulline: Development of a phenylglyoxal-based probe to visualize protein citrullination 
Journal of the American Chemical Society  2012;134(41):17015-17018.
The Protein Arginine Deiminases (PADs) catalyze the hydrolysis of peptidyl-arginine to form peptidylcitrulline. Abnormally high PAD activity is observed in a host of human diseases, however, the exact role of protein citrullination in these diseases, as well as the identities of specific citrullinated disease biomarkers, remain unknown, largely due to the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.
doi:10.1021/ja308871v
PMCID: PMC3572846  PMID: 23030787
7.  A Key Role of microRNA-29b for the Suppression of Colon Cancer Cell Migration by American Ginseng 
PLoS ONE  2013;8(10):e75034.
Metastasis of colon cancer cells increases the risk of colon cancer mortality. We have recently shown that American ginseng prevents colon cancer, and a Hexane extract of American Ginseng (HAG) has particularly potent anti-inflammatory and anti-cancer properties. Dysregulated microRNA (miR) expression has been observed in several disease conditions including colon cancer. Using global miR expression profiling, we observed increased miR-29b in colon cancer cells following exposure to HAG. Since miR-29b plays a role in regulating the migration of cancer cells, we hypothesized that HAG induces miR-29b expression to target matrix metalloproteinase-2 (MMP-2) thereby suppressing the migration of colon cancer cells. Results are consistent with this hypothesis. Our study supports the understanding that targeting MMP-2 by miR-29b is a mechanism by which HAG suppresses the migration of colon cancer cells.
doi:10.1371/journal.pone.0075034
PMCID: PMC3794036  PMID: 24130681
8.  A hexane fraction of american ginseng suppresses mouse colitis and associated colon cancer: anti-inflammatory and pro-apoptotic mechanisms 
Ulcerative colitis (UC) is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American Ginseng (AG) extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of AG that suppress colitis and prevent colon cancer. Among five different fractions of AG (Butanol, Hexane, Ethylacetate, Dicholoromethane and Water), a Hexane Fraction has particularly potent anti-oxidant and pro-apoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex-vivo model (CD4+/CD25− primary effector T cells). A key in vivo finding was that compared with the whole AG extract, the Hexane Fraction of AG was more potent in treating colitis in a dextran sulfate sodium (DSS) mouse model, as well as suppressing azoxymethane (AOM)/DSS-induced colon cancer. Furthermore, TUNEL labeling of inflammatory cells within the colonic mesenteric lymph nodes (MLN) was elevated in mice consuming DSS + the Hexane Fraction of AG. Results are consistent with our in vitro data, and with the hypothesis that the Hexane Fraction of AG has antiinflammatory properties, and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of AG that suppress colitis, and prevent colon cancer associated with colitis.
doi:10.1158/1940-6207.CAPR-11-0421
PMCID: PMC3324646  PMID: 22293630
Inflammation; Ginseng; Colitis; Hexane; Colon; Apoptosis
9.  Role of resveratrol-induced CD11b+ Gr-1+ myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3+ T cells and amelioration of chronic colitis in IL-10−/− mice 
Brain, behavior, and immunity  2011;26(1):72-82.
