Anti-cancer effect of dung beetle glycosaminoglycans on melanoma.

BACKGROUND: Dung beetle glycosaminoglycan is known to possess anti-aging activities. However, its anti-cancer mechanisms are not fully elucidated yet. The objective of this study was to evaluate the anti-cancer effect of insect-derived polymer dung beetle glycosaminoglycan (GAG) after intraperitoneally injecting it to melanoma mice induced by B16F10 cells. METHODS: To determine molecular mechanism involved in the anti-cancer effect of dung beetle GAG, its origin N-glycan under 3KD Dalton was assayed for melanoma cell cytotoxicity. Quantitative comparisons of adhesive molecule on extracellular matrix and activities of tissue inhibitor of metalloprotease 2 (TIMP-2) were also investigated. In vivo anti-cancer effect of dung beetle GAG on solid tumor size, survival time and gene-expression profiles was also assayed using B10F10 melanoma mice model. Mice with induced melanoma were then treated with Catharsius molossus (dung beetle) GAG (CaG) at 5 mg/kg for 8 weeks to investigate its anti-cancer effects compared to bumblebee (Bombus ignitus) queen glycosaminoglycan (IQG) and Huechys sanguinea glycosaminoglycan (HEG). RESULTS: These N-glycans derived from these GAG were composed of many linear heparinoid polysaccharides, polymers with hexose and N-acetylhexose. Adminstration with these GAGs increased survival time and decreased melanoma sizes in mice, in accordance with their inhibitory effects on cell growth ratio of melanoma B16F10. In addition, treatment with N-glycans derived from theses glycosaminoglycan increased activities of TIMP-2 in HMVEC cells pretreated with TNF-alpha and in melanoma cells, suggesting that they had anti-inflammatory and anticancer activities. In DNA microarray results, compared to control, CaG treated mouse group showed upregulation of 192 genes including collagen,typeI,alpha1 (Col1a1), consistent with the highly increased in vitro extracellular matrix (ECM) adhesion on collagen 1 and up-regulation of heparanase (Hpse). After treatment with CaG, a total of 152 genes were down-regulated, including nuclear RNA export factor (Nxf3) and hyaluronan proteoglycan link protein1 (Hapln1). CONCLUSIONS: Glycosaminoglycan, CaG can strengthen ECM by increasing activity of TIMP-2 and adhesion activity on collagen known to inhibit changes of ECM, leading to tumor cell invasion and progression.

Anti-Obesity Effect of Bombus ignitus Queen Glycosaminoglycans in Rats on a High-Fat Diet.

The mechanism of functional insect glycosaminoglycan (GAG) on obesity caused a high fat diet has not yet been elucidated. Therefore, insect glycosaminoglycans derived from Isaria sinclairii, Bombus ignitus (a type of bumblebee) queen, and Gryllus bimaculatus were purified and investigated as a potential functional food. 14-week old male Wistar rats were fed a high-fat diet (HFD) for 6 weeks. There were five groups that received daily intraperitoneal administration of phosphate buffered saline (PBS, control), GbG (GAG from Gryllus bimaculatus) 10 mg/kg, ISG (GAG from Isaria sinclairii) 10 mg/kg, IQG (GAG from Bombus ignites) 10 mg/kg, or Pravastatin (2 mg/kg). All treatments were performed for one month. IQG produced a potential anti-inflammatory effect with the inhibition of c-reactive protein and sero-biochemical parameters of phospholipids and free fatty acids indicative of an anti-hyperlipidemic effect. Abdominal and epididymidal fat weight were reduced in conjunction with a mild increase in body weight. The level of laminin in HMVEC-C cells or fibronectin in HFD rat hepatocytes was significantly affected by these GAG treatments, which regulated adipogenesis and adipocyte function. Compared to the control rats, IQG-treated rats displayed up-regulation of 87 genes (test:control ratio >2.0) including fatty acid synthase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase, with the down-regulation of 47 genes including the uridine diphosphate (UDP) glycosyltransferase 2 families, polypeptidase B, and insulin-like growth factor binding protein 1. The data suggest that IQG could potentially prevent or treat fatty liver or hyperlipidemia.

