E-Cadherin truncation and sialyl Lewis-X overexpression in oral squamous cell carcinoma and oral precancerous conditions.

The present study aimed to determine significance of E-cadherin, a cell adhesion molecule, and sialyl Lewis-X (sLeX), a cell surface antigen, in oral carcinogenesis. Expressions of E-cadherin and sLeX were detected using western blot analysis from oral malignant (n=25), and oral precancerous tissues (OPC, n=20) and their adjacent normal tissues. An altered expression of E-cadherin (E-cad) and sLeX was observed in malignant and precancerous tissues. E-cad western blot revealed presence of two bands, a 120 kDa (native, E-cad120) and a 97 kDa (known as truncated E-cad97). The accumulation of truncated E-cad97 and sLeX in malignant and OPC tissues compared to their adjacent normal tissues was observed. Receiver's Operating Characteristics (ROC) curve analysis showed good discriminatory efficacy of E-cad97, E-cad97:120 ratio and sLeX between the malignant and adjacent. normal tissues. Further, a positive correlation of E-cad97 and sLeX overexpression with advanced stage of the disease and lymphnode metastasis was observed. The data suggest that E-cadherin truncation and sLeX overexpression are early events which may facilitate the tumor cells to metastasize. Also, overexpression E-cad97 and sLeX in OPC tissues may be useful to predict metastatic potentials of tumors at an early stage of oral carcinogenesis. Key words: Oral cancer, oral precancerous conditions, E-cadherin, sialyl Lewis-X, metastasis.

Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support.

In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase.

Neoplastic cells transport large amounts of glucose in order to produce anabolic precursors and energy within the inhospitable environment of a tumor. The ras signaling pathway is activated in several cancers and has been found to stimulate glycolytic flux to lactate. Glycolysis is regulated by ras via the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFK2/FBPase), which modulate the intracellular concentration of the allosteric glycolytic activator, fructose-2,6-bisphosphate (F2,6BP). We report herein that sequential immortalization and ras-transformation of mouse fibroblasts or human bronchial epithelial cells paradoxically decreases the intracellular concentration of F2,6BP. This marked reduction in the intracellular concentration of F2,6BP sensitizes transformed cells to the antimetabolic effects of PFK2/FBPase inhibition. Moreover, despite co-expression of all four mRNA species (PFKFB1-4), heterozygotic genomic deletion of the inducible PFKFB3 gene in ras-transformed mouse lung fibroblasts suppresses F2,6BP production, glycolytic flux to lactate, and growth as soft agar colonies or tumors in athymic mice. These data indicate that the PFKFB3 protein product may serve as an essential downstream metabolic mediator of oncogenic ras, and we propose that pharmacologic inhibition of this enzyme should selectively suppress the high rate of glycolysis and growth by cancer cells.

Activity and stability of alkaline phosphatase (ALP) immobilized onto magnetic nanoparticles (Fe3O4).

Direct binding of alkaline phosphatase (ALP) on magnetic nanoparticles (Fe(3)O(4)) in the presence of carbodiimide (CDI) using two different methods is described. The activity and stability of immobilized ALP with both shaking and sonication method were compared. The results indicated the ALP binding efficiency to be in the range of 80-100% with both the immobilization techniques. The activities retained were in the range of 20-38% with shaking method and 30-43% with sonication method, respectively. The activities of the immobilized ALP preparations were found to be stable compared to the free (unbound) ALP for at least 16-week storage period. Moreover, ALP immobilized on magnetic nanoparticles was successfully used for dephosphorylation of plasmid DNA before it was used for ligation reaction. The use of immobilized ALP for plasmid dephosphorylation allows easy manipulation, reduces the procedural time, and also avoids exposure of reaction mixture to high temperature.

MRI study of the lumbar spine in achondroplasia. A morphometric analysis for the evaluation of stenosis of the canal.

We carried out an MRI study of the lumbar spine in 15 patients with achondroplasia to evaluate the degree of stenosis of the canal. They were divided into asymptomatic and symptomatic groups. We measured the sagittal canal diameter, the sagittal cord diameter, the interpedicular distance at the mid-pedicle level and the cross-sectional area of the canal and spinal cord at mid-body and mid-disc levels. The MRI findings showed that in achondroplasia there was a significant difference between the groups in the cross-sectional area of the body canal at the upper lumbar levels. Patients with a narrower canal are more likely to develop symptoms of spinal stenosis than others.

Comparison of lactoferrin activity in fresh and stored human milk.

