An In Vivo Mouse Model of Pelvic Recurrence of Human Colorectal Cancer.

Pelvic recurrence of colorectal cancer is a crucial problem because radical surgery can lead to excessive invasion. Novel therapeutic strategies are required instead of surgery. However, there are few suitable models because of the difficulty in transplanting and observing tumors in the pelvis. We have established an appropriate injection site suitable for the establishment of colorectal cancer pelvic recurrence that allows for the observation of tumor growth. DLD-1 cells stably expressing luciferase (DLD-1 clone#1-Luc) were inoculated into various points of female BALB/c nude mice and the engrafted cells were analyzed with an imaging system employing bioluminescent signals and computed tomography. Weekly analysis with the imaging system showed that a triangular area defined by the vagina, the anus, and the ischial spine was suitable for the engraftment of pelvic tumors. The imaging system was able to detect the engrafted tumor 7 days after the inoculation of cells. Weight loss was observed in our model, and overall survival was 21-42 days. Tumor involvement of adjacent organs was detected histopathologically, as is the case in the clinical situation. These findings suggest that this model is valid for evaluations of the therapeutic effects of novel treatments under development. It is hoped that this model will be used in preclinical research.

Comparative proteomic analysis of paclitaxel resistance-related proteins in human breast cancer cell lines.

Paclitaxel is widely used to treat various cancers; however, resistance to this drug is a major obstacle to breast cancer chemotherapy. To identify the proteins involved in paclitaxel resistance, the present study compared the proteomes of MCF-7 human breast cancer cells and its paclitaxel-resistant subclone MCF-7/PTX. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry, 11 upregulated and 12 downregulated proteins were identified in MCF-7/PTX cells compared with the parental cell line. These 23 proteins were functionally classified as stress-induced chaperones, metabolic enzymes and cytoskeletal proteins. The anti-apoptotic proteins, stress-70 protein, 78-kD glucose-regulated protein, peptidyl-prolyl cis-trans isomerase A (PPIA) and heterogeneous nuclear ribonucleoprotein H3, were also upregulated in MCF-7/PTX cells. Notably, knockdown of the stress-response chaperone PPIA using small interfering RNA in MCF-7/PTX cells restored their sensitivity to paclitaxel. These findings indicated that PPIA may have an important role in paclitaxel resistance in MCF-7/PTX cells.

Microglia contact induces synapse formation in developing somatosensory cortex.

Microglia are the immune cells of the central nervous system that play important roles in brain pathologies. Microglia also help shape neuronal circuits during development, via phagocytosing weak synapses and regulating neurogenesis. Using in vivo multiphoton imaging of layer 2/3 pyramidal neurons in the developing somatosensory cortex, we demonstrate here that microglial contact with dendrites directly induces filopodia formation. This filopodia formation occurs only around postnatal day 8-10, a period of intense synaptogenesis and when microglia have an activated phenotype. Filopodia formation is preceded by contact-induced Ca(2+) transients and actin accumulation. Inhibition of microglia by genetic ablation decreases subsequent spine density, functional excitatory synapses and reduces the relative connectivity from layer 4 neurons. Our data provide the direct demonstration of microglial-induced spine formation and provide further insights into immune system regulation of neuronal circuit development, with potential implications for developmental disorders of immune and brain dysfunction.

Microglial Contact Prevents Excess Depolarization and Rescues Neurons from Excitotoxicity.

Microglia survey and directly contact neurons in both healthy and damaged brain, but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons and, in some cases, the removal of axonal debris that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia-axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance: detecting, wrapping, and rescuing neuronal soma from damage due to excessive activity.

Pharmacokinetics and pharmacodynamics of FSK0808 and Gran after single intravenous drip administration or single subcutaneous administration: comparative study in healthy Japanese adult male subjects.

FSK0808 is a recombinant human granulocyte colony-stimulating factor developed by Fuji Pharma Co., Ltd and Mochida Pharmaceutical Co., Ltd. as a biosimilar product of Gran®. We verified the pharmacokinetic/pharmacodynamic equivalence of FSK0808 and commercially available Gran® by a randomized crossover study of single intravenous dose (200 µg/m(2)) and single subcutaneous dose (400 µg/m(2)) in healthy Japanese adult male subjects. According to the bioequivalence guidelines, the area under the blood concentration - time curve by 48 hours after administration (AUC0-48) in a single intravenous drip (IVD) study, and AUC0-48 and maximum blood concentration (Cmax) in a single subcutaneous (SC) dose study were used as primary endpoints, and the pharmacodynamic parameters including absolute neutrophil count (ANC) or number of CD34 positive cells (CD34(+) cells) as secondary endpoints. The safety was evaluated based on the characteristics and incidence of adverse reactions. As a result, the 90% confidence interval (CI) of the difference in mean value for AUC0-48 among drugs ranged from log(0.8) to log(1.25), in the IVD study, and those for Cmax and AUC0-48 were within the range of log(0.8)-log(1.25) in the SC study. Those for secondary endpoints were all within the range of log(0.8)-log(1.25). Thus, the pharmacokinetics/pharmacodynamics of both drugs were considered equivalent for all routes of administration, and the profiles of adverse reactions were also very similar.

Microglia and synapse interactions: fine tuning neural circuits and candidate molecules.

Brain function depends critically on the interactions among the underlying components that comprise neural circuits. This includes coordinated activity in pre-synaptic and postsynaptic neuronal elements, but also in the non-neuronal elements such as glial cells. Microglia are glial cells in the central nervous system (CNS) that have well-known roles in neuronal immune function, responding to infections and brain injury and influencing the progress of neurodegenerative disorders. However, microglia are also surveyors of the healthy brain, continuously extending and retracting their processes and making contacts with pre- and postsynaptic elements of neural circuits, a process that clearly consumes considerable energy. Pruning of synapses during development and in response to injury has also been documented, and we propose that this extensive surveillance of the brain parenchyma in adult healthy brain results in similar "fine-tuning" of neural circuits. A reasonable extension is that a dysfunction of such a homeostatic role of microglia could be a primary cause of neuronal disease. Indeed, neuronal functions including cognition, personality, and information processing are affected by immune status. In this review we focus on the interactions between microglia and synapses, the possible cellular and molecular mechanisms that mediate such contacts, and the possible implications these interactions may have in the fine tuning of neural circuits that is so important for physiological brain function.

Microglia: actively surveying and shaping neuronal circuit structure and function.

The traditional role of microglia has been in brain infection and disease, phagocytosing debris and secreting factors to modify disease progression. Recent evidence extends their role to healthy brain homeostasis, including the regulation of cell death, synapse elimination, neurogenesis, and neuronal surveillance. These actions contribute to the maturation and plasticity of neural circuits that ultimately shape behavior. Here we review microglial contributions to the development, plasticity, and maintenance of neural circuits with a focus on interactions with synapses. We introduce this topic by reviewing recent studies on the migration and proliferation of microglia within the brain, and conclude with the proposal that microglia dysfunction may adversely affect brain function, and thereby contribute to the development of psychiatric and neurological disorders.

Enhanced GABAergic activity in the mouse primary somatosensory cortex is insufficient to alleviate chronic pain behavior with reduced expression of neuronal potassium-chloride cotransporter.

The correct balance between excitation and inhibition is crucial for brain function and disrupted in several pathological conditions. Excitatory neuronal circuits in the primary somatosensory cortex (S1) are modulated by local inhibitory neurons with the balance of this excitatory and inhibitory activity important for function. The activity of excitatory layer 2/3 neurons (L2/3) in the S1 cortex is increased in chronic pain, but it is not known how the local interneurons, nor the balance between excitation and inhibition, may change in chronic pain. Using in vivo two-photon calcium imaging and electrophysiology, we report here that the response of L2/3 local inhibitory neurons to both sensory stimulation and to layer 4 electrical stimulation increases in inflammatory chronic pain. Local application into L2/3 of a GABA(A) receptor blocker further enhanced the activity of S1 excitatory neurons and reduced pain thresholds, whereas local application of the GABA(A) receptor modulators (muscimol and diazepam) transiently alleviated the allodynia. This illustrates the importance of the local inhibitory pathways in chronic pain sensation. A reduction in the expression and function of the potassium-chloride cotransporter 2 occurred during chronic pain, which reduces the efficacy of the inhibitory inputs to L2/3 excitatory neurons. In summary, both excitatory and inhibitory neuronal activities in the S1 are enhanced in the chronic pain model, but the increased inhibition is insufficient to completely counterbalance the increased excitation and alleviate the symptoms of chronic pain.

Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.

To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 ß, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.

Inhibition of nitric oxide synthase in hyperdynamic circulation of rats with early or late cirrhosis secondary to common bile duct ligation.

BACKGROUND/AIMS: Preventing internal hemorrhage extends the lifespan of rats with chronic bile duct ligation (CBDL), a common animal model of portal hypertension. We investigated hemodynamics during the early and late stages of cirrhosis caused by CBDL. We also evaluated the hemodynamic influence of NO, which is the chief vasodilator in hyperdynamic syndrome, by administration of an NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester: L-NAME; 10 mg/kg). ANIMALS/METHODS: In 24 Sprague-Dawley rats (9 sham rats and 15 CBDL rats), hemodynamics were assessed under conscious and unrestrained conditions 4 and 8 weeks after surgery. Before and 30 minutes after L-NAME administration, the cardiac index (CI) and regional blood flow were measured with the reference sample method using (141)Ce- and (113)Sn-microspheres (15 µm in diameter). RESULTS: A hyperdynamic systemic circulation and splanchnic hyperemia were observed after CBDL, and these changes increased with the progression of cirrhosis. L-NAME significantly diminished the hyperdynamic circulation and also reduced splanchnic hyperemia. In 4-week CBDL rats, a low hemoglobin concentration made an important contribution to the hyperdynamic circulation, and the portal collateral system collapsed when inflow to the portal territory was reduced by L-NAME treatment. In 8-week CBDL rats, systemic hemodynamics were closely linked to both the splanchnic circulation and the renal circulation before and after L-NAME administration, apart from hepatic artery blood flow. CONCLUSION: The distinctive hemodynamic changes of portal hypertension were found in 8-week CBDL rats. Thus, 8-week CBDL rats may be a better animal model of human portal hypertension than 4-week CBDL rats.

Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABA(A) receptors in vitro.

BACKGROUND AND PURPOSE: Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA(A) receptors (GABA(A)Rs). We previously reported that trans-6,4'-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA(A)Rs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4'-methoxyretrochalcone (Rc-Br) and 4,3'-dimethoxychalcone (Ch-OMe) on GABA(A)Rs. EXPERIMENTAL APPROACH: Whole-cell patch-clamp recordings were made to determine the effects of these derivatives on GABA(A)Rs expressed in HEK-293 cells and in hippocampal CA1 pyramidal and thalamic neurones from rat brain. KEY RESULTS: Rc-OMe strongly potentiated GABA-evoked currents at recombinant α(1-4)ß(2)γ(2s) and α(4)ß(3)δ receptors but much less at α(1)ß(2) and α(4)ß(3). Rc-Br and Ch-OMe potentiated GABA-evoked currents at α(1)ß(2)γ(2s). The potentiation by Rc-OMe was only reduced at α(1)H101Rß(2)γ(2s) and α(1)ß(2)N265Sγ(2s), mutations known to abolish the potentiation by diazepam and loreclezole respectively. The modulation of Rc-OMe and pentobarbital as well as by Rc-OMe and the neurosteroid 3α,21-dihydroxy-5α-pregnan-20-one was supra-additive. Rc-OMe modulation exhibited no apparent voltage-dependence, but was markedly dependent on GABA concentration. In neurones, Rc-Br slowed the decay of spontaneous inhibitory postsynaptic currents and both Rc-OMe and Rc-Br positively modulated synaptic and extrasynaptic diazepam-insensitive GABA(A)Rs. CONCLUSIONS AND IMPLICATIONS: The trans-retrochalcones are powerful positive allosteric modulators of synaptic and extrasynaptic GABA(A)Rs. These novel modulators act through an original mode, thus making them putative drug candidates in the treatment of GABA(A)-related disorders in vivo.