Gene expression profiling and inhibition of adipose tissue accumulation of G. bimaculatus extract in rats on high fat diet.

BACKGROUND: Molecular genetic mechanisms underlying the anti-inflammatory effects of ethanol extract (GB) from G. bimaculatus, a type of cricket, are not fully elucidated. G. bimaculatus was reported to be rich in unsaturated fatty acid and to decrease the omega-6/omega-3 fatty acid ratio when fed to chickens. GB may reduce the amount of fat or increase the unsaturated fatty acid ratio. METHODS: Male Wistar rats fed a high-fat diet (HFD) were orally administered with 5 groups: phosphate buffered saline (PBS, control), GB (100 mg/kg or 200 mg/kg), Pravastatin or Isaria sinclairii (IS) extract, which is reported to have fat-reducing effects, for either 1 or 2 months. GB's sero-biochemial, hematological and anti-oxidizing hepato-cellular biomarker levels were evaluated to dertermine their antilipidemic, anti-inflammatory, and anti-coagulant effect in rats after 1 or 2 month GB treatments on HFD (fat 60 %) Wistar rat. The abdominal and epididymidal fat weight were measured and the composition of fatty acid was analyzed by GC/MS. Microarray analyses were performed with a rat 28 K cDNA clone set array to identify the gene-expression profiles for the GB exposed high fat dieted Wistar rat. RESULTS: The weight and fatty acid composition of abdominal fat and epididymidal fat, total cholesterol, LDL-cholesterol, and triglyceride in GB treated rats were at lower levels than those of the control group. The anti-oxidant hepato-cellular biomarker levels, protein carbonyl content and malondialdehyde concentration in GB treated rats were significantly decreased. Compared to the control, the GB treated rat group (treated at a dose of 100 and 200 mg/kg), had 190 up-regulated genes including Gpm6a (glycoprotein m6a), Tmem14a (transmembrane protein 14A) and Fasin (fatty acid synthase), with down-regulated 235 genes including Cc121b (chemokine ligand 21b), Glycan1 (glycosylation dependent cell adhesion moleule, Serpinb1a (serine proteinase inhibitor) and Tcrb (T-cell receptor beta chain). CONCLUSION: The data suggest Fasin-related fatty acid synthesis and adipose differentiation related protein (Adfp), which is related to obesity, were upregulated by GB treatment, indicating their potential therapeutic markers for anti-atheriosclerosis or inflammation.

The role of noble bumblebee (Bombus terrestris) queen glycosaminoglycan in aged rat and gene expression profile based on DNA microarray.

Glycosaminoglycans (GAGs) have been used to diminish the deleterious effects associated with aging by preventing the destruction of cartilage, bone, discs, and skin. The objective of this study was to evaluate the anti-aging effect of a newly prepared GAG derived from bumblebee (Bombus terrestris) queen (BTQG, 10 mg/kg). Gryllus bimaculatus (Gb, cricket) GAG (GbG, 10 mg/kg) or glucosamine sulfate (GS) was used as a positive control. N-glycans derived from BTQG contained hexose polymers including Hex4HexNAc3Pen1, Hex9, and Hex5HexNAc3dHex2 as the primary components. The GAGs were intraperitoneally administered to 14-month-old aged rats for 1 month. BTQG reduced the serum levels of free fatty acid, alkaline phosphatase (ALP), glutamate pyruvate transaminase (GPT), creatinine, and blood urea nitrogen (BUN), showing hepato-and renal-protective effects with anti-lipidemic activities comparable to GS. The changes of gene expression profile of liver tissue by cDNA microarray showed the simultaneous upregulation of 36 genes in the BTQG-treated rat group compared to the control group, including secretogranin II (Scg2), Activator (AP)-1-regulated protein-related reactive oxygen species (ROS) DNA damage repair, metallothionein 1a, and alpha-2 macroglobulin. The BTQG-treated group also showed 417 downregulated genes, including vimentin, moesin, and mitochondrial carbonic anhydrase. Insect glycosaminoglycan from the bumblebee (B. terrestris) queen may help decelerate the aging stage by ameliorating the aging effects on circulation, and liver and kidney function.

