Considerable attention has been paid to the absorption mechanisms of plasmalogen (Pls) because its intake has been expected to have preventive effects on brain-related diseases. Possible structural changes of Pls during absorption (i.e., preferential arachidonic acid re-esterification at the sn-2 position and base conversion of ethanolamine Pls (PE-Pls) into choline Pls (PC-Pls)) have previously been proposed. Since the physiological functions of Pls differ according to its structure, further elucidation of such structural changes during absorption is important to understand how Pls exerts its physiological effects in vivo. Hence, the absorption mechanism of Pls was investigated using the lymph-cannulation method and the everted jejunal sac model, with a focus on Pls molecular species. In the lymph-cannulation method, relatively high amounts of PE-Pls 18:0/20:4 and PC-Pls 18:0/20:4 were detected from the lymph even though these species were minor in the administered emulsion. Moreover, a significant increase of PE-Pls 18:0/20:4 and PC-Pls 18:0/20:4 in the intestinal mucosa was also confirmed by the everted jejunal sac model. Therefore, structural changes of PE-Pls in the intestinal mucosa were strongly suggested. The results of this study may provide an understanding of the relationship between intestinal absorption of Pls and exertion of its physiological functions in vivo.

Mulberry leaves are rich in aza-sugars, particularly 1-deoxynojirimycin (DNJ), fagomine, and 2-O-α-d-galactopyranosyl-1-deoxynojirimycin (GAL-DNJ), which have antidiabetes and antiobesity properties. To help us understand the mechanisms of action of aza-sugars, pharmacokinetic studies are necessary. Therefore, in this study, we evaluated and compared the absorption and organ distribution of these aza-sugars in rats. Following oral intake, DNJ exhibited the highest plasma concentration followed by fagomine and GAL-DNJ. Meanwhile, similar amounts of DNJ and fagomine were present in organs, while GAL-DNJ was hardly detected, suggesting the diversity in absorption and distribution characteristics of these aza-sugars. We then investigated the role of the sodium-glucose cotransporter and the glucose transporter (GLUT) in the transport of aza-sugars and found that both are involved in DNJ transport, while transport of fagomine is solely facilitated by the GLUT. These findings provide insight into the bioavailability and bioactive mechanisms of these aza-sugars.

The Amami rabbit (Pentagulus furnessi) is a dark brown-furred rabbit classified as an endangered species and only found in the Amami Islands of Japan. They are often called living fossils because they retain primitive characteristics of ancient rabbits that lived approximately 1 million years ago, such as short feet and hind legs and small ears. Although the ancient rabbit has disappeared due to the competition with European rabbit (Oryctolagus cuniculus) in the most of the Asian area, Amami rabbit survived since Amami Islands has isolated from Japan and Taiwan. Although Amari rabbit is one of the protected animals, their population decreases each year due to human activities, such as deforestation and roadkill. In this study, we collected roadkill samples of Amami rabbits and established primary and immortalized fibroblast cell lines. Combined expression of human-derived mutant Cyclin-dependent kinase 4, Cyclin D1, and hTERT allowed us to immortalize fibroblasts successfully in three individuals of Amami rabbits. The immortalized fibroblasts dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. Furthermore, the immortalized cells maintained their normal chromosomal pattern (2n = 46). Our results suggest that cellular senescence which mainly regulated by p16-RB signaling pathway is conserved in animal evolution at least from 1 million years ago.

1-Deoxynojirimycin (DNJ) has been known for its functional properties, such as its anti-hyperglycemic and anti-obesity activities. Previously, we developed a sustainable procedure to produce culture broth powder (CBP) containing DNJ using Bacillus amyloliquefaciens AS385 and demonstrated its regulatory effect on the blood glucose and lipid parameters in C57BL/6J mice. The present study was aimed to determine the molecular mechanism underlying the physiological effects of CBP intake in different concentrations (low, medium and high) towards the development of high-fat diet (HFD)-induced metabolic disorders. Ten-week consumption of CBP-supplemented diets ameliorated HFD-induced adiposity, glucose intolerance, and reduced insulin sensitivity in C57BL/6J mice. To investigate how these physiological events could take place, we analyzed the expression of genes involved in lipid metabolism and insulin signaling in epididymal white adipose tissue and found that CBP had a regulatory effect on the expression of genes related to lipid metabolism (Pparγ, Srebf1c, Acc, Scd, Hsl, Lpl), adiponectin secretion (Foxo1 and Sirt1), and insulin signaling (Irs1 and Akt2). Next, we confirmed that DNJ acted as the main active component in CBP and detected the dose-dependent DNJ uptake in vital metabolic tissues, which may explain the dose-dependent alteration in the metabolic parameters and related gene expressions following the CBP intake in this study. Collectively, our results suggested that DNJ intake in the form of CBP prevented the progression of HFD-induced metabolic disorders through regulation of adipocyte gene expression involved in lipid metabolism and insulin signaling.

Androgenetic alopecia (AGA) is the most common type of hair loss, and is mainly caused by the biological effects of testosterone on dermal papilla cells (DPCs). In vitro culturing of DPCs might be a useful tool for the screening of target molecule of AGA. However, primary DPCs cannot continuously proliferate owing to cellular senescence and cell culture stress. In this study, we introduced mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) into DPCs. We confirmed protein expression of CDK4 and Cyclin D1, and enzymatic activity of TERT. Furthermore, we found the established cell line was free from cellular senescence. We also introduced the androgen receptor gene using a recombinant retrovirus, to compensate the transcriptional suppressed endogenous androgen receptor in the process of cell proliferation. Furthermore, we detected the efficient nuclear translocation of androgen receptor into the nucleus after the treatment of dihydrotestosterone, indicating the functionality of our introduced receptor. Our established cell line is a useful tool to identify the downstream signaling pathway, which activated by the testosterone.

