Ryeo-Eun Go

Methoxychlor and triclosan stimulates ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an estrogen receptor-dependent pathway.

Methoxychlor and triclosan are emergent or suspected endocrine-disrupting chemicals (EDCs). Methoxychlor [MXC; 1,1,1-trichlor-2,2-bis (4-methoxyphenyl) ethane] is an organochlorine pesticide that has been primarily used since dichlorodiphenyltrichloroethane (DDT) was banned. In addition, triclosan (TCS) is used as a common component of soaps, deodorants, toothpastes, and other hygiene products at concentrations up to 0.3%. In the present study, the potential impact of MXC and TCS on ovarian cancer cell growth and underlying mechanism(s) was examined following their treatments in BG-1 ovarian cancer cells. As results, MXC and TCS induced BG-1 cell growth via regulating cyclin D1, p21 and Bax genes related with cell cycle and apoptosis. A methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay confirmed that the proliferation of BG-1 ovarian cancer cells was stimulated by MXC (10(-6), 10(-7), 10(-8), and 10(-9)M) or TCS (10(-6), 10(-7), 10(-8), and 10(-9)M). Treatment of BG-1 cells with MXC or TCS resulted in the upregulation of cyclin D1 and downregulation of p21 and Bax transcriptions. In addition, the protein level of cyclin D1 was increased by MXC or TCS while p21 and Bax protein levels appeared to be reduced in these cells. Furthermore, MXC- or TCS-induced alterations of these genes were reversed in the presence of ICI 182,780 (10(-7)M), suggesting that the changes in these gene expressions may be regulated by an ER-dependent signaling pathway. In conclusion, the results of our investigation indicate that two potential EDCs, MXC and TCS, may stimulate ovarian cancer growth by regulating cell cycle- and apoptosis-related genes via an ER-dependent pathway.

Cytochrome P450 1 family and cancers.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to xenobiotics response element (XREs), and then starts the expressions of downstream genes including cytochrome P450 (CYP) 1 family members: CYP1A1, CYP1A2 and CYP1B1. Role of CYP1 family is involved in the metabolism of endogenous hormones, xenobiotics and drug. The expression of CYP1 family is regulated by estradiol (E2) or xenobiotics in diverse cancers. In breast cancers expressing estrogen receptors (ERs), level of CYP1B1 is increased by E2 and reversed by an estrogen receptor antagonist, ICI 182,780 or 4-hydrotamoxifen, which indicates that the expression of CYP1 family in downstream region of AhR is regulated by an activation of ERα. In metabolic pathways, E2 is converted into 4-hydroxyestradiol by CYP1B1, which can be converted into mainly estradiol-3,4-quinone, a potential carcinogen, by peroxidase. Increased expression of CYP1 family indicates the possibility of carcinogenesis by exposure of xenobiotics in endometrial and ovarian cancers. Apart from roles of CYP1 family in relation with ER pathway, CYP1 family is over-expressed in ER independent cancers. CYP1A1 exhibits hydroxylase activity in oxidation of arachidonic acid, which has been transformed to 12(R)-hydrxyeicosatetraenoic (HETEs), a potent activator of AhR activity. On the basis of results, phytoestrogens and dexamethasone are provided as cancer therapy regulating the expression of CYP1 family. Thus, this review focuses on the role(s) of CYP1 family in ER-dependent or ER-independent cancers and the potential for cancer therapy to target CYP1 family in these cancers.

Chemopreventive and chemotherapeutic effects of genistein, a soy isoflavone, upon cancer development and progression in preclinical animal models.

Genistein is one of isoflavones mostly derived in a leguminous plant. It is well known as one of phytoestrogens that have structures similar to the principal mammalian estrogen. It has diverse biological functions including chemopreventive properties against cancers. Anticancer efficacies of genistein have been related with the epidemiological observations indicating that the incidence of some cancers is much lower in Asia, where diets are rich in soyfoods, than Western countries. This review deals with in vivo anticancer activities of genistein identified in animal studies being divided into its effects on carcinogenesis and cancer progression. Because animal studies have advantages in designing the experiments to suit the goals, they imply diverse information on the anticancer activity of genistein. The in vivo animal studies have adopted the specific animal models according to a developmental stage of cancer to prove the anticancer efficacies of genistein against diverse types of cancer. The numerous previous studies insist that genistein effectively inhibits carcinogenesis in the DMBA-induced animal cancer models by reducing the incidence of adenocarcinoma and cancer progression in the transgenic and xenograft animal models by suppressing the tumor growth and metastatic transition. Although the protective effect of genistein against cancer has been controversial, genistein may be a candidate for chemoprevention of carcinogenesis and cancer progression and may deserve to be the central compound supporting the epidemiological evidence.