Antidiabetic effects and gene expression profiling in obese mice treated with Isaria sinclairii over a 6-month period.

The molecular mechanisms underlying the glucose-lowering effects of Isaria sinclairii (Cicada Dongchunghacho), a fungus cultured on silkworm, are not fully elucidated. Thus the glucose-lowering effects of I. sinclairii as potential an antidiabetic agent were investigated in C57BL/6 obese (ob/ob) mice over a 6-mo period. For a period of 26 wk, ob mice were administered either 5 or 10% (w/w) I. sinclairii powder (IS), 10% dry mulberry leaf powder (ML), or 10% silkworm (SW) powder in the standard diet while a control group received only standard diet. The ML and SW preparations served as positive controls. Isaria sinclairii at 10% in the diet was more effective in reducing body weight compared to 10% ML, 10% SW, or 5% I. sinclairii. The fall in blood glucose levels in the groups treated for 26 wk was greater in both IS groups at 1 mo compared to ML or SW but equal in all groups at 6 mo. Microarray analyses were performed with a mouse 7.4K cDNA clone set array to identify the gene-expression profiles for the IS-, ML-, and SW-exposed ob mouse liver. The 10% IS group, compared to control, showed that 15 genes including glucokinase (Gk-rs1) and LDL receptor relating protein 1 were upregulated and 12 genes including cell translocation gene2 (antiproliferative) and hydroxyprostaglandin dehydrogenase (Hpgd 15) were downregulated. Upregulation of Gk-rs 1 and downregulation of Hpgd 15 were previously shown to occur in drug-induced suppression of diabetes. With ML, Lepr (leptin receptor), Pik3cb (phosphatidylinositol 3-kinase), and Prodh (proline dehydrogenase), related to suppression of diabetes, were upregulated. In the case of SW, the enzymes (G2an, alpha glucosidase 2) and Mmp9 (matrix metalloproteinase 9) involved in elevation of blood glucose levels were both downregulated. Data suggest that I. sinclarii is effective in lowering blood glucose due to the upregulation of glucokinase (Gk-rs1) and downregulation of hydroxyprostaglandin dehydrogenase (Hpgd 15), both associated with suppression of diabetes, indicating that microarray analysis is a useful tool to assess pharmacological potency of therapeutic compounds.

Erratum to "Anti-Diabetic Effect of Dung Beetle Glycosaminoglycan on db Mice and Gene Expression Profiling" [Toxicol. Res. 34 (2018) 151-162].

[This corrects the article on p. 151 in vol. 34, PMID: 29686777.].

Anti-Diabetic Effects of Dung Beetle Glycosaminoglycan on db Mice and Gene Expression Profiling.

Anti-diabetes activity of Catharsius molossus (Ca, a type of dung beetle) glycosaminoglycan (G) was evaluated to reduce glucose, creatinine kinase, triglyceride and free fatty acid levels in db mice. Diabetic mice in six groups were administrated intraperitoneally: Db heterozygous (Normal), Db homozygous (CON), Heuchys sanguinea glycosaminoglycan (HEG, 5 mg/kg), dung beetle glycosaminoglycan (CaG, 5 mg/kg), bumblebee (Bombus ignitus) queen glycosaminoglycan (IQG, 5 mg/kg) and metformin (10 mg/kg), for 1 month. Biochemical analyses in the serum were evaluated to determine their anti-diabetic and anti-inflammatory actions in db mice after 1 month treatment with HEG, CaG or IQG treatments. Blood glucose level was decreased by treatment with CaG. CaG produced significant anti-diabetic actions by inhiting creatinine kinase and alkaline phosphatase levels. As diabetic parameters, serum glucose level, total cholesterol and triglyceride were significantly decreased in CaG5-treated group compared to the controls. Dung beetle glycosaminoglycan, compared to the control, could be a potential therapeutic agent with anti-diabetic activity in diabetic mice. CaG5-treated group, compared to the control, showed the up-regulation of 48 genes including mitochondrial yen coded tRNA lysine (mt-TK), cytochrome P450, family 8/2, subfamily b, polypeptide 1 (Cyp8b1), and down-regulation of 79 genes including S100 calcium binding protein A9 (S100a9) and immunoglobulin kappa chain complex (Igk), and 3-hydroxy-3-methylglutaryl-CoenzymeAsynthase1 (Hmgcs1). Moreover, mitochondrial thymidine kinase (mt-TK), was up-regulated, and calgranulin A (S100a9) were down-regulated by CaG5 treatment, indicating a potential therapeutic use for anti-diabetic agent.