Resveratrol, a naturally occurring polyphenol has received significant attention as a potent anti-inflammatory agent. Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population characterized by the co-expression of CD11b+ and Gr-1+ and have long been known for their immunosuppressive function. We report that resveratrol effectively attenuated overall clinical scores as well as various pathological markers of colitis in IL-10−/− mice by down regulating Th1 responses. Resveratrol lessened the colitis-associated decrease in body weight and increased levels of serum amyloid A (SAA), CXCL10 and colon TNF-α, IL-6, RANTES, IL-12 and IL-1β concentrations. After resveratrol treatment, the percentage of CXCR3 expressing T cells was decreased in the spleen, mesenteric lymph nodes (MLN), and intestinal lamina propria (LP). However, the percentage and absolute numbers of CD11b+ and Gr-1+cells in the lamina propria (LP) and spleen were increased after resveratrol treatment as compared with the vehicle treatment. Co-culture of resveratrol induced CD11b+ Gr-1+ cells with T cells, attenuated T cell proliferation, and most importantly reduced IFN-γ and GM-CSF production by LP derived T cells from vehicle treated IL-10−/− mice with chronic colitis. The current study suggests that administration of resveratrol into IL-10−/− mice induces immunosuppressive CD11b+ Gr-1+ MDSCs in the colon, which correlates with reversal of established chronic colitis, and down regulation of mucosal and systemic CXCR3+ expressing effector T cells as well as inflammatory cytokines in the colon. The induction of immunosuppressive CD11b+ Gr-1+ cells by resveratrol during colitis is unique, and suggests an as-yet-unidentified mode of anti-inflammatory action of this plant polyphenol.
doi:10.1016/j.bbi.2011.07.236
PMCID: PMC3506001  PMID: 21807089
Inflammation; Resveratrol; Colitis; CXCR3; CD11b+ and Gr-1+ and MDSCs
10.  A Limited Role of p53 on the Ability of a Hexane Fraction of American Ginseng to Suppress Mouse Colitis 
Ulcerative colitis (UC) is debilitating and carries a high colon cancer risk. Apoptosis of inflammatory cells is a key mechanism regulating UC. We have recently shown that American ginseng (AG), and to a greater extent, a Hexane fraction of AG (HAG) can cause apoptosis and suppress mouse colitis through a p53-mediated mechanism. Here, we tested the hypothesis that HAG suppresses colitis through a p53 mechanism. We found only a limited impact of p53 in the ability of HAG to induce inflammatory cell apoptosis and suppress mouse colitis in vitro and in vivo. Finally, we asked whether HAG could cause cell cycle arrest of HCT116 colon cancer cells in vitro. Interestingly, HAG caused a G1 arrest of such cells independent of p53 status. Findings are significant because HAG suppresses colitis and associated colon cancer, and mutation in p53 is observed in most colitis-driven colon cancers. Therefore, HAG might be very effective in targeting the inflammatory cells and cancer cells since it induces apoptosis of inflammatory cells and cell cycle arrest in both p53−/− and WT p53 colon cancer cells.
doi:10.1155/2012/785739
PMCID: PMC3414200  PMID: 22899889
11.  Suppression of colitis-driven colon cancer in mice by a novel small molecule inhibitor of sphingosine kinase 
Carcinogenesis  2010;31(10):1787-1793.
Sphingolipid metabolism is driven by inflammatory cytokines. These cascade of events include the activation of sphingosine kinase (SK), and subsequent production of the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). Overall, S1P is one of the crucial components in inflammation, making SK an excellent target for the development of new anti-inflammatory drugs. We have recently shown that SK inhibitors suppress colitis and hypothesize here that the novel SK inhibitor, ABC294640, prevents the development of colon cancer. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model, there was a dose-dependent decrease in tumor incidence with SK inhibitor treatment. The tumor incidence (number of animals with tumors per group) in the vehicle, ABC294640 (20 mg/kg) and ABC294640 (50 mg/kg) groups were 80, 40 and 30%, respectively. Tumor multiplicity (number of tumors per animal) also decreased from 2.1 ± 0.23 tumors per animal in the AOM + DSS + vehicle group to 1.2 ± 0 tumors per animal in the AOM + DSS + ABC294640 (20 mg/kg) and to 0.8 ± 0.4 tumors per animal in the AOM + DSS + ABC294640 (50 mg/kg) group. Importantly, with ABC294640, there were no observed toxic side effects. To explore mechanisms, we isolated cells from the colon (CD45−, representing primarily colon epithelial cells) and (CD45+, representing primarily colon inflammatory cells) then measured known targets of SK that control cell survival. Results are consistent with the hypothesis that the inhibition of SK activity by our novel SK inhibitor modulates key pathways involved in cell survival and may be a viable treatment strategy for the chemoprevention colitis-driven colon cancer.