When α-tocopherol (α-Toc) exerts its antioxidative effect, a portion of α-Toc is converted to certain oxidation products. Although accumulation of such oxidation products is considered to cause a deterioration in the quality of foods, their distribution and generation in food samples have been still unknown. In this study, we tried to analyze α-Toc hydroperoxide (Toc-OOH) stereoisomers and tocopherylquinone (TQ) in extra virgin olive oil (EVOO) using liquid chromatography-tandem mass spectrometry. Photo-irradiation (5000 lx) to EVOO increased Toc-OOH stereoisomers but not TQ. In contrast, thermal oxidation (150 °C) of EVOO increased TQ but not Toc-OOH. We considered that the generation of Toc-OOH and TQ were due to the [4+2]-cycloaddition reaction and proton donation from the phenolic hydrogen, respectively. Our data and method would be helpful for understanding of α-Toc oxidation mechanisms in edible oil samples or the estimation of food quality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

[This corrects the article DOI: 10.1371/journal.pone.0221364.].

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.

Squalene is a terpenoid found in human skin surface lipids (SSLs) and foods that possesses beneficial properties. However, since oxidation of squalene causes various complications, it is necessary to identify the mechanisms by which squalene is oxidized. In this study, we aimed to determine the oxidation mechanisms of squalene in SSLs and shark liver oil (SLO) supplements by the analysis of squalene monohydroperoxide (SQOOH) isomers, on the basis of our previous finding that different oxidation mechanisms yield different SQOOH isomers. Liquid chromatography-tandem mass spectrometry analysis of SQOOH isomers revealed that squalene in human SSLs was oxidized by singlet oxygen oxidation, whereas squalene in SLO was oxidized mainly by free radicals. As a result, we have presented the first evidence suggesting that the analysis of SQOOH isomers enables estimation of oxidation mechanisms. Estimating oxidation mechanisms by analyzing SQOOH isomers may provide a foundation for the prevention of skin diseases and food deterioration via regulation of squalene oxidation.

γ-Oryzanol (OZ), which has a lot of beneficial effects, is a mixture of ferulic acid esters of triterpene alcohols (i.e., triterpene alcohol type of OZ (TTA-OZ)) and ferulic acid esters of plant sterols (i.e., plant sterol type of OZ (PS-OZ)). Although it has been reported that OZ is found in several kinds of cereal typified by rice, TTA-OZ (e.g., 24-methylenecycloartanyl ferulate (24MCA-FA)) has been believed to be characteristic to rice and has not been found in other cereals. In this study, we isolated a compound considered as a TTA-OZ (i.e., 24MCA-FA) from barley and determined the chemical structure using by HPLC-UV-MS, high-resolution MS, and NMR. Based on these results, we proved for the first time that barley certainly contains 24MCA-FA (i.e., TTA-OZ). During the isolation and purification of 24MCA-FA from barley, we found the prospect that a compound with similar properties to OZ (compound-X) might exist. To confirm this finding, the compound-X was also isolated, determined the chemical structure, and identified as a caffeic acid ester of 24-methylenecycloartanol (24MCA-CA), which has rarely been reported before. We also quantified these compounds in various species of barley cultivars and found for the first time the existence of 24MCA-FA and 24MCA-CA in various barley. Through these findings, it opens the possibility to use barley as a new source of 24MCA-FA and 24MCA-CA.

BACKGROUND: Curcuminoids, mainly present in the plant rhizomes of turmeric (Curcuma longa), consist of mainly three forms (curcumin (CUR), bisdemethoxycurcumin (BDMC) and demethoxycurcumin (DMC)). It has been reported that different forms of curcuminoids possess different biological activities. However, the mechanisms associated with these differences are not well-understood. Recently, our laboratory found differences in the cellular uptake of these curcuminoids. Therefore, it has been inferred that these differences contribute to the different biological activities. PURPOSE: In this study, we investigated the mechanisms of differential cellular uptake of these curcuminoids. METHOD: Based on our previous study, we hypothesized the differential cellular uptake is caused by (I) polarity, (II) transporters, (III) metabolism rate of curcuminoids and (IV) medium components. These four hypotheses were each investigated by (I) neutralizing the polarities of curcuminoids by encapsulation into poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs), (II) inhibition of polyphenol-related absorption transporters, (III) analysis of the cellular curcuminoids and their metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and (IV) use of different mediums in cell study. RESULTS: The differential cellular uptake was not affected by (I-III). However, when investigating (IV), not only CUR but also BDMC and DMC were incorporated into cells when serum free media was used. Furthermore, when we used the serum free medium containing bovine serum albumin (BSA), only CUR was taken up but BDMC and DMC were not. Therefore, we identified that the differential cellular uptake of curcuminoids is caused by the medium components, especially BSA. Also, the fluorescence quenching study suggested that differential cellular uptake is due to the different interaction between BSA and each curcuminoid. CONCLUSION: The differential cellular uptake of curcuminoids was caused by the different interaction between curcuminoids and BSA. The results from this study might give clues on the mechanisms by which curcuminoids exhibit different physiological activities.

γ-Oryzanol (OZ), a bioactive phytochemical abundant in cereals such as rice, has been reported to be mainly hydrolyzed to ferulic acid (FA) in the body. Meanwhile, in our previous study, we revealed that a part of OZ is absorbed into the body and exists in its intact form. However, the comprehensive absorption profile of OZ and its metabolites (e.g., FA) after OZ intake has not been fully elucidated yet. Therefore, in this study, we measured the concentrations of OZ, FA, and FA conjugates (i.e., FA sulfate and glucuronide) in the blood of rats with the use of HPLC-MS/MS after a single oral administration of 300 µmol/kg body weight of rice bran OZ (RBOZ). As a result, intact OZ along with FA and FA conjugates existed in the blood, which implied that these constituents may all contribute to the physiological effects under OZ intake. Additionally, when an equimolar amount of FA (300 µmol/kg body weight) was administered, it was found that the absorption profile of FA was significantly different from that when RBOZ was administered.

