Sin-Aye Park

15-Deoxy-Δ12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS

Recent studies suggest that inflammation is causally linked to carcinogenesis. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandins, is inappropriately expressed in various cancers and hence recognized as one of the hallmarks of chronic inflammation-associated malignancies. However, the mechanistic role of COX-2 as a link between inflammation and cancer remains undefined. Here, we report that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), one of the final products of COX-mediated arachidonic acid metabolism, upregulates the expression of COX-2 in the human breast cancer MCF-7 cell line. 15d-PGJ(2)-induced COX-2 expression was mediated by activation of Akt and subsequently activator protein-1 (AP-1). Furthermore, 15d-PGJ(2) formed reactive oxygen species, which led to increased phosphorylation of Akt, DNA binding of AP-1 and expression of COX-2. In contrast to 15d-PGJ(2), 9,10-dihydro-15d-PGJ(2) did not elicit any of effects induced by 15d-PGJ(2) in this study, suggesting that an electrophilic carbon center present in 15d-PGJ(2) is critical for COX-2 expression as well activation of upstream signal transduction induced by this cyclopentenone prostaglandin. Taken together, these observations suggest that 15d-PGJ(2) produced by COX-2 overexpression may function as a positive regulator of COX-2 in human breast cancer MCF-7 cells.

4-Hydroxyestradiol Induces Anchorage-Independent Growth of Human Mammary Epithelial Cells via Activation of IκB Kinase: Potential Role of Reactive Oxygen Species

Estrogen is converted by cytochrome P450 1B1 to 4-hydroxyestradiol (4-OHE(2)), a putative carcinogenic metabolite of estrogen. This catechol estrogen metabolite is oxidized further to produce a reactive quinone via semiquinone. Redox cycling between 4-OHE(2) and its quinoid generates reactive oxygen species (ROS). ROS not only causes oxidative DNA damage but also promotes neoplastic transformation of initiated cells. In the present study, 4-OHE(2) induced anchorage-independent colony formation in human mammary epithelial cells (MCF-10A). MCF-10A cells treated with 4-OHE(2) exhibited increased accumulation of intracellular ROS. The antioxidant N-acetyl-l-cysteine inhibited the neoplastic transformation induced by 4-OHE(2). ROS overproduced by 4-OHE(2) increased the nuclear translocation of nuclear factor-kappaB (NF-kappaB) and its DNA binding through induction of IkappaB kinase alpha (IKKalpha) and IKKbeta activities. The inhibition of the IKK activities with Bay 11-7082 significantly reduced the anchorage-independent growth induced by 4-OHE(2). The 4-OHE(2)-induced activation of extracellular signal-regulated kinase and Akt resulted in enhanced IKK activities and phosphorylation of IkappaBalpha, thereby inducing NF-kappaB activation and anchorage-independent growth of MCF-10A cells. In conclusion, ROS, concomitantly overproduced during redox cycling of 4-OHE(2), activates IKK signaling, which may contribute to neoplastic transformation of MCF-10A cells.

Keap1 Cysteine 288 as a Potential Target for Diallyl Trisulfide-Induced Nrf2 Activation

Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.

KG-135 inhibits COX-2 expression by blocking the activation of JNK and AP-1 in phorbol ester-stimulated human breast epithelial cells.

Abstract:  Ginsenosides, ingredients of ginseng, have a wide array of pharmacologic effects. Especially, ginsenosides Rk1, Rg3, and Rg5 derived from heat‐processed ginseng have been shown to possess substantial anti‐tumor‐promoting effects. KG‐135 is a formulated complex that contains several antitumorigenic ginsenosides, such as Rk1, Rg3, Rg5, Rk2, Rk3, Rs3, Rs4, Rs5, Rs6, Rs7, etc. The present article was aimed at evaluating the chemopreventive as well as anti‐inflammatory effects of KG‐135 in the human breast epithelial cell line (MCF‐10A). One of the well‐recognized molecular targets for chemoprevention is cyclooxygenase‐2 (COX‐2) that is abnormally upregulated in many premalignant and malignant tissues and cells. In this study, we found that KG‐135 inhibited COX‐2 expression in MCF‐10A cells stimulated with a prototype tumor promotor 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Since the transcription factor activator protein‐1 (AP‐1) plays a role in tumor promotion and is also known to regulate COX‐2 induction, we attempted to determine the effect of KG‐135 on TPA‐induced activation of AP‐1. Cotreatment with KG‐135 resulted in a decrease in TPA‐induced DNA binding of AP‐1. In addition, KG‐135 inhibited TPA‐induced phosphorylation of c‐Jun N‐terminal kinases (JNK) that regulates COX‐2 expression in MCF‐10A cells. The JNK inhibitor SP600125 attenuated COX‐2 expression in TPA‐treated MCF‐10A cells. Taken together, the above findings suggest that KG‐135 inhibits TPA‐induced COX‐2 expression in MCF‐10A cells by blocking the JNK/AP‐1 signaling pathway.

Helicobacter pylori Activates IL-6-STAT3 Signaling in Human Gastric Cancer Cells: Potential Roles for Reactive Oxygen Species.

Recent studies have shown that Helicobacter pylori (H. pylori) activates signal transducer and activator of transcription 3 (STAT3) that plays an important role in gastric carcinogenesis. However, the molecular mechanism underlying H. pylori‐mediated STAT3 activation is still not fully understood. In this study, we investigated H. pylori‐induced activation of STAT3 signaling in AGS human gastric cancer cells and the underlying mechanism.

