A putative novel protein, DEPDC1B, is overexpressed in oral cancer patients, and enhanced anchorage-independent growth in oral cancer cells that is mediated by Rac1 and ERK.

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.

Lipid-lowering effects of curcumin in patients with metabolic syndrome: a randomized, double-blind, placebo-controlled trial.

Human studies of curcumin extract on lipid-lowering effect have not been completely investigated and have had controversy results. This study tested the effect of daily curcumin extract for 12 weeks on weight, glucose, and lipid profiles in patients with metabolic syndrome. Sixty-five patients were randomized into two groups; 33 patients taking curcumin extract capsule (630 mg thrice daily) and 32 patients taking a placebo capsule thrice daily for 12 weeks. At 12 weeks after the curcumin extract consumption, the level of high-density lipoprotein cholesterol (HDL-C) significantly increased from 40.96 ± 8.59 to 43.76 ± 2.79 mg/dL (p < 0.05), and the level of low-density lipoprotein cholesterol (LDL) was significantly reduced (120.55 ± 36.81 to 106.51 ± 25.02 mg/dL, p < 0.05). The triglyceride-lowering effect, a reduction of 65 mg/dL, was also found in this study. In subgroups analysis, the consumption of curcumin may have a lowering cholesterol effect in male patients and an increasing HDL-C effect in female patients, both of which result in a decrease of T-Chol/HDL-C ratio. The intake of the curcumin extract of 1890 mg/day for 12 weeks was associated with lipid-lowering effect but did not improve weight and glucose homeostasis in the patients with metabolic syndrome. Daily curcumin consumption may be an alternative choice to modify cholesterol-related parameters, especially in metabolic syndrome patients.

EGFR-mediated hyperacetylation of tubulin induced docetaxel resistance by downregulation of HDAC6 and upregulation of MCAK and PLK1 in prostate cancer cells.

Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for castration-resistant prostate cancer (PCa). However, clinical treatment with docetaxel often encounters a number of undesirable effects, including drug resistance. Tubulin isoforms have been previously examined for their resistance to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, a series of docetaxel-resistant PC/DX cell sublines were established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Western blotting results showed significantly higher expression of acetyl-tubulin, α-tubulin, ß-tubulin, γ-tubulin, and ßIII-tubulin in PC/DX25 than in parental PC3 cells. PC/DX25 with greater resistance to docetaxel had higher levels of acetyl-tubulin and mitotic centromere-associated kinesin (MCAK) than PC3 cells. This study found that docetaxel induced the expression of acetyl-tubulin and MCAK in PC3 cells at a dose- and time-dependent manner. Both mRNA and protein levels of histone deacetylase 6 (HDAC6) were significantly decreased in PC/DX25 compared with PC3 cells. PC3 increased the resistance to docetaxel by HDAC6 knockdown and Tubastatin A (HDAC6 inhibitor). Conversely, PC/DX25 reversed the sensitivity to docetaxel by MCAK knockdown. Notably, flow cytometry analysis revealed that MCAK knockdown induced significantly sub G1 fraction in PC/DX cells. Overexpression of polo-like kinase-1 increased the cell survival rate and resistance to docetaxel in PC3 cells. Moreover, epidermal growth factor receptor (EGFR) activation induced the upregulation of acetyl-tubulin in docetaxel-resistant PCa cells. These findings demonstrated that the EGFR-mediated upregulated expression of acetyl-tubulin played an important role in docetaxel-resistant PCa.

Kaempferol-Enhanced Migration and Differentiation of C2C12 Myoblasts via ITG1B/FAK/Paxillin and IGF1R/AKT/mTOR Signaling Pathways.

SCOPE: Kaempferol (KMP), a bioactive flavonoid compound found in fruits and vegetables, contributes to human health in many ways but little is known about its relationship with muscle mass. The effect of KMP on C2C12 myoblast differentiation and the mechanisms that might underlie that effect are studied. METHODS AND RESULTS: This study finds that KMP (1, 10 µM) increases the migration and differentiation of C2C12 myoblasts in vitro. Studying the possible mechanism underlying its effect on migration, the study finds that KMP activates Integrin Subunit Beta 1 (ITGB1) in C2C12 myoblasts, increasing p-FAK (Tyr398) and its downstream cell division cycle 42 (CDC42), a protein previously associated with cell migration. Regarding differentiation, KMP upregulates the expression of myosin heavy chain (MHC) and activates IGF1/AKT/mTOR/P70S6K. Interestingly, pretreatment with an AKT inhibitor (LY294002) and siRNA knockdown of IGF1R leads to a decrease in cell differentiation, suggesting that IGF1/AKT activation is required for KMP to induce C2C12 myoblast differentiation. CONCLUSION: Together, the findings suggest that KMP enhances the migration and differentiation of C2C12 myoblasts through the ITG1B/FAK/paxillin and IGF1R/AKT/mTOR pathways. Thus, KMP supplementation might potentially be used to prevent or delay age-related loss of muscle mass and help maintain muscle health.