OBJECTIVE: Lactoferrin (Lf), the dominant protein in human milk (HM), has been shown to have anti-inflammatory and anti-microbial activity in the neonatal gut. Previous studies indicate that freezing significantly decreases the concentration of Lf in HM. The objective of our study was to compare the activity of Lf in fresh and frozen HM over time. STUDY DESIGN: HM samples were examined fresh and after storage at -20 °C for 3 and 6 months. Lf concentration was determined by enzyme-linked immunoassay, and the activity was measured by examination of nitric oxide (NO) production and tumor necrosis factor-α secretion from rat macrophages exposed to HM samples. RESULT: After 3 and 6 months at -20 °C, the average decrease in Lf concentrations was 55% and 65%, respectively. The bioactivity of Lf also decreased significantly over 6 months. CONCLUSION: Freezing HM for 3 or more months significantly decreases Lf levels and activity. Periodically providing fresh HM may benefit vulnerable preterm neonates.

Clinical development of cancer therapeutics that target metabolism.

Glucose and glutamine metabolism in cancer cells are markedly elevated relative to non-transformed normal cells. This metabolic reprogramming enables the production of adenosine triphosphate and the anabolic precursors needed for survival, growth and motility. The recent observations that mutant oncogenic proteins and the loss of tumor suppressors activate key metabolic enzymes suggest that selective inhibition of these enzymes may yield effective cancer therapeutics with acceptable toxicities. In support of this concept, pre-clinical studies of small molecule antagonists of several metabolic enzymes in tumor-bearing mice have demonstrated reasonable therapeutic indices. We will review the rationale for targeting metabolic enzymes as a strategy to treat cancer and will detail the results of several recent clinical trials of metabolic inhibitors in advanced cancer patients.

Inhibition of intercellular communication between liver cells by the liver tumor promoter 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane.

A dose dependent inhibition of intercellular communication (metabolic cooperation) between primary cultures of rat liver hepatocytes and an established adult rat liver epithelial cell 6-thioguanine resistant strain by the liver tumor promoter 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is demonstrated. This in vitro assay is proposed to evaluate the tumor promoting activity of oncogenic agents shown to be non-genotoxic in the liver culture systems.

Effect of corticosterone treatment on energy metabolism in rat liver mitochondria.

1. Effect of in vivo treatment (40 mg/kg body wt) with corticosterone on energy metabolism in rat liver mitochondria was examined under acute and chronic conditions in 20-, 35- and 60-day-old rats. 2. Acute treatment did not affect body or liver weight. However, chronic treatment caused increased liver weight in the former two age groups; in the 60-day-old animals the liver weight decreased. 3. Acute treatment resulted in a generalized decrease in state 3 respiration rates and state 4 respiration rates without having any significant effect on ADP/O ratios with glutamate, succinate and ascorbate + TMPD as substrates. However, rates of ATP synthesis decreased significantly. The effect was age-dependent, older animals showed increased resistance. 4. Chronic treatment resulted in uncoupling of oxidative phosphorylation without having significant effects on respiration rates. Once again, the effects were age-dependent. Consequently, the ATP synthesis rates were significantly lowered. However, it was apparent that the underlying mechanisms were entirely different. 5. With succinate as the substrate the state 3 respiration rates increased with age to reach adult values by day 60. The coupling efficiency was also exhibited via maturational changes.

Association of the Ban I dimorphic site at the human cytosolic phospholipase A2 gene with schizophrenia.

There is evidence of increased phospholipid breakdown in cell membranes of patients suffering from schizophrenia. This may be related to increased levels of the enzyme cytosolic phospholipase A2 (cPLA2) which have been reported in schizophrenic subjects. We have identified a Ban I dimorphic site on the first intron of the cPLA2 gene. Schizophrenic subjects were found to have a significant excess of the A2/A2 homozygote relative to healthy control subjects. Genetically determined alterations in phospholipase activity may thus underlie the reported abnormalities of phospholipid metabolism in schizophrenia.

Differences in responses of 4 adult rat-liver epithelial cell lines to a spectrum of chemical mutagens.

An assay for mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in adult rat-liver epithelial cell cultures (ARL) has been developed to take advantage of the capacity of this cell type to metabolically activate promutagens/procarcinogens. A survey of the effect of 5 types of activation-dependent mutagens/carcinogens on 4 ARL lines indicates that the ARL/HGPRT mutagenesis assay with the 4 target cell lines is able to detect a spectrum of activation-dependent carcinogens. Individual ARL lines, however, responded quite differently to a given carcinogen. The ARL/HGPRT mutagenesis assay system thus offers distinct possibilities for the study of the control of chemical biotransformation processes. However, in light of the specificity of the various cell lines to respond to a particular class of mutagens under the current assay condition, this particular assay system cannot be readily applied to routine screening of suspected environmental mutagens of unknown requirements for metabolic activation. Nevertheless, for agents with a structure related to those activated by a specific line, this system can be used to study mutagenesis resulting from intact cellular metabolism.

