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Although various advancements in radical surgery and neoadjuvant chemotherapy have been developed in treating osteosarcoma (OS), their clinical prognosis remains poor. A synthetic chemical compound, 3-hydroxylflavone, that is reported to regulate ROS production is known to inhibit human bone osteosarcoma cells. However, its role and mechanism in human OS cells remains unclear. In this study, we have determined the potential of 3-Hydroxy-2-phenylchromone (3-HF) against OS using human osteosarcoma (HOS) cells. Our previous studies showed that Zipper sterile-alpha-motif kinase (ZAK), a kinase member of the MAP3K family, was involved in various cellular events such as cell proliferation and cell apoptosis, and encoded two transcriptional variants, ZAK&alpha, and &beta, In this study, we show that 3-HF induces the expression of ZAK and thereby enhances cellular apoptosis. Using gain of function and loss of function studies, we have demonstrated that ZAK activation by 3-HF in OS cells is confined to a ZAK&beta, form that presumably plays a leading role in triggering ZAK&alpha, expression, resulting in an aggravated cancer apoptosis. Our results also validate ZAK&beta, as the predominant form of ZAK to drive the anticancer mechanism in HOS cells.

ZAK is a novel mixed lineage kinase-like protein that contains a leucine-zipper and a sterile-alpha motif as a protein-protein interaction domain, and it is located in the cytoplasm. There are 2 alternatively spliced forms of ZAK: ZAKα and ZAKβ. Previous studies showed that ZAKα is involved in various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy, but the molecular mechanism of ZAKβ is not yet known. In a recent study in our laboratory, we found that ZAKβ can ameliorate the apoptotic effect induced by ZAKα in H9c2 cells. We further hypothesized that ZAKβ could also improve the apoptotic effect induced by ZAKα in human osteosarcoma cells. The results of this study show that ZAKβ can induce apoptosis and decrease cell viability similar to the effects of ZAKα. Interestingly, our ZAKα-specific inhibitor assay shows that the expression of ZAKβ is highly dependent on ZAKα expression. However, ZAKβ expression effectively induces ZAKα expression and results in synergistic enhancement of apoptosis in human osteosarcoma cells. Furthermore, co-immunoprecipitation results revealed that ZAKα can directly interact with ZAKβ, and this interaction may contribute to the enhanced apoptotic effects. Significance of the study ZAK is a mixed lineage kinase involved in cell differentiation, proliferation, and hypertrophic growth. ZAKα isoform of ZAK is associated with tumorigenesis, but the function of ZAKβ is not yet known. In H9c2 cells, ZAKβ was found to ameliorate the apoptotic effect induced by ZAKα. However, in osteosarcoma cells, ZAKβ elevates the apoptotic effect induced by ZAKα. In this study, we show that similar to ZAKα, the ZAKβ induces apoptosis and decreases cell viability. Interestingly, the expression of ZAKβ is dependent on ZAKα expression, and ZAKβ further enhances ZAKα expression and results in synergistic enhancement of apoptosis in osteosarcoma cells.

Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX-treated human OS cells.

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

Cardiomyopathy involves changes in the myocardial ultra-structure, hypertrophy, apoptosis, fibrosis and inflammation. Angiotensin II (AngII) stimulates the expression of insulin like-growth factors (IGF-2) and IGF-2 receptor (IGF-2R) in H9c2 cardiomyoblasts and subsequently leads to apoptosis. Estrogen receptors protect cardiomyocytes from apoptosis and fibrosis. Tanshinone IIA (TSN), a main active ingredient from Danshen, has been shown to protect cardiomyocytes from death caused by different stress signals. Estrogen receptor α (ER) is required for the rapid activation of the IGF-1R signaling cascade. This study aimed to investigate whether TSN protected H9c2 cardiomyocytes from AngII-induced activation of IGF-2R pathway and hypertrophy via ERs. We found that AngII caused the reduction in IGF-1R phosphorylation and the elevation of β-catenin and IGF-2R levels. This was reversed by increasing doses of TSN and of caspase-3 and ERK1/2 phosphorylation mediated by ERs. The phytoestrogen significantly attenuated AngII-induced apoptosis and suppressed the subsequent cardiac remodeling effect. Therefore, TSN reduced the AngII-induced activation of β-catenin and IGF-2R pathways, apoptosis and cardiac remodeling via ERs in H9c2 cardiomyoblasts.

ZAK (sterile alpha motif and leucine zipper containing kinase AZK), a serine/threonine kinase with multiple biochemical functions, has been associated with various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy. In our previous reports, we found that the activation of ZAKα signaling was critical for cardiac hypertrophy. In this study, we show that the expression of ZAKα activated apoptosis through both a FAS-dependent pathway and a mitochondria-dependent pathway by subsequently inducing caspase-3. ZAKβ, an isoform of ZAKα, is dramatically expressed during cardiac hypertrophy and apoptosis. The interaction between ZAKα and ZAKβ was demonstrated here using immunoprecipitation. The results show that ZAKβ has the ability to diminish the expression level of ZAKα. These findings reveal an inherent regulatory role of ZAKβ to antagonize ZAKα and to subsequently downregulate the cardiac hypertrophy and apoptosis induced by ZAKα.

