The Accomplishments of KoCVAM in the Development and Implementation of Alternative Methods in Korea.

The Korean Center for the Validation of Alternative Methods (KoCVAM), which promotes the Three Rs principles and the use of alternative methods in Korea, has been operating within the Toxicological Screening and Testing Division of the Ministry of Food and Drug Safety (MFDS) since 2009. KoCVAM has exchanged opinions and information on the development and validation of non-animal alternative test methods as part of the International Cooperation on Alternative Test Methods (ICATM), and provided input into draft OECD Test Guidelines (TGs). Several Korean laws (e.g. the Cosmetics Act) encourage the use of alternative test methods for chemical testing and assessment. To promote and support the use of alternative test methods in the country, KoCVAM has published information and provided training on the national guidelines, which are based on the OECD TGs. In addition, KoCVAM has held annual training workshops on alternative test methods, to help Korean research institutions (including GLP test facilities) to implement them. In addition, by helping to develop and validate alternative test methods that were adopted in OECD TG 442B, TG 492 and TG 439, KoCVAM has contributed to the enhanced competitiveness of Korean industry on the worldwide stage.

KoCVAM-led development of phototoxicity alternative test method using reconstructed human epidermis model (KeraSkin™).

Phototoxicity is an acute toxic reaction induced by topical skin exposure to photoreactive chemicals followed by exposure to environmental light and thus chemicals that absorb UV are recommended to be evaluated for phototoxic potential. There are currently three internationally harmonized alternative test methods for phototoxicity. One of them is the in vitro Phototoxicity: RhE Phototoxicity test method (OECD TG498). Korean center for the Validation of Alternative Methods (KoCVAM) developed an in vitro phototoxicity test method using a KeraSkin™ reconstructed human epidermis model (KeraSkin™ Phototoxicity Assay) as a 'me-too' test method of OECD TG498. For the development and optimization of KeraSkin™ Phototoxicity Assay, the following test chemicals were used: 6 proficiency chemicals in OECD TG498 (3 phototoxic and 3 non-phototoxic), 6 reference chemicals in OECD Performance Standard No. 356 (excluding the proficiency test chemicals, 3 phototoxic and 3 non-phototoxic) and 13 additional chemicals (7 phototoxic and 6 non-phototoxic). Based on the test results generated from the test chemicals above, the overall predictive capacity of KeraSkin™ Phototoxicity Assay was calculated. In particular, the assay exhibited 100 % accuracy, 100 % sensitivity, and 100 % specificity. Therefore, it fulfills the requirements to be included as a 'me-too' test method in OECD TG498.

Soy milk digestion extract inhibits progression of prostate cancer cell growth via regulation of prostate cancer­specific antigen and cell cycle-regulatory genes in human LNCaP cancer cells.

Soy milk, which is produced from whole soybeans, contains a variety of biologically active components. Isoflavones are a class of soy-derived phytoestrogens with beneficial effects, among which genistein (GEN) has been previously indicated to reduce the risk of prostate cancer. The present study evaluated the effects of soy milk digestion extract (SMD) on the progression of prostate cancer via the estrogen receptor (ER)ß in human LNCaP prostate cancer cells. To evaluate the effects of SMD (daizein, 1.988 mg/100g, glycitein, 23.537 mg/100 g and GEN, 0.685 mg/100g) on cell proliferation, LNCaP cells were cultured in media containing vehicle (0.1% dimethyl sulfoxide), 17ß­estradiol (E2; 2.7x10­7 mg/ml), GEN (2.7x10-2 mg/ml) of SMD (total aglycon concentration, 0.79 mg/ml), after which the cell viability was examined using an MTT assay. The cell viability was significantly elevated by E2 (by 45±0.18%), while it was markedly reduced by GEN (73.2±0.03%) or SMD (74.8±0.09%). Semi­quantitative reverse transcription polymerase chain reaction analysis was performed to assess the mRNA expression levels of target genes, including ERß, prostate cancer­specific antigen (PSA) and cell cycle regulators p21, Cyclin D1 and cyclin-dependent kinase (CDK)4. The expression of ERß was almost completely diminished by E2, whereas it was significantly elevated by SMD. In addition, the expression levels of PSA were considerably reduced by SMD. The expression of p21 was significantly elevated by SMD, while it was markedly reduced by E2. Of note, the expression levels of Cyclin D1 and CDK4 were considerably elevated by E2, while being significantly reduced by GEN and SMD. All of these results indicated that SMD may inhibit the proliferation of human prostate cancer cells via regulating the expression of ERß, PSA, p21, Cyclin D1 and CDK4 in an ER-dependent manner.