Although immortalized cultured cells are useful for various functional assays or transcriptome analysis, highly efficient and reproducible immortalization methods have not been developed in avian-derived cells. We introduced the simian virus 40 T antigen (SV40T) and human papillomavirus (HPV)-E6E7 to chick and Okinawa rail (endangered species)derived fibroblast. As a result, neither the SV40T nor E6E7 genes could induce avian cell immortality. Accordingly, we attempted to use a recently developed immortalization method, which involved the coexpression of mutant cyclin-dependent kinase 4 (CDK4), Cyclin D, and TERT (K4DT method) in these avian cells. Although the K4DT method could not efficiently induce the efficient immortalization in mass cell population, cellular division until the senescence was significantly extended by K4DT, we succeeded to obtain the immortalized avian cells (chick K4DT: one clone, Okinawa rail K4DT: three clones, Okinawa rail K4DT + telomerase RNA component: one clone) with K4DT expression. We conclude that K4DT expression is used to extend the cell division and immortalization of avian-derived cells.

1-Deoxynojirimycin (DNJ) has been known as a potent α-glucosidase inhibitor from mulberry leaves and considered beneficial in prevention of type 2 diabetes. Due to limited amount of DNJ in mulberry leaves, recent studies have focused in finding alternative source that can produce higher amount of DNJ. Previously, we produced a high DNJ-containing culture medium from Bacillus amyloliquefaciens AS385 and constructed a concentration method of bacterial culture medium using cation exchange column. However, less complicated concentration procedure is necessary to save time and cost during the large-scale production. Therefore, we developed a simpler concentration method using anion exchange resin to yield B. amyloliquefaciens AS385 culture broth powder (CBP; 1% DNJ) and evaluated the physiological effects of 5-wk dietary CBP intake in C57BL/6J mice. CBP intake tended to suppress the elevation of blood glucose level during oral glucose tolerance test. Moreover, CBP intake significantly lowered the fasting plasma glucose level and white adipose tissue mass. Next, we evaluated the absorption and distribution of DNJ in mice organs after daily CBP intake. We found detectable amount of DNJ in organs with intestine and kidney as the major targeted organs. We concluded that the DNJ content in CBP is absorbed from digestive tract, distributed and accumulated in organs, which most likely to contribute to the alteration of blood glucose regulation and adiposity in C57BL/6J mice. Our study was the first to report the physiological effects of CBP produced from B. amyloliquefaciens AS385 and the organ distribution of DNJ from CBP.

Many functional foods or physiologically active ingredients derived from plants and animals are actively being investigated for their role in chronic disease prevention. Squalene (SQ) is found as active ingredient in the functional foods predominantly present in olive oil and shark liver oil. It is known that during chemotherapy anticancer drugs induce inflammation. SQ has been thought to prevent and suppress inflammation; however, there is little direct evidence available. We examined the adjuvant effect of SQ on tumor-transplanted mice along with anticancer drug doxorubicin (DOX). SQ significantly suppressed the DOX-induced increase in prostaglandin E2 (PGE2) concentration (P < 0.05) in plasma of tumor-bearing mice. SQ inhibited the numbers of writhing response (P < 0.05), formalin-induced pain and decreased COX-2 and substance P expression in the tumor tissue compared to control mice and also enhanced the antitumor efficacy of DOX in allograft mice. Thus, SQ reduces inflammation through modulation of PGE2 production indicating its potential as an adjuvant during chemotherapy in tumor-bearing mice.

The Draize test has been used on rabbits since the 1960s to evaluate the irritation caused by commercial chemicals in products such as cosmetics or hairdressing products. However, since 2003, such tests, including the Draize test for cosmetics, have been prohibited in European countries because they are considered problematic to animal welfare. For this reason, replacement of in vivo methods with the alternative in vitro methods has become an important goal. In this study, we established a corneal epithelial cell line co-expressing a mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original morphology and had an enhanced proliferation rate. Furthermore, the cells showed a significant, dose-dependent decrease in viability in an irritation test using glycolic acid and Benzalkonium chloride. These cells can now be shared with toxicology scientists and should contribute to increasing the reproducibility of chemical testing in vitro.

The Bonin flying fox (Pteropus pselaphon) is one of the most critically endangered species of animals. The number of this species is estimated to be around 150; being classified at the top rank in the list by International Union of Animal Conservation. Our group previously showed that expression of CDK4, CYCLIN D1, and telomerase reverse transcriptase (TERT) efficiently induce immortalization of human, bovine, swine, monkey, and buffalo-derived cells. In this manuscript, we successfully established the primary cells from Bonin flying fox. We introduced CDK4, CYCLIN D1, and TERT into the primary cells. The established cells showed efficient expression of introduced genes at the protein level. Furthermore, the established cells were free from senescence, indicating it reached to immortalization. Moreover, we showed that interspecies somatic cell nuclear transfer of Bonin flying fox derived cell into bovine embryo allowed the development of the embryo to 8 cell stages. Our established cell has the potential to contribute to species conservation.

As an intermediate metabolite during the biosynthesis of sterols, squalene is found ubiquitously in plants and animals. In rice, squalene is contained in rice bran, and consequently, squalene in rice bran oil has gained attention. Studies have shown that the intake of squalene from food sources demonstrate various physiological benefits such as the prevention of cancer and cardiovascular disease. Squalene is also known as an effective antioxidant in edible oils. However, due to its chemical structure, squalene is susceptible to oxidation, which may cause a decline in the nutraceutical and antioxidative effects of squalene in edible oils. Oxidative degradation of squalene also results in the formation of scission products (i.e., aldehydes and ketones) which may lead to off-flavor. Since the rate of squalene oxidation depends on the factors that induce its oxidation (i.e., light or heat), emphasis on oxidation mechanisms is necessary. It has been demonstrated in previous studies that the oxidation products formed by the singlet oxygen oxidation and free radical oxidation of squalene are different, and more recently, we demonstrated that different squalene monohydroperoxide isomers are formed by each oxidation mechanism. We herein discuss the significance of squalene in rice bran oil as well as the oxidative degradation of squalene in edible oils with focus on oxidation mechanisms.