// Wei-Jen Ting 1,2 , Jaw-Ji Yang 3 , Chia-Hua Kuo 4 , Zi-Jun Xiao 5 , Xin-Ze Lu 5 , Yu-Lan Yeh 6,7 , Cecilia-Hsuan Day 8 , Su-Ying Wen 9,10 , Vijaya PadmaViswanadha 11 , Chong-He Jiang 1 , Wei-Wen Kuo 12,* and Chih-Yang Huang 2,13,14,* 1 The Sixth Affiliated Hospital of Guangzhou Medical University, Guangdong, China 2 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan 3 Institute of Medicine, School of Dentistry, Chung-Shan Medical University, Taichung, Taiwan 4 Department of Sports Sciences, University of Taipei, Taipei, Taiwan 5 National Taichung First Senior High School, Taichung, Taiwan 6 Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan 7 Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan 8 Department of Nursing, Mei Ho University, Pingtung, Taiwan 9 Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan 10 Department of Dermatology, Taipei City Hospital, Renai Branch, Taipei, Taiwan 11 Department of Biotechnology, Bharathiar University, Coimbatore, India 12 Department of Biological Science and Technology, China Medical University, Taichung, Taiwan 13 Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan 14 Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan * These authors have contributed equally to this paper Correspondence to: Chih-Yang Huang, email: // Keywords : environmental tobacco smoke (ETS); Sirt-1; autophagy; Gerotarget Received : November 02, 2015 Accepted : April 16, 2016 Published : May 04, 2016 Abstract Recently, a survey by the Centers for Disease Control and Prevention (CDC) reported that nearly 90% of U.S. adult smokers began smoking at the age of 18. This demonstrates that the exposure to environmental tobacco smoke (ETS) of youngsters today is changing from passive smoking to active smoking (direct inhalation of tobacco). In the current study, an investigation of ETS exposure in young C57BL mice was conducted. After 6 weeks of ETS exposure, the Sirt-1 protein level was decreased and cardiac autophagy was increased in C57BL mice. Furthermore, the IGF2R cardiac hypertrophy signaling pathway was also triggered, although cardiac apoptosis and hypertrophy were not induced. Youngsters’ desire to look more mature is one of the psychological factors that impacts smoking amongst young people. Our results suggest that though ETS exposure might cause cardiac autophagy amongst youngsters, the loss of the longevity Sirt-1 protein and the increase in IGF2R cardiac hypertrophy signaling could still promote heart diseases that are age-specific.

Osteosarcoma (OS) is a tumor entity that can cause a large number of cancer-related deaths. Although chemotherapy can decrease proliferation and increase apoptosis of human OS cells, the clinical prognosis remains poor. Fisetin is a flavonol found in fruits and vegetables and is reported to inhibit cell growth in numerous cancers. But the molecular mechanism underlying fisetin in human OS cells is not clear. It is known that sterile-alpha motif and leucine zipper containing kinase (ZAK), a kinase in the MAP3K family, is involved in various cell processes, including proliferation and apoptosis. In our lab, we have demonstrated that overexpression of ZAK can induce apoptosis in human OS cells. In the previous studies, MAP4K, the upstream of MAP3K, can act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway. Turning on the Hippo pathway can decrease proliferation and otherwise cause cell apoptosis in cancer cells. In this study, we found that fisetin can upregulate ZAK expression to induce the Hippo pathway and mediate the activation of JNK/ERK, the downstream of ZAK, to trigger cell apoptosis via AP-1 dependent manner in human OS cells. These findings reveal a novel molecular mechanism underlying fisetin effect on human OS cells.

Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKβ, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKβ proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKβ proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKβ:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKβ protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKβ or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and β-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKβ and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKβ, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKβ had powerful antitumor properties and that ZAKα could promote tumor growth.

Introduction Mineral trioxide aggregate (MTA) has been successfully used in clinical applications in endodontics. Studies show that the antibacterial effects of CO 2 laser irradiation are highly efficient when bacteria are embedded in biofilm because of a photothermal mechanism. The aim of this study was to confirm the effects of CO 2 laser irradiation on MTA with regard to both material characterization and cell viability. Methods MTA was irradiated with a dental CO 2 laser using directly mounted fiber optics in the wound healing mode with a spot area of 0.25 cm 2 and then stored in an incubator at 100% relative humidity and 37°C for 1 day to set. The human dental pulp cells cultured on MTA were analyzed along with their proliferation and odontogenic differentiation behaviors. Results The results indicate that the setting time of MTA after irradiation by the CO 2 laser was significantly reduced to 118 minutes rather than the usual 143 minutes. The maximum diametral tensile strength and x-ray diffraction patterns were similar to those obtained without CO 2 laser irradiation. However, the CO 2 laser irradiation increased the amount of Ca and Si ions released from the MTA and regulated cell behavior. CO 2 laser–irradiated MTA promoted odontogenic differentiation of hDPCs, with the increased formation of mineralized nodules on the substrate's surface. It also up-regulated the protein expression of multiple markers of odontogenic and the expression of dentin sialophosphoprotein protein. Conclusions The current study provides new and important data about the effects of CO 2 laser irradiation on MTA with regard to the decreased setting time and increased ion release. Taking cell functions into account, the Si concentration released from MTA with laser irradiation may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue.

Background: In addition to releasing a pool of growth factors during activation, platelets have many features that indicate their role in the anti-infective host defense. The antimicrobial activities of platelet-rich plasma (PRP) and related plasma preparations against periodontal disease–associated bacteria were evaluated.Methods: Four distinct plasma fractions were extracted in the formulation used commonly in dentistry and were tested for their antibacterial properties against three periodontal bacteria: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. The minimum inhibitory concentration of each plasma preparation was determined, and in vitro time-kill assays were used to detect their abilities to inhibit bacterial growth. Bacterial adhesion interference and the susceptibility of bacterial adherence by these plasma preparations were also conducted.Results: All plasma preparations can inhibit bacterial growth, with PRP showing the superior activity. Bacteria...