Impact of activator type on the immobilisation of lead in fly ash-based geopolymer.

Immobilisation of heavy metals in geopolymers has attracted attention as a potential means of treating toxic wastes. Lead is known to be effectively immobilised in a geopolymer matrix, but detailed explanation for the mechanisms involved and the specific chemical form of lead are not fully understood. To reveal the effect of the activator types on the immobilisation of lead in geopolymers, 0.5 and 1.0wt% lead in the form of lead nitrate was mixed with fly ash and alkaline activators. Different alkaline activators (either combined sodium hydroxide and sodium silicate or sodium aluminate) were used to achieve the target Si:Al ratios 2.0 and 5.0 in geopolymers. Zeolite was formed in aluminate-activated geopolymers having a Si:Al ratio of 2.0, but the zeolite crystallization was suppressed as lead content increased. No specific crystalline phase of lead was detected by X-ray diffraction, electron diffraction or FT-IR spectrometry. In fact, double Cs corrected TEM analysis revealed that lead was evenly distributed with no evidence of formation of a specific lead compound. A sequential extraction procedure for fractionation of lead showed that lead did not exist as an exchangeable ion in geopolymers, regardless of activator type used. Aluminate activation is shown to be superior in the immobilisation of lead because about 99% of extracted lead existed in the oxidizing and residual fractions.

Anticancer effect of genistein on BG-1 ovarian cancer growth induced by 17 ß-estradiol or bisphenol A via the suppression of the crosstalk between estrogen receptor α 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.

Resveratrol regulates the cell viability promoted by 17ß-estradiol or bisphenol A via down-regulation of the cross-talk between estrogen receptor α and insulin growth factor-1 receptor in BG-1 ovarian cancer cells.

Endocrine disrupting chemicals (EDCs) and estrogens appear to promote development of estrogen-dependent cancers, including breast and ovarian carcinomas. In this study, we evaluated the cell viability effect of BPA on BG-1 human ovarian cancer cells, along with the growth inhibitory effect of resveratrol (trans-3,4,5-trihydroxystilbene; RES), a naturally occurring phytoestrogen. In addition, we investigated the underlying mechanism(s) of BPA and RES in regulating the interaction between estrogen receptor alpha (ERα) and insulin-like growth factor-1 receptor (IGF-1R) signals, a non- genomic pathway induced by 17ß-estradiol (E2). BPA induced a significant increase in BG-1 cell growth and up-regulated mRNA levels of ERα and IGF-1R. In parallel with its mRNA level, the protein expression of ERα was induced, and phosphorylated insulin receptor substrate-1 (p-IRS-1), phosphorylated Akt1/2/3, and cyclin D1 were increased by BPA or E2. However, RES effectively reversed the BG-1 cell proliferation induced by E2 or BPA by inversely down-regulating the expressions of ERα, IGF-1R, p-IRS-1, and p-Akt1/2/3, and cyclin D1 at both transcriptional and translational levels. Taken together, these results suggest that RES is a novel candidate for prevention of tumor progression caused by EDCs, including BPA via effective inhibition of the cross-talk of ERα and IGF-1R signaling pathways.

Suppression of the growth of human colorectal cancer cells by therapeutic stem cells expressing cytosine deaminase and interferon-ß via their tumor-tropic effect in cellular and xenograft mouse models.

Genetically engineered stem cells (GESTECs) exhibit a potent therapeutic efficacy via their strong tumor tropism toward cancer cells. In this study, we introduced the human parental neural stem cells, HB1.F3, with the human interferon beta (IFN-ß) gene which is a typical cytokine gene that has an antitumor effect and the cytosine deaminase (CD) gene from Escherichia coli (E. coli) that could convert the non-toxic prodrug, 5-fluorocytosine (5-FC), to a toxic metabolite, 5-fluorouracil (5-FU). Two types of stem cells expressing the CD gene (HB1.F3.CD cells) and both the CD and human IFN-ß genes (HB1.F3.CD.IFN-ß) were generated. The present study was performed to examine the migratory and therapeutic effects of these GESTECs against the colorectal cancer cell line, HT-29. When co-cultured with colorectal cancer cells in the presence of 5-FC, HB1.F3.CD and HB1.F3.CD.IFN-ß cells exhibited the cytotoxicity on HT-29 cells via the bystander effect. In particular, HB1.F3.CD.IFN-ß cells showed the synergistic cytotoxic activity of 5-FU and IFN-ß. We also confirmed the migration ability of HB1.F3.CD and HB1.F3.CD.IFN-ß cells toward HT-29 cells by a modified migration assay in vitro, where chemoattractant factors secreted by HT-29 cells attracted the GESTECs. In a xenograft mouse model, the volume of tumor mass was decreased up to 56% in HB1.F3.CD injected mice while the tumor mass was greatly inhibited about 76% in HB1.F3.CD.IFN-ß injected mice. The therapeutic treatment by these GESTECs is a novel strategy where the combination of the migration capacity of stem cells as a vector for therapeutic genes towards colorectal cancer and a synergistic antitumor effect of CD and IFN-ß genes can selectively target this type of cancer.

Risk of cardiovascular disease is suppressed by dietary supplementation with protamine and chitooligosaccharide in Sprague-Dawley rats.

Protamine from salmon spermaries is a novel dietary protein. Chitooligosaccharide (COS) is an oligosaccharide derived from chitin or chitosan, a long-chain polymer, by chemical or enzymatic hydrolysis. These two compounds are known to enhance lipid metabolism by interrupting the digestion and absorption of fat in the body. Cardiovascular disease (CVD) refers to any type of specific disease that affects the heart and circulatory system. Dyslipidemia, a condition involving high levels of low-density lipoprotein (LDL) cholesterol and low levels of high-density lipoprotein (HDL) cholesterol, is generally known to be a primary cause of CVD development. The risk of CVD is usually associated with the atherogenic index (AI) and cardiac risk factor (CRF). The CVD risk is also closely associated with serum levels of total cholesterol (T-CHO), LDL cholesterol and HDL cholesterol. In the present study, we evaluated alterations in serum lipid contents following the administration of protamine, COS and mixtures of these two compounds to male Sprague-Dawley (SD) rats, and their ability to reduce CVD risk. Based on the results of a serum lipid assay, protamine, COS and their mixtures were found to significantly reduce AI, CRF and CVD risk by decreasing serum levels of TG, T-CHO and LDL cholesterol and increasing serum HDL cholesterol levels. By contrast, TG and T-CHO concentrations in feces were markedly increased. Accumulation of lipids in the liver tissues of the SD rats fed high-fat diets was also inhibited by the intake of protamine and COS. Our findings suggest that protamine, COS and combinations of the two compounds may be used as a dietary therapy for preventing CVD due to their suppressive effects on hyperlipidemia and hypercholesterolemia.

Genistein, a soy phytoestrogen, prevents the growth of BG-1 ovarian cancer cells induced by 17ß-estradiol or bisphenol A via the inhibition of cell cycle progression.

