Normal Rat Liver Research Articles

Antidiabetic effect of Ruta montana L. in streptozotocin-induced diabetic rats.

Ruta montana (L.) is known as a medicinal plant with many beneficial effects, including those that can be used in the treatment of diabetes. The objective of the study was to investigate the antidiabetic effect of this plant in diabetic rat. This study investigated the effects of an aerial part aqueous extract (APAE) of Ruta montana (L.) (RM) at a dose of 5 mg/kg on blood glucose levels in normal and streptozotocin (STZ)-induced diabetic rats. Histopathological changes were also evaluated in liver and pancreas both in normal and STZ-induced rats. The effect of this aqueous extract on glucose tolerance was demonstrated in normal rats. Furthermore, the relative organ weight (ROW) of liver, kidney, pancreas, and brown adipose tissue were evaluated after 15 days of daily oral administration of the aqueous extract. Both single and repeated oral doses of APAE (5 mg/kg) produced significant reductions in the blood glucose levels in normal and STZ-induced rats. Oral glucose tolerance test results showed that, after the administration of 3 g/kg of glucose, RM APAE (5 mg/kg) improved the increase in blood glucose levels in normal rats at the 30th min (p<0.01) and 90th min (p<0.001). RM APAE exhibits a potent hypoglycemic effect in normal rats and an antidiabetic effect in STZ-induced rats. This finding supports the use of this plant in traditional Moroccan medicine for diabetes management.

Tithonia diversifolia saponin-blood lipid interaction and its influence on immune system of normal wistar rats.

This study investigated the effect of saponins (20-100mg/kg) from Tithonia diversifolia leaf (STD) on the liver, kidney, heart, lipid profile and hematological parameters of normal rats. The results showed that STD (20-100mg/kg) though caused a slight increase in the liver, heart and kidney activity of ALT, AST, ALP and GGT (p<0.05), did not result in corresponding increase in the serum level of these enzymes. A significant reduction in the level of triglycerides, LDL and cholesterol, creatinine, urea, LDH, PCV and hemoglobin were observed with a concomitant increase in HDL, white blood cell and lymphocyte. These study demonstrated the role of STD in enhancing immune response and in reducing cholesterol and triglycerides in normal rats at studied dosages.

Study on morphological changes induced by aspartame on liver of normal and diabetic male albino rats

Abstract Background: This work was designed to study the histological and ultrastructure abnormalities in the liver induced by high dose of aspartame (ASP) in normal and diabetic rats. Methods:

Investigation of an antidiabetic potential of a phenolic compound isolated from the leaves of mempelas paya

Introduction: Leaves of mempelas paya (Tetracera indica) (family: Dilleniaceae) is traditionally used in diabetes management in Malaysia. However, no attempt has ever been made to discover an active principle responsible for its blood glucose lowering effect. Hence, this research deals with the isolation, in vivo antidiabetic and toxicological evaluation of an active principle. Methods: Chemical investigation of methanol extract derived from the powdered leaves resulted in the isolation of a phenolic compound through repeated silica gel column chromatography. The structure was elucidated by spectroscopic (1H-NMR, 13C-NMR, MS, IR and UV) analysis. In vivo antidiabetic and toxicological studies were carried out using Sprague-Dawley rats (diabetic and normal) at three different concentrations (1, 5 and 25 mg/kg b.w.). Results: 5,7-dihydroxy-8methoxyflavone was found to exhibit significant anti-hyperglycaemic activity (p&lt;0.05) in diabetic rats at 5 and 25 mg/kg b.w. without showing hypoglycaemic effect in normal rats. The anti-hyperglycaemic activity was found to be comparable with glibenclamide (GLBC) (p&lt;0.05). In vivo toxicological study for continuous 21 days showed no abnormal activity and mortality. Histopathology of kidney, liver and pancreas of both normal and diabetic treated rats demonstrated normal and improved condition when compared to diabetic control group. There were no significant changes seen in the kidney (serum urea and creatinine) and lipid (TC, TG, LDL-C and HDL-C) functional parameters tested. Conclusions: 5,7-dihydroxy-8-methoxyflavone isolated from the leaves of mempelas paya coud be a safe antidiabetic agent.

Liver Perfusion Modifies Gd-DTPA and Gd-BOPTA Hepatocyte Concentrations Through Transfer Clearances Across Sinusoidal Membranes.

Gadobenate dimeglumine (Gd-BOPTA) is a commercialised hepatobiliary contrast agent used during liver magnetic resonance imaging (MRI) to detect liver diseases. It enters into human hepatocytes through organic anion transporting polypeptides (OATP1B1/B3) and crosses the canalicular transporter multiple resistance-associated protein 2 (MRP2) to be excreted into bile canaliculi. Gd-BOPTA can return to sinusoids via the sinusoidal transporters MRP3/MRP4. Hepatocyte concentrations of Gd-BOPTA depend on three clearances: the sinusoidal clearance or volume of sinusoidal blood cleared of drugs per unit of time and two hepatocyte clearances (into bile canaliculi or back to sinusoids) or volume of hepatocytes cleared of drugs per unit of time in the respective liver compartments. The present study investigates whether changing liver blood flow modifies hepatocyte concentrations when plasma concentrations do not change. We perfused normal rat livers at various portal flow rates (24, 30, and 36ml/min) with 200µM Gd-BOPTA and measured sinusoidal clearances, hepatocyte clearances, and hepatocyte concentrations of Gd-BOPTA. We showed that varying portal flow rates changes the sinusoidal clearance of Gd-BOPTA despite its low extraction ratio. Portal flow rates do not modify Gd-BOPTA clearance from hepatocytes into bile canaliculi but can change hepatocyte clearance back to sinusoids. At a given perfused concentration, portal flow rates modify Gd-BOPTA hepatocyte concentrations, a result important to consider when interpreting liver imaging.

Babao Dan attenuates hepatic fibrosis by inhibiting hepatic stellate cells activation and proliferation via TLR4 signaling pathway.

Babao Dan (BBD), a traditional Chinese medicine, has been widely used as a complementary and alternative medicine to treat chronic liver diseases. In this study, we aimed to observe the protective effect of BBD on rat hepatic fibrosis induced by diethylnitrosamine (DEN) and explore it possible mechanism. BBD was administrated while DEN was given. After eight weeks, values of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicated that BBD significantly protected liver from damaging by DEN and had no obvious side effect on normal rat livers. Meanwhile, BBD attenuated hepatic inflammation and fibrosis in DEN-induced rat livers through histopathological examination and hepatic hydroxyproline content. Furthermore, we found that BBD inhibited hepatic stellate cells activation and proliferation without altering the concentration of lipopolysaccharide (LPS) in portal vein. In vitro study, serum from BBD treated rats (BBD-serum) could also significantly suppress LPS-induced HSCs activation through TLR4/NF-κB pathway. In addition, BBD-serum also inhibited the proliferation of HSCs by regulating TLR4/ERK pathway. Our study demonstrated that BBD may provide a new therapy strategy of hepatic injury and hepatic fibrosis.

Ex vivo study of acoustic radiation force impulse imaging elastography for evaluation of rat liver with steatosis

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in developed countries. Accurate, noninvasive tests for diagnosing NAFLD are urgently needed. The goals of this study were to evaluate the utility of acoustic radiation force impulse (ARFI) elastography for determining the severity grade of steatosis in rat livers, and to investigate the changes in various histologic and biochemical characteristics. Steatosis was induced in the livers of 57 rats by gavage feeding of a high fat emulsion; 12 rats received a standard diet only and served as controls. Liver mechanics were measured ex vivo using shear wave velocity (SWV) induced by acoustic radiation force. The measured mean values of liver SWV ranged from 1.33 to 3.85m/s for different grades of steatosis. The area under the receiver operative characteristic curve (⩾S1) was equal to 0.82 (95% CI=0.69, 0.96) between the steatosis group and the normal group, and the optimal cutoff value was 2.59 with sensitivity of 88% and specificity of 76%. However, there are no significant differences in SWV measurements between the steatosis grades. SWV values did not correlate with the early grade of inflammation. In conclusion, ARFI elastography is a promising method for differentiating normal rat liver from rat liver with steatosis, but it cannot reliably predict the grade of steatosis in rat livers. The early grade of inflammation activity did not significantly affect the SWV measurements.