doi:10.1093/carcin/bgq158
PMCID: PMC2981458  PMID: 20688834
12.  Resveratrol suppresses colitis and colon cancer associated with colitis 
Resveratrol is a naturally occurring polyphenol that exhibits pleiotropic health beneficial effects including anti-inflammatory, cardio- and cancer-protective activities. It is recognized as one of the more promising natural molecules in the prevention and treatment of chronic inflammatory and autoimmune disorders. Ulcerative Colitis (UC) is an idiopathic, chronic inflammatory disease of the colon associated with a high colon cancer risk. Here, we used a Dextran Sulfate Sodium (DSS) mouse model of colitis, which resembles human UC pathology. Resveratrol mixed in food ameliorates DSS-induced colitis in mice in a dose-dependent manner. Resveratrol significantly improves inflammation score, down regulates the percentage of neutrophils in the mesenteric lymph nodes and lamina propiria, and modulates CD3+ T cells that express tumor necrosis factor-alpha and interferon gamma. Markers of inflammation and inflammatory stress (p53 and p53-Phospho-Serine 15), are also down regulated by resveratrol. Since chronic colitis drives colon cancer risk, we carried out experiments to determine the chemopreventive properties of resveratrol. Tumor incidence is reduced from 80% in mice treated with Azoxymethane (AOM) + DSS to 20% in AOM + DSS + Resveratrol (300 p.p.m.) treated mice. Tumor multiplicity also decreased with resveratrol treatment. AOM + DSS treated mice had 2.4 ± 0.7 tumors per animal compared with AOM + DSS + 300 p.p.m. resveratrol, which had 0.2 ± 0.13 tumors per animal. The current study indicates that resveratrol is a useful, non-toxic complementary and alternative strategy to abate colitis and potentially colon cancer associated with colitis.
doi:10.1158/1940-6207.CAPR-09-0117
PMCID: PMC2853724  PMID: 20332304
Inflammation; Resveratrol; Colitis; Colon Cancer
13.  American ginseng suppresses colitis through p53 mediated apoptosis of inflammatory cells 
Ulcerative colitis (UC) is a dynamic, chronic inflammatory condition associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism regulating UC. American ginseng (AG) is a putative anti-oxidant that can suppress hyperactive immune cells. We have recently shown that AG can prevent and treat mouse colitis. Because p53 levels are elevated in inflammatory cells in both mouse and human colitis, we tested the hypothesis that AG protects from colitis by driving inflammatory cell apoptosis through a p53 mechanism. We used isogenic p53+/+ and p53−/− inflammatory cell lines, as well as primary CD4+/CD25− effector T cells from p53+/+ and p53−/− mice to show that AG drives apoptosis in a p53-dependent manner. Moreover, we used a dextran sulfate sodium (DSS) model of colitis in C57BL/6 p53+/+ and p53−/− mice to test whether the protective effect of AG against colitis is p53-dependent. Data indicate AG induces apoptosis in p53+/+, but not in isogenic p53−/− cells in vitro. In vivo, C57BL/6 p53+/+ mice are responsive to the protective effects of AG against DSS-induced colitis, while AG fails to protect from colitis in p53−/− mice. Furthermore, TUNEL labeling of inflammatory cells within the colonic mesenteric lymph nodes is elevated in p53+/+ mice consuming DSS + AG, but not in p53−/− mice consuming DSS + AG. Results are consistent with our in vitro data, and with the hypothesis that AG drives inflammatory cell apoptosis in vivo, providing a mechanism by which AG protects from colitis in this DSS mouse model.
doi:10.1158/1940-6207.CAPR-09-0116
PMCID: PMC2833220  PMID: 20179294
p53; Inflammation; Ginseng; Colitis; Colon Cancer
14.  Differential effects of reactive nitrogen species on DNA base excision repair initiated by the alkyladenine DNA glycosylase 
Carcinogenesis  2009;30(12):2123-2129.