Tsushima leopard cat is the subspecies of Amur cats, and it is classified as the most highest class of critically endangered animals. Although the protection activity is highly recognized, the number of animals is decreasing due to the human activity and invasion of domestic cats and infectious disease. In this study, we succeeded primary culture of normal fibroblasts derived from the Tsushima leopard cat (Prionailurus bengalensis euptilurus). Furthermore, we introduced the human derived mutant Cyclin Dependent Kinase 4, Cyclin D1, and telomere reverse transcriptase. We showed that the expression of these three genes efficiently immortalized cells derived from Tsushima leopard cat. Furthermore, we showed that the chromosome pattern of the established cells is identical with the original one. These data indicate that our method of immortalization is useful to establish cell lines from critically endangered cats, which potentially contributes to the re-generation of critically endangered animals from cultured cell with reproductive technique, such as somatic cloning.

Luteolin is a flavonoid present in plants in the form of aglycone or glucosides. In this study, luteolin glucosides (i.e., luteolin-7- O-β-d-glucoside, luteolin-7- O-[2-(β-d-apiosyl)-β-d-glucoside], and luteolin-7- O-[2-(β-d-apiosyl)-6-malonyl-β-d-glucoside]) prepared from green pepper leaves as well as luteolin aglycone were orally administered to rats. Regardless of the administered luteolin form, luteolin glucuronides were mainly detected from plasma and organs. Subsequently, luteolin aglycone, the most absorbed form of luteolin in rats, was orally administered to humans. As a result, luteolin-3'- O-sulfate was mainly identified from plasma, suggesting that not only luteolin form but also animal species affect the absorption and metabolism of luteolin. When LPS-treated RAW264.7 cells were treated with luteolin glucuronides and luteolin sulfate (the characteristic metabolites identified from rats and humans, respectively), the different luteolin conjugates were metabolized in different ways, suggesting that such difference in metabolism results in their difference in anti-inflammatory effects.

Conservation of the genetic resources of endangered animals is crucial for future generations. The loggerhead sea turtle (Caretta caretta) is a critically endangered species, because of human hunting, hybridisation with other sea turtle species, and infectious diseases. In the present study, we established primary fibroblast cell lines from the loggerhead sea turtle, and showed its species specific chromosome number is 2n = 56, which is identical to that of the hawksbill and olive ridley sea turtles. We first showed that intensive hybridization among multiple sea turtle species caused due to the identical chromosome number, which allows existence of stable hybridization among the multiple sea turtle species. Expressions of human-derived mutant Cyclin-dependent kinase 4 (CDK4) and Cyclin D dramatically extended the cell culture period, when it was compared with the cell culture period of wild type cells. The recombinant fibroblast cell lines maintained the normal chromosome condition and morphology, indicating that, at the G1/S phase, the machinery to control the cellular proliferation is evolutionally conserved among various vertebrates. To our knowledge, this study is the first to demonstrate the functional conservation to overcome the negative feedback system to limit the turn over of the cell cycle between mammalian and reptiles. Our cell culture method will enable the sharing of cells from critically endangered animals as research materials.

Oxidation of squalene (SQ) causes a decline in the nutritional value of SQ in foods, as well as an accumulation of SQ oxidation products in skin lipids which lead to adverse skin conditions. However, mechanistic insights as to how SQ is oxidized by different oxidation mechanisms have been limited, and thus effective measures towards the prevention of SQ oxidation have not been identified. In this study, we oxidized SQ by either singlet oxygen oxidation or free radical oxidation, and monitored the formation of the six SQ monohydroperoxide (SQOOH) isomers, the primary oxidation products of SQ, at the isomeric level. While singlet oxygen oxidation of SQ resulted in the formation of similar amounts of the six SQOOH isomers, free radical oxidation of SQ mainly formed two types of isomers, 2-OOH-SQ and 3-OOH-SQ. The addition of β-carotene during singlet oxygen oxidation, and the addition of α-tocopherol during free radical oxidation lead to a dose-dependent decrease in the formation of SQOOH isomers. Such results suggest that the analysis of SQOOH at the isomeric level allows for the determination of the cause of SQ oxidation in various samples, and provides a foothold for future studies concerning the prevention of SQ oxidation.

Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

Triacylglycerol (TG), the main component of edible oil, is oxidized by thermal- or photo- oxidation to form TG hydroperoxide (TGOOH) as the primary oxidation product. Since TGOOH and its subsequent oxidation products cause not only the deterioration of oil quality but also various toxicities, preventing the oxidation of edible oils is essential. Therefore understanding oxidation mechanisms that cause the formation of TGOOH is necessary. Since isomeric information of lipid hydroperoxide provides insights about oil oxidation mechanisms, we focused on dioleoyl-(hydroperoxy octadecadienoyl)-TG (OO-HpODE-TG) isomers, which are the primary oxidation products of the most abundant TG molecular species (dioleoyl-linoleoyl-TG) in canola oil. To secure highly selective and sensitive analysis, authentic OO-HpODE-TG isomer references (i.e., hydroperoxide positional/geometrical isomers) were synthesized and analyzed with HPLC-MS/MS. With the use of the method, photo- or thermal- oxidized edible oils were analyzed. While dioleoyl-(10-hydroperoxy-8E,12Z-octadecadienoyl)-TG (OO-(10-HpODE)-TG) and dioleoyl-(12-hydroperoxy-9Z,13E-octadecadienoyl)-TG (OO-(12-HpODE)-TG) were characteristically detected in photo-oxidized oils, dioleoyl-(9-hydroperoxy-10E,12E-octadecadienoyl)-TG and dioleoyl-(13-hydroperoxy-9E,11E-octadecadienoyl)-TG were found to increase depending on temperature in thermal-oxidized oils. These results prove that our methods not only evaluate oil oxidation in levels that are unquantifiable with peroxide value, but also allows for the determination of oil oxidation mechanisms. From the analysis of marketed canola oils, photo-oxidized products (i.e., OO-(10-HpODE)-TG and OO-(12-HpODE)-TG) were characteristically accumulated compared to the oil analyzed immediately after production. The method described in this paper is valuable in the understanding of oil and food oxidation mechanisms, and may be applied to the development of preventive methods against food deterioration.