Zebrafish Dkk3a protein regulates the activity of myf5 promoter through interaction with membrane receptor integrin α6b.

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the ß-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.

dickkopf-3-related gene regulates the expression of zebrafish myf5 gene through phosphorylated p38a-dependent Smad4 activity.

Myf5 is a myogenic regulatory factor that functions in myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through the targeting of dickkopf-3-related gene (dkk3r). However, the molecular mechanism underlying the control of myf5 expression by dkk3r is unknown. By injecting dkk3r-specific morpholino-oligonucleotide (dkk3r-MO) to knock down Dkk3r, we found that the phosphorylated p38a protein was reduced. Knockdown of p38a resulted in malformed somites and reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3r-MO. To block the MAPK pathway, phosphorylation of p38 was inhibited by introduction of SB203580, which caused the down-regulation of myf5 expression. The GFP signal was dramatically decreased in somites when we injected p38a-MO into embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by the myf5 promoter. Although these p38a-MO-induced defects were rescued by co-injection with p38a mRNA, they were not rescued with p38a mRNA containing a mutation at the phosphorylation domain. Moreover, overexpression of Smad2 or Smad3a enhanced myf5 expression, but the defects induced by the dominant negative form of either Smad2 or Smad3a equaled those of embryos injected with either dkk3r-MO or p38a-MO. These results support the involvement of Smad2·Smad3a in p38a mediation. Overexpression of Smad4 enabled the rescue of myf5 defects in the dkk3r-MO-injected embryos, but knockdown of either dkk3r or p38a caused Smad4 protein to lose stability. Therefore, we concluded that Dkk3r regulates p38a phosphorylation to maintain Smad4 stability, in turn enabling the Smad2·Smad3a·Smad4 complex to form and activate the myf5 promoter.

The Promotion of Migration and Myogenic Differentiation in Skeletal Muscle Cells by Quercetin and Underlying Mechanisms.

Aging and muscle disorders frequently cause a decrease in myoblast migration and differentiation, leading to losses in skeletal muscle function and regeneration. Several studies have reported that natural flavonoids can stimulate muscle development. Quercetin, one such flavonoid found in many vegetables and fruits, has been used to promote muscle development. In this study, we investigated the effect of quercetin on migration and differentiation, two processes critical to muscle regeneration. We found that quercetin induced the migration and differentiation of mouse C2C12 cells. These results indicated quercetin could induce myogenic differentiation at the early stage through activated p-IGF-1R. The molecular mechanisms of quercetin include the promotion of myogenic differentiation via activated transcription factors STAT3 and the AKT signaling pathway. In addition, we demonstrated that AKT activation is required for quercetin induction of myogenic differentiation to occur. In addition, quercetin was found to promote myoblast migration by regulating the ITGB1 signaling pathway and activating phosphorylation of FAK and paxillin. In conclusion, quercetin can potentially be used to induce migration and differentiation and thus improve muscle regeneration.

Chlorogenic Acid Inhibition of Esophageal Squamous Cell Carcinoma Metastasis via EGFR/p-Akt/Snail Signaling Pathways.

BACKGROUND/AIM: Chlorogenic acid (CGA) is a polyphenol compound found in a variety of foods, including coffee, tea, cherries, and apples. It has been found by a number of studies to affect the viability of human cancer cells. No study has investigated its effect on esophageal squamous cell carcinoma (ESCC) metastasis or the molecular mechanism underlying its effect on this disease. MATERIALS AND METHODS: We first used the Taiwanese ESCC cell line CE81T/VGH to create CE81T-M4 cells. Treatment of higher motility cells with chlorogenic acid for 24 h led to inhibition of cell migration and invasion as shown by scratch migration and transwell assays. RESULTS: Western blotting showed that chlorogenic acid halted the activation of EGFR/p-Akt/Snail pathway and suppressed the expression of MMP-2 and MMP-9. Knockdown of either EGFR or Akt inhibited Snail, MMP2, and MMP9 activity as well as cell migration and invasion. CONCLUSION: Chlorogenic acid inhibited cancer cell motility via the EGFR/p-Akt/Snail pathway and could potentially be used to develop an antimetastatic agent for ESCC in the future.