An endocrine disrupting chemical (EDC) is a global health concern. In this study, we examined the effects of genistein (GEN) on bisphenol A (BPA) or 17ß-estradiol (E2)-induced cell growth and gene alterations of BG-1 ovarian cancer cells expressing estrogen receptors (ERs). In an in vitro cell viability assay, E2 or BPA significantly increased the growth of BG-1 cells. This increased proliferative activity was reversed by treatment with ICI 182,780, a well-known ER antagonist, while cell proliferation was further promoted in the presence of propyl pyrazole triol (PPT), an ERα agonist. These results imply that cell proliferation increased by E2 or BPA was mediated by ERs, particularly ERα. BPA clearly acted as a xenoestrogen in BG-1 ovarian cancer cells by mimicking E2 action. In contrast, GEN effectively suppressed BG-1 cell proliferation promoted by E2 or BPA by inhibiting cell cycle progression. E2 and BPA increased the expression of cyclin D1, a factor responsible for the G1/S cell cycle transition. They also decreased the expression of p21, a potent cyclin-dependent kinase (CDK) inhibitor that arrests the cell cycle in G1 phase, and promoted the proliferation of BG-1 cells. As shown by its repressive effect on cell growth, GEN decreased the expression of cyclin D1 augmented by E2 or BPA. On the other hand, GEN increased the p21 expression downregulated by E2 or BPA. Collectively, our findings suggest that GEN, a dietary phytoestrogen, has an inhibitory effect on the growth of estrogen-dependent cancers promoted by E2 or BPA.

Induced growth of BG-1 ovarian cancer cells by 17ß-estradiol or various endocrine disrupting chemicals was reversed by resveratrol via downregulation of cell cycle progression.

Resveratrol (trans-3,4',5-trihydroxystilbene; RES), a phytoestrogen, exists in grape skin and red wine. Endocrine disrupting chemicals (EDCs) appear to promote the development and progression of estrogen-dependent cancers. In this study, we evaluated the inhibitory effect of RES on the cell proliferation induced by various EDCs in BG-1 ovarian cancer cells. Various EDCs, such as bisphenol A (BPA), nonylphenol (NP), octylphenol (OP), methoxychlor (MXC) and hexabromocyclododecane (HBCD), were employed in this study. In the in vitro experiments, treatment of BG-1 cells with E2, BPA, NP, OP, MXC or HBCD resulted in an increase of cell growth. By contrast, increased cell viability induced by these EDCs was reversed when co-cultured with RES. In addition, we examined the regulation of cell cycle-related genes by RT-PCR and western blot analysis. Treatment with each EDC was found to decrease only the gene expression of p21 and increase the expression of cell cycle-dependent kinase 2 (CDK2). However, co-treatment with RES and each EDC resulted in an increased gene expression of p21 and a decreased expression of CDK2. Cyclin D1 was increased by downregulating p21 only when treated with each EDC in the absence of RES, while co-treatment with RES and each EDC decreased the gene expression of cyclin D1 by upregulating p21. Taken together, RES appears to be an inhibitor of cyclin D1 and CDK2 and is responsible for the cell cycle arrest at the G1 phase. In addition, when co-treated with each EDC, RES increased the expression of p21 and resulted in the growth inhibition of BG-1 ovarian cancer cells. Taken together, these results indicate the antiproliferative effect of RES, a dietary phytoestrogen, on estrogen-dependent ovarian cancer cells activated by EDCs.

Modulation of lipid metabolism by mixtures of protamine and chitooligosaccharide through pancreatic lipase inhibitory activity in a rat model.

Overweight and obesity are usually related with high fat and calorie intake, and seriously causative of lifestyle-related diseases such as cardiovascular disorders, arteriosclerosis, and colon cancer. In this study, we propose a novel dietary therapy against overweight and obesity using mixtures of protamine and chitooligosaccharide (COS), which are known to interrupt the lipid metabolism in the body. Protamine is a dietary protein originated from salmon reproductive organ, and COS is an oligosaccharide made from chitin or chitosan by chemical or enzymatic hydrolysis. In the enzyme activity analysis in vitro, protamine and COS strongly suppressed the activity of pancreatic lipase, which is the primary enzyme for the digestion and absorption of lipids in the intestine. In in vivo animal test, the mixtures of protamine and COS significantly reduced the serum levels of triglyceride (TG), total cholesterol (T-CHO), and low density lipoprotein-cholesterol (LDLC) and inhibited the accumulation of lipids in liver tissue of Sprague Dawley (SD) rats fed high fat diets. On the other hand, they increased fecal TG and T-CHO contents. From these alterations in lipid metabolism, we verified that protamine and COS mixtures could effectively interrupt the digestion and absorption of dietary lipids in the body by inhibiting pancreatic lipase activity. In addition, protamine and COS mixtures increased the serum level of high density lipoprotein-cholesterol (HDLC), responsible for removing cholesterol from cells and protecting atherosclerosis, and therefore decreased the potential risks of cardiovascular diseases by lowering values of the atherogenic index (AI) and cardiac risk factor (CRF). Taken together, we suggest protamine and COS mixtures as a prominent dietary therapy for the prevention of overweight, obesity, and further cardiovascular diseases related with hyperlipidemia.