Resveratrol suppresses 4-hydroxyestradiol-induced transformation of human breast epithelial cells by blocking IκB kinaseβ-NF-κB signalling

Abstract Excess estrogen stimulates the proliferation of mammary epithelial cells and hence represents a major risk factor for breast cancer. Estrogen is subjected to cytochrome P450-catalysed oxidative metabolism to produce an oncogenic catechol estrogen, 4-hydroxyestradiol (4-OHE2). 4-OHE2 undergoes redox cycling during which reactive oxygen species (ROS) as well as the chemically reactive estrogen semiquinone and quinone intermediates are produced, thereby contributing to hormonal carcinogenesis. Resveratrol (3,4′,5-trihydroxy stilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the inhibitory effects of resveratrol on 4-OHE2-induced transformation of human breast epithelial MCF-10A cells. Resveratrol inhibited migration and anchorage-independent growth of MCF-10A cells treated with 4-OHE2. Resveratrol treatment suppressed the 4-OHE2-induced activation of IκB kinaseβ (IKKβ) and phosphorylation of IκBα, and consequently NF-κB DNA binding activity and cyclooxygenase-2 (COX-2) expression. Resveratrol suppressed ROS production and phosphorylation of Akt and ERK induced by 4-OHE2 treatment. In conclusion, resveratrol blocks activation of IKKβ-NF-κB signalling and induction of COX-2 expression in 4-OHE2-treated MCF-10A cells, thereby suppressing migration and transformation of these cells.

4-Hydroxyestradiol induces mammary epithelial cell transformation through Nrf2-mediated heme oxygenase-1 overexpression

Estrogen (17β-estradiol, E2) undergoes oxidative metabolism by CYP1B1 to form 4-hydroxyestradiol (4-OHE2), a putative carcinogenic metabolite of estrogen. Our previous study showed that 4-OHE2-induced production of reactive oxygen species contributed to neoplastic transformation of human breast epithelial (MCF-10A) cells. In this study, 4-OHE2, but not E2, increased the expression of heme oxygenase-1 (HO-1), a sensor and regulator of oxidative stress, in MCF-10A cells. Silencing the HO-1 gene in MCF-10A cells suppressed 4-OHE2-induced cell proliferation and transformation. In addition, subcutaneous administration of 4-OHE2 markedly enhanced the growth of the MDA-MB-231 human breast cancer xenografts, which was retarded by zinc protoporphyrin, a pharmacological inhibitor of HO-1. 4-OHE2-induced HO-1 expression was mediated by NF-E2-related factor 2 (Nrf2). We speculate that an electrophilic quinone formed as a consequence of oxidation of 4-OHE2 binds directly to Kelch-like ECH-associated protein 1 (Keap1), an inhibitory protein that sequesters Nrf2 in the cytoplasm. This will diminish association between Nrf2 and Keap1. 4-OHE2 failed to interrupt the interaction between Keap1 and Nrf2 and to induce HO-1 expression in Keap1-C273S or C288S mutant cells. Lano-LC-ESI-MS/MS analysis in MCF-10A-Keap1-WT cells which were treated with 4-OHE2 revealed that the peptide fragment containing Cys288 gained a molecular mass of 287.15 Da, equivalent to the addition of a single molecule of 4-OHE2-derived ortho-quinones.

Hypoxia induces epithelial-mesenchymal transition in colorectal cancer cells through ubiquitin-specific protease 47-mediated stabilization of Snail: A potential role of Sox9

During the metastatic phase, cancer cells require the dissolution of cadherin-mediated cell-cell adhesion and a dramatic re-organization of the cytoskeleton through epithelial-mesenchymal transition (EMT), thereby acquiring migratory and invasive capabilities. In most tumors, EMT is accompanied by hypoxia. However, the intracellular signaling molecule that mediates hypoxia-induced EMT remained overlooked. By utilizing the microarray database system of the Cancer Genome Atlas, we identified ubiquitin-specific protease 47 (USP47), a deubiquitinating enzyme, as a potential mediator of hypoxia-induced EMT. Immunofluorescence staining of human colorectal tissue microarrays revealed that USP47 is overexpressed in colorectal adenocarcinoma tissues compared with normal adjacent tissues. The expression of USP47 was found to be elevated in three different human colorectal cancer cell lines. The enhancement of USP47 in colorectal cancer cells under hypoxic conditions induced the disassembly of E-cadherin and promoted EMT through deubiquitination of Snail. Silencing of USP47 accelerated the proteasomal degradation of Snail and inhibited EMT. Notably, hypoxia-induced USP47 upregulation was mediated by Sox9. These results demonstrate, for the first time, the role for USP47, as a novel target of Sox9, in the regulation of EMT and metastasis of colorectal cancer cells.

Modulation of tumor microenvironment by chemopreventive natural products

The tumor microenvironment provides a niche in which cancer cells and their surrounding stromal cells reside and in which their interactions occur. The cross talk between cancer and stromal cells in the tumor microenvironment promotes many biological processes to support cancer cell growth, invasion, angiogenesis, and metastasis. Recently, not only cancer cells but also multiple types of surrounding stromal cells, including endothelial cells, immune cells, and fibroblasts in the tumor microenvironment, have been recognized to be attractive targets for reducing resistance to anticancer therapy and tumor recurrence. Many natural products present in fruits, vegetables, herbs, spices, and some marine organisms have been reported to inhibit, delay, or reverse multistage carcinogenesis and to inhibit the proliferation of cancerous cells and the self‐renewal capacity of preexisting cancer stem‐like cells. Some of these naturally occurring chemopreventive and anticarcinogenic substances can modulate the signal transduction involved in maintaining the activities/functions of stromal cells and their interactions with cancer cells within the tumor microenvironment.

Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells

Docosahexaenoic acid (DHA), an ω-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)δ and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKCδ inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-l-cysteine and Trolox attenuated DHA-induced activation of PKCδ, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKCδ-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression.