Novel iontophoretic administration method for local therapy of breast cancer.

Ductal drug therapy is a novel therapeutic approach for primary breast cancers, particularly those involving ductal carcinoma in situ lesions. Total or partial mastectomy with or without radiotherapy is the standard local therapy for primary breast cancer. Here, we propose a novel drug administration method for ductal drug therapy based on a drug delivery system (DDS) for primary breast cancer. This DDS was designed to deliver miproxifen phosphate (TAT-59), an antiestrogen drug, to ductal lesions via the milk duct, where carcinomas originate, more efficiently than systemic administration, using an iontophoretic technique applied to the nipple (IP administration). Autoradiography imaging confirmed that TAT-59 was directly delivered to the milk duct using IP administration. The plasma concentrations of TAT-59 and its active metabolite DP-TAT-59 were quite low with IP administration. The area under the curve value of DP-TAT-59 in the mammary tissue was approximately 3 times higher with IP administration than with oral administration, at a 6-fold lower dose, indicating higher availability of the drug delivered via DDS than via systemic administration. The low plasma concentrations would limit adverse effects to minor ones. These characteristics show that this DDS is suitable for the delivery of active DP-TAT-59 to ductal lesions.

Preparation and properties of inhalable nanocomposite particles for treatment of lung cancer.

Nanoparticles have widely been studied in drug delivery research for targeting and controlled release. The aim of this article is application of nanoparticles as an inhalable agent for treatment of lung cancer. To deposit effectively deep the particles in the lungs, the PLGA nanoparticles loaded with the anticancer drug 6-{[2-(dimethylamino)ethyl]amino}-3-hydroxyl-7H-indeno[2,1-c]quinolin-7-one dihydrochloride (TAS-103) were prepared in the form of nanocomposite particles. The nanocomposite particles consist of the complex of drug-loaded nanoparticles and excipients. In this study, the anticancer effects of the nanocomposite particles against the lung cancer cell line A549. Also, the concentration of TAS-103 in blood and lungs were determined after administration of the nanocomposite particles by inhalation to rats. TAS-103-loaded PLGA nanoparticles were prepared with 5% and 10% of loading ratio by spray drying method with trehalose as an excipient. The 5% drug-loaded nanocomposite particles were more suitable for inhalable agent because of the sustained release of TAS-103 and higher FPF value. Cytotoxicity of nanocomposite particles against A549 cells was higher than that of free drug. When the nanocomposite particles were administered in rats by inhalation, drug concentration in lung was much higher than that in plasma. Furthermore, drug concentration in lungs administered by inhalation of nanocomposite particles was much higher than that after intravenous administration of free drug. From these results, the nanocomposite particle systems could be promising for treatment of lung cancer.

A synthetic retinoid, TAC-101 (4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid), plus cisplatin: potential new therapy for ovarian clear cell adenocarcinoma.

OBJECTIVE: A novel retinoid, TAC-101 (4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid), induces apoptosis of ovarian clear cell adenocarcinoma. The antitumor effect of TAC-101 alone or combined with cisplatin was tested using human ovarian carcinoma. METHODS: Induction of genes related to apoptosis by TAC-101 or cisplatin was assessed by DNA microarray analysis. TAC-101 (8 mg/kg/day orally for 21 days), cisplatin (7 mg/kg intravenously on day 1), or a combination of both drugs at the same dosages was administered to nude mice implanted subcutaneously with RMG-I or RMG-II clear cell adenocarcinoma cells. The antitumor effect was evaluated by calculating the treated/control tumor volume ratio at 21 days after implantation. The histoculture drug response assay was also performed using fresh surgical specimens of human ovarian cancer to determine the 50% inhibitory concentration (IC50). RESULTS: Different apoptosis-related genes were induced by TAC-101 and cisplatin. Compared with control mice, the volume of both RMG-I and RMG-II tumors was significantly reduced (p<0.05) by either drug. The IC50 values of cisplatin and TAC-101 showed a significant correlation (p<0.01). CONCLUSION: These in vitro findings suggest that a combination of TAC-101 and cisplatin may be a potential new treatment for ovarian clear cell adenocarcinoma.

Gene expression analysis using human cancer xenografts to identify novel predictive marker genes for the efficacy of 5-fluorouracil-based drugs.

The development of a diagnostic method for predicting the therapeutic efficacy or toxicity of anticancer drugs is a critical issue. We carried out a gene expression analysis to identify genes whose expression profiles were correlated with the sensitivity of 30 human tumor xenografts to 5-fluorouracil (5-FU)-based drugs (tegafur + uracil [UFT], tegafur + gimeracil + oteracil [S-1], 5'-deoxy-5-fluorouridine [5'-DFUR], and N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine [capecitabine]), as well as three other drugs (cisplatin [CDDP], irinotecan hydrochloride [CPT-11], and paclitaxel) that have different modes of action. In the present study, we focused especially on the fluoropyrimidines. The efficacy of all anticancer drugs was assayed using human tumor xenografts in nude mice. The mRNA expression profile of each of these xenografts was analyzed using a Human Focus array. Correlation analysis between the gene expression profiles and the chemosensitivities of seven drugs identified 39 genes whose expression levels were correlated significantly with multidrug sensitivity, and we suggest that the angiogenic pathway plays a pivotal role in resistance to fluoropyrimidines. Furthermore, many genes showing specific correlations with each drug were also identified. Among the candidate genes associated with 5-FU resistance, the dihydropyrimidine dehydrogenase mRNA expression profiles of the tumors showed a significant negative correlation with chemosensitivity to all of the 5-FU based drugs except for S-1. Therefore, the administration of S-1 might be an effective strategy for the treatment of high dihydropyrimidine dehydrogenase-expressing tumors. The results of the present study may enhance the prediction of tumor response to anticancer drugs and contribute to the development of tailor-made chemotherapy.

Excess HDM2 impacts cell cycle and apoptosis and has a selective effect on p53-dependent transcription.

Mutational inactivation of p53 is only one of the ways that tumors lose p53 function. An alternate route is through overexpression of HDM2, the negative regulator of p53. To further understand how excess HDM2 regulates p53-mediated functions, we generated H1299 cell clones that constitutively express both ectopic HDM2 and tetracycline-regulated inducible p53. We found that over a range of p53 concentrations constitutively expressed HDM2 did not affect the levels of p53 protein. Nevertheless, cells with excess HDM2 displayed numerous changes in their response to p53. After DNA damage, such cells had both increased p53-mediated G2 arrest and reduced cell death. They also showed selective impairment of p53 target gene induction in that some p53 targets were unaffected whereas others were markedly less well induced in the presence of extra HDM2 protein. We also found that excess HDM2 was correlated with reduced p53 acetylation but did not affect p53 association with target promoters in vivo. Indeed, there was no significant difference in the amount of HDM2 associated with p53 at target promoters that differed in their expression depending on the presence of extra HDM2. Thus, HDM2 can selectively down-regulate the transcription function of p53 without either degrading p53 or affecting the interaction of p53 with target promoters.

Correlations between antitumor activities of fluoropyrimidines and DPD activity in lung tumor xenografts.

The purposes of this study were to evaluate the antitumor activity of S-1 (1 M tegafur, 0.4 M 5-chloro-2,4-dihydroxypyridine and 1 M potassium oxonate) on human lung tumor xenografts, as compared with other fluoro-pyrimidines, and to investigate the relationships between fluoropyrimidine antitumor activities and four distinct enzymatic activities involved in the phosphorylation and degradation pathways of 5-fluorouracil (5-FU) metabolism. S-1, UFT (1 M tegafur-4 M uracil), 5'-deoxy-5-fluorouridine (5'-DFUR), capecitabine and 5-FU were administered for 14 consecutive days to nude mice bearing lung tumor xenografts. S-1 showed stronger tumor growth inhibition in four of the seven tumors than the other drugs. Cluster analysis, on the basis of antitumor activity, indicated that S-1/UFT and 5'-DFUR/capecitabine/5-FU could be classified into another group. We investigated tumor thymidylate synthase content, dihydropyrimidine dehydrogenase (DPD) activity, thymidine phosphorylase (TP) activity and orotate phosphoribosyl transferase activity in seven human lung tumor xenografts and performed regression analyses for the antitumor activities of fluoropyrimidines. There were inverse correlations between antitumor and DPD activities for 5'-DFUR (r=-0.79, P=0.034), capecitabine (r=-0.56, P=0.19) and 5-FU (r=-0.86, P=0.013). However, no such correlations were observed for S-1 and UFT. These findings suggest that S-1 containing a potent DPD inhibitor may have an antitumor effect on lung tumors, with high basal DPD activity, superior to those of other fluoropyrimidines.

Napthalimidobenzamide DB-51630: a novel DNA binding agent inducing p300 gene expression and exerting a potent anti-cancer activity.