Aberrant expression of leucine zipper- and sterile ɑ motif-containing kinase (ZAK) observed in pathological human myocardial tissue is associated with the progression and elevation of hypertrophy. Our previous reports have correlated high levels of estrogen (E2) and abundant estrogen receptor (ER) α with a low incidence of pathological cardiac-hypertrophy and heart failure in the premenopause female population. However, the effect of elevated ERβ expression is not well known yet. Therefore, in this study, we have analyzed the cardioprotective effects and mechanisms of E2 and/or ERβ against ZAK overexpression-induced cellular hypertrophy. We have used transient transfection to overexpress ERβ into the ZAK tet-on H9c2 cells that harbor the doxycycline-inducible ZAK plasmid. The results show that ZAK overexpression in H9c2 cells resulted in hypertrophic effects, which was correlated with the upregulation of p-JNK and p-p38 MAPKs and their downstream transcription factors c-Jun and GATA-4. However, ERβ and E2 with ERβ overexpressions totally suppressed the effects of ZAK overexpression and inhibited the levels of p-JNK, p-p38, c-Jun, and GATA-4 effectively. Our results further reveal that ERβ directly binds with ZAK under normal conditions; however, ZAK overexpression reduced the association of ZAK-ERβ. Interestingly, increase in ERβ and E2 along with ERβ overexpression both enhanced the binding strengths of ERβ and ZAK and reduced the ZAK protein level. ERβ overexpression also suppressed the E3 ligase-casitas B-lineage lymphoma (CBL) and attenuated CBL-phosphoinositide 3-kinase (PI3K) protein association to prevent PI3K protein degradation. Moreover, ERβ and/or E2 blocked ZAK nuclear translocation via the inhibition of small ubiquitin-like modifier (SUMO)-1 modification. Taken together, our results further suggest that ERβ overexpression strongly suppresses ZAK-induced cellular hypertrophy and myocardial damage.

ZAK is a novel mixed lineage kinase-like protein that contains a leucine-zipper and a sterile-alpha motif as a protein-protein interaction domain, and it is located in the cytoplasm. There are 2 alternatively spliced forms of ZAK: ZAKα and ZAKβ. Previous studies showed that ZAKα is involved in various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy, but the molecular mechanism of ZAKβ is not yet known. In a recent study in our laboratory, we found that ZAKβ can ameliorate the apoptotic effect induced by ZAKα in H9c2 cells. We further hypothesized that ZAKβ could also improve the apoptotic effect induced by ZAKα in human osteosarcoma cells. The results of this study show that ZAKβ can induce apoptosis and decrease cell viability similar to the effects of ZAKα. Interestingly, our ZAKα-specific inhibitor assay shows that the expression of ZAKβ is highly dependent on ZAKα expression. However, ZAKβ expression effectively induces ZAKα expression and results in synergistic enhancement of apoptosis in human osteosarcoma cells. Furthermore, co-immunoprecipitation results revealed that ZAKα can directly interact with ZAKβ, and this interaction may contribute to the enhanced apoptotic effects.ZAK is a mixed lineage kinase involved in cell differentiation, proliferation, and hypertrophic growth. ZAKα isoform of ZAK is associated with tumorigenesis, but the function of ZAKβ is not yet known. In H9c2 cells, ZAKβ was found to ameliorate the apoptotic effect induced by ZAKα. However, in osteosarcoma cells, ZAKβ elevates the apoptotic effect induced by ZAKα. In this study, we show that similar to ZAKα, the ZAKβ induces apoptosis and decreases cell viability. Interestingly, the expression of ZAKβ is dependent on ZAKα expression, and ZAKβ further enhances ZAKα expression and results in synergistic enhancement of apoptosis in osteosarcoma cells.

β-Tricalcium phosphate (β-TCP) is an osteoconductive material in clinical. In this study, we have doped silica (Si) into β-TCP and enhanced its bioactive and osteostimulative properties. To check its effectiveness, a series of Si-doped with different ratios were prepared to make new bioactive and biodegradable biocomposites for bone repair. Formation of the diametral tensile strength, ions released and weight loss of cements was considered after immersion. In addition, we also examined the behavior of human dental pulp cells (hDPCs) cultured on Si-doped β-TCP cements. The results showed that setting time and injectability of the Si-doped β-TCP cements were decreased as the Si content was increased. At the end of the immersion point, weight losses of 30.1%, 36.9%, 48.1%, and 55.3% were observed for the cement doping 0%, 10%, 20%, and 30% Si into β-TCP cements, respectively. In vitro cell experiments show that the Si-rich cements promote human dental pulp cell (hDPC) proliferation and differentiation. However, when the Si-doped in the cement is more than 20%, the amount of cells and osteogenesis protein of hDPCs was stimulated by Si released from Si-doped β-TCP cements. The degradation of β-TCP and osteogenesis of Si gives a strong reason to believe that these Si-doped β-TCP cements may prove to be promising bone repair materials.

Puerh tea has been proposed to promote weight loss and favorably modify glucose, insulin and blood lipids. This study tested the effect of daily Puerh tea consumption for 3 months on weight and body mass index (BMI), and select metabolic parameters. The effect of daily Puerh tea intake on weight, BMI and changes in glucose, HbA1c and lipids was evaluated in patients with metabolic syndrome. The patients (N = 70) were randomized into two groups: those taking Puerh tea extract capsule (333 mg Puerh tea extract) three times a day and those taking a placebo tea for 3 months. There was a decrease in body weight of 1.3 kg in the Puerh tea group (p = 0.077) versus 0.23 kg in the placebo arm (p = 0.186). There was also a slight decrease in BMI 0.47 kg/m2 in the Puerh tea group (p = 0.076) versus 0.09 kg/m2 in the placebo arm (p = 0.185), suggesting a trend of weight change, but without statistical significance. Subgroup analysis of the male patients demonstrated statistically significant improvements in body weight reduction (p = 0.004) and BMI (p = 0.004). However, the change in other metabolic parameters (cholesterol or triglyceride) or HbA1c was not statistically significant. Intake of Puerh tea for 3 months was associated with a slight reduction in body weight and BMI, especially in the male patients. Therefore, daily Puerh tea consumption may be an alternative choice to modify body weight. Copyright © 2014 John Wiley & Sons, Ltd.

Background: Coffee is a major dietary source of polyphenols. Previous research found that coffee had a protective effect on periodontal disease. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis (P. gingivalis). Methods: Coffee extract and chlorogenic acid were prepared by a two-fold serial dilution. The turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. The time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0&ndash, 24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. Results: As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer-lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. The coffee extract showed antibacterial activity as observed by the disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. Conclusions: Collectively, our novel findings indicate that chlorogenic acid not only has antimicrobial activity but also reduced the protease activity of P. gingivalis. In addition, coffee extract inhibits the proliferation of P. gingivalis, which may partly be attributed to the effect of chlorogenic acid.

