15-Keto prostaglandin E2 induces heme oxygenase-1 expression through activation of Nrf2 in human colon epithelial CCD 841 CoN cells

Prostaglandin E-2 (PGE(2)) plays a key role in inflammation-associated carcinogenesis. NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S)-hydroxyl group of PGE(2) to generate 15-keto PGE(2). 15-PGDH has been known as a tumor suppressor in various malignancies including colon cancer. However, the molecular mechanisms underlying the tumor-suppressive function of 15-PGDH remain largely unresolved. In this study, we found that 15-keto PGE(2) upregulated the expression of heme oxygenase-1 (HO-1), a representative antioxidative and anti-inflammatory enzyme, at both transcriptional and translational levels, in human colon epithelial CCD 841 CoN cells. A redox-sensitive transcription factor, NF-E2-related factor (Nrf2) plays a critical role in the regulation of HO-1 and other cytoprotective proteins. 15-Keto PGE(2) induced translocation of Nrf2 into the nucleus and antioxidant response element-driven luciferase activity. Furthermore, the silencing of the Nrf2 gene abolished 15-keto PGE(2)-induced HO-1 expression in CCD 841 CoN cells. 15-Keto PGE(2) activated AKT signaling, and the pharmacological AKT inhibitor, LY294002 suppressed the 15-keto PGE(2)-induced HO-1 expression. 15-Keto PGE(2) generates the reactive oxygen species which is suppressed by the general antioxidant N-acetyl-L-cysteine. N-acetyl-L-cysteine treatment attenuated the 15-keto PGE(2)-induced phosphorylation of GSK3 beta, transcriptional activity of Nrf2, and subsequently HO-1 expression. However, 13,14-dihydro-15-keto PGE(2) lacking the alpha,beta-unsaturated carbonyl moiety failed to induce intracellular production of reactive oxygen species, HO-1 expression and nuclear translocation of Nrf2. In conclusion, 15-keto PGE(2) induces HO-1 expression through Nrf2 activation in human colon epithelial cells.

Effects of Genetic and Pharmacologic Inhibition of COX-2 on Colitis-associated Carcinogenesis in Mice

COX-2 has been inappropriately overexpressed in various human malignancies, and is considered as one of the representative targets for the chemoprevention of inflammation-associated cancer. In order to assess the role of COX-2 in colitis-induced carcinogenesis, the selective COX-2 inhibitor celecoxib and COX-2 null mice were exploited in an azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted murine colon carcinogenesis model. The administration of 2% DSS in drinking water for 1 week after a single intraperitoneal injection of AOM produced colorectal adenomas in 83% of mice, whereas only 27% of mice given AOM alone developed tumors. Oral administration of celecoxib significantly lowered the incidence as well as the multiplicity of colon tumors. The expression of COX-2 and inducible nitric oxide synthase (iNOS) was upregulated in the colon tissues of mice treated with AOM and DSS, and this was inhibited by celecoxib administration. Likewise, celecoxib treatment abrogated the DNA binding of NF-kappa B, a key transcription factor responsible for regulating expression of aforementioned pro-inflammatory enzymes, which was associated with suppression of I kappa B alpha degradation. In the COX-2 null (COX-2(-/-)) mice, there was about 30% reduction in the incidence of colon tumors, and the tumor multiplicity was also markedly reduced (7.7 +/- 2.5 vs. 2.43 +/- 1.4, P < 0.01). As both pharmacologic inhibition and genetic ablation of COX- 2 gene could not completely suppress colon tumor formation following treatment with AOM and DSS, it is speculated that other pro-inflammatory mediators, including COX-1 and iNOS, should be additionally targeted to prevent inflammation-associated colon carcinogenesis.

Fibroblast growth factor-2, derived from cancer-associated fibroblasts, stimulates growth and progression of human breast cancer cells via FGFR1 signaling