Lutein, a type of xanthophyll, possesses antioxidative properties that contribute to the prevention of various diseases. Preliminary screening has shown that Japanese mugwort (Artemisia princeps Pamp.) contains high amounts of lutein. In this study, we evaluated the lutein concentration in a processed mugwort product (mugwort paste). By using high-performance liquid chromatography coupled with visible light detection or mass spectrometry, the lutein concentration in mugwort paste was determined as 38 mg/100 g dry weight, which indicates that mugwort is a potentially valuable natural food source of lutein. We also investigated the effects of the manufacturing process and found that the lutein content was significantly increased by the boiling and dehydrating processes during the production of mugwort paste. Mugwort paste that is rich in lutein may therefore serve as an effective nutraceutical.

Previous studies have demonstrated that tocotrienol (T3) has antiatherogenic effects. However, the T3 preparations used in those studies contained considerable amounts of tocopherol (Toc), which might affect the biological activity of T3. There is little information on the effect of highly purified T3 on atherosclerosis formation. This study investigated the effect of high-purity T3 on atherosclerotic lesion formation and the underlying mechanisms. Male apolipoprotein E knockout (apoE-KO) mice were fed a cholesterol-containing diet either alone or supplemented with T3 concentrate (Toc-free T3) or with alpha-Toc for 12 weeks. ApoE-KO mice fed the 0.2% T3-supplemented diet showed reduced atherosclerotic lesion formation in the aortic root. The 0.2% T3 diet induced Slc27a1 and Ldlr gene expression levels in the liver, whereas the alpha-Toc-supplemented diet did not affect those expression levels. T3 was predominantly deposited in fat tissue in the T3 diet-fed mice, whereas alpha-Toc was preferentially accumulated in liver in the alpha-Toc diet-fed mice. Considered together, these data demonstrate that dietary T3 exerts anti-atherosclerotic effect in apoE-KO mice. The characteristic tissue distribution and biological effects of T3, that are substantially different from those of Toc, may contribute to the antiatherogenic properties of T3. (C) 2017 Elsevier Inc. All rights reserved.

In this study, we created porcine-induced pluripotent stem (iPS) cells with the expression of six reprogramming factors (Oct3/4, Klf4, Sox2, c-Myc, Lin28, and Nanog). The resulting cells showed growth dependent on LIF (leukemia inhibitory factor) and expression of multiple stem cell markers. Furthermore, the iPS cells caused teratoma formation with three layers of differentiation and had both active X chromosomes (XaXa). Our iPS cells satisfied the both of important characteristics of stem cells: teratoma formation and activation of both X chromosomes. Injection of these iPS cells into morula stage embryos showed that these cells participate in the early stage of porcine embryogenesis. Furthermore, the RNA-Seq analysis detected that expression levels of endogenous pluripotent related genes, NANOG, SOX2, ZFP42, OCT3/4, ESRRB, and ERAS were much higher in iPS with six factors than that with four reprogramming factors. We can conclude that the expression of six reprogramming factors enables the creation of porcine iPS cells, which is partially close to naive iPS state. J. Cell. Biochem. 118: 537-553, 2017. (c) 2016 Wiley Periodicals, Inc.

Squalene (SQ), a main component of human sebum, is readily photooxidized by exposure to sunlight, producing six squalene monohydroperoxide (SQ-OOH) isomers. Despite its known connection to various skin conditions, few studies have sought to analyze SQ-OOH at the isomeric level. In this study, we aimed to develop a method to discriminate each SQ-OOH isomer with the use of tandem mass spectrometry (MS/MS). The six standard SQ-OOH isomers were prepared by photooxidizing SQ in the presence of rose bengal, a photosensitizer, and isolated by semipreparative high-performance liquid chromatography (HPLC). To purify each isomer, 2-methoxypropene, which reversibly reacts with the hydroperoxide group of SQ-OOH, was utilized. Product ion scanning was then performed on the standard SQ-OOH isomers in the absence and presence of the sodium ion. In the absence of the sodium ion, the fragmentation patterns produced by atmospheric pressure chemical ionization were similar between the isomers, whereas in the presence of the sodium ion by electrospray ionization, unique fragmentation patterns were achieved. Based on these fragment ions, HPLC-MS/MS multiple reaction monitoring analysis was conducted on a mixture of the standard SQ-OOH isomers. We achieved discrimination of SQ-OOH isomers with high selectivity and detected SQ-OOH isomers at nanogram levels. These results may improve our understanding of the effect of SQ-OOH on skin conditions as well as the mechanism behind SQ peroxidation.

Rice bran is a rich source of functional compounds, including tocotrienol (T3) and ferulic acid (FA). We previously investigated the anti-cancer properties of T3, and reported on the potent inhibitory effects of delta-T3 on angiogenesis and telomerase activity. In this study, we examined the synergistic suppressive effects of the combination of delta-T3 and FA on telomerase activity in DLD-1 human colorectal adenocarcinoma cells. Co-treatment with delta-T3 and FA significantly decreased cellular telomerase activity compared to treatment with delta-T3 alone, whereas FA alone had no inhibitory effect. Co-treatment with delta-T3 and FA also synergistically down-regulated the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, indicating that the enzymatic activity of telomerase is controlled at the transcriptional level. FA significantly increased the intracellular concentration of delta-T3, suggesting that FA improved the bioavailability of delta-T3, thereby increasing the inhibitory potency of delta-T3 on telomerase. FA may be a promising candidate for augmenting the anti-cancer activity of delta-T3.