Mitomycin C (MMC) is an active antineoplastic agent and is suggested to induce apoptosis in a caspase- dependent manner in human gastric, bladder, and breast cancer cells. In this study, the death mode of human cervical cancer cells (HeLa) induced by MMC and the cellular localization of MMC-induced P-glycoprotein (P-gp) were investigated. The results of caspase-3 activity, Annexin V binding, and DNA fragmentation suggested that the degree of caspase-dependent apoptosis induced by MMC was in a dose-, but not time-dependent, manner. Further, in low-dose (0.0299 microM) and long-term (2 months) treatment with MMC, P-gp is itself extruded from the cells and colocalized with nuclear DNA and the overexpression was achieved.

Leucine-zipper and sterile-alpha motif kinase (ZAK) is the key intra-cellular mediator protein in cardiomyocyte hypertrophy induction by transforming growth factor beta 1 (TGF-beta1) which has also been identified as a profibrotic cytokine involved in cardiac fibrosis progression. We hypothesized whether ZAK over-expression causes cardiac scar formation due to the extra-cellular matrix (ECM) degraded enzyme regulation in this paper. Using immuno-histochemical analysis of the human cardiovascular tissue array, we found a positively significant association between ZAK over-expression and myocardial scars. ZAK over-expression in H9c2 cardiomyoblast cells increases the metalloproteinase tissue inhibitor 1/2 (TIMP-1/2) protein level, which reduces matria metalloproteinase-9 (MMP-9) activity and also activates c-JNK N-terminal kinase 1/2 (JNK1/2) and p38 signaling, which induces MMP-2, possibly resulting in cardiac fibrosis. Taken together, ZAK activity inhibition may be a good strategy to prevent the cardiac fibrosis progression.

It is unclear whether cardiac hypertrophy and hypertrophy-related pathways will be induced by long-term intermittent hypoxia. Thirty-six Sprague–Dawley rats were randomly assigned into three groups: normoxia, and long-term intermittent hypoxia (12% O2, 8 h per day) for 4 weeks (4WLTIH) or for 8 weeks (8WLTIH). Myocardial morphology, trophic factors and signalling pathways in the three groups were determined by heart weight index, histological analysis, Western blotting and reverse transcriptase-polymerase chain reaction from the excised left ventricle. The ratio of whole heart weight to body weight, the ratio of left ventricular weight to body weight, the gross vertical cross-section of the heart and myocardial morphological changes were increased in the 4WLTIH group and were further augmented in the 8WLTIH group. In the 4WLTIH group, tumour necrosis factor-α(TNFα), insulin-like growth factor (IGF)-II, phosphorylated p38 mitogen-activated protein kinase (P38), signal transducers and activators of transcription (STAT)-1 and STAT-3 were significantly increased in the cardiac tissues. However, in the 8WLTIH group, in addition to the above factors, interleukin-6, mitogen-activated protein kinase (MEK)5 and extracellular signal-regulated kinase (ERK)5 were significantly increased compared with the normoxia group. We conclude that cardiac hypertrophy associated with TNFα and IGF-II was induced by intermittent hypoxia. The longer duration of intermittent hypoxia further activated the eccentric hypertrophy-related pathway, as well as the interleukin 6-related MEK5–ERK5 and STAT-3 pathways, which could result in the development of cardiac dilatation and pathology.

The chemopreventive property of garlic is related in part to its induction of phase II detoxification enzymes. In the present study, we investigated the modulatory effect of 3 garlic organosulfur compounds, i.e., diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), which differ in their number of sulfur atoms, on the gene expression of the class of glutathione S-transferase (GSTP). Hepatocytes isolated from male Sprague-Dawley rats were cultured with 50 -200 mol/L of DAS, DADS, or DATS for 24 h. DADS and DATS increased GST activity toward ethacrynic acid by 40 and 66%, respectively (P 0.05). Moreover, both garlic allyl sulfides dose dependently induced GSTP mRNA and protein expression. DATS increased the protein level more than DADS (P 0.05). In contrast, DAS did not affect the activity or the protein or mRNA levels of this phase II drug-metabolizing enzyme. In Clone 9 liver cells, the pTA-luciferase reporter assay showed that luciferase activity in DADS- and DATS-treated cells was 2.8- and 3.9-fold higher than that in control cells, respectively (P 0.05). Again, luciferase activity was not affected by treatment with DAS. Deletion of 2.7 to 2.6 kb in the GSTP promoter region, which contains the GSTP enhancer (GPE) I element, abolished the upregulation of GSTP transcription by DADS and DATS. Deletion of GPE II, however, did not affect the induction of reporter activity. In conclusion, the effectiveness of 3 garlic allyl sulfides on GSTP expression was related to the number of sulfur atoms in the molecules, and GPE I was responsible for this upregulation. J. Nutr. 135: 2560 -2565, 2005.

The regulation of genes by amino acids is attracting increasing attention. In the present study, we investigated the restriction of expression of the pi class of glutathione S-transferase (GST Yp) by sulfur amino acids. Hepatocytes isolated from male Sprague-Dawley rats were cultured with L-15-based medium containing low (LSAA; 0.1 mmol/L L-methionine and 0.1 mmol/L L-cysteine) or high (HSAA; 0.5 mmol/L L-methionine and 0.2 mmol/L L-cysteine) amounts of sulfur amino acids for up to 6 d. Cellular protein contents did not differ between LSAA- and HSAA-treated cells over the entire period. In contrast, glutathione concentrations were suppressed by the LSAA medium and on d 6 were only 20% of those of HSAA-treated cells (P < 0.05). As shown by immunoblot analysis, GST Yp protein levels were greater in LSAA-treated cells than in HSAA-treated cells (P < 0.05). The induction of GST Yp by L-methionine and L-cysteine restriction was not affected by insulin and dexamethasone, but the latter suppressed GST Yp expression (P < 0.05). LSAA increased GST Yp mRNA levels and GST activity toward ethacrynic acid (P < 0.05). GST Yp induction occurred only in cells with a limited supply of L-methionine; restriction of L-isoleucine, L-leucine, L-lysine, and L-phenylalanine had no significant effect. In contrast with the induction of GST Yp, the expression of the GST isoforms Ya and Yb was not changed by amino acid restriction. In conclusion, hepatic GST Yp gene expression is upregulated by a limited availability of sulfur amino acids.