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. In the present study, we investigated the role for CAFs in breast cancer progression and underlying molecular mechanisms. Human breast cancer MDA-MB-231 cells treated with the CAF-conditioned media manifested a more proliferative phenotype, as evidenced by enhanced messenger RNA (mRNA) expression of Cyclin D1, c-Myc, and proliferating cell nuclear antigen. Analysis of data from The Cancer Genome Atlas revealed that fibroblast growth factor-2 (FGF2) expression was well correlated with the presence of CAFs. We noticed that the mRNA level of FGF2 in CAFs was higher than that in normal fibroblasts. FGF2 exerts its biological effects through interaction with FGF receptor 1 (FGFR1). In the breast cancer tissue array, 42% estrogen receptor-negative patients coexpressed FGF2 and FGFR1, whereas only 19% estrogen receptor-positive patients exhibited coexpression. CAF-stimulated MDA-MB-231 cell migration and invasiveness were abolished when FGF2-neutralizing antibody was added to the conditioned media of CAFs. In a xenograft mouse model, coinjection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced tumor growth, and this was abrogated by silencing of FGFR1 in cancer cells. In addition, treatment of MDA-MB-231 cells with FGF2 enhanced expression of Cyclin D1, a key molecule involved in cell cycle progression. FGF2-induced cell migration and upregulation of Cyclin D1 were abolished by siRNA-mediated FGFR1 silencing. Taken together, the above findings suggest that CAFs promote growth, migration and invasion of MDA-MB-231 cells via the paracrine FGF2-FGFR1 loop in the breast tumor microenvironment.

Changes in Microbial Community Composition Related to Sex and Colon Cancer by Nrf2 Knockout

The frequency of azoxymethane/dextran sulfate sodium (AOM/DSS)-induced carcinogenesis in male mice is higher than that in female mice. Previous studies have reported that 17β-estradiol inhibits tumorigenesis in males by modulating nuclear factor-erythroid 2-related factor 2 (Nrf2). This study aimed to investigate the changes in mouse gut microbiome composition based on sex, AOM/DSS-induced colorectal cancer (CRC), and Nrf2 genotype. The gut microbiome composition was determined by 16S rRNA gene sequencing fecal samples obtained at week 16 post-AOM administration. In terms of sex differences, our results showed that the wild-type (WT) male control mice had higher alpha diversity (i.e. Chao1, Shannon, and Simpson) than the WT female control mice. The linear discriminant analysis effect size (LEfSe) results revealed that the abundances of Akkermansia muciniphila and Lactobacillus murinus were higher in WT male control mice than in WT female controls. In terms of colon tumorigenesis, the alpha diversity of the male CRC group was lower than that of the male controls in both WT and Nrf2 KO, but did not show such changes in females. Furthermore, the abundance of A. muciniphila was higher in male CRC groups than in male controls in both WT and Nrf2 KO. The abundance of Bacteroides vulgatus was higher in WT CRC groups than in WT controls in both males and females. However, the abundance of L. murinus was lower in WT female CRC and Nrf2 KO male CRC groups than in its controls. The abundance of A. muciniphila was not altered by Nrf2 KO. In contrast, the abundances of L. murinus and B. vulgatus were changed differently by Nrf2 KO depending on sex and CRC. Interestingly, L. murinus showed negative correlation with tumor numbers in the whole colon. In addition, B. vulgatus showed positive correlation with inflammatory markers (i.e. myeloperoxidase and IL-1β levels), tumor numbers, and high-grade adenoma, especially, developed mucosal and submucosal invasive adenocarcinoma at the distal part of the colon. In conclusion, Nrf2 differentially alters the gut microbiota composition depending on sex and CRC induction

17β-estradiol reduces inflammation and modulates antioxidant enzymes in colonic epithelial cells

Background/Aims: Estrogen is known to have protective effect in colorectal cancer development. The aims of this study are to investigate whether estradiol treatment reduces inflammation in CCD841CoN, a female human colonic epithelial cell line and to uncover underlying mechanisms of estradiol effects. Methods: 17 beta-Estradiol (E2) effect was measured by Western blot after inducing inflammation of CCD841CoN by tumor necrosis factor alpha (TNF-alpha). Expression levels of estrogen receptor alpha (ER alpha) and beta (ER beta), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-kappa B), heme oxygenase-1 (HO-1), and NAD(P)H-quinone oxidoreductase-1 (NQO-1) were also evaluated. Results: E2 treatment induced expression of ERO but did not increase that of ER alpha. E2 treatment for 48 hours significantly elevated the expression of anti-oxidant enzymes, HO-1 and NQO-1. TNF-alpha treatment significantly increased the level of activated NF-kappa B (p < 0.05), and this increase was significantly suppressed by treatment of to nM of E2 (p < 0.05). E2 treatment ameliorated TNF-alpha-induced COX-2 expression and decrease of HO-1 expression. 4-(2-phenyl-5,7-bis(trifluoromethyl) pyrazolo(1,5-a)pyrimidin-3-yl)phenol (PHTPP), antagonist of ER beta, removed the inhibitory effect of E2 in the TNF-alpha-induced COX-2 expression (p = 0.05). Conclusions: Estrogen seems to inhibit inflammation in female human colonic epithelial cell lines, through down-regulation of NF-kappa B and COX-2 expression and induction of anti-oxidant enzymes such as HO-1 and NQO-1.