Control of gene expression by small molecule compounds is a novel therapeutic strategy for cancer and usually it requires the presence of specific molecular recognition. The development of the compounds preferentially binding to the specific DNA sequence is one of the potential but very difficult approaches in this strategy. We designed and synthesized novel napthalimidobenzamide derivatives and analyzed their binding preferences to oligonucleotides by EtBr-displacement assay with DNA sequences, being essential fragments of the genes. To test whether these compounds modify the expression of specific genes, we analyzed the effect on the gene expression in AZ521 cells by differential display analysis using the compounds showing different characteristics in the recognition of specific DNA sequence. Among them, DB-51630, which showed approximately 350 times higher preferential binding to GC-repeats than to the AT and AA-repeating oligomers, caused the induction of a specific mRNA. The genetic sequence was identified to be the p300 gene by sequencing of the cloned cDNA. The p300 is a transcriptional co-activator protein that acts with other nuclear proteins in various cell differentiation and signal transduction pathways. This protein has intrinsic histone acetyltransferase activity and may act on chromatin directly to facilitate transcription. The increase of the amount of p300 mRNA increased after DB-51630 treatment by real time RT-PCR and Northern blot analysis. DB-51630 inhibited cell growth in various cancer cell lines at nanomolar range of concentrations, whereas p300 mRNA induction was observed at sub-nanomolar concentrations and the maximal induction occurred 8h after DB-51630 treatment. In contrast, anti-cancer drugs such as doxorubicin, vincristine, cisplatin, etoposide, and actinomycin D did not increase p300 transcription. DB-51630 revealed potent anti-cancer activity against human solid tumor xenografts. Thus, we demonstrated the anti-cancer activity of DB-51630, which interacts with a specific DNA sequence, thereby inducing p300 gene expression and exhibited significant anti-cancer activity in human tumor xenografts. Furthermore, such compounds that bind to specific DNA sequences may not only control the expression of specific genes but also exert other mechanisms in the anti-cancer effect than those of classical DNA binding drugs.

Antimetastatic and anticancer activity of S-1, a new oral dihydropyrimidine-dehydrogenase-inhibiting fluoropyrimidine, alone and in combination with paclitaxel in an orthotopically implanted human breast cancer model.

To evaluate the antitumor and antimetastatic efficacy of oral fluoropyrimidines, alone and combined with taxane on human breast cancer xenografts model, we developed a breast cancer model that spontaneously metastasizes to the lung by orthotopic implantation of MDA-MB-435S-HM tumors into the mammary fat pad (mfp) of SCID mice. The activity of the 5-fluorouracil (5-FU)-degrading enzyme dihydropyrimidine dehydrogenase (DPD) was significantly higher in the metastatic tumors than in the primary tumors. Based on this enzymatic characteristic of pulmonary metastases of breast cancer in regard to 5-FU metabolism, we investigated the antitumor activity of two types of oral 5-FU prodrugs, with and without paclitaxel, on both orthotopically implanted breast tumors and metastatic lung tumors in mice. The drugs and doses used were: S-1, a new oral DPD-inhibiting fluoropyrimidine (DIF) 8.3 mg/kg/day, capecitabine 360 mg/kg/day as a non-DIF, and paclitaxel 50 mg/kg, all of which display minimal toxicity in mice. In the primary tumors, paclitaxel and S-1 displayed a significant antitumor activity, with 57 and 41%, respectively inhibition of tumor growth (p < 0.01), but capecitabine had no effect. When S-1 and paclitaxel were combined, they synergistically caused tumor regression (tumor growth inhibition ratio 94%, p < 0.01) in mice compared to capecitabine plus paclitaxel, without any toxicity. In the pulmonary metastasis model, paclitaxel, and both S-1 alone and combined with paclitaxel, but not capecitabine alone or combined with paclitaxel, diaplayed almost complete antimetastatic activity. These results strongly suggest that combination of S-1, as a DIF with taxanes will show a potent high antitumor and antimetastatic effect on refractory human breast cancers, especially those expressing strong DPD activity.

A novel anticancer ribonucleoside, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine, enhances radiation-induced cell death in tumor cells.

