Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRß) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRß. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

A Novel Mucidosphaerium sp. Downregulates Inflammatory Gene Expression in Skin and Articular Cells.

CONTEXT: Hot-spring therapy is occasionally used for the treatment of inflammatory diseases. Microorganisms might contribute to the anti-inflammatory functions seen in thermal mud therapies. Natural microorganisms, derived from traditional spa resorts, could be useful as a preventive strategy for alternative medical applications. OBJECTIVE: The aim of the study was to find effective microalgae from prominent hot springs to use for the treatment of inflammatory diseases. DESIGN: The research team performed an in-vitro study. Microalgae, derived from Beppu hot springs, were isolated and homogeneously cultured. SETTING: The study took place at the Saravio Central Institute at Saravio Cosmetics in Oita, Japan and the Department of Bioscience and Biotechnology in the Graduate School of Agriculture at Shinshu University in Nagano, Japan. INTERVENTION: For identification, the 18S ribosomal RNA genes of microalgae were investigated by DNA sequencing and homology search, together with microscopic observation. OUTCOME MEASURES: To examine the pharmacological activities of the algal extracts, real-time polymerase chain reactions were performed, using either primary dermal fibroblasts (DFs), dermal papilla cells (DPCs), or fibroblast-like synoviocytes (FLSs). To test the antioxidant activity, both the oxygen radical absorbance capacity and the generation of intracellular reactive oxygen species (ROS) were evaluated. RESULTS: A novel strain of green algae, Mucidosphaerium sp., was isolated from a Beppu hot spring. The algal extract downregulated gene-expression levels of pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor- alpha (TNF-α), in various primary cells pre-exposed to IL-1ß. The protein level of the risk factors was concomitantly reduced. In addition, the algal extract suppressed the IL-1ß-induced upregulation of cyclooxygenase-2, nerve growth factor, and matrix metalloproteinase-1 (MMP-1) and MMP-3 in DFs. It also inhibited that of MMP-1, -3, and -9 in FLSs. Moreover, the extract inhibited total MMP protease activities. The microalgae decreased the intracellular reactive oxygen species (ROS) level in FLSs with an antioxidant activity of 178.3 ± 0.9 µmol of trolox equivalent/g. CONCLUSIONS: The present study showed that the novel Mucidosphaerium sp., derived from a Beppu hot spring, suppressed inflammatory reactions in both cutaneous and articular cells, partly due to its antioxidative properties. The novel algal strain may be a useful tool as an alternative medicine for skin and joint inflammatory disorders.

JOIN trial: treatment outcome and recovery status of peripheral sensory neuropathy during a 3-year follow-up in patients receiving modified FOLFOX6 as adjuvant treatment for stage II/III colon cancer.

PURPOSE: Adjuvant FOLFOX therapy is an established standard-of-care for resected colon cancer. Peripheral sensory neuropathy (PSN) is regarded as the major toxicity issue related to FOLFOX therapy. There have been a few reports on the recovery status from PSN thereafter. JOIN trial investigated the tolerability and efficacy of adjuvant modified FOLFOX6 (mFOLFOX6) in Japanese patients with stage II/III colon cancer. METHODS: Twelve cycles of mFOLFOX6 were given to patients with stage II/III curatively resected colon cancer. Treatment outcomes, including disease-free survival (DFS), relapse-free survival (RFS), overall survival (OS), and recovery status of PSN during 3-year follow-up, were investigated. RESULTS: Of the 882 patients enrolled from 2010 to 2012, 864 were eligible for the efficacy analyses. Three-year DFS, RFS, and OS were favorable in 92.1, 92.8, and 97.4% of stage II patients; 76.4, 77.9, and 93.8% of stage IIIA/B; and 61.6, 62.7, and 85.9% of stage IIIC, respectively. The cumulative incidence of PSN during treatment was 47.8% in grade 1 (G1), 30.3% in G2, and 5.8% in G3. For those with G3 PSN during treatment, there was gradual recovery in 1.1% of patients at 12 months after enrollment, 0.5% at 24 months, and 0.2% at 36 months. However, G1 or G2 residual PSN after 3 years was observed in 21.0% (18.7%, G1; 2.3%, G2). CONCLUSIONS: Adjuvant mFOLFOX6 therapy was effective and well tolerated in patients with stage II/III colon cancer. Most patients recovered from G3 PSN related to oxaliplatin, but approximately 20% of patients had G1 or G2 PSN at 3-year follow-up.