A novel growth hormone 1 gene-derived probe for Oncorhynchus masou formosanus distinguished from the Oncorhynchus subspecies.

Traditional mitochondrial 16S rRNA is commonly used in many species identification studies. However, it is difficult to apply to the phylogenetic studies among the Oncorhynchus subspecies, which is a crucial need for management purposes for Oncorhynchus masou formosanus, Taiwan salmon. In this study, we have developed an improved species identification method for Taiwan salmon distinguished with other Oncorhynchus subspecies tested by exploiting PCR for growth hormone (GH) 1 gene. By comparing DNA sequences for GH1 from 11 species of Oncorhynchus subspecies we designed novel PCR primers that exploit differences between Taiwan salmon and other Oncorhynchus subspecies. Therefore, the technique is an important tool in the management of populations of the endangered land-locked Taiwan salmon preventing from their possible hybrids with other Oncorhynchus subspecies once tested.

The upregulation of matrix metalloproteinase-9 in inflamed human dental pulps.

The matrix metalloproteinases (MMPs) participate in a wide variety of extracellular matrix degradation. Detailed knowledge of MMPs may be important for understanding the pathogenesis of pulpal inflammation. The purpose of this study was to compare MMP-9 expression in clinically healthy human pulp and inflamed human pulp tissue specimens. We compared the levels of MMP-9 between clinically healthy pulp and inflamed pulp tissues by using the semi-quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of MMP-9 expression in pulp specimens. MMP-9 mRNA gene was found to be increased in inflamed pulps as compared with clinically healthy pulp tissues (p < 0.05). The results from immunohistochemistry demonstrated that MMP-9 expression was significantly higher in the inflamed pulps than clinically healthy pulps (p < 0.05). MMP-9 stain was detected in the odontoblasts, fibroblasts, inflammatory infiltrates, and endothelial cells. Taken together, MMP-9 may play an important role in the pathogenesis of pulpal inflammation.

Induction of vascular endothelial growth factor gene expression by proinflammatory cytokines in human pulp and gingival fibroblasts.

Vascular endothelial growth factor (VEGF) enhances the permeability of blood vessels, which is an important vascular change observed during inflammatory processes. The purpose of this in vitro study was to investigate the effect of proinflammatory cytokines on the expression of VEGF mRNA gene in human pulp and gingival fibroblasts. Interlukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were used to evaluate VEGF mRNA gene expression in human pulp and gingival fibroblasts. The levels of mRNAs were measured by quantitative reverse-transcriptase polymerase chain reaction analysis. Both IL-1alpha and TNF-alpha induced significantly high levels of VEGF mRNA gene expression in human pulp and gingival fibroblasts (p < 0.05). In addition, TNF-alpha was found to be more effective in the induction of VEGF mRNA gene expression in pulp than gingival fibroblasts (p < 0.05). Moreover, IL-1alpha was found to be more effective in the induction of VEGF mRNA gene expression than TNF-alpha in gingival fibroblast cultures (p < 0.05). These results indicate that proinflammatory cytokines can induce VEGF mRNA gene expression, and such an effect may partially contribute to the destruction of pulpal and periapical tissues through promoting expansion of the vascular network coincident to progression of the inflammation.

Osteogenesis and angiogenesis properties of dental pulp cell on novel injectable tricalcium phosphate cement by silica doped.

ß-Tricalcium phosphate (ß-TCP) is an osteoconductive material in clinical. In this study, we have doped silica (Si) into ß-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 ß-TCP cements. The results showed that setting time and injectability of the Si-doped ß-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 ß-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 ß-TCP cements. The degradation of ß-TCP and osteogenesis of Si gives a strong reason to believe that these Si-doped ß-TCP cements may prove to be promising bone repair materials.

The expression of ribonucleotide reductase M2 in the carcinogenesis of uterine cervix and its relationship with clinicopathological characteristics and prognosis of cancer patients.