Comparative proteomic analysis of the ribosomes in 5-fluorouracil resistance of a human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

Many auxiliary functions of ribosomal proteins (r-proteins) have received considerable attention in recent years. However, human r-proteins have hardly been examined by proteomic analysis. In this study, we isolated ribosomal particles and subsequently compared the proteome of r-proteins between the DLD-1 human colon cancer cell line and its 5-fluorouracil (5-FU)-resistant sub-line, DLD-1/5-FU, using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability to separate basic proteins, and we discuss the role of r-proteins in 5-FU resistance. Densitometric analysis was performed to quantify modulated proteins, and protein spots showing significant changes were identified by employing matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Three basic proteins (L15, L37 and prohibitin) which were significantly modulated between DLD-1 and DLD-1/5-FU were identified. Two proteins, L15 and L37, showed down-regulated expression in DLD-1/5-FU in comparison to DLD-1. Prohibitin, which is not an r-protein and is known to be localized in the mitochondria, showed up-regulated expression in DLD-1/5-FU. These 3 proteins may be related to 5-FU resistance.

Depolarizing shift in the GABA-induced current reversal potential by lidocaine hydrochloride.

Lidocaine hydrochloride (LC-HCl) is widely used as a local anesthetic, while various adverse effects of LC-HCl, such as seizures have also been reported. Lidocaine is reported to inhibit various channels and receptors including GABA(A) receptors. Although the GABA(A) receptor-mediated response depends on Cl(-) equilibrium potential (E(Cl)), little is known about the effect of LC-HCl on E(Cl). In the present study, we investigated the effect of LC-HCl on GABA-induced currents in cultured rat hippocampal neurons with gramicidin-perforated patch-clamp recording which is known to keep the intracellular Cl(-) concentration intact. LC-HCl inhibited outward GABA-induced currents with depolarizing shift of the GABA reversal potential (E(GABA)). The LC-HCl-induced positive E(GABA) shift was not observed with conventional whole-cell patch-clamp method which cannot retain intact intracellular Cl(-) concentration. The LC-HCl action on E(GABA) was inhibited by either furosemide, a blocker of both Na(+)-K(+)-Cl(-) cotransporter (NKCC) and K(+)-Cl(-) cotransporter (KCC), or an increase in extracellular K(+) concentrations. Neither bumetanide, a specific inhibitor of NKCC, nor Na(+)-free external solution had any effect on the LC-HCl-induced E(GABA) shift. QX-314, a membrane impermeable lidocaine derivative, failed to shift E(GABA) to positive potential. Furthermore, LC-HCl caused a depolarizing shift of E(GABA) in cultured GT1-7 cells expressing KCC2 but failed to change E(GABA) in GT1-7 cells without expression of KCC2. These results suggest that the LC-HCl-induced positive E(GABA) shift is due to a blockade of KCC2. Together with the direct LC-HCl action to GABA(A) receptors, the positive E(GABA) shift induced by LC-HCl reduces the GABAergic inhibition in the central nervous system.

Effect of chronic methylene blue administration on hypoxemia in rats with common bile duct ligation.

AIM: Acute administration of methylene blue (MB) can reverse hypoxemia in patients with hepatopulmonary syndrome (HPS). We evaluated the effect of chronic MB administration in common bile duct-ligated rats, which develop HPS by 5 weeks after surgery. METHODS: A total of 96 Sprague-Dawley rats were used, including 63 rats with common bile duct ligation (CBDL), 22 sham-operated rats and 11 normal control rats. MB (6 mg/kg) was injected s.c. once a day for 4 weeks. Evaluation of hemodynamics and intrapulmonary vascular dilatation (IPVD), as well as blood sampling for arterial blood gas analysis, were done under conscious and unrestrained conditions. Hemodynamics were assessed by the reference sample method using (141)Ce-microspheres (15 microm in diameter), and IPVD was also determined by i.v. injection of these microspheres. Histological examination of the lungs was done with hematoxylin-eosin staining and immunohistochemical staining for von Willebrand factor or vascular endothelial growth factor. RESULTS: Both the arterial oxygen tension and alveolar-arterial oxygen difference were significantly improved in MB-treated CBDL rats. The hyperdynamic circulation and splanchnic hyperemia seen in untreated CBDL rats were also alleviated by MB treatment. However, IPVD was not affected by MB. Histological examination of the lungs indicated that MB treatment reduced the proliferation of alveolar capillary vessels and angiogenesis, leading to improvement of arterial dysoxygenation. Hepatic synthetic and detoxification functions, as well as renal function, were not altered by MB treatment. CONCLUSION: Methylene blue may be a candidate treatment for HPS that does not cause deterioration of hepatic or renal function.