Aqueous Extract of Glucoraphanin-Rich Broccoli Sprouts Inhibits Formation of Advanced Glycation End Products and Attenuates Inflammatory Reactions in Endothelial Cells.

We have previously shown that sulforaphane not only inhibits formation of advanced glycation end products (AGEs) but also exerts anti-inflammatory effects on AGE-exposed human umbilical vein endothelial cells (HUVECs) and AGE-injected rat aortae. Here we examined the effects of aqueous extract of glucoraphanin-rich broccoli sprouts on formation of AGEs and then investigated whether the extract could attenuate inflammatory or oxidative stress reactions in tumor necrosis factor-alpha (TNF-α)- or AGE-exposed HUVECs. Fresh broccoli sprouts were homogenized in phosphate-buffered saline and filtered through a gauze. After centrifugation, clear extract was obtained. AGE formation was measured by enzyme-linked immunosorbent assay. Gene expression was evaluated by real-time reverse transcription-polymerase chain reaction. Reactive oxygen species (ROS) generation were measured using a fluorescent dye. Five percent broccoli sprout extract inhibited the formation of AGEs, reduced basal gene expressions of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1,) and receptor for AGEs (RAGE), and upregulated endothelial nitric oxide synthase (eNOS) mRNA levels in HUVECs. TNF-α upregulated MCP-1, ICAM-1, and RAGE mRNA levels in HUVECs, all of which were attenuated by the treatment with 1% broccoli sprout extract. Pretreatment of 1% broccoli sprout extract prevented the ROS generation in HUVECs evoked by AGEs. The present study demonstrates that sulforaphane-rich broccoli sprout extract could inhibit the AGE-RAGE axis and exhibit anti-inflammatory actions in HUVECs. Supplementation of sulforaphane-rich broccoli sprout extract may play a protective role against vascular injury.

Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

N-butanol extracts of Morinda citrifolia suppress advanced glycation end products (AGE)-induced inflammatory reactions in endothelial cells through its anti-oxidative properties.

BACKGROUND: Advanced glycation end products (AGEs), senescent macroprotein derivatives formed during a normal aging process and acceleratedly under diabetic conditions, play a role in atherosclerotic cardiovascular disease. AGEs cause endothelial cell (EC) damage, an initial trigger for atherosclerosis through the interaction with a receptor for AGEs (RAGE). We have previously shown that n-butanol extracts of Morinda citrifolia (noni), a plant belonging to the family Rubiaceae, block the binding of AGEs to RAGE in vitro. In this study, we examined the effects of n-butanol extracts of noni on reactive oxygen species (ROS) generation and inflammatory reactions on AGE-exposed human umbilical vein ECs (HUVECs). METHODS: HUVECs were treated with 100 µg/ml AGE-bovine serum albumin (AGE-BSA) or non-glycated BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni for 4 h. Then ROS generation and inflammatory and gene expression in HUVECs were evaluated by dihydroethidium staining and real-time reverse transcription-polymerase chain reaction analyses, respectively. THP-1 cell adhesion to HUVECs was measured after 2-day incubation of AGE-BSA or BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni. RESULTS: N-butanol extracts of noni at 670 ng/ml significantly inhibited the AGE-induced ROS generation and RAGE, intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 gene expressions in HUVECs. AGEs significantly increased monocytic THP-1 cell adhesion to HUVECs, which was also prevented by 670 ng/ml n-butanol extracts of noni. CONCLUSIONS: The present study demonstrated for the first time that N-butanol extracts of noni could suppress the AGE-induced inflammatory reactions in HUVECs through its anti-oxidative properties via blocking of the interaction of AGEs with RAGE. Inhibition of the AGE-RAGE axis by n-butanol extracts of noni may be a novel nutraceutical strategy for the treatment of cardiovascular disease.