Gene-expression profiling of human mononuclear cells from welders using cDNA microarray.

A toxicogenomic chip developed to detect welding-related diseases was tested and validated for field trials. To verify the suitability of the microarray, white blood cells (WBC) or whole blood was purified and characterized from 20 subjects in the control group (average work experience of 7 yr) and 20 welders in the welding-fume exposed group (welders with an average work experience of 23 yr). Two hundred and fifty-three rat genes homologous to human genes were obtained and spotted on the chip slide. Meanwhile, a human cDNA chip spotted with 8600 human genes was also used to detect any increased or decreased levels of gene expression among the welders. After comparing the levels of gene expression between the control and welder groups using the toxicogenomic chips, 103 genes were identified as likely to be specifically changed by welding-fume exposure. Eighteen of the 253 rat genes were specifically changed in the welders, while 103 genes from the human cDNA chip were specifically changed. The genes specifically expressed by the welders were associated with inflammatory responses, toxic chemical metabolism, stress proteins, transcription factors, and signal transduction. In contrast, there was no significant change in the genes related to short-term welding-fume exposure, such as tumor necrosis factor (TNF)-alpha and interleukin. In conclusion, if further validation studies are conducted, the present toxicogenomic gene chips could be used for the effective monitoring of welding-fume-exposure-related diseases among welders.

Anti-aging effect and gene expression profiling of dung beetle glycosaminoglycan in aged rats.

BACKGROUND: This study aimed to evaluate the anti-aging effect of a newly prepared insect-derived compound, dung beetle glycosaminoglycan (GAG), given intraperitoneally to old SD rats as part of their diet for 1 month. Insect GAG administration was found to be related to a reduction in oxidative damage, hepato-cellular biomarker levels, protein carbonyl content, and malondialdehyde concentration. The anti-aging-related molecular genetic mechanisms of dung beetle GAG are not yet fully elucidated. RESULTS: Catharsius molossus (a type of dung beetle) GAG (CaG) possessed anti-aging activities; it reduced the serum level of creatinine kinase, had aortic vasorelaxant activities and cardioprotective actions, and maintained a normal glucose level in treated rats. Microarray analysis was performed with a rat 30 K cDNA clone set array to identify the gene-expression profiles of 14-month-old SD rats treated with dung beetle glycosaminoglycan 5 mg/kg (CaG5) over a 1-month period, which was done to investigate its anti-aging effect as compared to that of either Bombus ignitus (a type of bumblebee) queen GAG 5 mg/kg (IQG5) or chondroitin sulfate 10 mg/kg. CaG5 and IQG5 had marked anti-inflammatory effects, bringing about inhibition of free fatty acid, uric acid, sGPT, IL-1 beta, and CK values. In addition, anticoagulant and antithrombotic effects were seen: the concentration of factor 1 (fibrinogen) was increased in CaG- treated rat plasma. The CaG5-treated rat group, compared to the control, displayed upregulation of 131 genes, including lipocalin 2 (Lbp) and a serine peptidase inhibitor, Kaszal type3 (Spink3), and 64 downregulated genes, including lysyl oxidase (Lox), serine dehydratase (sds), and retinol saturase (Retsat). CONCLUSION: Our data suggest that dung beetle glycosaminoglycan may be a helpful treatment for aged rats, which indicates its potential as a therapeutic biomaterial for aging.

Taurine-responsive genes related to signal transduction as identified by cDNA microarray analyses of HepG2 cells.