1-(3-C-Ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106) is a newly developed anti-tumor agent that targets RNA synthesis. We report here that a low dose of ECyd induces radiosensitization of caspase-dependent apoptosis and reproductive cell death in cells of the gastric tumor cell lines MKN45 and MKN28 and murine rectum adenocarcinoma Colon26. Flow cytometry demonstrated that TAS106 induced the abrogation of the X-ray-induced G(2)/M checkpoint. Western blot analysis showed that X rays increased the expression of cyclin B1, phospho-Cdc2 and Wee1, whereas co-treatment with X rays and TAS106 decreased the expression of these cell cycle proteins associated with the G(2)/M checkpoint. Furthermore, TAS106 was shown to decrease the radiation-induced expression of survivin but not Bcl2 and BclX(L) regardless of TP53 status and cell type. Overexpression of wild-type survivin in MKN45 cells inhibited the induction of apoptosis induced by co-treatment with X rays and TAS106. These results suggest that TAS106 enhances X-ray-induced cell death through down-regulation of survivin and abrogation of the cell cycle machinery.

A novel retinoid, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), induces apoptosis of human ovarian carcinoma cells and shows potential as a new antitumor agent for clear cell adenocarcinoma.

OBJECTIVES: A novel retinobenzoic acid derivative, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), was reported to suppress the growth and invasion of human gastric cancer or hepatocellular carcinoma by induction of apoptosis. We examined the antitumor activity of TAC-101 against human ovarian carcinoma cell lines. METHODS: Apoptosis of human epithelial ovarian carcinoma-derived cell lines (RMG-I, RMG-II, RTSG, RMUG-S, RMUG-L, and KF) was investigated by detecting DNA laddering and was quantified by an enzyme-linked immunosorbent assay. Inhibition of apoptosis was also examined using a caspase inhibitor. Furthermore, TAC-101 (8 mg kg(-1) day(-1) orally for 30 days) was investigated in nude mice with subcutaneous RMG-II tumors. A prominent apoptotic response to TAC-101 was observed. The antitumor effects of cisplatin (7 mg/kg intravenously on day 1) and paclitaxel (36 mg/kg intravenously on days 1 and 5) were also assessed for comparison. RESULTS: Apoptosis occurred in all of the cell lines (except KF) in a concentration-dependent manner after exposure to TAC-101 and was markedly induced in RMG-I and RMG-II cells (derived from ovarian clear cell adenocarcinomas). A caspase inhibitor blocked the induction of apoptosis by TAC-101. The maximum inhibition of RMG-II tumor growth in nude mice by TAC-101, cisplatin, and paclitaxel was 45%, 34%, and 47%, respectively. CONCLUSION: Oral TAC-101 shows potential as a novel antitumor agent for ovarian carcinoma, especially ovarian clear cell adenocarcinoma.

A novel antimetabolite, TAS-102 retains its effect on FU-related resistant cancer cells.

TAS-102 is a new oral anti-tumor drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-tri-fluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). TAS-102 is currently undergoing clinical trials, and has been demonstrated to have at least 2 mechanisms; inhibition of thymidylate synthase (TS) and incorporation into DNA. 5-FU is widely used in the treatment of solid tumor, but the inherent or acquired resistance of certain tumors to 5-FU therapy is a major clinical problem. In the present study, we investigated FTD in vitro and in vivo comparing with 5-FU and using FU-resistant cells. There was no relationship between FTD and 5-FU growth inhibition effect in vitro. A different sensitivity pattern was observed by the log-mean graph. We next investigated the anti-tumor activity of TAS-102 in a FU-resistant xenograft model. Comparative efficacy was observed between FU-resistant cell and its parent cell. We also studied the influence of TAS-102 on liver metastasis in a mouse model of human colorectal cancer, because liver metastasis of colorectal cancer is associated with patient survival. Human cancer DNA was detected by PCR, and TAS-102 markedly inhibited the number of liver metastasis. A novel angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), was shown to be identical to a previously characterized intracellular enzyme, thymidine phosphorylase, TAS-102 can be expected to have not only anti-tumor cytocidal effects but also antiangiogenesis activity and may inhibit liver metastasis. Our findings suggested that TAS-102 is a promising candidate for clinical use and can be expected to decrease minimal residual disease.

Thymidine kinase and thymidine phosphorylase level as the main predictive parameter for sensitivity to TAS-102 in a mouse model.

