Bo-Rim Yi

Cell growth of BG-1 ovarian cancer cells is promoted by di-n-butyl phthalate and hexabromocyclododecane via upregulation of the cyclin D and cyclin-dependent kinase-4 genes

Endocrine-disrupting chemicals (EDCs) are environmentally persistent exogenous compounds released from various industrial products such as plastics, pesticides, drugs, detergents and cosmetics. They can cause a variety of adverse effects to the reproductive, developmental, immune and nervous systems in humans and wildlife. Di-n-butyl phthalate (DBP) is the main compound of phthalates and is reported to inhibit estrogen receptor (ER)-mediated gene expression and to interfere with normal fetal development of the male reproductive system. Hexabromocyclododecane (HBCD or HBCDD) is one of the brominated flame retardants (BFRs) which have been widely used in plastic, electronic and textile applications and are known to cause endocrine disruption with toxicity of the nervous system. In the present study, the estrogenic effects of DBP and HBCD were examined in an ovarian cancer cell line, BG-1, expressing high levels of ER via MTT assay and semi-quantitative reverse-transcription PCR. Treatment with DBP (10(-8)-10(-5) M) or HBCD (2 x 10(-8) -2 x 10(-6) M) resulted in increased cell proliferation of BG-1 cells as observed with 17-β estradiol (E2). In addition, both DBP and HBCD upregulated the expression levels of cell cycle-regulatory genes, such as cyclin D and cyclin-dependent kinase-4 (cdk-4), which are downstream target genes of ER, at 6 h after treatment. However, the expression of the p21 gene was not altered by DBP or HBCD at any time as with E2. Taken together, these results suggest that DBP and HBCD are EDCs which have apparent estrogenic activities by stimulating the cell proliferation of BG-1 cells and by inducing the expression of cyclin D and cdk-4. Our results suggest that DBP and HBCD have sufficient potency to disrupt the endocrine system and to stimulate cell growth in ER-positive cancer cells.

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.

Molecular Mechanisms and In Vivo Mouse Models of Skin Aging Associated with Dermal Matrix Alterations

Skin is the most superficial body organ and plays an important role in protecting the body from environmental damage and in forming social relations. With the increase of the aging population in our society, dermatological and cosmetic concerns of skin aging are rapidly increasing. Skin aging is a complex process combined with intrinsic and extrinsic factors. Intrinsic or chronological skin aging results from the passage of time and is influenced by genetic factors. Extrinsic skin aging is mainly determined by UV irradiation, also called photoaging. These two types of aging processes are superimposed on sun-exposed skin, and have a common feature of causing dermal matrix alterations that mostly contribute to the formation of wrinkles, laxity, and fragility of aged skin. The dermal matrix contains extracellular matrix proteins such as collagen, elastin, and proteoglycans that confer the strength and resiliency of skin. Skin aging associated with dermal matrix alterations and atrophy can be caused by cellular senescence of dermal cells like fibroblasts, and decreased synthesis and accelerated degradation of dermal matrix components, especially collagen fibers. Both intrinsic aging and photoaging exert influence during each step of dermal matrix alteration via different mechanisms. Mouse models of skin aging have been extensively developed to elucidate intrinsic aging and photoaging processes, to validate in vitro biochemical data, and to test the effects of pharmacological tools for retarding skin aging because they have the advantages of being genetically similar to humans and are easily available.

Anticancer effect of genistein on BG-1 ovarian cancer growth induced by 17 β-estradiol or bisphenol A via the suppression of the crosstalk between estrogen receptor alpha and insulin-like growth factor-1 receptor signaling pathways

The interaction between estrogen receptor (ER) and insulin-like growth factor-1 receptor (IGF-1R) signaling pathway plays an important role in proliferation of and resistance to endocrine therapy to estrogen dependent cancers. Estrogen (E2) upregulates the expression of components of IGF-1 system and induces the downstream of mitogenic signaling cascades via phosphorylation of insulin receptor substrate-1 (IRS-1). In the present study, we evaluated the xenoestrogenic effect of bisphenol A (BPA) and antiproliferative activity of genistein (GEN) in accordance with the influence on this crosstalk. BPA was determined to affect this crosstalk by upregulating mRNA expressions of ERα and IGF-1R and inducing phosphorylation of IRS-1 and Akt in protein level in BG-1 ovarian cancer cells as E2 did. In the mouse model xenografted with BG-1 cells, BPA significantly increased a tumor burden of mice and expressions of ERα, pIRS-1, and cyclin D1 in tumor mass compared to vehicle, indicating that BPA induces ovarian cancer growth by promoting the crosstalk between ER and IGF-1R signals. On the other hand, GEN effectively reversed estrogenicity of BPA by reversing mRNA and protein expressions of ERα, IGF-1R, pIRS-1, and pAkt induced by BPA in cellular model and also significantly decreased tumor growth and in vivo expressions of ERα, pIRS-1, and pAkt in xenografted mouse model. Also, GEN was confirmed to have an antiproliferative effect by inducing apoptotic signaling cascades. Taken together, these results suggest that GEN effectively reversed the increased proliferation of BG-1 ovarian cancer by suppressing the crosstalk between ERα and IGF-1R signaling pathways upregulated by BPA or E2.