Tocotrienol (T3), unsaturated vitamin E, is gaining a lot of attention owing to its potent anticancer effect, since its efficacy is much greater than that of tocopherol (Toc). Various factors are known to be involved in such antitumor action, including cell cycle arrest, apoptosis induction, antiangiogenesis, anti-metastasis, nuclear factor-kappa B suppression, and telomerase inhibition. Owing to a difference in the affinity of T3 and Toc for the alpha-tocopherol transfer protein, the bioavailability of orally ingested T3 is lower than that of Toc. Furthermore, cellular uptake of T3 is interrupted by coadministration of alpha-Toc in vitro and in vivo. Based on this, several studies are in progress to screen for molecules that can synergize with T3 in order to augment its potency. Combinations of T3 with chemotherapeutic drugs (e.g., statins, celecoxib, and gefitinib) or dietary components (e.g., polyphenols, sesamin, and ferulic acid) exhibit synergistic actions on cancer cell growth and signaling pathways. In this review, we summarize the current status of synergistic effects of T3 and an array of agents on cancer cells, and discuss their molecular mechanisms of action. These combination strategies would encourage further investigation and application in cancer prevention and therapy.

Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD) induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism associated gene expression. (C) 2016 Elsevier Inc. All rights reserved.

Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, dephosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (gamma IR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in gamma IR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.

Tocotrienol (T3) has recently gained increasing interest due to its anti-cancer effect. Here, we investigated the modulating effect of delta and gamma T3 on the Ras-Raf-MEK-ERK oncogenic upstream signaling pathway in human hepatocellular carcinoma HepG2 cells. The results indicated that T3 regulated the upstream signaling cascades of this pathway.

In this study, we investigated cellular uptake and metabolism of phosphatidylcholine hydroperoxide (PCOOH) in human hepatoma HepG2 cells by high performance liquid chromatography-tandem mass spectrometry, and then evaluated whether PCOOH or its metabolites cause pathophysiological effects such as cytotoxicity and apoptosis. Although we found that most PCOOH was reduced to PC hydroxide in HepG2 cells, the remaining PCOOH caused cytotoxic effects that may be mediated through an unusual apoptosis pathway. These results will enhance our fundamental understanding of how PCOOH, which is present in oxidized low density lipoproteins, is involved in the development of atherosclerosis. (C) 2015 Elsevier Inc. All rights reserved.

Nonhuman primates are useful animal models for the study of human diseases. However, the number of established cell lines from nonhuman primates is quite limited compared with the number established from other experimental animals. The establishment of nonhuman primate cell lines would allow drug testing on those cell lines before moving experiments into primates. In this study, we established nonhuman primate primary cell lines by introducing the genes for CDK4R24C, cyclin D1, and hTERT. These cell lines proliferated more rapidly than primary cells and bypassed cellular senescence. Karyotype analysis showed that the chromosome patterns were intact in the immortalized cell lines. Furthermore, we showed that the expression of introduced genes could be precisely controlled through the Tet-Off system with the addition of doxycycline. The present study shows that introduction of the CDK4R24C, cyclin D1, and hTERT genes are effective methods of establishing nonhuman primate cell lines. (C) 2014 Wiley Periodicals, Inc.

Rice bran consists of many functional compounds and thus much attention has been focused on the health benefits of its components. Here, we investigated the synergistic inhibitory effects of its components, particularly delta-tocotrienol (delta-13) and ferulic acid (FA), against the proliferation of an array of cancer cells, including DU-145 (prostate cancer), MCF-7 (breast cancer), and PANC-1 (pancreatic cancer) cells. The combination of delta-T3 and FA markedly reduced cell proliferation relative to delta-T3 alone, and FA had no effect when used alone. Although 6-T3 induced G1 arrest by up-regulating p21 in PANC-1 cells, more cells accumulated in G1 phase with the combination of delta-T3 and FA. This synergistic effect was attributed to an increase in the cellular concentration of delta-T3 by FA. Our results suggest that the combination of delta-T3 and FA may present a new strategy for cancer prevention and therapy. (C) 2014 Elsevier Inc. All rights reserved.

Cattle and pigs comprise the most economically important livestock. Despite their importance, cultured cells from these species, which are useful for physiological analyses, are quite limited in cell banks. One of the reasons for the limited number of cell lines is the difficulty in their establishment. To overcome limitations in cell-line establishment, we attempted to immortalize bovine and porcine fibroblasts by transduction of multiple cell cycle regulators (mutant cyclin dependent kinase 4, cyclin D and telomerase reverse transcriptase). The transduced cells continued to display a stable proliferation rate and did not show cellular senescence. Furthermore, cell cycle assays showed that induction of these exogenous genes enhanced turnover of the cell cycle, especially at the G1-S phase. Furthermore, our established cell lines maintained normal diploid karyotypes at 98-100%. Our study demonstrated that bypassing p16/Rb-mediated cell arrest and activation of telomerase activity enabled efficient establishment of immortalized bovine- and porcine-derived fibroblasts. The high efficiency of establishing cell lines suggests that the networks of cell cycle regulators, especially p16/Rb-associated cell cycle arrest, have been conserved during evolution of humans, cattle, and pigs. (C) 2014 Elsevier B.V. All rights reserved.