TAS-102 is a new oral anti-cancer drug preparation, composed of a 1:0.5 mixture (on a molar basis) of alpha,alpha,alpha-trifluorothymidine (FTD) and 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). TAS-102 currently undergoing clinical trials, has been demonstrated to have at least two mechanisms, inhibition of TS and incorporation into DNA. We hypothesized that the thymidine metabolism enzyme may be a crucial factor that affects the antitumor activity of TAS-102. In the present study, we measured the enzyme activity of thymidine kinase (TK), thymidine phosphorylase (TP) and thymidilate synthase (TS) in human cancer xenografts to investigate the contribution of these enzymes to the sensitivity of TAS-102. Antitumor activity of TAS-102 appears to be associated with TK, tumor growth and TS. However, the most related factors in this study were the TK and TP ratio. There was a significant correlation (p=0.04) between tumor growth inhibition and this ratio. These results suggested that the activation and degradation pattern of FTD plays an important role in the efficacy of TAS-102 and that it is possible to use the TK/TP ratio to predict response to TAS-102 therapy. We also studied the influence of TPI on the capacity of exogenous dThd to reverse FTD-dependent growth inhibition. Thymidine (dThd) levels rescued the effect of FTD in vitro and significantly increased in serum after administration of TAS-102 or TPI alone but not FTD alone. This may suggest the possibility of a decrease in antitumor effect. However, our study indicated that the therapeutic index was clearly increased by FTD combined with TPI, compared with FTD alone, suggesting FTD-induced toxicity to sensitive host tissue can be selectively reversed with dThd. In conclusion, TK and TPI effects on TP play important roles in the cytotoxic action of TAS-102, and it is possible to use the TK/TP ratio to predict more precisely individual resistance or sensitivity.

An optimal dosing schedule for a novel combination antimetabolite, TAS-102, based on its intracellular metabolism and its incorporation into DNA.

TAS-102 is a combination drug consisting of alpha,alpha,alpha-trifluorothymidine (FTD) and thymidine phosphorylase inhibitor (TPI). FTD is converted to F3TMP by thymidine kinase and inhibits the thymidylate synthetase (TS) activity by binding to TS. In addition, FTD triphosphate form, F3TTP is incorporated into DNA, which leads to cytocidal effects. Therefore, the incorporation of FTD into DNA is expected to be an important factor, discriminating it from 5-FU showing TS inhibitory activity as their main mechanism of action. To assess a clinically more effective regimen protocol, the intracellular metabolism and the incorporation of FTD into DNA were investigated using human cancer cell lines in vitro and in vivo. FTD was incorporated into DNA in a time-dependent manner, but not in a concentration-dependent manner. FTD was the most efficiently incorporated into DNA after treatment with a several-micro molar level of FTD for around 8 h. The intracellular F3TTP was rapidly eliminated from tumor cells, as soon as FTD was washed out from the culture medium, whereas the FTD incorporated into DNA was retained by 80% or more even at 24 h after a washing-out procedure. When TAS-102 was administered into tumor-bearing mice once daily or three times daily at 3-h intervals at a dose of 150 mg/kg/day for one or 3 consecutive days, incorporation of FTD into tumor DNA by divided dosing significantly higher than that of single dosing. Based on our findings, the antitumor effects of TAS-102 against 3 different human cancer cell xenografts into mice were examined. The divided daily dosing resulted in enhancement of the antitumor effects of TAS-102 without any additional side effects. It is concluded that multiple daily dosing may result in better clinical benefits of TAS-102, when compared with single daily dosing and TAS-102 is a promising candidate for not only FU-sensitive but also FU-resistant cancer patients.

Integrin-linked kinase regulates vascular morphogenesis induced by vascular endothelial growth factor.

Integrin-linked kinase (ILK) is one of the signaling moieties that interact with the cytoplasmic domains of integrin beta1 and beta3 subunits. Integrin-mediated outside-in signals cooperate with vascular endothelial growth factor (VEGF) receptor to promote morphological changes, cell proliferation and motility in endothelial cells. In this report we demonstrate that VEGF-induced vessel morphogenesis of human umbilical vein endothelial cells (HUVEC) was inhibited by the transfection of a dominant negative, kinase-deficient ILK (ILK-KD), as well as by treatment with the phosphatidylinositol 3-kinase inhibitor LY294002. VEGF induced phosphorylation of protein kinase B (PKB/Akt), a regulator of cell survival and apoptosis, on serine 473, but not on threonine 308, in an ILK-dependent manner. Furthermore, transfection of antisense ILK (ILK-AS) blocked the survival effect of VEGF in annexin-V binding assays, and a VEGF-mediated decrease in caspase activity was reversed by both ILK-KD and ILK-AS as measured by a homogeneous caspase-3/7 assay. We also demonstrate that both chemotactic migration and cell proliferation of HUVEC induced by VEGF were suppressed by the inhibition of ILK. We conclude that ILK plays an important role in vascular morphogenesis mediated by VEGF.