Polyphenols contribute to the antioxidant and antiproliferative activity of Phyllanthus debilis plant in-vitro.

BackgroundPhyllanthus debilis (Elapitawakka) is a medicinal plant used in traditional systems of medicine in Sri Lanka. Present study was carried out to evaluate in-vitro anti-oxidant and anti-proliferative activity of the water extracts of aerial parts (AP) and roots (RP) of P.debilis plant and the role of polyphenolic compounds in view of its medicinal use.MethodTotal polyphenols, flavonoids and proanthocyanidin content of the extracts were quantified. DPPH, hydroxyl radical, nitric oxide and hydrogen peroxide scavenging potentials and the total antioxidant capacity, ferric ion reducing power were determined to evaluate antioxidant capacity. Anti-proliferative activity was assessed with MTT assay for Human Rhabdomyosarcoma (RD) and normal rat liver cells (CC1) after 24 h exposure to the plant extracts. DPPH and MTT assays were carried out for AP and RP extracts after removal of polyphenols to assess the contribution of polyphenols on antioxidant and anti-proliferative activity of Phyllanthus debilis.ResultsFlavonoid content of the AP extract was significantly lower than that of RP (P < 0.001) while no significant difference was observed in polyphenolic as well as in proanthocyanidin contents. All the assays except for phosphomolybdate assay demonstrated that the RP extract had higher antioxidant capacity (p < 0.001) compared to AP. Further, antioxidant capacity and anti-proliferative activity were lower (p < 0.001) in AP and RP in the absence of polyphenols compared to the crude extract.ConclusionRoot contains higher levels of flavonoids than the aerial part. Moreover, the presence of polyphenols is required for antioxidant and anti-proliferative activities of both AP and RP.

Abstract 4264: Engineered in vitro models of cancer metastasis using decellularized biomatrix

Abstract Background: Metastasis contributes to majority of death in cancer patients. However, cancer metastasis is difficult to study due to the lack of experimental models that can fully recapitulate the biological process, especially the organ specificity of cancer metastasis. Recent advances in tissue engineering demonstrated that decellularized tissue, organs that are denuded of cells, are excellent scaffolds for tissue engineering of complex organs, such as liver and lung. These decellularized organs preserve the organ microenvironment, which is critical in cancer metastasis. We hypothesized that we can utilize decellularized organ and engineer in vitro models of cancer metastasis that can recapitulate the organ specificity of cancer metastasis. Here we report the proof-of-principle of this approach by employing colorectal cancer as a disease model. Since the primary cause of morbidity and mortality in colorectal patients is liver and lung metastasis, we aimed to engineer in vitro models of colorectal cancer lung and liver metastasis. Methods: Decellularized biomatrix was prepared by sodium deoxycholate based perfusion decellularization of rat liver or lung. We compared the growth factors on the biomatrix scaffold to that of normal rat liver and lung using semi-quantitative ELISA. We cultured colorectal cancer cell lines: HT-29, SW480 and Caco2, on tissue culture dishes coated with liver and lung decellularized biomatrix scaffolds. Cell colony morphology and structure was examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and hematoxylin and eosin stain. Therapeutic response (chemotherapy and radiation) for cells grown on biomatrix was also examined. Results: Decellularized organs preserved 92% growth factors bound to biomatrix scaffold at near physiological levels. HT-29, SW480 and Caco2 were able to proliferate on dishes coated with biomatrix scaffold and spontaneously formed three-dimensional (3D) colonies when maintained at higher levels. These tumor colonies are millimeter in size and are spherical in shape, similar to in vivo metastases. SEM showed cells on the tumor surface appeared to form tight junctions with each other. TEM images further confirmed that these 3D tumor colonies contain areas of necrosis, consistent existing literature 3D colonies and in vivo metastasis. Importantly, we identified signet ring cell formation in the engineered 3D metastases. We also found the cells in engineered liver and lung metastasis responded to chemotherapy treatment differently than the cells cultured under standard conditions or on collagen or on matrigel. Interestingly, cells in both engineered liver and lung metastasis are more sensitive to radiotherapy than standard condition. Conclusions: Our engineered in vitro tumor metastasis models closely mimic the in vivo metastasis phenotypically, histologically, and biologically. Citation Format: XI TIAN, Michael E. Werner, Henry P. Foote, Ariel D. Hanson, Lola M. Reid, Andrew Z. Wang. Engineered in vitro models of cancer metastasis using decellularized biomatrix. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4264.