Role of the epithelial–mesenchymal transition and its effects on embryonic stem cells

The epithelial–mesenchymal transition (EMT) is important for embryonic development and the formation of various tissues or organs. However, EMT dysfunction in normal cells leads to diseases, such as cancer or fibrosis. During the EMT, epithelial cells are converted into more invasive and active mesenchymal cells. E-box-binding proteins, including Snail, ZEB and helix–loop–helix family members, serve as EMT-activating transcription factors. These transcription factors repress the expression of epithelial markers, for example, E-cadherin, rearrange the cytoskeleton and promote the expression of mesenchymal markers, such as vimentin, fibronectin and other EMT-activating transcription factors. Signaling pathways that induce EMT, including transforming growth factor-β, Wnt/glycogen synthase kinase-3β, Notch and receptor tyrosine kinase signaling pathways, interact with each other for the regulation of this process. Although the mechanism(s) underlying EMT in cancer or embryonic development have been identified, the mechanism(s) in embryonic stem cells (ESCs) remain unclear. In this review, we describe the underlying mechanisms of important EMT factors, indicating a precise role for EMT in ESCs, and characterize the relationship between EMT and ESCs.

Gene alterations of ovarian cancer cells expressing estrogen receptors by estrogen and bisphenol a using microarray analysis.

Since endocrine disrupting chemicals (EDCs) may interfere with the endocrine system(s) of our body and have an estrogenicity, we evaluated the effect(s) of bisphenol A (BPA) on the transcriptional levels of altered genes in estrogen receptor (ER)-positive BG-1 ovarian cancer cells by microarray and real-time polymerase-chain reaction. In this study, treatment with 17β-estradiol (E2) or BPA increased mRNA levels of E2-responsive genes related to apoptosis, cancer and cell cycle, signal transduction and nucleic acid binding etc. In parallel with their microarray data, the mRNA levels of some altered genes including RAB31_MEMBER RAS ONCOGENE FAMILY (U59877), CYCLIN D1 (X59798), CYCLIN-DEPENDENT KINASE 4 (U37022), IGF-BINDING PROTEIN 4 (U20982), and ANTI-MULLERIAN HORMONE (NM_000479) were significantly induced by E2 or BPA in this cell model. These results indicate that BPA in parallel with E2 induced the transcriptional levels of E2-responsive genes in an estrogen receptor (ER)-positive BG-1 cells. In conclusion, these microarray and real-time polymerase-chain reaction results indicate that BPA, a potential weak estrogen, may have estrogenic effect by regulating E2-responsive genes in ER-positive BG-1 cells and BG-1 cells would be the best in vitro model to detect these estrogenic EDCs.

Human amniotic membrane-derived epithelial stem cells display anticancer activity in BALB/c female nude mice bearing disseminated breast cancer xenografts

Breast cancer is one of the most common malignant tumors and the leading cause of mortality among women. In this study, we propose a human stem cell transplantation strategy, an important method for treating various cancers, as a potential breast cancer therapy. To this end, we used human amniotic membrane-derived epithelial stem cells (hAECs) as a cell source for performing human stem cell transplantation. hAECs have multipotent differentiation abilities and possess high proliferative potential. We transplanted hAECs into female BALB/c nude mice bearing tumors originating from MDA-MB-231 breast cancer cells. Co-culturred hAECs and MDA-MB-231 cells at a ratio of 1:4 or 1:8 (tumor cells to stem cells) inhibited breast cancer cell growth by 67.29 and 67.33%, respectively. In the xenograft mouse model, tumor volumes were significantly decreased by 5-flurouracil (5-FU) treatment and two different ratios of hAECs (1:4 and 1:8) by 84.33, 73.88 and 56.89%, respectively. Treatment of nude mice with hAECs (1:4) produced remarkable antitumor effects without any side-effects (e.g., weight loss, death and bruising) compared to the mice that received only 5-FU treatment. Tumor progression was significantly reduced by hAEC treatment compared to the xenograft model. On the other hand, breast tissues (e.g., the epidermis, dermis and reticular layer) appeared to be well-maintained following treatment with hAECs. Taken together, these results provide strong evidence that hAECs can be used as a safe and effective cancer-targeting cytotherapy for treating breast cancer.

Benzophenone-1 stimulated the growth of BG-1 ovarian cancer cells by cell cycle regulation via an estrogen receptor alpha-mediated signaling pathway in cellular and xenograft mouse models.