Taurine-induced changes in the expression profiles of HepG2 cells were assessed using a cDNA microarray technology, and confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses. Of 8,298 human genes on the microarray, 128 genes (87 known genes) were up-regulated, and 349 (206 known genes) were down-regulated more than 2.0-fold by taurine. Among the 293 known genes regulated by taurine, a total of 44 genes were involved in signal transduction; 16 genes were up-regulated greater than 2.0-fold, and 28 genes were down-regulated more than 2.0-fold by taurine. The results of RT-PCR analyses for the five genes selected were consistent with our microarray data, although the fold changes in the expression level differed somewhat between the two analytical methods. Among signal transduction-related genes affected by taurine, four genes--mitogen-activated protein kinase (MAPK) kinase kinase 7, p21-activated kinase 4, sprouty homolog 2, and MAPK kinase 1--are implicated in the MAPK signaling pathway. Taurine also regulated the expression of signal transducer and activator of transcription (STAT) 3 gene involved in the Janus kinase-STAT pathway, and diacylglycerol kinase, zeta 104 kDa, the downstream mediator of the protein kinase C transmembrane signaling pathway. In conclusion, gene expression profiling of HepG2 cells treated with taurine provided us with new insights into the novel aspects of taurine as a possible regulator of MAPK signaling cascades and protein kinase C signaling pathways involved in cellular processes such as cell growth, differentiation, and apoptosis.

Antilipidemic effects and gene expression profiling of the glycosaminoglycans from cricket in rats on a high fat diet.

Glycosaminoglycan (GAG) from cricket (Gryllus bimaculatus) was studied as a potential health supplement. Antiatherosclerotic and antilipidemic effects of the GAG of G. bimaculatus (GbG, 5 or 10 mg/kg) were investigated in 15-week old Wistar rats treated with GbG for over a month. GbG produced a meaningful anti-edema effect with inhibition of C-reactive protein (CRP). Also, the weights of abdominal and epididymidal fat were also reduced in conjunction with a mild increase in body weight. Furthermore, the sero-biochemical parameters showed an antihyperlipidemic effect with decreased levels of phospholipid, AST, ALT, total cholesterol and glucose in a dose-dependent manner. In addition anticoagulant and antithrombotic effects were seen: platelet, thrombin time, prothrombin time and Factor I were increased with GbG treatment. Furthermore, the GbG treated rat group (at 10 mg/kg) compared to control, showed that 588 genes (test/control ratio >2.0) including lipocalin 2 (Lcn2) and alpha 2-macroglobulin (A2 m) were up-regulated, and 569 genes (test/control ratio >0.5) including stearoyl-coenzyme A desaturase 1 (Scd1) were down-regulated. Based on these results, GbG could potentially prevent or treat fatty liver or hyperlipidemia in rats on a high-fat diet.

Anti-aging Effect and Gene Expression Profiling of Aged Rats Treated with G. bimaculatus Extract.

Extract from Gryllus bimaculatus crickets inhibits oxidation at the DNA level, with reduced production of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Microarray analyses were performed with a rat 28K cDNA clone set array to identify the gene expression profiles of aged (10 months old) Wistar Kyoto rats treated for one month with 100 mg/kg G. bimaculatus ethanol extract to assess the effects. The extract produced a meaningful anti-edema effect, evident by the inhibition of creatinine phosphokinase activity. The weights of abdominal and ovarian adipose tissues were reduced and the proportion of unsaturated fatty acids in adipose tissues was increased in an extract dose-dependent manner. Compared with untreated control rats, rats treated with the extract displayed the upregulation of 1053 genes including Fas (tumor necrosis factor receptor superfamily, member 6), Amigo3 (adhesion molecule with an immunoglobulin-like domain), Reticulon 4, 3-hydroxy-3-methylglutaryl-coenzyme (Hmgcr; a reductase), related anti-fatigue (enzyme metabolism), and Rtn antioxidant, and the downregulation of 73 genes including Ugt2b (UDP glycosyltransferase 2 family), Early growth response 1, and Glycoprotein m6a. Data suggest that G. bimaculatus extract may have value in lessening the effects of aging, resulting in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes.