Cytochrome P-450 is an important bioactivation-detoxification system in vivo. Its expression is regulated by foreign chemicals and dietary factors, and lipids have been found to regulate its gene expression. We showed previously that prostaglandin E(2) (PGE(2)), a fatty acid metabolite, down-regulates cytochrome P-450 2B1 (CYP 2B1) expression induced by phenobarbital. The objective of the present study was to determine whether PGE(2) type 2 receptor (EP(2))-which is coupled to Gs-protein when bound by PGE(2), leading to cAMP production-is involved in this down-regulation. We also determined the possible roles of EP(2) downstream pathways in this down-regulation. We used a primary rat hepatocyte culture model in which EP(2) was shown to be present to study this question. The intracellular cAMP concentration in primary rat hepatocytes was significantly higher after treatment with 1microM PGE(2) than after treatment with 0, 0.01, or 0.1microM PGE(2). Butaprost, an EP(2) agonist, down-regulated CYP 2B1 expression in a dose-dependent manner. SQ22536, an adenylate cyclase inhibitor, reversed the down-regulation by PGE(2) as did H-89, a protein kinase A inhibitor. These results suggest that EP(2) and the downstream pathways of cAMP and protein kinase A are involved in the down-regulation of CYP 2B1 expression by PGE(2) in the presence of phenobarbital.

Transforming growth factor-beta (TGF-beta) has been associated with the onset of cardiac cell hypertrophy, but the mechanisms underlying this dissociation are not completely understood. By a previous study, we investigated the involvement of a MAP3K, ZAK, which in cultured H9c2 cardiac cells is a positive mediator of cell hypertrophy. Our results showed that expression of a dominant-negative form of ZAK inhibited the characteristic TGF-beta-induced features of cardiac hypertrophy, including increased cell size, elevated expression of atrial natriuretic factor (ANF), and increased organization of actin fibers. Furthermore, dominant-negative MKK7 effectively blocked both TGF-beta-and ZAK-induced ANF expression. In contrast, a JNK/SAPK specific inhibitor, sp600125, had little effect on TGF-beta- or ZAK-induced ANF expression. Our findings suggest that a ZAK mediates TGF-beta-induced cardiac hypertrophic growth via a novel TGF-beta signaling pathway that can be summarized as TGF-beta>ZAK>MKK7>ANF.

Background The DEP domain is a globular domain containing approximately 90 amino acids, which was first discovered in 3 proteins: Drosophila disheveled, Caenorhabditis elegans EGL-10, and mammalian Pleckstrin; hence the term, DEP. DEPDC1B is categorized as a potential Rho GTPase-activating protein. The function of the DEP domain in signal transduction pathways is not fully understood. The DEPDC1B protein exhibits the characteristic features of a signaling protein, and contains 2 conserved domains (DEP and RhoGAP) that are involved in Rho GTPase signaling. Small GTPases, such as Rac, CDC42, and Rho, regulate a multitude of cell events, including cell motility, growth, differentiation, cytoskeletal reorganization and cell cycle progression. Results In this study, we found that it was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B plays a role in regulating Rac1 translocated onto cell membranes, suggesting that DEPDC1B exerts a biological function by regulating Rac1. We examined oral cancer tissue; 6 out of 7 oral cancer tissue test samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue. Conclusions DEPDC1B was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B exerts a biological function by regulating Rac1. We found that oral cancer samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue. Suggest that DEPDC1B plays a role in the development of oral cancer. We revealed that proliferation was linked to a novel DEPDC1B-Rac1-ERK1/2 signaling axis in oral cancer cell lines.

The modulation of cytochrome P-450 2B1 expression by alpha-tocopheryl succinate and whether prostaglandin E2 is involved in this modulation in primary rat hepatocytes in the presence of phenobarbital were investigated. A primary rat hepatocyte culture model that faithfully reproduces the phenobarbital response observed in vivo was used. Intracellular alpha-tocopherol content was dose dependently increased by alpha-tocopheryl succinate incubation. Hepatocytes were demonstrated to have prostaglandin E2-synthesizing capability. alpha-Tocopheryl succinate inhibited prostaglandin E2 synthesis by hepatocytes and increased cytochrome P-450 2B1 expression in the presence of phenobarbital; however, it had little effect on intracellular cAMP level. To mimic the exogenous source of prostaglandin E2 from nonparenchymal cells, various concentrations of prostaglandin E2 were added to the cell culture. High doses of exogenous prostaglandin E2 (100 and 1,000 nM) inhibited the cytochrome P-450 2B1 expression in the presence of phenobarbital compared with low doses (1 and 10 nM); however, the presence of high doses of prostaglandin E2 had no effect on intracellular cAMP level. Forskolin significantly increased intracellular cAMP level and inhibited cytochrome P-450 2B1 expression in the presence of phenobarbital. The results of this study indicate that alpha-tocopheryl succinate increases cytochrome P-450 2B1 expression via its inhibition of prostaglandin E2 synthesis in the presence of phenobarbital; however, changes in intracellular cAMP level are not related to cytochrome P-450 2B1 expression.