Chronic generation of reactive nitrogen species (RNS) can cause DNA damage and may also directly modify DNA repair proteins. RNS-modified DNA is repaired predominantly by the base excision repair (BER) pathway, which includes the alkyladenine DNA glycosylase (AAG). The AAG active site contains several tyrosines and cysteines that are potential sites for modification by RNS. In vitro, we demonstrate that RNS differentially alter AAG activity depending on the site and type of modification. Nitration of tyrosine 162 impaired 1,N6-ethenoadenine (εA)-excision activity, whereas nitrosation of cysteine 167 increased εA excision. To understand the effects of RNS on BER in vivo, we examined intestinal adenomas for levels of inducible nitric oxide synthase (iNOS) and AAG. A striking correlation between AAG and iNOS expression was observed (r = 0.76, P = 0.00002). Interestingly, there was no correlation between changes in AAG levels and enzymatic activity. We found AAG to be nitrated in human adenomas, suggesting that this RNS modification is relevant in the human disease. Expression of key downstream components of BER, apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (POLβ), was also examined. POLβ protein was increased in nearly all adenomas compared with adjacent non-tumor tissues, whereas APE1 expression was only increased in approximately half of the adenomas and also was relocalized to the cytoplasm in adenomas. Collectively, the results suggest that BER is dysregulated in colon adenomas. RNS-induced posttranslational modification of AAG is one mechanism of BER dysregulation, and the type of modification may define the role of AAG during carcinogenesis.
doi:10.1093/carcin/bgp256
PMCID: PMC2792317  PMID: 19864471
15.  Circadian Disruption, Per3, and Human Cytokine Secretion 
Integrative cancer therapies  2009;8(4):329-336.
Circadian disruption has been linked with inflammation, an established cancer risk factor. Per3 clock gene polymorphisms have also been associated with circadian disruption and with increased cancer risk. Patients completed a questionnaire and provided a blood sample prior to undergoing a colonoscopy (n = 70). Adjusted mean serum cytokine concentrations (IL-6, TNF-alpha, gamma-INF, IL-I ra, IL-I-beta, VEGF) were compared among patients with high and low scores for fatigue (Multidimensional Fatigue Inventory), depressive symptoms (Beck Depression Inventory II), or sleep disruption (Pittsburgh Sleep Quality Index), or among patients with different Per3 clock gene variants. Poor sleep was associated with elevated VEGF, and fatigue-related reduced activity was associated with elevated TNF-alpha concentrations. Participants with the 4/5 or 5/5 Per3 variable tandem repeat sequence had elevated IL-6 concentrations compared to those with the 4/4 genotype. Biological processes linking circadian disruption with cancer remain to be elucidated. Increased inflammatory cytokine secretion may playa role.
doi:10.1177/1534735409352029
PMCID: PMC2959170  PMID: 19926609
circadian rhythm; clock gene; cytokine; inflammation
16.  American ginseng preferentially suppresses STAT/iNOS signaling in activated macrophages 
Journal of ethnopharmacology  2009;125(1):145-150.
Aim of the study
Ginseng has been used as general tonic for thousands of years in Asia and becomes a popular herbal medicine all over the world. However, the cellular and molecular mechanisms underlying its benefit effects are less explored. Thus, we investigated the effect of a crude extract from Panax quinquefolius (American ginseng) on suppression of pro-inflammatory responses in macrophages with a focus on signal transducer and activator of transcription (STAT) signaling.