Proteomics-based identification of a tumor-associated antigen and its corresponding autoantibody in gastric cancer.

Identification of novel tumor-related antigens and autoantibodies will lead to early diagnosis of cancer and the development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens from the gastric cancer cell lines MkN-1, MkN-45 and KATOIII, and their related autoantibodies in sera of patients with gastric cancer using a proteomics-based approach. Proteins from the gastric cancer cell lines (MkN-1, MkN-45 and KATOIII) were separated by two-dimensional polyacrylamide gel electrophoresis, followed by Western blotting and antibody reaction with sera from patients with gastric cancer, healthy individuals and patients with other cancers. Positive spots were excised from Coomassie blue stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Sera from patients with gastric cancer yielded multiple spots, one of which was identified as the 78 kDa glucose-regulated protein (GRP78) by MALDI-TOF/TOF MS. Western blots against recombinant GRP78 showed reactivity in sera from 17/60 (28.3%) patients with gastric cancer and 0/20 (0.0%) of healthy individuals. Autoantibodies against GRP78 were found in 4/15 (26.7%) and 3/15 (20.0%) patients with esophageal and colon cancer, respectively. We identified for the first time an autoantibody against GRP78 in gastric cancer patients. The proteomic approach implemented in this study offers a powerful tool for identifying novel serum markers that may display clinical usefulness in cancer.

Histoculture drug response assay predicts the postoperative prognosis of patients with esophageal cancer.

Predicting response to chemotherapy would provide patients suffering from malignant tumor with not only more favorable outcomes, but also reduction of adverse events, and would enable chemotherapy tailored to individual patients. The purpose of this retrospective study was to evaluate the utility of histoculture drug response assay (HDRA) with the MTT endpoint. Subjects comprised 53 consecutive patients diagnosed with esophageal cancer, with 15 patients receiving preoperative chemoradiotherapy (CRT) followed by surgery (CRT group; n=15) and 38 patients undergoing surgery (surgery group; n=38), including 17 patients with histological lymph node metastasis who received postoperative chemotherapy. Tumor samples obtained from patients were used for HDRA with MTT endpoint and correlations of sensitivity from HDRA with MTT endpoint to clinical response to preoperative CRT, accuracy of in vitro sensitivity test, and clinical outcomes based on HDRA sensitivity were analyzed. HDRA was able to evaluate 379 of 424 assays (89.3%). In the CRT group, no significant correlation was confirmed between efficacy rate of 5-fluorouracil or cisplatin and histological findings in resected specimens after CRT. Efficacy rates of several anticancer agents using HDRA in the surgery group were observed in the range of 0.0-44.8%. On examination of clinical outcomes in the surgery group, in which patients with stage III received adjuvant chemotherapy, chemosensitivity-negative patients tended to display worse prognosis than chemosensitivity-positive patients. HDRA with MTT endpoint probably predicts the postoperative prognosis of patients with esophageal cancer.

Proteomic analysis of the basic proteins in 5-fluorouracil resistance of human colon cancer cell line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis.

5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers and it is the mainstay of chemotherapy. However, the acquisition of resistance to 5-FU is one of the most prominent obstacles to successful chemotherapy. The purpose of this study was to identify the novel biological basis of 5-FU resistance in colon cancer cells. This study is the first comparative proteomic analysis of basic proteins between the DLD-1 human colon cancer cell line and DLD-1/5-FU its 5-FU resistant sub-line using the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis, which has a superior ability in the separation of basic proteins and the quantification of post-translational modification. A densitometric analysis was performed to quantify the modulated proteins, and protein spots showing significant changes were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Six basic proteins significantly modulated between DLD-1 and DLD-1/5-FU were identified. All of them showed up-regulated expression in DLD-1/5-FU in comparison to DLD-1. The six identified spots, corresponding to five different proteins included heterogeneous nuclear ribonucleoprotein G, mitochondrial transcription factor A, histone H2B, histone H4 and ribosomal protein L3. Among the 5 basic proteins, several proteins are potentially related to 5-FU resistance by protecting the cells from DNA damage.

Proteomics-based identification of autoantibody against heat shock protein 70 as a diagnostic marker in esophageal squamous cell carcinoma.