Protective role of sulphoraphane against vascular complications in diabetes.

Context Diabetes is a global health challenge. Although large prospective clinical trials have shown that intensive control of blood glucose or blood pressure reduces the risk for development and progression of vascular complications in diabetes, a substantial number of diabetic patients still experience renal failure and cardiovascular events, which could account for disabilities and high mortality rate in these subjects. Objective Sulphoraphane is a naturally occurring isothiocyanate found in widely consumed cruciferous vegetables, such as broccoli, cabbage and Brussels sprouts, and an inducer of phase II antioxidant and detoxification enzymes with anticancer properties. We reviewed here the protective role of sulphoraphane against diabetic vascular complications. Methods In this review, literature searches were undertaken in Medline and in CrossRef. Non-English language articles were excluded. Keywords [sulphoraphane and (diabetes, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, diabetic complications, vascular, cardiomyocytes, heart or glycation)] have been used to select the articles. Results There is accumulating evidence that sulphoraphane exerts beneficial effects on vascular damage in both cell culture and diabetic animal models via antioxidative properties. Furthermore, we have recently found that sulphoraphane inhibits in vitro formation of advanced glycation end products (AGEs), suppresses the AGE-induced inflammatory reactions in rat aorta by reducing receptor for AGEs (RAGE) expression and decreases serum levels of AGEs in humans. Conclusion These findings suggest that blockade of oxidative stress and/or the AGE-RAGE axis by sulphoraphane may be a novel therapeutic strategy for preventing vascular complications in diabetes.

Initial safety report on the tolerability of modified FOLFOX6 as adjuvant therapy in patients with curatively resected stage II or III colon cancer (JFMC41-1001-C2: JOIN trial).

PURPOSE: Adjuvant FOLFOX is a widely accepted standard therapy for resected colon cancer. The incidence of grade 3-4 peripheral sensory neuropathy (PSN) was 12.4 and 5.7 % in the MOSAIC and Eastern MASCOT trials, while that of grade 3-4 allergic reactions (AR) was 2.9 and 3.1 %, respectively. The JFMC41-1001-C2 trial (JOIN trial) investigated the tolerability of modified FOLFOX6 (mFOLFOX6) in Japanese colon cancer patients. METHODS: Twelve cycles of mFOLFOX6 were given to patients with the same eligibility criteria as in the MOSAIC study: stage II or III curatively resected colon cancer, performance status of 0-1, aged 20 years or older, starting mFOLFOX6 within 7 weeks of surgery, and adequate organ function. The primary endpoints were the incidence of PSN persisting for ≥8 days that interfered with daily activities and the incidence of grade 3-4 AR. The target sample size was 800. RESULTS: From November 2010 to March 2012, 882 patients were enrolled at 198 institutions. Safety was analyzed in 828 patients with finalized data out of 848 patients receiving mFOLFOX6. The incidence of PSN persisting ≥8 days was 3.3 % [95 % confidence interval (CI) 2.2-4.7], while that of grade 3-4 AR was 1.7 % (95 % CI 0.9-2.8). The treatment completion rate was 67.0 %. The median total dosage of oxaliplatin was 811.1 mg/m(2). The overall incidence of grade 3-4 PSN was 5.8 %. Interstitial pneumonitis occurred in one patient. There were no treatment-related deaths. CONCLUSIONS: Adjuvant mFOLFOX6 is tolerable for Japanese patients with colon cancer.

Sulforaphane inhibits advanced glycation end product-induced pericyte damage by reducing expression of receptor for advanced glycation end products.