The individual and combined effects of dietary fat and garlic oil on two drug-metabolizing enzymes, cytochrome P 450 2B1 and the placental form of glutathione (GSH) S-transferase (PGST), in rat liver were examined in this study. Rats were fed a low corn oil, high corn oil or high fish oil diet and received various amount of garlic oil (0, 30, 80, 200 mg/kg body) orally three times per week for 6 wk. The fat energy in the low and high fat diets accounted for 11.6 and 45.7% of total energy, respectively. Final body weights did not differ among the three dietary fat groups and were not affected by garlic oil treatment. The fatty acid profile in hepatic phospholipids revealed higher eicosapentaenoic acid [20:5(n-3)] and docosahexaenoic acid [22:6(n-3)] levels in the fish oil-fed group than in the low and high corn oil-fed groups (P < 0.05). In contrast, the corn oil-fed groups had greater hepatic phospholipid arachidonic acid [20:4(n-6)] levels (P < 0.05). Both dietary fat and garlic oil significantly affected hepatic cytochrome 7-pentoxyresorufin O-dealkylase (PROD) activity and GST activity toward ethacrynic acid. Rats fed the high fish oil diet had 85 and 51 % higher PROD activity compared with those fed the low or the high corn oil diet, respectively (P < 0.05). The GST activity in the high fish oil and the high corn oil groups was 33 and 18% higher than that in the low corn oil group (P < 0.05), respectively, and the GST activity in rats fed the high fish oil diet was higher than in those fed the high corn oil diet (P < 0.05). Garlic oil dose-dependently increased GST activity. No interaction between dietary fat and garlic oil on PROD or GST activity was noted. Northern and Western blot analysis revealed that dietary fish oil increased both cytochrome P 450 2B1 and PGST mRNA and protein levels. Cytochrome P 450 2B1 and PGST mRNA and protein levels were also dose-dependently increased by garlic oil treatment. The effects of garlic oil and dietary fat on P 450 2B1 and PGST mRNA and protein expression were independent. These results indicate that dietary fat and garlic oil independently modulate P 450 2B1 and PGST expression at transcriptional and/or post-transcriptional stages.

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.

Anchorage-independent growth is characteristic of neoplastic cells, but the signal transduction pathways that mediate this phenotype are poorly understood. Several important cell cycle events are dependent on cell-substratum adhesion in non-transformed cells, including activation of G1 cyclin-dependent kinases and expression of cyclin A; the adhesion requirement of these events is abrogated in Ras-transformed cells. The ER-1-2 mutant rat fibroblast cell line is: 1) resistant to Ras-mediated, anchorage-independent growth; 2) defective in Ras-mediated, adhesion-independent expression of cyclin A, but not adhesion-independent activation of cyclin-dependent kinases; and 3) rescued for Ras-induced, anchorage-independent growth by ectopic expression of cyclin A. We report here that extracellular ATP induces adhesion-independent expression of cyclin A and rescues growth in soft agar by ER-1-2 cells that express Ras. ADP, AMP and the non-hydrolyzable analog adenosine 5'-(beta, gamma-iminodiphosphate) are also effective, but adenosine is not. Adenine nucleotide-induced growth in soft agar is inhibited by reactive blue 2, an antagonist of some P2 purinoceptors. ATP does not induce adhesion-independent expression of cyclin A in ER-1-2 or control rat fibroblasts that do not express Ras, indicating a requirement for additional Ras-regulated signals for expression of this gene; one such signal may lead to phosphorylation of the retinoblastoma protein, pRB, and related proteins. These results suggest that extracellular ATP could play a role in the multistage carcinogenic process in vivo.

Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. Consistent with this function, tNOX knockdown by RNA interference leads to a significant reduction in cell proliferation and migration in HeLa cells, whereas tNOX overexpression confers an aggressive phenotype. Here, for the first time, we report that tNOX is phosphorylated by protein kinase Cδ (PKCδ) both in vitro and in vivo. Replacement of serine-504 with alanine significantly reduces phosphorylation by PKCδ. Co-immunoprecipitation experiments reveal an interaction between tNOX and PKCδ. Moreover, whereas overexpression of wild-type tNOX in NIH3T3 cells increases cell proliferation and migration, overexpression of the S504A tNOX mutant leads to diminished cell proliferation and migration, reflecting reduced stability of the unphosphorylatable tNOX mutant protein. Collectively, these results suggest that phosphorylation of serine-504 by PKCδ modulates the biological function of tNOX.

Our previous study shows that caveolin-1 colocalizes and interacts with ATP-binding cassette transporter A1 (ABCA1), which is intimately involved in cellular cholesterol efflux. In this study, we further clarified the region of caveolin-1 that interacts with ABCA1. We also examined the interaction between mutant caveolin-1 and ABCA1 in HDL-mediated cholesterol efflux. We constructed a panel of mutant caveolin-1 proteins and co-transfected them into rat aortic endothelial and human embryonic kidney 293 (HEK293) cells. The co-immunoprecipitation shows that mutant oligomerization domain of caveolin-1, caveolin-1(Δ62-100), is required for the interaction of caveolin-1 with ABCA1. Caveolin-1(Δ62-100) did not colocalize with ABCA1 in the cholesterol-loaded cells after HDL incubation as observed by immunofluorescence confocal microscopy. Concomitantly, caveolin-1(Δ62-100) suppressed HDL-mediated cholesterol efflux. The results suggest that the region of caveolin-1 between amino acids 62 and 100 is an oligomerization domain as well as an attachment site for ABCA1 interaction that regulates HDL-mediated cholesterol efflux.