Curcumin induces expression of 15-hydroxyprostaglandin dehydrogenase in gastric mucosal cells and mouse stomach in vivo: AP-1 as a potential target

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the conversion of oncogenic prostaglandin E-2 to non-tumerigenic 15-keto prostaglandin E-2. In the present study, we found that curcumin, a yellow coloring agent present in the rhizome of Curcuma Tonga Linn (Zingiberaceae), induced expression of 15-PGDH at the both transcriptional and translational levels in normal rat gastric mucosal cells. By using deletion constructs of 15-PGDH promoter, we were able to demonstrate that activator protein-1 (AP-1) is the principal transcription factor responsible for regulating curcumin-induced 15-PGDH expression. Curcumin enhanced the expression of c-jun and cFos that are functional subunits of AP-1, in the nuclear fraction of cells. Silencing of c-jun suppressed curcumin-induced expression of 15-PGDH. Moreover, the chromatin immunoprecipitation assay revealed curcumin-induced binding of c-Jun to the AP-1 consensus sequence present in the 15-PGDH promoter. Curaimin increased phosphorylation of ERK1/2 and JNK. and pharmacologic inhibition of these kinases abrogated the curcumin-induced phosphorylation of clun and 15-PGDH expression. In contrast, tetrahydrocurcumin which lacks the alpha,beta-unsaturated carbonyl group failed to induce 15-PGDH expression, suggesting that the electrophilic carbonyl group of curcumin is essential for its induction of 15-PGDH expression. Curcumin restored the expression of 15-PGDH which is down-regulated by Helicobater pylori through suppression of DNA methyltransferase 1. In addition, oral administration of curcumin increased the expression of 15-PGDH and its regulators such as p-ERK1/2, p-JNK and c-Jun in the mouse stomach. Taken together, these findings suggest that curcumin-induced upregulation of 15-PGDH may contribute to chemopreventive effects of this phytochemical on inflammation-associated gastric carcinogenesis. (C) 2020 Elsevier Inc. All rights reserved.

17β-Estradiol supplementation changes gut microbiota diversity in intact and colorectal cancer-induced ICR male mice

The composition of the gut microbiota is influenced by sex hormones and colorectal cancer (CRC). Previously, we reported that 17 beta -estradiol (E2) inhibits azoxymethane/dextran sulfate sodium (AOM/DSS)-induced tumorigenesis in male mice. Here, we investigated whether the composition of the gut microbiota is different between male and female, and is regulated by estrogen as a secondary outcome of previous studies. We established four groups of mice based on the sex and estrogen status [ovariectomized (OVX) female and E2-treated male]. Additionally, three groups of males were established by treating them with AOM/DSS, and E2, after subjecting them to AOM/DSS treatment. The mice were sacrificed at 21 weeks old. The composition of the gut microbiota was analyzed using 16S rRNA metagenomics sequencing. We observed a significant increase in the microbial diversity (Chao1 index) in females, males supplemented with E2, and males treated with AOM/DSS/E2 compared with normal males. In normal physiological condition, sex difference and E2 treatment did not affect the ratio of Firmicutes/Bacteroidetes (F/B). However, in AOM/DSS-treated male mice, E2 supplementation showed significantly lower level of the F/B ratio. The ratio of commensal bacteria to opportunistic pathogens was higher in females and E2-treated males compared to normal males and females subjected to OVX. Unexpectedly, this ratio was higher in the AOM/DSS group than that determined in other males and the AOM/DSS/E2 group. Our findings suggest that estrogen alters the gut microbiota in ICR (CrljOri:CD1) mice, particularly AOM/DSS-treated males, by decreasing the F/B ratio and changing Shannon and Simpson index by supply of estrogen. This highlights another possibility that estrogen could cause changes in the gut microbiota, thereby reducing the risk of developing CRC.

Nuclear Factor Erythroid-Derived 2-Like 2-Induced Reductive Stress Favors Self-Renewal of Breast Cancer Stem-Like Cells via the FoxO3a-Bmi-1 Axis