Amphipathic silica nanoparticles induce cytotoxicity through oxidative stress mediated and p53 dependent apoptosis pathway in human liver cell line HL-7702 and rat liver cell line BRL-3A

The aim of this study was to evaluate the potential cytotoxicity and the underlying mechanism of amphipathic silica nanoparticles (SiO2 NPs) exposure to human normal liver HL-7702 cells and rat normal liver BRL-3A cells. Prior to the cellular studies, transmission electron microscopy (TEM), dynamic light scattering (DLS), and X ray diffraction (XRD) were used to characterize SiO2 NPs, which proved the amorphous nature of SiO2 NPs with TEM diameter of 19.8±2.7nm. Further studies proved that exposure to SiO2 NPs dose-dependently induced cytotoxicity as revealed by cell counting kit (CCK-8) and lactate dehydrogenase (LDH) assays, with more severe cytotoxicity in HL-7702 cells than BRL-3A cells. Reactive oxygen species (ROS) and glutathione (GSH) assays showed elevated oxidative stress in both cells. Morphological studies by microscopic observation, Hochest 33258 and AO/EB staining indicated significant apoptotic changes after the cells being exposed to SiO2 NPs. Further studies by western blot indicated that SiO2 NPs exposure to both cells up-regulated p53, Bax and cleaved caspase-3 expression and down-regulated Bcl-2 and caspase-3 levels. Activated caspase-3 activity detected by colorimetric assay kit and caspase-3/7 activity detected by fluorescent real-time detection kit were significantly increased by SiO2 NPs exposure. In addition, antioxidant vitamin C significantly attenuated SiO2 NPs-induced caspase-3 activation, which indicated that SiO2 NPs-induced oxidative stress was involved in the process of HL-7702 and BRL-3A cell apoptosis. Taken together, these results suggested that SiO2 NPs-induced cytotoxicity in HL-7702 and BRL-3A cells was through oxidative stress mediated and p53, caspase-3 and Bax/Bcl-2 dependent pathway and HL-7702 cells were more sensitive to SiO2 NPs-induced cytotoxicity than BRL-3A cells.