Previously, we reported that near-infrared irradiation that simulates solar near-infrared irradiation with pre- and parallel-irradiational cooling can non-thermally induce cytocidal effects in cancer cells. To explore these effects, we assessed cell viability, DNA damage response pathways, and the percentage of mitotic cancer cells after near-infrared treatment. Further, we evaluated the anti-cancer effects of near-infrared irradiation compared with doxorubicin in xenografts in nude mice by measuring tumor volume and assessing protein phosphorylation by immunoblot analysis. The cell viability of A549 lung adenocarcinoma cells was significantly decreased after three rounds of near-infrared irradiation at 20 J / cm2. Apoptotic cells were observed in near-infrared treated cells. Moreover, near-infrared treatment increased the phosphorylation of ataxia-telangiectasia mutated (ATM) at Ser1981, H2AX at Ser139, Chk1 at Ser317, structural maintenance of chromosome (SMC) 1 at Ser966, and p53 at Ser15 in A549 cells compared with control. Notably, near-infrared treatment induced the formation of nucleic foci of ?H2AX. The percentage of mitotic A549 cells, as measured by histone H3 phosphorylation, decreased significantly after three rounds of near-infrared irradiation at 20 J /cm2. Both near-infrared and doxorubicin inhibited the tumor growth of MDA-MB435 melanoma cell xenografts in nude mice and increased the phosphorylation of p53 at Ser15, Chk1 at Ser317, SMC1 at Ser966, and H2AX at Ser139 compared with control mice. These results indicate that near-infrared irradiation can non-thermally induce cytocidal effects in cancer cells as a result of activation of the DNA damage response pathway. The near-infrared irradiation schedule used here reduces discomfort and side effects. Therefore, this strategy may have potential application in the treatment of cancer. (Cancer Sci 2012; 103: 14671473)

The pig is an important animal for both agricultural and medical purposes. However, the number of pig-derived cell lines is relatively limited when compared with mouse- and human-derived lines. We established in this study a retroviral conditional expression system for the Simian vacuolating virus 40 large T fragment (SV40T) which allowed us to efficiently establish pig embryonic fibroblast cell lines. The established cell lines showed high levels of cell proliferation and resistance to cellular senescence. A chromosome analysis showed that 84% of the cells had the normal karyotype. Transient expression of the Cre recombinase allowed us to excise the SV40T fragment from the genome. The development of this research tool will enable us to quickly establish new cell lines derived from various animals.

Several lines of experimental data have highlighted a key role of Amadori-glycated phosphatidylethanolamine (Amadori-PE) in the development of diabetic complications. Recent epidemiological studies suggest that diabetes mellitus could be a risk factor for some cancers. A characteristic of cancer cells is their immortal phenotype, and the enzyme telomerase contributes to the infinite replicative potential of cancer cells. The purpose of this study was to obtain new information about the effect of Amadori-PE on the regulation of telomerase in PANC-1 human pancreatic carcinoma cells. Amadori-PE enhanced cellular telomerase in a time- and dose-dependent manner by up-regulating hTERT expression through induction of c-myc. These results provide experimental evidence for a novel role of Amadori-PE in linking diabetes and cancer. (c) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Several lines of evidence support the beneficial effect of locotrienol (T3; an unsaturated vitamin E) on inhibition of tumor development. Many factors, including decrease in oxidative stress and modulation of cell signaling pathways in tumor and cridotliclial cells, have been implicated in such anticancer action of T3, while the in vivo potency and exact intracellular mechanisms anticancer properties of T3 remain not fully understood. We have hypothesized that the inhibitory effect of T3 on cancer may be attributable to the antiangiogenic activity of T3, and we found that T3 acts as a potent regulator of growth-factor-dependent signaling in endothelial cells and as an antiaingiogenic agent minimizing tumor growth. In this work, we review the history and biological action (i.e., anticancer) of vitamin E and describe current research on the antiangiogenic effects of T3 and its mechanisms. (C) 2009 Elsevier Inc. All rights reserved.

As high telomerase activity is detected in most cancer cells, inhibition of telomerase by drug or dietary food components is a new strategy for cancer prevention. Here, we investigated the inhibitory effect of vitamin E, with particular emphasis on tocotrienol (unsaturated vitamin E), on human telomerase in cell-culture study. As results, tocotrienol inhibited telomerase activity of DLD-1 human colorectal adenocarcinoma cells in time- and dose-dependent manner, interestingly, with 5-tocotrienol exhibiting the highest inhibitory activity. Tocotrienol inhibited protein kinase C activity, resulting in down-regulation of delta-myc and human telomerase reverse transcriptase (hTERT) expression, thereby reducing telomerase activity. In contrast to tocotrienol, tocopherol showed very weak telomerase inhibition. These results provide novel evidence for the first time indicating that tocotrienol acts as a potent candidate regulator of telomerase and supporting the anti-proliferative function of tocotrienol. (c) 2006 Elsevier Inc. All rights reserved.

As high telomerase activity is detected in most cancer cells, telomerase represents a promising cancer therapeutic target. We investigated the inhibitory effect of various fatty acids on telomerase, with particular emphasis on those with antitumor properties, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To evaluate the direct effect of fatty acids on telomerase, cell lysates of DLD-1 human colorectal adenocarcinoma cells were mixed with sample fatty acids, and the telomerase activity was determined. Saturated fatty acids and trans-fatty acids showed very weak or no inhibition of telomerase. In contrast, cis-unsaturated fatty acids significantly inhibited the enzyme, and the inhibitory potency was elevated with an increase in the number of double bonds. Accordingly, polyunsaturated fatty acids (PUFAs), like EPA and DHA, appeared to be powerful telomerase inhibitors. To assess the transcriptional effect, DLD-1 cells were cultured in the presence of sample fatty acids, and telomerase activity and gene expression were subsequently evaluated. Culturing DLD-1 cells with either EPA or DHA resulted in a remarkable decrease in telomerase activity. EPA and DHA inhibited telomerase by down-regulating human telomerase reverse transcriptase (hTERT) and c-myc expression via protein kinase C inhibition. These results indicate that PUFAs can directly inhibit the enzymatic activity of telomerase as well as modulate the telomerase at the transcriptional level. (c) 2005 Elsevier B.V. All rights reserved.