Materials and methods
The crude extract of American ginseng that was supplied by the National Research Council of Canada, Institute for National Measurement Standards (NRCC-INMS) was freshly solvated in Dulbecco’s Modified Eagle Medium (DMEM) prior to each experiment. RAW264.7 cells, a murine macrophage cell line, were exposed to lipopolysaccharide (LPS) to induce inflammatory responses such as expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR (Q-PCR). Activator protein 1 (AP-1)-, nuclear factor-κB (NF-κB)- and STAT-mediated transcriptional activities were investigated using luciferase reporter constructs.
Results
American ginseng inhibited LPS-induced iNOS expression; however, it did not affect LPS-induced COX2 expression. While American ginseng had no impact on LPS-induced activation of AP-1 or NF-κB pathways, it dramatically inhibited LPS-induced activation of STAT signaling. Moreover, American ginseng and AG490, an inhibitor of STAT cascade, synergistically suppressed the LPS-induced iNOS expression.
Conclusion
American ginseng selectively inhibits the expression of iNOS via suppression of STAT cascade but not NF-κB and AP1 pathways in inflamed macrophages. Such a preferential suppression of STAT/iNOS cascade by American ginseng might have therapeutic potential for inflammatory diseases with over-activation of iNOS.
doi:10.1016/j.jep.2009.05.032
PMCID: PMC2790430  PMID: 19505555
Panax. quinquefolius; American ginseng; STAT; Macrophages; Inflammation; iNOS; COX2
17.  Attenuation of Experimental Autoimmune Hepatitis by Exogenous and Endogenous Cannabinoids: Involvement of Regulatory T Cells 
Molecular pharmacology  2008;74(1):20-33.
Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Δ9-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.
doi:10.1124/mol.108.047035
PMCID: PMC2828293  PMID: 18388242
18.  Nitric Oxide Is a Key Component in Inflammation-Accelerated Tumorigenesis 
Cancer research  2008;68(17):7130-7136.
Nitric oxide (NO•), an important signaling molecule and a component of inflammatory response, is involved in tumorigenesis. However, the quantity of NO• and the cellular micro-environment influences the role of NO• in tumor development. We used a genetic strategy to test the hypothesis that an inflammatory microenvironment with an enhanced level of NO• accelerates spontaneous tumor development. C. parvum–induced inflammation and increased NO• synthase-2 (NOS2) expression coincided with accelerated spontaneous tumor development, mostly lymphomas, in p53−/−NOS2+/+ C57BL6 mice when compared with the controls (P = 0.001). However, p53−/−NOS2−/− mice did not show any difference in tumor latency between C. parvum–treated and control groups. In C. parvum–treated p53−/−NOS2+/+ mice, tumor development was preceded by a higher expression of NOS2 and phosphorylated Akt-Ser473 (pAkt-Ser473) in spleen, increased cell proliferation measured by Ki-67 IHC in spleen and thymus, and a lower apoptotic index and CD95-L expression in spleen and thymus. C. parvum–treated p53−/−NOS2+/+ mice showed an increase in the number of Foxp3(+) T-reg cells, dendritic cells (DC), as well as increased CD80+, CD86+, CD40+, and CD83+ on DC in the spleen. Regulatory T-cells (T-reg) and the maturation of DC may modulate tumorigenesis. An increase in the FoxP3(+)T-reg cells in C. parvum–treated p53−/−NOS2+/+ mice indicates a role of NO• in the regulation of T-reg cells that may contribute to a protumor shift of the immune environment favoring an accelerated tumor development. These data provide genetic and mechanistic evidence that an inflammatory microenvironment and an increased level of NO• can accelerate tumor development.
doi:10.1158/0008-5472.CAN-08-0410
PMCID: PMC2576291  PMID: 18757428
19.  Ginkgo biloba extract EGb 761 has anti-inflammatory properties and ameliorates colitis in mice by driving effector T cell apoptosis 
Carcinogenesis  2008;29(9):1799-1806.