COMPLETE INHIBITION OF REGENERATION IN MICE FOLLOWING SIMULTANEOUS ELIMINATION OF MET‐EGFR SIGNALING

The Liver has an amazing ability to regenerate following loss of mass. Regeneration is a complex process that involves integration of multiple growth signaling pathways. Liver regeneration has been experimentally studied by surgical removal of 2/3 rd of liver in rodents. Following a partial hepatectomy (PHx), more than 100 genes exhibit differential expression and multiple signaling pathways are activated. Among the growth factors that are associated with hepatocyte proliferation, only Hepatocyte growth factor (HGF) and Epidermal growth factor (EGF) are complete mitogens that stimulate DNA synthesis in hepatocyte cultures maintained in chemically defined media. They are also the only ones that stimulate DNA synthesis in the liver of normal mice and rats. EGFR and Met phosphorylation increases over basal levels by 60 minutes after a PHx. Cross talk between these two pathways has been documented and integration of these two pathways may be required for regeneration to proceed efficiently. We decided to study the effect on regeneration in mice following complete elimination of EGFR‐MET signaling pathway. To this end, a post natal elimination of c‐met by using ER‐Cre‐Tamoxiphen‐inducible system was utilized. Mice were injected with Tamoxiphen (1mg/ml) and injected i.p everyday for 5 days. Controls were injected with corn oil alone. This treatment deleted exon 16 that contains a critical ATP‐binding site in the intracellular tyrosine kinase (TK) domain, essential for the activation of c‐met. PhosphoEGFR was inhibited by using Canertinib, an irreversible pan‐pEGFR inhibitor that was administered i.p at 80 mg/kg. Mice were injected a day before PHx and then everyday for 7 days. Treatment of Met‐KO mice with Canertinib inhibited EGFR signaling significantly. Treatment resulted in global changes in signaling pathways that seemed to promote block in proliferation and liver damage at time 0 Hrs. post PHx. A significant reduction in Ki67 labeling was also evident, as was a reduction in liver to body weight ratio. The size of the lobules and the hepatocytes was decreased in Met KO mice treated with Canertinib. Met KO mice treated with Canertinib had increased morbidity and had elevated AST, ALT values, and lower albumin levels. Combined elimination of both MET and EGFR signaling resulted in sustained inhibition of liver regeneration up to 14 days suggesting a liver regeneration deficit. The AKT‐mTOR and the ERK pathways associated with cellular proliferation were down regulated in MET‐EGFR inhibited mice. Suppression of MET‐EGFR signaling caused profound alterations in expression of many cell cycle associated genes and genes associated with fatty acid biosynthesis pathway. There appears to be is no redundancy and compensatory scheme for liver regeneration following complete elimination of EGFR‐MET signaling network.Support or Funding InformationNIH Grant No. CA 35373, CA 103958 &amp; Rangos Fund for Enhancement of Research in Pathology.

Capillary microfluidics-derived doxorubicin-containing human serum albumin microbeads for transarterial chemoembolization of hepatic cancer

In this study, we prepared doxorubicin (DOXO)-loaded albumin microbeads (DOXO-MBs) using a capillary microfluidic device for transarterial chemoembolization of hepatic cancer. Albumin droplets were fabricated using the capillary microfluidic device and solidified by addition of glutaraldehyde. The acquired DOXO-MBs were homogeneous and the size was adjustable from 183.2±12.2μm to 351.5±7.9μm by changing the flow rate of fluidic solutions. The loading amount of DOXO was 9.7±1.5mg/g, and over 15.7% of DOXO was released over one month in pH7.2 buffer. Intra-portal injection of DOXO-MBs on normal liver of rats proved microbeads efficiently embolized hepatic vessels. Hepatic lobes, recovered 24days after intra-portal injection, showed that the DOXO-MBs remained in hepatic vessels and released DOXO to surrounding hepatic tissues. In the hepatic tumor xenograft mouse model, DOXO-MBs inhibited tumor growth more efficiently than intravenous (I.V.) injection of free DOXO (p<0.01).