Aims: A subpopulation of cancer cells, termed cancer stem cells (CSCs), has stemness properties, such as self-renewal and differentiation, which drive cancer recurrence and tumor resistance. CSCs possess enhanced protection capabilities to maintain reduced intracellular levels of reactive oxygen species (ROS) compared with nonstem-like cancer cells. This study investigated whether reductive stress could regulate self-renewal activity in breast CSCs. Results: We found that manifestation of stemness in breast cancer stem-like cells was associated with an elevated production of reduced glutathione (GSH) maintained by upregulation of glutamate cysteine ligase catalytic subunit (GCLC) and consequently, lowered ROS levels. This was accompanied by upregulation of phospho-AMP-activated protein kinase, FoxO3a, and Bmi-1. Notably, expression of nuclear factor erythroid-derived 2-like 2 (Nrf2) protein was substantially increased in cells undergoing sphere formation. We noticed that expression of Bmi-1 was inhibited after introduction of Nrf2 short interfering RNA into MCF-7 mammosphere cells. Silencing of Nrf2 expression suppressed the xenograft growth of subcutaneously or orthotopically injected human breast cancer cells. Innovation: Association between Nrf2 and self-renewal signaling in CSCs has been reported, but the underlying molecular mechanism remains largely unresolved. This study demonstrates the Nrf2-mediated signaling pathway in maintenance of reductive stress in breast CSCs. Conclusion: Nrf2 overactivation in breast CSCs upregulates GCLC expression and consequently enhances GSH biosynthesis with concurrent reduction in intracellular ROS accumulation, thereby provoking the reductive stress. The consequent upregulation of nuclear FoxO3a and its binding to the promoter of the gene encoding Bmi-1 account for the self-renewal activity of breast cancer stem-like cells and their growth in a xenograft mouse model.

An Electrophilic Deguelin Analogue Inhibits STAT3 Signaling in H-Ras-Transformed Human Mammary Epithelial Cells: The Cysteine 259 Residue as a Potential Target

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signals that are often constitutively activated in many cancerous or transformed cells and some stromal cells in the tumor microenvironment. Persistent STAT3 activation in malignant cells stimulates proliferation, survival, angiogenesis, invasion, and tumor-promoting inflammation. STAT3 undergoes activation through phosphorylation on tyrosine 705, which facilitates its dimerization. Dimeric STAT3 translocates to the nucleus, where it regulates the transcription of genes involved in cell proliferation, survival, etc. In the present study, a synthetic deguelin analogue SH48, discovered by virtual screening, inhibited the phosphorylation, nuclear translocation, and transcriptional activity of STAT3 in H-ras transformed human mammary epithelial MCF-10A cells (MCF10A-ras). We speculated that SH48 bearing an alpha,beta-unsaturated carbonyl group could interact with a thiol residue of STAT3, thereby inactivating this transcription factor. Non-electrophilic analogues of SH48 failed to inhibit STAT3 activation, lending support to the above supposition. By utilizing a biotinylated SH48, we were able to demonstrate the complex formation between SH48 and STAT3. SH48 treatment to MCF10A-ras cells induced autophagy, which was verified by staining with a fluorescent acidotropic probe, LysoTracker Red, as well as upregulating the expression of LC3II and p62. In conclusion, the electrophilic analogue of deguelin interacts with STAT3 and inhibits its activation in MCF10A-ras cells, which may account for its induction of autophagic death.

The Role of Nutrition in Influencing Mechanisms Involved in Environmentally Mediated Diseases

Human exposure to environmental contaminants such as persistent chlorinated organics, heavy metals, pesticides, phthalates, flame retardants, electronic waste and airborne pollutants around the world, and especially in Southeast Asian regions, are significant and require urgent attention. Given this widespread contamination and abundance of such toxins as persistent organic pollutants (POPs) in the ecosystem, it is unlikely that remediation alone will be sufficient to address the health impacts associated with this exposure. Furthermore, we must assume that the impact on health of some of these contaminants results in populations with extraordinary vulnerabilities to disease risks. Further exacerbating risk; infectious diseases, poverty and malnutrition are common in the Southeast Asian regions of the world. Thus, exploring preventive measures of environmental exposure and disease risk through new paradigms of environmental toxicology, optimal and/or healthful nutrition and health is essential. For example, folic acid supplementation can lower blood arsenic levels, and plant-derived bioactive nutrients can lower cardiovascular and cancer risks linked to pollutant exposure. Data also indicate that diets enriched with bioactive food components such as polyphenols and omega-3 polyunsaturated fatty acids can prevent or decrease toxicant-induced inflammation. Thus, consuming healthy diets that exhibit high levels of antioxidant and anti-inflammatory properties, is a meaningful way to reduce the vulnerability to non-communicable diseases linked to environmental toxic insults. This nutritional paradigm in environmental toxicology requires further study in order to improve our understanding of the relationship between nutrition or other lifestyle modifications and toxicant-induced diseases. Understanding mechanistic relationships between nutritional modulation of environmental toxicants and susceptibility to disease development are important for both cumulative risk assessment and the design and implementation of future public health programs and behavioral interventions