Benzophenone-1 and Nonylphenol Stimulated MCF-7 Breast Cancer Growth by Regulating Cell Cycle and Metastasis-Related Genes Via an Estrogen Receptor α-Dependent Pathway

Endocrine-disrupting chemicals (EDC) are defined as environmental compounds that produce adverse health manifestations in mammals by disrupting the endocrine system. Benzophenone-1 (2,4-dihydroxybenzophenone, BP1) and nonylphenol (NP), which are discharged from numerous industrial products, are known EDC. The aim of this study was to examine the effects of BP1 and NP on proliferation and metastasis of MCF-7 human breast cancer cells expressing estrogen receptors (ER). Treatment with BP1 (10−5-10−7M) and NP (10−6-10−7 M) promoted proliferation of MCF-7 cells similar to the positive control 17 -beta-estradiol (E2). When ICI 182,780, an ER antagonist, was co-incubated with E2, BP1, or NP, proliferation of MCF-7 cells returned to the level of a control. Addition of BP1 or NP markedly induced migration of MCF-7 cells similar to E2. To elucidate the underlying molecular mechanisms produced by these EDC, alterations in transcriptional and translational levels of proliferation and metastasis-related markers, including cyclin D1, p21, and cathepsin D, were determined. Data showed increase in expression of cyclin D1 and cathepsin D and decrease in p21 at both transcriptional and translational levels. However, BP1- or NP-induced alterations of these genes were blocked by ICI 182,780, suggesting that changes in expression of these genes may be regulated by an ERα-dependent pathway. In conclusion, BP1 and NP may accelerate growth of MCF-7 breast cancer cells by regulating cell cycle-related genes and promote cancer metastasis through amplification of cathepsin D.

Cigarette smoke extracts induced the colon cancer migration via regulating epithelial mesenchymal transition and metastatic genes in human colon cancer cells

There was considerable evidence that exposure to cigarette smoke is associated with an increased risk for colon cancer. Nevertheless, the mechanism underlying the relationship between cigarette smoking and colon cancer remains unclear. Moreover, there were only a few studies on effects of complexing substance contained in cigarette smoke on colon cancer. Thus, we further investigated whether cigarette smoke extract (CSE) affects the cell cycle, apoptosis and migration of human metastatic colon cancer cells, SW‐620. MTT assay revealed that SW‐620 cell proliferation was significantly inhibited following treatments with all CSEs, 3R4F, and two‐domestic cigarettes, for 9 days in a concentration‐dependent manner. Moreover, CSE treatments decreased cyclin D1 and E1, and increased p21 and p27 proteins by Western blot analysis in SW‐620 cells. Additionally, the treatment of the cells with CSE contributed to these effects expressing by apoptosis‐related proteins. An increased migration or invasion ability of SW‐620 cells following CSE treatment was also confirmed by a scratch or fibronectin invasion assay in vitro. In addition, the protein levels of E‐cadherin as an epithelial maker were down‐regulated, while the mesenchymal markers, N‐cadherin, snail, and slug, were up‐regulated in a time‐dependent manner. A metastatic marker, cathepsin D, was also down‐regulated by CSE treatment. Taken together, these results indicate that CSE exposure in colon cancer cells may deregulate the cell growth by altering the expression of cell cycle‐related proteins and pro‐apoptotic protein, and stimulate cell metastatic ability by altering epithelial‐mesenchymal transition (EMT) markers and cathepsin D expression.

Treatment of BG-1 Ovarian Cancer Cells Expressing Estrogen Receptors with Lambda-cyhalothrin and Cypermethrin Caused a Partial Estrogenicity Via an Estrogen Receptor-dependent Pathway.

Synthetic pyrethroids (SPs) are the most common pesticides which are recently used for indoor pest control. The widespread use of SPs has resulted in the increased exposure to wild animals and humans. Recently, some SPs are suspected as endocrine disrupting chemicals (EDCs) and have been assessed for their potential estrogenicity by adopting various analyzing assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LC) and cypermethrin (CP), the most commonly used pesticides in Korea, using BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To evaluate the estrogenic activities of two SPs, LC and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT-PCR) in LC or CP treated BG-1 ovarian cancer cells. In MTT assay, LC (10−6 M) and CP (10−5 M) significantly induced the growth of BG-1 cancer cells. LC or CP-induced cell growth was antagonized by addition of ICI 182,720 (10−8 M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT-PCR results showed that transcriptional level of cyclin D1, a cell cycle-regulating gene, was significantly up-regulated by LC and CP, while these effects were reversed by co-treatment of ICI 182,780. However, p21, a cyclin D-ckd-4 inhibitor gene, was not altered by LC or CP. Moreover, ERα expression was not significantly changed by LC and CP, while downregulated by E2. Finally, in xenografted mouse model transplanted with human BG-1 ovarian cancer cells, E2 significantly increased the tumor volume compare to a negative control, but LC did not. Taken together, these results suggest that LC and CP may possess estrogenic potentials by stimulating the growth of BG-1 ovarian cancer cells via partially ER signaling pathway associated with cell cycle as did E2, but this estrogenic effect was not found in in vivo mouse model.