Glycogen content in hepatocytes is related with their size in normal rat liver but not in cirrhotic one

Hepatocytes differ from one another by the degree of the ploidy, size, position in the liver lobule, and level of the DNA-synthetic processes. It is believed, that the cell size exerts substantial influence on the metabolism of the hepatocytes and the glycogen content in them. The aim of the present study was to test this hypothesis. Dry weight of hepatocytes, their ploidy and glycogen content were determined in the normal and the cirrhotic rat liver. Liver cirrhosis in rats was produced by chronic inhalation of CCl4 vapours in the course of 6 months. A combined cytophotometric method was used. Dry weight of the cell, its glycogen and DNA content were successively measured on a mapped preparation. Hepatocytes of each ploidy class in the normal and the cirrhotic rat liver accumulated glycogen at the same rate. In the normal liver, there was a distinct correlation between the size of hepatocytes and glycogen content in them. This correlation was observed in each ploidy class, and was especially pronounced in the class of mononucleate tetraploid hepatocytes. In the cirrhotic liver, there was no correlation between the size of the cells and their glycogen content. The impairment of liver lobular structure probably explains the observed lack of correlation between hepatocyte size and their glycogen content in the cirrhotic liver. © 2016 International Society for Advancement of Cytometry.

Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location.

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs.

Normal and Fibrotic Rat Livers Demonstrate Shear Strain Softening and Compression Stiffening: A Model for Soft Tissue Mechanics.

Tissues including liver stiffen and acquire more extracellular matrix with fibrosis. The relationship between matrix content and stiffness, however, is non-linear, and stiffness is only one component of tissue mechanics. The mechanical response of tissues such as liver to physiological stresses is not well described, and models of tissue mechanics are limited. To better understand the mechanics of the normal and fibrotic rat liver, we carried out a series of studies using parallel plate rheometry, measuring the response to compressive, extensional, and shear strains. We found that the shear storage and loss moduli G’ and G” and the apparent Young's moduli measured by uniaxial strain orthogonal to the shear direction increased markedly with both progressive fibrosis and increasing compression, that livers shear strain softened, and that significant increases in shear modulus with compressional stress occurred within a range consistent with increased sinusoidal pressures in liver disease. Proteoglycan content and integrin-matrix interactions were significant determinants of liver mechanics, particularly in compression. We propose a new non-linear constitutive model of the liver. A key feature of this model is that, while it assumes overall liver incompressibility, it takes into account water flow and solid phase compressibility. In sum, we report a detailed study of non-linear liver mechanics under physiological strains in the normal state, early fibrosis, and late fibrosis. We propose a constitutive model that captures compression stiffening, tension softening, and shear softening, and can be understood in terms of the cellular and matrix components of the liver.

Caveolin-1 is upregulated in hepatic stellate cells but not sinusoidal endothelial cells after liver injury.

Sinusoidal endothelial cells (SEC) and hepatic stellate cells (HSC) are closely associated specialized vascular cells residing in the hepatic sinusoid. These cells have been shown to play important roles in many different pathophysiologic processes, in particular in liver fibrosis/cirrhosis and portal hypertension. Caveolin-1 functions as a scaffolding protein, and has a variety of functions including in many disease states, such as liver cirrhosis. Although previous studies have shown that in the injured rat liver, caveolin-1 is upregulated, the precise cells in which remains unclear. Therefore, the purpose of this study was to clarify the cell type (or types) in which caveolin-1 is expressed in normal and injured rat liver. We have utilized both detailed immunohistochemical labeling with cell specific markers as well as cell isolation techniques (isolating sinusoidal endothelial cells, HSCs, and hepatocytes) in normal and injured (bile duct ligation) rat liver. We show here that in the normal liver caveolin-1 is expressed predominantly in HSCs and SECs but after liver injury there is upregulation of caveolin-1 in HSCs, but not in SECs. These data have functional implications for the cells in which caveolin-1 is regulated.

In Vivo Anti-diabetic and Biological Activities of Milk Protein and Milk Protein Hydrolyaste

The effect of milk protein (MP) and milk protein hydrolysate (MPH) as Anti-diabetic agent were investigated in vivo using six groups of normal and type 2 diabetic rats. The results of this study showed that the treatments of diabetic rats by oral intake of MP or MPH significantly reduced the concentrations of plasma glucose, total lipids of blood plasma, triglycerides, total cholesterol, LDL and VLDL in rat plasma. Also, the treatments of diabetic rats by oral intake of MP or MPH significantly increased the globulin value and HDL, while the concentration of urea, creatinine and bilirubin were reduced. In addition, oral intake of MPH has no affective on acid phosphatase (ACP), alkaline phosphatase (ALP), alanine transaminase (ALT) and aspartate transaminase (AST) activities in blood plasma and liver of normal rats and protective its concentrations in diabetic rats. The present results concluded that MP and MPH could be used as anti-diabetic agents.