Corticosterone administration and lipid metabolism in brain regions during development.

Effect of corticosterone on lipid contents of different brain regions and the effect of age on the sensitivity of these regions to corticosterone have been studied. Corticosterone administration (40 mg/kg body wt, sc) to 17-day-old rat for 3 days led to significant decrease in phospholipid content of cerebellum and increase in cholesterol contents of hippocampus and striatum. However, there was no effect on cerebral cortex and brain stem lipids. This alteration in lipids was associated with decrease in [U-14C] glucose incorporation into cholesterol and phospholipids, decrease in plasma beta-hydroxy butyrate levels and increase in beta-hydroxy butyrate dehydrogenase activity in hippocampus and striatum, thereby suggesting that suppression of glucose utilization by corticosterone was compensated by higher utilization of ketone bodies for lipid synthesis in these regions. The sensitivity to corticosterone appears to be age-specific as, at 20-day, cerebellum, hippocampus and striatum were susceptible, at 10-day only hippocampus and at 40- and 90-day none of these regions responded to the treatment.

Lipid composition of brain regions during chronic maternal alcohol treatment and withdrawal in the rat.

The lipid composition of whole brain, cerebrum, cerebellum and brain stem was studied in rat pups exposed to alcohol during prenatal and postnatal period and subsequent withdrawal or continuation during postweaning period. The concentrations of cholesterol and galactolipids were increased in the whole brain and brain regions of the pups exposed to alcohol. Even after 6 weeks of withdrawal from alcohol during postweaning period, the lipid levels were significantly higher compared to the controls. These observations suggest possible alterations in the functions of CNS related to membrane integrity.

6-Phosphofructo-2-kinase (PFKFB3) promotes cell cycle progression and suppresses apoptosis via Cdk1-mediated phosphorylation of p27.

The control of glucose metabolism and the cell cycle must be coordinated in order to guarantee sufficient ATP and anabolic substrates at distinct phases of the cell cycle. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are well established regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent allosteric activator of 6-phosphofructo-1-kinase (Pfk-1). PFKFB3 is overexpressed in human cancers, regulated by HIF-1α, Akt and PTEN, and required for the survival and growth of multiple cancer types. Although most functional studies of the role of PFKFB3 in cancer progression have invoked its well-recognized function in the regulation of glycolysis, recent observations have established that PFKFB3 also traffics to the nucleus and that its product, F2,6BP, activates cyclin-dependent kinases (Cdks). In particular, F2,6BP stimulates the Cdk-mediated phosphorylation of the Cip/Kip protein p27 (threonine 187), which in turn results in p27's ubiquitination and proteasomal degradation. As p27 is a potent suppressor of the G1/S transition and activator of apoptosis, we hypothesized that the known requirement of PFKFB3 for cell cycle progression and prevention of apoptosis may be partly due to the ability of F2,6BP to activate Cdks. In this study, we demonstrate that siRNA silencing of endogenous PFKFB3 inhibits Cdk1 activity, which in turn stabilizes p27 protein levels causing cell cycle arrest at G1/S and increased apoptosis in HeLa cells. Importantly, we demonstrate that the increase in apoptosis and suppression of the G1/S transition caused by siRNA silencing of PFKFB3 expression is reversed by co-siRNA silencing of p27. Taken together with prior publications, these observations support a model whereby PFKFB3 and F2,6BP function not only as regulators of Pfk-1 but also of Cdk1 activity, and therefore serve to couple glucose metabolism with cell proliferation and survival in transformed cells.

Regulation of glycolytic and mitochondrial metabolism by ras.

High glucose uptake is a characteristic of most metastatic tumors and activation of Ras signaling in immortalized cells increases glycolytic flux into lactate, de novo nucleic acid synthesis and the tricarboxylic acid cycle, and increases NADH shuttling, oxygen consumption and uncoupling of ATP synthase from the proton gradient. Fructose-2,6- bisphosphate, C-Myc, HIF1α and AKT each have been found to be key regulators of glycolysis and to be controlled by Ras signaling, and there is abundant evidence for cross-talk between these regulators. The reprogramming of glycolytic and mitochondrial metabolism by Ras enables an integrated activation of energetic and anabolic pathways via the redox state of NADH that is required for the survival and growth of neoplastic cells in poorly vascularized tumors. Several small molecule antagonists specific for essential metabolic enzymes have been found to be selectively toxic to Ras-transformed cells as opposed to wild-type cells, indicating that this metabolic reprogramming and addiction may have utility for the development of anti-neoplastic agents.

A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/AKT signaling.

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling.

Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.