Novel mixed-lineage kinase protein zipper sterile-alpha-motif kinase (ZAK) was first cloned by our laboratory. Lung cancer is the leading cause of cancer-related death in the world, including in Taiwan. Here, we wanted to investigate whether ZAK plays a potential role in lung cancer development. First, Western blot analysis results demonstrated that four cell lines expressed high levels of ZAK from among a panel of 10 lung cancer cell lines, and two of three normal lung cells expressed ZAK. ZAK gene expressions were down-regulated in lung cancers by real-time PCR analysis. Overexpression of ZAK suppressed cell proliferation in parallel with increased phosphorylated levels of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In contrast, ZAK silencing cells inhibited the expressions of phosphorylated ERK and JNK without affecting the expression of phosphorylated p38. The effect of the decreased cell growth rate was significantly but incompletely reversed when ZAK-overexpressing cells were treated with a specific ERK or JNK inhibitor. Moreover, c-Fos and c-Jun, the major downstream components of MAPKs, were up-regulated by ERK and JNK, respectively. When ZAK-overexpressing cells introduced with c-Jun RNA interference (RNAi), the activator protein-1 (AP-1) transcription activity detected by a secreted alkaline phosphatase (SEAP) assay was suppressed and the decreased cell number was reversed compared with the control RNAi-treated group. More importantly, ZAK significantly depressed tumor growth in in vivo study. Taken together, results from both in vitro and in vivo studies indicated that the decrease of lung cancer cell proliferation by ZAK may involve the ERK and JNK pathways via an AP-1 transcription factor.

In this report, we identified the novel protein heart protein phosphatase 1-binding protein (Hepp1), encoded by FLJ23654. Hepp1 associated with protein phosphatase 1 (PP1) by yeast two-hybrid, GST pull-down, co-immunoprecipitation, and far Western blotting assays. Northern blot analysis revealed that Hepp1 mRNA was only expressed in human heart and testis. Recombinant Hepp1 slightly enhanced the enzymatic activity of PP1 and antagonized the ability of phospho-inhibitor-1 or inhibitor-2 to inhibit PP1. Hepp1 protein in human heart tissues was detected by Western blot analysis. Together, our data suggest that Hepp1 can play a role in cardiac functions by working in concert with PP1.

We found that overexpression of RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIβ expression by RNA interference blocked hypertrophic growth. We further demonstrated that RhoGDIβ physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIβ functions. Moreover, the ZAK-RhoGDIβ interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIβ functions through phosphorylation and RhoGDIβ counteracts the effects of ZAK by physical interaction. Knockdown of ZAK expression in ZAK- and RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIβ.

tNOX, a tumor-associated NADH oxidase, is a growth-related protein present in transformed cells. In this study, we employed RNA interference (RNAi)-mediated down-regulation of tNOX protein expression to explore the role of tNOX in regulating cell growth in human cervical adenocarcinoma (HeLa) cells. In this first reported use of RNAi to decrease tNOX expression, we found that HeLa cell growth was significantly inhibited by shRNA-knockdown of tNOX. Furthermore, cell migration and membrane association of Rac were decreased concomitantly with the reduction in tNOX protein expression. These results indicate that shRNA targeting of tNOX inhibits the growth of cervical cancer cells, and reduces cell migration via a decrease in the membrane association of Rac. We propose that tNOX is a potential upstream mediator of Rho activation that plays a role in regulating cell proliferation, migration, and invasion.

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 microM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

We have identified a novel protein, protein phosphatase 1 F-actin cytoskeleton targeting subunit (phostensin). This protein is encoded by KIAA1949 and was found to associate with protein phosphatase 1 (PP1) in the yeast two-hybrid assay, co-immunoprecipitation, and GST pull-down assay. Northern blot analysis revealed that phostensin mRNA was predominantly distributed in leukocytes and spleen, and phostensin protein was present in crude extracts of human peripheral leukocytes. Immunofluorescence microscopic analysis revealed that the phostensin/PP1 complex was conspicuously localized with the actin cytoskeleton at the cell periphery in Madin–Darby canine kidney (MDCK) epithelial cells. Taken together, our data shows that phostensin targets PP1 to F-actin cytoskeleton. The phostensin/PP1 complex may play a vital role in modulation of actin rearrangements.

Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I.

Calcium silicate-based material (CS) has been successfully used in dental clinical applications. Some researches show that the antibacterial effects of CO2 laser irradiation are highly efficient when bacteria are embedded in biofilm, due to a photo-thermal mechanism. The purpose of this study was to confirm the effects of CO2 laser irradiation on CS, with regard to both material characterization and human periodontal ligament cell (hPDLs) viability. CS was irradiated with a dental CO2 laser using directly mounted fiber optics in wound healing mode with a spot area of 0.25 cm2, and then stored in an incubator at 100% relative humidity and 37 °C for 1 d to set. The hPDLs cultured on CS were analyzed, along with their proliferation and odontogenic differentiation behaviors. The results indicate that the CO2 laser irradiation increased the amount of Ca and Si ions released from the CS, and regulated cell behavior. CO2 laser-irradiated CS promoted cementogenic differentiation of hPDLs, with the increased formation of mineralized nodules on the substrate's surface. It also up-regulated the protein expression of multiple markers of cementogenic and the expression of cementum attachment protein. The current study provides new and important data about the effects of CO2 laser irradiation on CS. Taking cell functions into account, the Si concentration released from CS with laser irradiated may be lower than a critical value, and this information could lead to the development of new regenerative therapies for dentin and periodontal tissue.

It is unclear whether cardiac hypertrophy and hypertrophy-related pathways will be induced by long-term intermittent hypoxia. Thirty-six Sprague-Dawley rats were randomly assigned into three groups: normoxia, and long-term intermittent hypoxia (12% O(2), 8 h per day) for 4 weeks (4WLTIH) or for 8 weeks (8WLTIH). Myocardial morphology, trophic factors and signalling pathways in the three groups were determined by heart weight index, histological analysis, Western blotting and reverse transcriptase-polymerase chain reaction from the excised left ventricle. The ratio of whole heart weight to body weight, the ratio of left ventricular weight to body weight, the gross vertical cross-section of the heart and myocardial morphological changes were increased in the 4WLTIH group and were further augmented in the 8WLTIH group. In the 4WLTIH group, tumour necrosis factor-alpha(TNFalpha), insulin-like growth factor (IGF)-II, phosphorylated p38 mitogen-activated protein kinase (P38), signal transducers and activators of transcription (STAT)-1 and STAT-3 were significantly increased in the cardiac tissues. However, in the 8WLTIH group, in addition to the above factors, interleukin-6, mitogen-activated protein kinase (MEK)5 and extracellular signal-regulated kinase (ERK)5 were significantly increased compared with the normoxia group. We conclude that cardiac hypertrophy associated with TNFalpha and IGF-II was induced by intermittent hypoxia. The longer duration of intermittent hypoxia further activated the eccentric hypertrophy-related pathway, as well as the interleukin 6-related MEK5-ERK5 and STAT-3 pathways, which could result in the development of cardiac dilatation and pathology.