2,4-Dihydroxybenzophenone (benzophenone-1; BP-1) is an UV stabilizer primarily used to prevent polymer degradation and deterioration in quality due to UV irradiation. Recently, BP-1 has been reported to bioaccumulate in human bodies by absorption through the skin and has the potential to induce health problems including endocrine disruption. In the present study, we examined the xenoestrogenic effect of BP-1 on BG-1 human ovarian cancer cells expressing estrogen receptors (ERs) and relevant xenografted animal models in comparison with 17-β estradiol (E2). In in vitro cell viability assay, BP-1 (10(-8)-10(-5)M) significantly increased BG-1 cell growth the way E2 did. The mechanism underlying the BG-1 cell proliferation was proved to be related with the up-regulation of cyclin D1, a cell cycle progressor, by E2 or BP-1. Both BP-1 and E2 induced cell growth and up-regulation of cyclin D1 were reversed by co-treatment with ICI 182,780, an ER antagonist, suggesting that BP-1 may mediate the cancer cell proliferation via an ER-dependent pathway like E2. On the other hand, the expression of p21, a regulator of cell cycle progression at G1 phase, was not altered by BP-1 though it was down-regulated by E2. In xenograft mouse models transplanted with BG-1 cells, BP-1 or E2 treatment significantly increased the tumor mass formation compared to a vehicle (corn oil) within 8 weeks. In histopathological analysis, the tumor sections of E2 or BP-1 group displayed extensive cell formations with high density and disordered arrangement, which were supported by the increased number of BrdUrd positive nuclei and the over-expression of cyclin D1 protein. Taken together, these results suggest that BP-1 is an endocrine disrupting chemical (EDC) that exerts xenoestrogenic effects by stimulating the proliferation of BG-1 ovarian cancer via ER signaling pathway associated with cell cycle as did E2.

Antitumor effects of genetically engineered stem cells expressing yeast cytosine deaminase in lung cancer brain metastases via their tumor-tropic properties.

Although mortality related with primary tumors is approximately 10%, metastasis leads to 90% of cancer-associated death. The majority of brain metastases result from lung cancer, but the metastatic mechanism remains unclear. In general, chemotherapy for treating brain diseases is disrupted by the brain blood barrier (BBB). As an approach to improve treatment of lung cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs), consisting of neural stem cells (NSCs) expressing a suicide gene. Cytosine deaminase (CD), one of the suicide genes, originating from bacterial (bCD) or yeast (yCD), which can convert the non-toxic prodrug, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU), can inhibit cancer cell growth. We examined the therapeutic efficacy and migratory properties of GESTECs expressing yCD, designated as HB1.F3.yCD, in a xenograft mouse model of lung cancer metastasis to the brain. In this model, A549 lung cancer cells were implanted in the right hemisphere of the mouse brain, while CM-DiI pre-stained HB1.F3.yCD cells were implanted in the contralateral brain. Two days after the injection of stem cells, 5-FC was administered via intraperitoneal injection. The tumor-tropic effect of HB1.F3.yCD was evident by fluorescent analysis, in which red-colored stem cells migrated to the lung tumor mass of the contralateral brain. By histological analysis of extracted brain, the therapeutic efficacy of HB1.F3.yCD in the presence of 5-FC was confirmed by the reduction in density and aggressive tendency of lung cancer cells following treatment with 5-FC, compared to a negative control or HB1.F3.yCD injection without 5-FC. Taken together, these results indicate that HB1.F3.yCD expressing a suicide gene may be a new therapeutic strategy for lung cancer metastases to the brain in the presence of a prodrug.

Stem cells with fused gene expression of cytosine deaminase and interferon-β migrate to human gastric cancer cells and result in synergistic growth inhibition for potential therapeutic use

Genetically engineered stem cells (GESTECs) producing suicide enzymes and immunotherapeutic cytokines have therapeutic effects on tumors, and may possibly reduce the side effects of toxic drugs used for treatments. Suicide enzymes can convert non-toxic pro-drugs to toxic metabolites that can reduce tumor growth. Cytosine deaminase (CD) is a suicide enzyme that metabolizes a non-toxic pro-drug, 5-fluorocytosine (5-FC), into the cytotoxic agent, 5-fluorouracil (5-FU). As an immunotherapeutic agent, human interferon-β (IFN-β) has anticancer effects. In this study, we used modified human neural stem cells (HB1.F3) expressing the Escherichia coli (E. coli) CD gene (HB1.F3.CD) or both the CD and human IFN-β genes (HB1.F3.CD.IFN-β) and evaluated their effectiveness on gastric carcinoma cells (AGS); migration of GESTECs to AGS was analyzed as well as formation of 5-FU and IFN-β. Reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm the expression of CD and IFN-β genes in GESTECs along with confirming the production of chemoattractant molecules such as stem cell factor (SCF), CXCR4, c-Kit, vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). In addition, by co-culturing GESTECs with AGS in the presence of 5-FC, we were able to confirm that cancer growth was inhibited, along with a synergistic effect when the CD and IFN-β genes (HB1.F3.CD.IFN-β) were co-expressed. Indeed a marked anticancer effect was demonstrated when the CD and IFN-β genes were expressed together compared to expression of the CD gene alone (HB1.F3.CD). According to a modified transwell migration assay, the migration of GESTECs toward AGS was confirmed. In conclusion, these data suggest potential application of GESTECs to gastric cancer therapy, due to a remarkable synergistic effect of CD and IFN-β genes in the presence of 5-FC. Additionally, the tumor-selective migration capability in vitro suggests that GESTECs are a potential anticancer therapy candidate that may result in minimal side effects compared to the conventional chemotherapy.