Effects of cigarette smoke extracts on the progression and metastasis of human ovarian cancer cells via regulating epithelial-mesenchymal transition

Cigarette smoke (CS) contains over 60 well-established carcinogens, and there are strong links between these carcinogens and smoking-induced cancers. In this study we investigated whether three types of cigarette smoke extracts (CSEs), 3R4F (standard cigarette), CSE1 and CSE2 (two commercial cigarettes), affect the proliferation, migration, and invasive activity of BG-1 human ovarian cancer cells. All three types of CSEs increased BG-1 cell proliferation at nicotine concentrations of 1.5μM-2.1μM in a cell viability assay. The protein expressions of cyclin D1 and cyclin E1 were increased, while p21 and p27 expression was decreased by Western blot assay. However, they did not show a consistent dose-dependent tendency. The protein expressions of Bax and p53, pro-apoptotic genes, were also decreased by CSEs. The expression of E-cadherin, an epithelial marker, was reduced in the treatment of CSEs while the expression of its reverse transition marker, N-cadherin, was slightly increased by CSEs containing 2.1μM of nicotine, but a statistical significance was not observed. Epithelial-mesenchymal transition (EMT)-associated transcriptional factors, Snail and Slug, were also up-regulated by treatment with CSEs, indicating that CSEs can increase the EMT process in BG-1 ovarian cancer cells. In addition, CSEs increased the migratory and invasive propensity of cancer cells. These functional alterations were associated with changes in metastasis-related gene expression. Upon exposure to CSEs, the expression of MMP-9 and cathepsin D was increased. Taken together, we confirmed that CSEs increased the growth, migration, and invasion of human ovarian cancer cells by regulating cell cycle, apoptosis, EMT, and metastasis related cellular markers and signaling proteins. Based on the results, cigarette smokers of women might be at a higher risk of ovarian cancer than non-smokers.

Effect of fenhexamid and cyprodinil on the expression of cell cycle- and metastasis-related genes via an estrogen receptor-dependent pathway in cellular and xenografted ovarian cancer models.

Fenhexamid and cyprodinil are antifungal agents (pesticides) used for agriculture, and are present at measurable amounts in fruits and vegetables. In the current study, the effects of fenhexamid and cyprodinil on cancer cell proliferation and metastasis were examined. Additionally, the protein expression levels of cyclin D1 and cyclin E as well as cathepsin D were analyzed in BG-1 ovarian cancer cells that express estrogen receptors (ERs). The cells were cultured with 0.1% dimethyl sulfoxide (DMSO; control), 17β-estradiol (E2; 10(-9)M), and fenhexamid or cyprodinil (10(-5)-10(-7)M). Results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that fenhexamid and cyprodinil increased BG-1 cell proliferation about 1.5 to 2 times similar to E2 (5 times) compared to the control. When the cells were co-treated with ICI 182,780 (10(-8)M), an ER antagonist, the proliferation of pesticide-treated BG-1 cells was decreased to the level of the control. A wound healing assay revealed that the pesticides reduced the disrupted area in the BG-1 cell monolayer similar to E2. Protein levels of cyclin D1 and E as well as cathepsin D were increased by fenhexamid and cyprodinil. This effect was reversed by co-treatment with ICI 182,780. In a xenograft mouse model with transplanted BG-1 cells, cyprodinil significantly increased tumor mass formation about 2 times as did E2 (6 times) compared to the vehicle (0.1% DMSO) over an 80-day period. In contrast, fenhexamid did not promote ovarian tumor formation in this mouse model. Cyprodinil also induced cell proliferation along with the expression of proliferating cell nuclear antigen (PCNA) and cathepsin D in tumor tissues similar to E2. Taken together, these results imply that fenhexamid and cyprodinil may have disruptive effects on ER-expressing cancer by altering the cell cycle- and metastasis-related gene expression via an ER-dependent pathway.

Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization.

INTRODUCTION Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. METHODS Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. RESULTS These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21(cip1), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells. DISCUSSION Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method.

Effects of microalgal polyunsaturated fatty acid oil on body weight and lipid accumulation in the liver of C57BL/6 mice fed a high fat diet

Abstract Dietary polyunsaturated fatty acids (PUFAs), which are abundant in marine fish oils, have recently received global attention for their prominent anti-obesogenic effects. Among PUFAs, eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which are n-3 long-chain PUFAs widely referred to as omega-3 oils, were reported to prevent the development of obesity in rodents and humans. In the present study, we evaluated the anti-obesity effects of microalgal oil on high-fat induced obese C57BL/6 mice, compared with commercial omega-3 fish oil and vegetable corn oil. Microalgal oil is an inherent mixture of several PUFAs, including EPA, DHA and other fatty acids produced from a marine microalgal strain of Thraustochytriidae sp. derived mutant. It was found to contain more PUFAs (>80%) and more omega-3 oils than commercial omega-3 fish oil (PUFAs >31%) and corn oil (PUFAs 59%). All three types of oils induced weight loss in high-fat-induced obese mice, with the loss induced by microalgal oil being most significant at 9 weeks (10% reduction). However, the oils tested did not improve blood lipid levels, although microalgal oil showed an apparent inhibitory effect on lipid accumulation in the liver. These findings may be attributed to the higher PUFA content, including omega-3 oils of microalgal oil than other oils. Collectively, these findings suggest that microalgal oil, derived from Thraustochytriidae sp. derived mutant, is a prominent candidate for replacement of omega-3 fish oils based on its apparent anti-obesity effect in vivo.