Advanced glycation end products (AGEs) not only inhibit DNA synthesis but also play a role in diabetic retinopathy by evoking apoptosis and inflammation in retinal pericytes via interaction with a receptor for AGE (RAGE). Similarly, sulforaphane, which is a naturally occurring isothiocyanate that is found in widely consumed cruciferous vegetables, protects against oxidative stress-induced tissue damage. Therefore, we hypothesized that sulforaphane could inhibit AGE-induced pericytes injury through its antioxidative properties. Advanced glycation end product stimulated superoxide generation as well as RAGE gene and protein expression in bovine-cultured retinal pericytes, and these effects were prevented by the treatment with sulforaphane. Antibodies directed against RAGE also blocked AGE-evoked reactive oxygen species generation in pericytes. Sulforaphane and antibodies directed against RAGE significantly inhibited the AGE-induced decrease in DNA synthesis, apoptotic cell death, and up-regulation of monocyte chemoattractant protein 1 messenger RNA levels in pericytes. For the first time, the present study demonstrates that sulforaphane could inhibit DNA synthesis, apoptotic cell death, and inflammatory reactions in AGE-exposed pericytes, partly by suppressing RAGE expression via its antioxidative properties. Blockade of the AGE-RAGE axis in pericytes by sulforaphane might be a novel therapeutic target for the treatment of diabetic retinopathy.

DNA aptamer raised against advanced glycation end products inhibits melanoma growth in nude mice.

Epidemiological studies have suggested that diabetes is associated with an increased risk of cancer. However, the underlying molecular mechanism remains unclear. We investigated here whether DNA aptamer directed against advanced glycation end products (AGE-aptamer) inhibited melanoma growth in nude mice. G361 melanoma cells were injected intradermally into the upper flank of athymic nude mice. Mice received continuous intraperitoneal infusion (0.136 µg/day) of either AGE-aptamer (n=9) or Control-aptamer (n=8) by an osmotic mini pump. Tumor volume was measured at 4-day interval, and G361 melanoma was excised at day 43 after the aptamer treatment. We further examined the effects of AGE-aptamer on proliferation of AGE-exposed endothelial cells and G361 cells. AGE-aptamer significantly inhibited the in vivo-tumor growth of G361 melanoma. Immunohistochemical and western blotting analyses of G361 melanoma revealed that AGE-aptamer decreased expression levels of proliferating nuclear antigen, CD31 and Mac-3, markers of endothelial cells and macrophages, respectively. AGE-aptamer significantly decreased the number of tumor-associated vessels. AGE, receptor for AGE (RAGE) and vascular endothelial growth factor levels were also reduced in AGE-aptamer-treated G361 melanoma. AGE-aptamer inhibited the AGE-induced proliferation and tube formation of endothelial cells as well as the growth of G361 cells in vitro. The present findings suggest that AGE-aptamer could inhibit the AGE-RAGE axis in G361 melanoma and resultantly suppress the tumor growth in nude mice by blocking the angiogenesis. AGE-aptamer might be a novel therapeutic strategy for preventing the progression of malignant melanoma in diabetes.

Goshajinkigan oxaliplatin neurotoxicity evaluation (GONE): a phase 2, multicenter, randomized, double­blind, placebo­controlled trial of goshajinkigan to prevent oxaliplatin­induced neuropathy.