Ulcerative colitis is a dynamic, chronic inflammatory condition of the colon associated with an increased colon cancer risk. Ginkgo biloba is a putative antioxidant and has been used for thousands of years to treat a variety of ailments. The aim of this study was to test whether the standardized G.biloba extract, EGb 761, is an antioxidant that can be used to prevent and treat colitis in mice. Here, we show that EGb 761 suppresses the activation of macrophages and can be used to both prevent and treat mouse colitis. Markers of inflammation (iNOS, Cox-2 and tumor necrosis factor-α) and inflammatory stress (p53 and p53-phospho-serine 15) are also downregulated by EGb 761. Furthermore, we show that EGb 761 reduces the numbers of CD4+/CD25−/Foxp3− effector T cells in the colon. Interestingly, EGb 761 drives CD4+ effector T cell apoptosis in vitro and in vivo, providing a mechanistic explanation to the reduction in numbers of this cell type in the colon. This current study is in agreement with previous studies supporting a use of EGb 761 as a complementary and alternative strategy to abate colitis and associated colon cancer.
doi:10.1093/carcin/bgn143
PMCID: PMC2527648  PMID: 18567620
20.  Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β 
PLoS ONE  2009;4(8):e6493.
Background
Altered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β over-expression has not yet been evaluated in a mouse model.
Methodology/Principal Findings
We have recently developed a novel transgenic mouse model that over-expresses Pol β. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β expression in esophageal adenocarcinomas and squamous carcinomas.
Conclusions/Significance
These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.
doi:10.1371/journal.pone.0006493
PMCID: PMC2716528  PMID: 19654874
21.  American ginseng suppresses inflammation and DNA damage associated with mouse colitis 
Carcinogenesis  2008;29(12):2351-2359.
Ulcerative colitis (UC) is a dynamic, idiopathic, chronic inflammatory condition associated with a high colon cancer risk. American ginseng has antioxidant properties and targets many of the players in inflammation. The aim of this study was to test whether American ginseng extract prevents and treats colitis. Colitis in mice was induced by the presence of 1% dextran sulfate sodium (DSS) in the drinking water or by 1% oxazolone rectally. American ginseng extract was mixed in the chow at levels consistent with that currently consumed by humans as a supplement (75 p.p.m., equivalent to 58 mg daily). To test prevention of colitis, American ginseng extract was given prior to colitis induction. To test treatment of colitis, American ginseng extract was given after the onset of colitis. In vitro studies were performed to examine mechanisms. Results indicate that American ginseng extract not only prevents but it also treats colitis. Inducible nitric oxide synthase and cyclooxygenase-2 (markers of inflammation) and p53 (induced by inflammatory stress) are also downregulated by American ginseng. Mucosal and DNA damage associated with colitis is at least in part a result of an oxidative burst from overactive leukocytes. We therefore tested the hypothesis that American ginseng extract can inhibit leukocyte activation and subsequent epithelial cell DNA damage in vitro and in vivo. Results are consistent with this hypothesis. The use of American ginseng extract represents a novel therapeutic approach for the prevention and treatment of UC.
doi:10.1093/carcin/bgn211
PMCID: PMC2639244  PMID: 18802031
22.  The p53 Tumor Suppressor Network Is a Key Responder to Microenvironmental Components of Chronic Inflammatory Stress 
Cancer research  2005;65(22):10255-10264.