Conjugated eicosapentaenoic acid (cEPA) selectively inhibited the activities of mammalian DNA polymerases (pols) and human DNA topoisomerases (topos) [Yonezawa Y, Tsuzuki T, Eitsuka T, Miyazawa T, Hada T, Uryu K, et al. Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II. Arch Biochem Biophys 2005;435:197-206]. In this report, we investigated the inhibitory effect of cEPA on a human promyelocytic leukemia cell line, HL-60, to determine which enzymes influence cell proliferation. cEPA inhibited the proliferation of HL-60 cells (LD50 = 20.0 mu M), and the inhibitory effect was stronger than that of nonconjugated EPA. cEPA arrested the cells at GI/S-phase, increased cyclin A and E protein levels, and prevented the incorporation of thymidine into the cells, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. This compound also induced apoptosis of the cells. These results suggested the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols. (c) 2005 Elsevier Inc. All rights reserved.

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism Such as replication reactions. We reported previously that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (ie., eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)) inhibited the activities of eukaryotic pols in vitro [Biochim. Biophys, Acta 1336 (1997) 509]. In the present study, we found that PUFA also inhibited human topos I and II activities and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger (ban that of normal EPA and DHA. cEPA and cDHA inhibited the activities of mammalian pols and human topos. but did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC50 values for mammalian pols and human topos of 11.0-31.8 and 0.5-2.5 muM, respectively. Therefore. the inhibitory effect of cEPA on topos was stronger than that on pols. Preincubation analysis suggested that cEPA directly bound both topos I and II, but did not bind or interact with substrate DNA. This is the first report that conjugated PUFA such as cEPA act as inhibitors of pols and topos. The results support the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols and topos. (C) 2004 Elsevier Inc. All rights reserved.

We reported previously that unsaturated linear-chain FA of the cis-configuration with a C-18-hydrocarbon chain such as linoleic acid (18:2Delta9c, 12c) could potently inhibit the activities of mammalian DNA polymerases and DNA topoisomerases, but their saturated forms could not. There are chemically two classes of unsaturated FA, normal and conjugated, but only the conjugated forms show potent antitumor activity. In this report, we study the inhibitory effects of chemically synthesized conjugated C-18-FA on mammalian DNA polymerases and DNA topoisomerases as compared with normal unsaturated FA. The conjugated alpha-eleostearic acid (18:3Delta9c, 11t, 13t) was the strongest of all the FA tested. For the inhibition, the conjugated form is crucially important. The energy-minimised 3-D structures of the FA were calculated, and both a length of less than 20 Angstrom and a width of 8.13-9.24 Angstrom in the C-18-FA structure were found to be important for enzyme inhibition. The 3-D structure of the active site of both DNA polymerases and topoisomerases must have had a pocket to join alpha-eleostearic acid, and this pocket was 12.03 Angstrom long and 9.24 Angstrom wide.

As high telomerase activity is detected in most cancer cells, telomerase represents a promising cancer therapeutic target. We investigated the inhibitory effect of sulfoquinovosyldiacylglycerol (SQDG), distributed in plants and seaweeds, on human telomerase in a cell-free system. SQDG inhibited telomerase activity dose-dependently with 50% inhibition at 22 muM, whereas monogalactosyldiacylglycerol and digalactosyldiacylglycerol did not, even at concentrations of 100 muM. Moreover, we confirmed that eicosapentaenoic acid, one of the fatty acid components of SQDG, is a potent telomerase inhibitor with 50% inhibition at 19 muM. We speculate that the structure of the sulfate group and fatty acid of SQDG is important for the potent telomerase-inhibitory effect. Our findings suggest that SQDG has potential use as a therapeutic dietary compound for telomerase inhibition. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Polyunsaturated fatty acids (PUFAs) have been reported to have antitumor activity. In this study, we have tested whether telomerase might be a target for the antitumor effect of fatty acids using DLD-1 colorectal adenocarcinoma cells. In a cell-free approach, fatty acids were added directly to cell lysates, and we confirmed that increasing fatty acid unsaturation correlates with increased inhibition of telomerase activity. Using a cell culture approach, DLD-1 cells were cultured with fatty acids. In a time and dose dependent manner, EPA and DHA suppressed cellular telomerase activity and the mRNAs encoding hTERT (human telomerase reverse transcriptase) and c-myc. Based on these observations, we suggest that PUFAs inhibit telomerase activity through dual mechanisms: direct inhibition of enzymatic activity and down regulation of hTERT, one of the telomerase components.

Inhibition of angiogenesis and telomerase activity with vitamin E compounds, especially for tocotrienol (T3), has been investigated. Nutrigenomic tools have been used for elucidating the bioactive mechanisms of T3. In the cell culture experiments, T3 reduced the vascular endothelial growth factor (VEGF)-stimulated tube formation by human umbilical vein endothelial cells (HUVEC). Among T3 isomers, delta-T3 appeared the highest activity. The T3 inhibited the new blood vessels formation on the growing chick embryo chorioallantoic membrane (CAM assay for an in vivo model of angiogenesis). In contrast, tocopherol did not. The findings suggested that the T3 has potential use for reducing angiogenic disorder. DNA chip analysis revealed that T3 specifically down-regulates the expression of VEGF receptor (VEGFR) in endothelial cells. It is well-known that VEGF regulates angiogenesis by binding to VEGFR. Therefore, T3 could block the intracellular signaling of VEGF via down-regulation of VEGFR, which resulted in the inhibition of angiogenesis. On the other hand, DNA chip analysis also revealed that T3 down-regulates the expression of protein kinase C (PKC) in the cultured HUVEC. Since PKC is involved with the control of telomerase activity, T3 has potential to act as anti-telomerase inhibitor via PKC inhibition. In this manner, DNA chip technology provides efficient access to genetic information regarding food function and its mechanism.