Identification of genes that are induced after cadmium exposure by suppression subtractive hybridization.

The heavy metal cadmium is a xenobiotic toxicant of environmental and occupational concern and it has been classified as a human carcinogen. Inhalation of cadmium has been implicated in the development of emphysema and pulmonary fibrosis, but, the detailed mechanism by which cadmium induces adverse biological effects is not yet known. Therefore, we undertook the investigation of genes that are induced after cadmium exposure to illustrate the mechanism of cadmium toxicity. For this purpose, we employed the polymerase chain reaction (PCR)-based suppression subtractive hybridization (SSH) technique. We identified 29 different cadmium-inducible genes in human peripheral blood mononuclear cells (PBMCs), such as macrophage migration inhibitory factor (MIF), lysophosphatidic acid acyltransferase-alpha, enolase-1alpha, VEGF, Bax, and neuron-derived orphan receptor-1 (Nor-1), which are known to be associated with inflammation, cell survival, and apoptosis. Induction of these genes by cadmium treatment was further confirmed by semi-quantitative reverse-transcription PCR. Further, we found that these genes were also induced after cadmium exposure in normal human lung fibroblast cell line, WI-38, suggesting potential use of this induction profile to monitor cadmium toxicity in the lung.

Myo-inositol restores the inflammation-induced down-regulation of taurine transport by the murine macrophage cell line, RAW 264.7.

The role of myo-inositol in the regulation of taurine transport in activated murine macrophage cell line, RAW 264.7, was studied. Challenge of RAW 264.7 murine macrophages for 24 hr with phorbol ester 12-myristate 13-acetate (PMA) (10 ng/ml), a PKC activator, resulted in a 62% decrease in taurine transport activity. Among the various monosaccharides (1 mM) tested in the presence of PMA, myo-inositol was most effective in restoring the PMA-induced down-regulation of taurine transport in murine macrophages (82% increase compared to the value for cells treated with PMA Alone, p < 0.01). The protective role of myo-inositol against stress-induced down-regulation of taurine transport by macrophages was further investigated in conditions mimicking bacterial infection, inflammation, and immune-suppressed circumstances. A challenge of murine macrophages with lipopolysaccharide (LPS) (0.1 and 10 microg/ml) resulted in a 60% decrease in taurine transport activity compared to the value for untreated control cells (p < 0.01). When cells were co-treated with myo-inositol (100 nM approximately 10 mM) in the presence of LPS for 24 hrs, taurine transport activity increased in a dose-dependent manner compared to the value for cells treated with LPS only. Taurine transport activity in cells treated with LPS (10 microg/ml) plus interferon-gamma (IFN-gamma) (150 unit/ml) for 24 hrs was 13% of the value for untreated control cells (p < 0.01). Again, this inflammation-induced down-regulation of taurine transport activity was completely antagonized with co-administration of 100 nM or higher levels of myo-inositol in the culture medium. Similarly, myo-inositol effectively restored the taurine transport activity suppressed by cyclosporin A (0.5 and 50 nM) in murine macrophages (p < 0.01). From these results, myo-inositol appears to be a common accelerator of taurine transport by murine macrophages in diverse conditions of down-regulated taurine transport.

Finding of TRE (TPA responsive element) in the sequence of human taurine transporter promoter.