Update on a tumor-associated NADH oxidase in gastric cancer cell growth.

Gastric cancer is one of the most common human malignancies, and its prevalence has been shown to be well-correlated with cancer-related deaths worldwide. Regrettably, the poor prognosis of this disease is mainly due to its late diagnosis at advanced stages after the cancer has already metastasized. Recent research has emphasized the identification of cancer biomarkers in the hope of diagnosing cancer early and designing targeted therapies to reverse cancer progression. One member of a family of growth-related nicotinamide adenine dinucleotide (NADH or hydroquinone) oxidases is tumor-associated NADH oxidase (tNOX; ENOX2). Unlike its counterpart CNOX (ENOX1), identified in normal rat liver plasma membranes and shown to be stimulated by growth factors and hormones, tNOX activity purified from rat hepatoma cells is constitutively active. Its activity is detectable in the sera of cancer patients but not in those of healthy volunteers, suggesting its clinical relevance. Interestingly, tNOX expression was shown to be present in an array of cancer cell lines. More importantly, inhibition of tNOX was well correlated with reduced cancer cell growth and induction of apoptosis. RNA interference targeting tNOX expression in cancer cells effectively restored non-cancerous phenotypes, further supporting the vital role of tNOX in cancer cells. Here, we review the regulatory role of tNOX in gastric cancer cell growth.

SPINK3: A novel growth factor that promotes rat liver regeneration

Serine peptidase inhibitor, Kazal type 3 (SPINK3) is a trypsin inhibitor, and also a growth factor that has an identical structure to epidermal growth factor (EGF), which could combine with epidermal growth factor receptor (EGFR) to promote cell proliferation. To shed light on the role and regulation mechanism of SPINK3 in rat liver regeneration (LR), Rat Genome 230 2.0 assay was used to detect the expression profiles of LR genes after partial hepatectomy (PH). The results showed that Spink3 was significantly up-regulated at 2-24 h and 72-168 h after PH. In the present study, RT-PCR and immunoblotting were used to validate the assay results. Ingenuity Pathway Analysis 9.0 (IPA) software was used to build the SPINK3 signaling regulating LR and analyze the possible mechanism. And then the expression of cell proliferation-associated gene Ccna2 was examined by RT-PCR in normal rat liver cell line BRL-3A in which Spink3 was overexpressed. The results showed that Ccna2 was significantly up-regulated in BRL-3A in which Spink3 was over-expressed. SPINK3 combining with EGFR accelerated cell proliferation during rat liver regeneration via P38, PKC, JAK-STAT and AKT pathways. Thus, SPINK3 was likely to promote hepatocytes proliferation in LR through P38, PKC, JAK-STAT and AKT.

HPLC determination of α-ketoglutaramate [5-amino-2,5-dioxopentanoate] in biological samples

α-Ketoglutaramate is an important glutamine metabolite in mammals, plants, and many bacteria. It is also a nicotine metabolite in certain bacteria. Previously published methods for the determination of α-ketoglutaramate in biological samples have considerable drawbacks. Here, we describe a relatively simple high-performance liquid chromatography (HPLC)-based method for measurement of α-ketoglutaramate in plasma and deproteinized tissues that overcomes these drawbacks. Concentrations of α-ketoglutaramate in normal rat liver, kidney, brain, and plasma were found to be approximately 216, 13, 6, and 19 μM, respectively. The HPLC method should be useful for studying the role of α-ketoglutaramate in eukaryotic glutamine metabolism and in bacterial nicotine metabolism.