PURPOSE: Oxaliplatin-induced peripheral neurotoxicity (OPN) is frequent and potentially severe, but successful treatment of this condition is still an unmet clinical need. We aimed to determine whether treatment with goshajinkigan (TJ-107), a traditional Japanese medicine, is better than placebo in preventing OPN in patients with advanced or recurrent colorectal cancer patients treated with standard FOLFOX regimens. METHODS: In this phase 2, randomized, double-blind, placebo-controlled study, patients undergoing oxaliplatin-based chemotherapy were randomized to receive either oral TJ-107 (7.5 g) or matching placebo daily. The severity of OPN was assessed according to the Common Toxicity Criteria for Adverse Events at baseline, every 2 weeks until the 8th cycle, and every 4 weeks thereafter until the 26th week. The primary endpoint was the incidence of grade 2 or greater OPN until the 8th cycle of chemotherapy. RESULTS: Analyses were done by intention to treat. Eighty-nine patients were randomly assigned to receive either TJ-107 (n = 44) or placebo (n = 45) between May 2009 and March 2010. The incidence of grade 2 or greater OPN until the 8th cycle was 39 and 51 % in the TJ-107 and placebo groups, respectively (relative risk (RR), 0.76; 95 % CI, 0.47­1.21). The incidence of grade 3 OPN was 7 % (TJ-107) vs. 13 % (placebo) (0.51, 0.14­1.92). No concerns regarding toxicity emerged with TJ-107 treatment. CONCLUSIONS: TJ-107 appears to have an acceptable safety margin and a promising effect in delaying the onset of grade 2 or greater OPN without impairing FOLFOX efficacy.

Glucagon-like peptide-1 receptor agonist inhibits asymmetric dimethylarginine generation in the kidney of streptozotocin-induced diabetic rats by blocking advanced glycation end product-induced protein arginine methyltranferase-1 expression.

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, contributes to diabetic nephropathy. We have found that glucagon-like peptide-1 (GLP-1) inhibits the AGE-induced inflammatory reactions in endothelial cells. However, effects of GLP-1 on the AGE-RAGE-ADMA axis are unknown. This study examined the effects of GLP-1 on reactive oxygen species (ROS) generation, gene expression of protein arginine methyltransfetase-1 (PRMT-1), an enzyme that mainly generates ADMA, and ADMA levels in human proximal tubular cells. Streptozotocin-induced diabetic rats received continuous i.p. infusion of 0.3 µg of vehicle or 1.5 µg of the GLP-1 analog exendin-4 per kilogram of body weight for 2 weeks. We further investigated whether and how exendin-4 treatment reduced ADMA levels and renal damage in streptozotocin-induced diabetic rats. GLP-1 inhibited the AGE-induced RAGE and PRMT-1 gene expression, ROS, and ADMA generation in tubular cells, which were blocked by small-interfering RNAs raised against GLP-1 receptor. Exendin-4 treatment decreased gene expression of Rage, Prmt-1, Icam-1, and Mcp-1 and ADMA level; reduced urinary excretions of 8-hydroxy-2'-deoxyguanosine and albumin; and improved histopathologic changes of the kidney in diabetic rats. Our present study suggests that GLP-1 receptor agonist may inhibit the AGE-RAGE-mediated ADMA generation by suppressing PRMT-1 expression via inhibition of ROS generation, thereby protecting against the development and progression of diabetic nephropathy.

Clinical utility of acarbose, an alpha-glucosidase inhibitor in cardiometabolic disorders.

Diabetes is associated with an increase risk for cardiovascular disease (CVD). Recently, macrovascular complications of diabetes have been shown to start before the development of diabetes. Indeed, several clinical studies have confirmed the increased risk of CVD in patients with impaired glucose tolerance (IGT). Since postprandial hyperglycemia and insulin resistance are thought to play a central role in the development and progression of CVD in patients with IGT, amelioration of postprandial hyperglycemia as well as insulin resistance is a therapeutic target for the prevention of CVD in these high-risk patients. Acarbose, an alpha-glucosidase inhibitor, delays the absorption of carbohydrate from the small intestine, thereby reducing postprandial hyperglycemia. Further, recently, acarbose has been shown to improve insulin resistance in vivo. These findings suggest that acarbose is a promising metabolic modifier that could reduce the risk of CVD in patients with the metabolic syndrome. In this paper, we review the clinical utility of acarbose in various cardiometabolic disorders.

Role of oxidative stress in the development of vascular injury and its therapeutic intervention by nifedipine.