Activity of the taurine transporter (TAUT) is regulated by signal transduction in response to diverse stimuli including tumor promoters such as phobol ester. Regulation of the transcription rate of TAUT appears to play an important role in exerting biological roles of taurine in mammalian tissues in adverse environments. Although cDNA of human TAUT has been cloned and sequenced in placenta, thyroid cells, and retinal pigment epithelial cells, the promoter region of TAUT has never been reported. In order to clone the upstream region of the human TAUT promoter, we have compared TAUT cDNA sequences with the entire human genome sequence. Polymerase chain reaction (PCR) was performed from genomic DNA prepared from a SK-Hep-1 cell line for the amplification of the TAUT promoter region including the partial exon (150 bp) and the 5' untranslated region (UTR, 380 bp). The PCR product of the promoter region, which was 1800 bp long, was ligated into the pGEM-T vector, and sequenced. The 5' flanking region of the TAUT promoter was analysed for the identification of enhancer and regulation motifs. Surprisingly we found the consensus TPA responsive element (TGAGTCAG) which is responsible for gene regulation by the protein kinase C (PKC)-mediated signal transduction pathway. The well known fact that proto-oncogene AP1 (cFos/cJun heterodimer or cJun/cJun homodimer) binds to TRE implies that TAUT expression might be closely linked to tumor promotion. Since AP1 activity is also tightly regulated in nerve cells, AP1-regulated TAUT transcription might be an important step in nerve cell function. Furthermore, the TFIID binding site, cap signal for transcription initiation, PEA3 motif, heat shock factor binding motif, and many other motifs were found in the TAUT promoter region, and require characterization.

Characterization of transcriptional activity of taurine transporter using luciferase reporter constructs.

Although taurine transporter (TAUT) activity has been known to be regulated by diverse intracellular and extracellular factors involved in the signal transduction pathway, such as protein kinase C, intracellular Ca concentration, and glucocorticoids, little is known concerning the underlying mechanisms. Evidence suggests that such stimulation-mediated changes in TAUT activity in mammalian cells are partly achieved through the modulation of TAUT transcription activity. In order to better understand the regulation of TAUT transcription activity and subsequently the role of taurine in the signal transduction pathway, we have cloned and sequenced the 5' flanking region of the human TAUT gene, and characterized the TAUT promoter region in human cells. For these reasons, the TAUT luciferase reporter vector was constructed using the 5' flanking region of the TAUT gene (1800 bp). The TAUT luciferase reporter vector was then transfected into SiHa cells, and luciferase activity was measured. The construct containing its own promoter of TAUT (pGL3 b TAU31) showed a 10 fold higher luciferase activity compared to the value found in the empty vector (pGL3 b). This implies a functional transcription of the homologous TAUT promoter. Similar results were obtained with the exon deleted construct [pGL3 b TAU31(-e)]. We also constructed the TAUT luciferase reporter gene (pGL3 pro TAU13) using a heterologous promoter. About 2.5 fold higher luciferase activity was observed in cells transfected with this construct containing the heterologous promoter compared to the value found in the control vector (PGL3 pro).

Anti-metastatic effects of fuzhengfangaitang on human fibrosarcoma cells HT1080.

Fuzhengfangaitang (FZFAT) is used to inhibit recurrence and metastasis of cancer in the clinic. By applying an in vitro invasion assay model, we examined the antimetastatic effect of FZFAT. In the 3H-thymidine incorporation assay, FZFAT-treated groups showed a decreased DNA synthesis rate compared with the control group (F-value 87.42981, P-value 2.02E-08, F0.05(3,12) 3.4903). Gelatin zymogram assay showed that FZFAT decreased the gelatinolytic activity of matrix metalloproteinases-9 (MMP-9) from human fibrosarcoma cell line (HT-1080), at concentrations of 200 and 400 microg/ml. In the MMPs dot blotting assay, FZFAT inhibited the expression of MMP-1 at concentration of 100 microg/ml, and MMP-9 at concentrations of 200 and 400 microg/ml. Western blots for AP-1 and its signal mediators Erk and JNK showed that expression of Fos and JNK were decreased by the addition of FZFAT at 300 microg/ml, whereas Erk was not. Therefore it was evident that FZFAT regulated the expression of MMP-9 through its transcription factor AP-1 and the signal mediator JNK. We examined whether FZFAT inhibited the invasion of HT-1080 cells through matrigel precoated transwell chambers. The results showed that FZFAT effectively inhibited the invasion of HT-1080 cells as compared with the control phorbol 12-myristate-13-acetate (+PMA) groups (t-value 5.871584, P-value 0.013901, t0.05(2) 2.919987). From our research, part of the mechanism underlying the antimetastatic effect of FZFAT has been elucidated in vitro.