BACKGROUND: To investigate the implication of ribonucleotide reductase M2 (RRM2) in the carcinogenesis of uterine cervix and its relationship with clinicopathological characteristics and prognosis of cancer patients. METHODOLOGY AND PRINCIPAL FINDINGS: The impact of RRM2 on cell viability was investigated in SiHa cervical cancer cells after RRM2 knockdown and the addition of cisplatin, which induces inter- and intra-strand DNA crosslinks. RRM2 immunoreactivity was evaluated by semi-quantitative H score among 29 normal, 30 low-grade dysplasia, 30 high-grade dysplasia and 103 invasive cancer tissue specimens of the uterine cervix, using tissue microarrays. RRM2 was then correlated with the clinicopathological variables of cervical cancer and patient survival. A greater toxic effect on cell viability using cisplatin was reflected by the greater reduction in RRM2 protein expression in SiHa cells. The RRM2 expression in cancer tissues was higher than that in high-grade dysplasia, low-grade dysplasia or normal cervical tissues. RRM2 upregulation was correlated with deep stromal invasion, large tumors and parametrial invasion and predicted poor survival. CONCLUSIONS: RRM2 is a new molecular marker for the diagnosis and clinical outcomes of cervical cancer. It is involved in cervical carcinogenesis and predicts poor survival, and may be a potential therapeutic target including in cisplatin treatment.

Aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of pentose phosphate pathway.

Obesity has become a major public health problem of global significance. Today, aspirin remains the most commonly used medication for the treatment of pyrexia, pain, inflammation and antiplatelet. The present study aims at evaluating the possible existence of a putative p53-dependent pathway underlying the aspirin-induced inhibition of adipogenesis. Cell migration assay was identified by the ability to migrate through Transwell insert. Oil Red O staining was employed to quantify adipose accumulation. The concentration of glucose and triglyceride were measured by using assay kits. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Aspirin significantly inhibited preadipocyte migration and adipose accumulation. The p53-p21 signaling and the expression of differentiation marker glycerol-3-phosphate dehydrogenase were increased in a dose-dependent manner. It indicated that aspirin induced adipocyte differentiation through p53-p21 pathway. The oncogenic ERK 1/2 MAPK signaling was induced, whereas, the expression of adipogenic markers peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (A-FABP) and inflammatory factors cyclooxygenase-2 (Cox-2), tumor necrosis factor α (TNFα) and inducible nitric oxide synthase (iNOS) were inhibited. Aspirin negatively regulated the pentose phosphate pathway (PPP) by inhibiting the expression of rate-limiting enzyme glucose-6-phosphate dehydrogenase. Knockdown the expression of oncogenic ERK 1/2 MAPK by using 10 µM PD98059 significantly increased triglyceride synthesis, adipose accumulation and activated PPP, however, decreased glucose uptake. Diverted the glucose flux to PPP, rather than increased glucose uptake, was associated with adipogenesis. Down-regulated the expression of tumor suppressor p53 by 10 µM pifithrin-α (PFTα) alone had no effect on adipose accumulation. However, administration of aspirin accompanied with PFTα abolished aspirin-induced inhibition of adipogenesis. We demonstrated that aspirin-induced inhibition of adipogenesis was p53-dependent and associated with inactivation of PPP. Blockade PPP may be a novel strategy for obesity prevention and therapy. Moreover, when use aspirin in therapeutic strategy, the p53 status should be considered.

High-throughput gender identification of three Columbidae species using melting curve analysis.

The objective was to perform high-throughput gender identification of three Columbidae species (Columba livia, Columba pulchricollis, and Streptopelia tranquebarica). Although the chromo-helicase-DNA binding protein (CHD)-based Griffiths P2/P8 primer set resolved the amplicon products of these species in 3% agarose gel electrophoresis, it was unsuitable for molecular gender identification using the melting curve analysis (MCA) curve for high-throughput analysis. After sequencing the CHD-Z and CHD-W genes for these species, we redesigned a female-specific CHD-W primer (dove-W) and a female/male (or CHD-Z/CHD-W)-common primer (dove-ZW) to combine with the Griffiths P2 primer to generate two PCR amplicons with different lengths (P2/dove-W and P2/dove-ZW for 252- and 104-bp, respectively). Melting temperature (Tm) values for P2/dove-W and P2/dove-ZW amplicons were determined and resolved in MCA at approximately 79.0∼79.5 and 77.5 °C, respectively. Accordingly, females contained two Tm peaks, whereas males contained one. In conclusion, melting curve analysis (MCA) using our proposed primer sets was a robust gender identification method for the three Columbidae species tested.