Detection of novel tumor-related antigens and autoantibodies in cancer patients is expected to facilitate the diagnosis of early-stage malignant tumor and establish effective new immunotherapies. The purpose of this study was to identify novel tumor antigens in an esophageal squamous cell carcinoma (ESCC) cell line (TE-2) and related autoantibodies in sera from patients with ESCC using a proteomics-based approach. TE-2 proteins were separated by two-dimensional polyacrylamide gel electrophoresis, followed by Western blot analysis in which sera from patients with ESCC, healthy controls and patients with other cancers were tested for primary antibodies. Positive spots were excised from silver-stained gels and analyzed by matrix-assisted laser disorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Sera from patients with ESCC yielded multiple spots, one of which was identified as heat shock protein 70 (Hsp70) by MALDI-TOF/TOF-MS. Concentrations of serum Hsp70 autoantibody were significantly higher for patients with ESCC (mean, 0.412+/-0.096 mg/ml) than for patients with gastric (0.236+/-0.112 mg/ml, P<0.001) or colon cancer (0.231+/-0.120 mg/ml, P<0.001) or healthy individuals (0.207+/-0.055 mg/ml, P<0.001) by enzyme-linked immunosorbent assay. We have identified an autoantibody against Hsp70 in ESCC patients. The proteomic approach implemented herein offers a powerful tool for identifying novel serum markers that may display clinical utility against cancer.

Correlation between thymidylate synthase and dihydropyrimidine dehydrogenase mRNA level and in vitro chemosensitivity to 5-fluorouracil, in relation to differentiation in gastric cancer.

PURPOSE: It has been suggested that the gene expression levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) help in the prediction of the response to 5-fluorouracil (5-FU) in vivo and in vitro in gastric cancers. METHODS: In this study, intratumoral TS and DPD gene expressions were evaluated with real time reverse transcriptional polymerase chain reaction technique to determine the correlation between the expression of these two genes and in vitro sensitivity to 5-FU, assessed by the histoculture drug response assay on 87 patients with gastric adenocarcinoma. RESULTS: The sensitivity to 5-FU did not show any difference in clinicopathological groups. DPD gene level was higher in undifferentiated (n = 39) than differentiated (n = 48) tumors (P = 0.043). In differentiated tumors, TS gene expression levels were higher in the tumors with relative resistance to 5-FU, while in undifferentiated cases, DPD mRNA levels were higher in tumors that showed resistance to 5-FU in vitro (P = 0.043 and 0.007, respectively). DPD also had significant predictive value for 5-FU sensitivity in undifferentiated cases [R(S) = -0.401, P = 0.011]. TS and DPD gene expression levels were more highly correlated in undifferentiated compared to differentiated cases [R(S) = 0.515 and 0.359, respectively]. CONCLUSIONS: Different gene expression might be responsible for 5-FU sensitivity in gastric cancers of different histologic origin.

Proteomics-based approach identifying autoantibody against peroxiredoxin VI as a novel serum marker in esophageal squamous cell carcinoma.

PURPOSE: Detection of novel tumor-related antigens and autoantibodies will aid in diagnosis of early-stage cancer and in development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens in an esophageal squamous cell carcinoma (ESCC) cell line (TE-2) and related autoantibodies in sera from patients with ESCC using a proteomics-based approach. EXPERIMENTAL DESIGN: TE-2 proteins were separated by two-dimensional PAGE, followed by Western blot analysis in which sera of patients with ESCC, healthy controls, and patients with other cancers were tested for primary antibodies. Positive spots were excised from silver-stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). RESULTS: Sera from patients with ESCC yielded multiple spots, one of which was identified as peroxiredoxin (Prx) VI by MALDI-TOF/TOF MS. Western blot analysis against recombinant Prx VI showed reactivity in sera from 15 of 30 (50%) patients with ESCC and 2 of 30 (6.6%) healthy individuals. Autoantibody against Prx VI was found in sera from 1 of 30 (3.3%) patients with other types of cancer (colon cancer). CONCLUSION: We have identified for the first time an autoantibody against Prx VI in ESCC patients. The proteomic approach implemented here offers a powerful tool for identifying novel serum markers that may display clinical usefulness against cancer.