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.

Stem cells with fused gene expression of cytosine deaminase and interferon-ß 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-ß (IFN-ß) 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-ß genes (HB1.F3.CD.IFN-ß) 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-ß. Reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm the expression of CD and IFN-ß 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-ß genes (HB1.F3.CD.IFN-ß) were co-expressed. Indeed a marked anticancer effect was demonstrated when the CD and IFN-ß 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-ß 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.

Antitumor therapeutic effects of cytosine deaminase and interferon-ß against endometrial cancer cells using genetically engineered stem cells in vitro.

Interest in gene therapy has recently increased; in particular, gene-directed enzyme/prodrug therapies have been found to be more advantageous compared to radiotherapy and/or chemotherapy. One of these, a cytosine deaminase (CD)/5-fluorocytosine (5-FC) system, is known to induce apoptosis by converting 5-FC, a prodrug, to its metabolically active form, 5-fluorouracil. The present study was designed to examine the migratory and therapeutic effects of engineered human stem cell lines against endometrial cancer using this strategy. The parental stem cells, HB1.F3, were modified to express Escherichia coli CD or human interferon-beta (IFN-ß), thereby producing HB1.F3.CD and HB1.F3.CD.IFN-ß cells, respectively. The parental and engineered stem cells (HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-ß) significantly migrated toward endometrial cancer cells (Ishikawa) more than primary bovine fibroblasts (bovine FB). In addition, important chemoattractant factors, including stem cell factor (SCF), vescular endothelial growth factor, vescular endothelial growth factor receptor 2, C-X-C chemokine receptor type 4, and c-KIT, involved in the tumor-tropic ability of stem cells were expressed in Ishikawa cells. In using a co-culture system and MTT assay, reduced viability of endometrial cancer cells was observed in the presence of HB1.F3.CD and HB1.F3.CD.IFN-ß cells with prodrug 5-FC. Taken together, these results suggest that gene therapy employing genetically modified stem cells expressing CD and IFN-ß may be effective for treating endometrial cancer.

Genetically engineered stem cells expressing cytosine deaminase and interferon-ß migrate to human lung cancer cells and have potentially therapeutic anti-tumor effects.

Recent studies have shown that genetically engineered stem cells (GESTECs) produce suicide enzymes that convert non-toxic pro-drugs to toxic metabolites which selectively migrate toward tumor sites and reduce tumor growth. In the present study, we evaluated whether these GESTECs are capable of migrating to lung cancer cells and examined the potential therapeutic efficacy of gene-directed enzyme pro-drug therapy against lung cancer cells in vitro. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to lung cancer cells. GESTECs [i.e., HB1.F3.CD or HB1.F3.CD.interferon-ß (IFN-ß)] engineered to express a suicide gene, cytosine deaminase (CD), selectively migrated toward lung cancer cells. Treatment of a human non-small cell lung carcinoma cell line (A549, a lung carcinoma derived from human lung epithelial cells) with the pro-drug 5-fluorocytosine (5-FC) in the presence of HB1.F3.CD or HB1.F3.CD.IFN-ß cells resulted in the inhibition of lung cancer cell growth. Based on the data presented herein, we suggest that GESTECs expressing CD may have a potent advantage for selective treatment of lung cancers. Furthermore, GESTECs expressing fusion genes (i.e., CD and IFN-ß) may have a synergic antitumor effect on lung cancer cells.