In mammals, polyunsaturated fatty acids (PUFAs) act not only as an important energy source, but also as substrates for cellular membrane and hormone formation. They also play key roles in cellular metabolism and gene regulation. The objective of the present study was to determine whether individual n-6 and n-3 PUFAs affect cytochrome P-450 2B1 (CYP 2B1) expression induced by phenobarbital (PB) in primary rat hepatocytes. We used 100-microM arachidonic acid (AA), linoleic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA) to test this hypothesis. Phenobarbital-induced CYP 2B1 expression was down-regulated by n-6 and n-3 PUFAs, especially AA and DHA. Prostaglandin (PG) E2 but not PGE3 was found to down-regulate PB-induced CYP 2B1 expression. The cyclooxygenase inhibitor indomethacin (20 microM) attenuated the down-regulation of CYP 2B1 gene expression by n-6 and n-3 PUFAs induced by PB, and maximal attenuation was found in the AA-treated group. We also studied the PGE2 downstream cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway to determine its role in the down-regulation of CYP 2B1 expression by AA with the use of 0.4 mM of the adenylate cyclase inhibitor 9-(tetrahydro-2'-furyl)adenine] (SQ22536) and 7.5 microM of the PKA inhibitor H-89. Both inhibitors attenuated the down-regulation of CYP 2B1 expression by AA. These results suggest that PB-induced CYP 2B1 expression is down-regulated by n-6 and n-3 PUFAs through different pathways. Prostaglandin E2 and the cAMP-dependent PKA pathway were involved in AA down-regulation of CYP 2B1 expression, whereas the down-regulation by n-3 PUFAs is not fully understood yet and the glucocorticoid receptor/constitutive androstane receptor/retinoid X receptor signal transduction cascade can be involved.

Background : Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. Objectives : To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. Study design : 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. Results : The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. Conclusions: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region. © 2004 Elsevier B.V. All rights reserved.

Background: Bleomycin A5 is an anti-neoplastic glycoprotein antibiotic used for the treatment of various cancers. Previous work has shown that bleomycin A5 exerts its apoptotic effects on tumor cells. This was to study the signal transduction pathways that might exert the apoptotic effects of bleomycin A5 on tumor cells, as well as to examine the possibility of lower dosing of such drug in combinative treatment with other compounds in vitro. Methods: Bleomycin A5 was applied on a human oral epidermoid carcinoma cell line, human oral epidermoid carcinoma (KB), and the apoptotic activity was determined by the presence of DNA fragmentation and 4,6-diamidino-2-phenylindole (DAPI) nuclear staining. The signal transduction pathway was measured through Western blotting and in vitro kinase assay. Results: The apoptotic effect was associated with the sustained activation of c-Jun N-terminal kinases (JNK) and the inhibition of extracellular signal-regulated kinases1 (ERK1) and -2 activities, suggesting that JNK plays a positive role in the death process. ERK1 and -2 might exert a protection pathway from cell death. Here, it was determined that a combination treatment of bleomycin A5 and the MAP kinase-ERK kinase (MEK) inhibitor, PD98059, could lead to enhanced apoptosis. The activities of ERK1 and -2 are required for cell survival signaling using stable cell clones expressing MEK1. Upon bleomycin A5 treatment, cells expressing MEK1 exhibited significant delays in the onset of apoptosis, where the presence of MEK1 inhibitor enhanced cell death. Moreover, the increased activity of ERK1 and -2 coincided with cell survival. The survival signals exerted by MEK1 most likely result from the activation of ERK1 and -2. Conclusions: The apoptosis enhancement through such combinative treatment in vitro has revealed new therapeutic opportunities and elucidated mechanisms contributing to the efficacy of existing anti-cancer treatments.

This present study was designed to investigate the combined modulatory effect of garlic oil (GO) and fish oil (FO) on the antioxidant and drug metabolism systems. Rats were fed either a low-maize oil (MO) diet (50 g MO/kg), high-MO diet (235 g MO/kg) or high-FO diet (205 g FO+30 g MO/kg) and received different doses of GO (0–200 mg/kg body weight) three times per week for 6 weeks. Fatty acid analysis showed that 20: 5n−3 and 22: 6n−3 were incorporated into serum lipid at the expense of 18: 2n−6 and 20: 4n−6 in rats fed the high-FO diet. GO dose-dependently increased hepatic glutathioneS-transferase (GST), glutathione reductase, superoxide dismutase (SOD) and ethoxyresorufinO-deethylase (EROD) activities, but decreased glutathione peroxidase andN-nitrosodimethylamine demethylase (NDMAD) activities (PP

ZAK has been implicated in cell cycle arrest regulation through its function on decreasing cyclin E expression. To explore the mechanistic basis for this regulation, the yeast two-hybrid system was used with a novel Kruppel-type C2H2 zinc finger member cloned. This cloned cDNA encodes a novel protein with Kruppel-type zinc fingers designed as ZZaPK (zinc finger and ZAK associated protein with KRAB domain) and is widely expressed. ZZaPK, when it is expressed in cells, is growth promoted and might lead to increasing E2F expression and induce cyclin E/CDK2 activity, which counteracts the ZAK function. The model proposed here is that ZAK might play a role as an upstream signal to suppress the ZZaPK function and decrease E2F expression.