Dihydropyridine-based calcium antagonists (DHPs) are widely used drugs for the treatment of hypertension and angina pectoris. We, along with others, have recently found that nifedipine, one of the most widely used DHPs, inhibits apoptotic cell death of endothelial cells (ECs) as well as vascular inflammation and subsequently improves endothelial function in patients with cardiovascular risk factors, including hypertension and/or diabetes, thus slowing the development and progression of atherosclerosis in these patients. Several papers have suggested that nifedipine exerts beneficial metabolic effects in vivo through its anti-inflammatory properties as well. However, the underlying molecular mechanisms for the cardiometabolic actions of nifedipine remain to be elucidated, because ECs do not possess voltage-operated L-type calcium channels. Meanwhile, we have very recently found that Bay w 9798, a dihydropyridine structurally related to nifedipine with no calcium antagonistic ability, has anti-oxidative and anti-inflammatory properties in vitro. In this paper, we review the role of oxidative stress in the development of vascular injury, especially focusing on the relationships between advanced glycation end products-receptor system, oxidized low-density lipoprotein and tumor necrosis factor-alpha and vasculopathy. We further discuss the potential clinical utility of anti-oxidative properties of nifedipine on various cardiometabolic disorders.

A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.

In order to determine the in vivo characteristics of huA33, an open label dose escalation biopsy-based phase I clinical trial and radioimmunolocalization study were conducted with a complement determinant region-grafted humanized monoclonal antibody against the A33 antigen in patients with gastric carcinoma. Thirteen patients were entered onto one of four dose levels (1.0, 2.0, 5.0 or 10.0 mg/m(2)). Patients with locally advanced (UICC-TNM [International Union Against Cancer-tumor, node, metastasis] stage over 2 but resectable at clinical diagnosis) gastric carcinoma received a single infusion of (131)I-huA33 1 week prior to surgery. Adverse events were monitored, and imaging studies with gamma camera plus ex vivo scintigraphy of the resected specimen, biodistribution study by dosimetry analysis of the biopsied and resected tissues, and immunohistochemical analysis were carried out and evaluated. No dose-limiting toxicity was observed during the trial. Therefore, the maximum tolerated dose was not reached. Although cancer tissues with + intensity and <25% extent by immunostaining in biopsied frozen sections did not show positive imaging or postoperative dosimetry findings, cancers with ++ or +++ intensity or wide (>25%) extent by frozen and paraffin sections in the biopsied specimen showed positive ex vivo tumor images and positive antigen expression in resected gastric cancer specimens, and the biodistribution analysis showed tumor uptake of (131)I-huA33. In conclusion, humanized monoclonal antibody huA33 demonstrated selective localization to gastric cancer that expressed A33 antigen strongly. These excellent targeting characteristics of huA33 indicate potential for targeted therapy of advanced gastric cancer that is refractory to cytotoxic therapy, and could also be exploitable for curatively resected early gastric cancer in an adjuvant setting.

Genotype of thymidylate synthase likely to affect efficacy of adjuvant 5-FU based chemotherapy in colon cancer.

Thymidylate synthase [TS, (EC 2.1.1.45)] is the target enzyme in 5-fluorouracil treatment. Recently, the DNA polymorphism of this gene has been found to affect TS protein (pTS) expression. However, no prospective studies have been performed to evaluate the influence of this polymorphism on the clinical efficacy of 5-FU-based adjuvant chemotherapy for colorectal cancer (CRC). In this study, we investigated the genotype of TS and immunopathological findings of pTS in 161 colon cancer specimens from patients who were registered in a prospective adjuvant immunochemotherapy clinical trial. The clinical course and prognosis of these patients were checked after the study had been completed. This study comprised 11 (6.8%) cases of 2R/2R, 40 (24.8%) of 2R/3R, and 110 (68.3%) of 3R/3R genotypes. All of the 2R/2R cases were still alive at the time of analysis although this finding was not statistically significant. In this prospective examination on a randomized controlled trial, the patients with colon cancer of the 2R/2R TS genotype may be good responders to 5-FU-based adjuvant chemotherapy. Furthermore, differences in the proportions of the TS genotypes can account for the interracial differences in the adverse effects of 5-FU-based chemotherapy.