Activation of the p53 network plays a central role in the inflammatory stress response associated with ulcerative colitis and may modulate cancer risk in patients afflicted with this chronic disease. Here, we describe the gene expression profiles associated with four microenvironmental components of the inflammatory response (NO•, H2O2, DNA replication arrest, and hypoxia) that result in p53 stabilization and activation. Isogenic HCT116 and HCT116 TP53−/− colon cancer cells were exposed to the NO• donor Sper/NO, H2O2, hypoxia, or hydroxyurea, and their mRNA was analyzed using oligonucleotide microarrays. Overall, 1,396 genes changed in a p53-dependent manner (P < 0.001), with the majority representing a “unique” profile for each condition. Only 14 genes were common to all four conditions. Included were eight known p53 target genes. Hierarchical sample clustering distinguished early (1 and 4 hours) from late responses (8, 12, and 24 hours), and each treatment was differentiated from the others. Overall, NO• and hypoxia stimulated similar transcriptional responses. Gene ontology analysis revealed cell cycle as a key feature of stress responses and confirmed the similarity between NO• and hypoxia. Cell cycle profiles analyzed by flow cytometry showed that NO• and hypoxia induced quiescent S-phase and G2-M arrest. Using a novel bioinformatic algorithm, we identified several putative p53-responsive elements among the genes induced in a p53-dependent manner, including four [KIAA0247, FLJ12484, p53CSV (HSPC132), and CNK (PLK3)] common to all exposures. In summary, the inflammatory stress response is a complex, integrated biological network in which p53 is a key molecular node regulating gene expression.
doi:10.1158/0008-5472.CAN-05-1714
PMCID: PMC1421332  PMID: 16288013
24.  The adaptive imbalance in base excision–repair enzymes generates microsatellite instability in chronic inflammation 
Journal of Clinical Investigation  2003;112(12):1887-1894.
Chronic infection and associated inflammation are key contributors to human carcinogenesis. Ulcerative colitis (UC) is an oxyradical overload disease and is characterized by free radical stress and colon cancer proneness. Here we examined tissues from noncancerous colons of ulcerative colitis patients to determine (a) the activity of two base excision–repair enzymes , AAG, the major 3-methyladenine DNA glycosylase, and APE1, the major apurinic site endonuclease; and (b) the prevalence of microsatellite instability (MSI). AAG and APE1 were significantly increased in UC colon epithelium undergoing elevated inflammation and MSI was positively correlated with their imbalanced enzymatic activities. These latter results were supported by mechanistic studies using yeast and human cell models in which overexpression of AAG and/or APE1 was associated with frameshift mutations and MSI. Our results are consistent with the hypothesis that the adaptive and imbalanced increase in AAG and APE1 is a novel mechanism contributing to MSI in patients with UC and may extend to chronic inflammatory or other diseases with MSI of unknown etiology.
doi:10.1172/JCI200319757
PMCID: PMC296999  PMID: 14679184
25.  The Induction of microRNA-16 in Colon Cancer Cells by Protein Arginine Deiminase Inhibition Causes a p53-Dependent Cell Cycle Arrest 
PLoS ONE  2013;8(1):e53791.
Protein Arginine Deiminases (PADs) catalyze the post-translational conversion of peptidyl-Arginine to peptidyl-Citrulline in a calcium-dependent, irreversible reaction. Evidence is emerging that PADs play a role in carcinogenesis. To determine the cancer-associated functional implications of PADs, we designed a small molecule PAD inhibitor (called Chor-amidine or Cl-amidine), and tested the impact of this drug on the cell cycle. Data derived from experiments in colon cancer cells indicate that Cl-amidine causes a G1 arrest, and that this was p53-dependent. In a separate set of experiments, we found that Cl-amidine caused a significant increase in microRNA-16 (miRNA-16), and that this increase was also p53-dependent. Because miRNA-16 is a putative tumor suppressor miRNA, and others have found that miRNA-16 suppresses proliferation, we hypothesized that the p53-dependent G1 arrest associated with PAD inhibition was, in turn, dependent on miRNA-16 expression. Results are consistent with this hypothesis. As well, we found the G1 arrest is at least in part due to the ability of Cl-amidine-mediated expression of miRNA-16 to suppress its' G1-associated targets: cyclins D1, D2, D3, E1, and cdk6. Our study sheds light into the mechanisms by which PAD inhibition can protect against or treat colon cancer.
doi:10.1371/journal.pone.0053791
PMCID: PMC3538596  PMID: 23308284

Results 1-25 (27)