Both N- and C-terminal transactivation functions of DNA-bound ERalpha are blocked by a novel synthetic estrogen ligand.

Estrogen receptors (ERs) play a central role in the diverse actions of estrogen. A number of synthetic ER ligands have been generated that can modulate various ER functions. Here we show that TAS-108, representing a novel class of synthetic ER ligands, blocked both ER transactivation functions without inhibiting DNA-binding activity. A transient expression assay showed that similar to ICI182,780, TAS-108 exhibited pure antagonistic activity as it blocked both the N-terminal AF-1 and C-terminal AF-2 transactivation functions. However, unlike ICI182,780, TAS-108 promoted the recruitment of the SMRT co-repressor that abolished ER transactivation function without inhibition of the ability of ERalpha to bind to its target DNA. Both TAS-108 and ICI182,780 acted as antagonists for the transactivation functions of the D351Y mutant, derived from tamoxifen-resistant breast cancer cells, while estrogen and known selective estrogen receptor modulators (SERMs), 4-OH tamoxifen and raloxifene, stimulated D351Y-mediated transcription. Thus, our findings indicated that TAS-108 acts as a novel estrogen antagonist that recruits co-repressors to ERs without AF-1 activation or prevention of DNA binding. Therefore, TAS-108 may be effective against tamoxifen-resistant breast cancer via a different mechanism than that for ICI182,780.

Prognostic significance of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-3 (GalNAc-T3) expression in patients with gastric carcinoma.

Aberrant glycosylation occurs during development of gastric carcinomas. The initiation of mucin-type O-glycosylation is regulated by GalNAc-T3 (UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase-3). However, the clinical significance of GalNAc-T3 expression in human gastric carcinoma has not yet been demonstrated. In the present study, we investigated the relationship between immunohistochemical GalNAc-T3 expression and various clinicopathologic factors, including prognosis, in 117 gastric carcinoma patients. Of 117 gastric carcinomas examined, 59 (50.4%) showed strong expression of GalNAc-T3. Strong expression was detected in 38 of 59 (64.4%) differentiated type and in 21 of 58 (36.2%) undifferentiated gastric carcinomas, indicating that the expression of GalNAc-T3 correlated significantly with tumor differentiation (P=0.0023, chi2 test). Overall 5-year survival rate in patients with strong GalNAc-T3 expression (71.0%) was significantly better than that of patients with weak expression (49.3%) (P=0.0197, log-rank test). Multivariate analysis identified GalNAc-T3 expression as an independent prognostic factor (P=0.0158, Cox proportional hazards model). Our data suggest that GalNAc-T3 expression may be a useful marker for prognosis and differentiation of gastric carcinomas.

Depolymerized holothurian glycosaminoglycan (DHG), a novel alternative anticoagulant for hemodialysis, is safe and effective in a dog renal failure model.

BACKGROUND: Depolymerized holothurian glycosaminoglycan (DHG) is a new agent with anticoagulant properties quite different from those of unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) in terms of antithrombin III-dependency, and exerts an antithrombotic effect with less bleeding than UFH and LMWH in vivo. In this study, the anticoagulant and hemorrhagic effects of DHG were investigated on hemodialysis in a dog model of renal failure and compared with those of UFH, LMWH, and nafamostat mesilate (FUT). METHODS: The dog renal failure model was prepared by 7/8 renal artery ligation. Effectiveness was based on completion of 3-hour hemodialysis, no marked clot deposition in the extracorporeal circuit, and permeability of blood urea nitrogen (BUN) and creatinine. Template bleeding was measured by determining the hemoglobin content of the blood from the wound. RESULTS: DHG induced no major bleeding or clot formation during 3-hour hemodialysis, in contrast to UFH and LMWH, each of which induced marked bleeding. These glycosaminoglycans (GAGs) were equally effective in decreasing plasma levels of BUN and creatinine. On the other hand, dogs treated with FUT failed to complete 3-hour hemodialysis. These anticoagulants prolonged activated partial thromboplastin time (APTT) to different extents and GAGs prolonged thrombin clotting time markedly but FUT did not. CONCLUSION: Our findings suggest that thrombin clotting time prolongation can contribute to prevention of clot formation in extracorporeal circuits, and the non-antithrombin III-dependent activities of DHG may be related to its low risk of hemorrhage for hemodialysis. DHG appears to be promising as an alternative anticoagulant with low risk of hemorrhage for hemodialysis.

Prolonged bleeding time induced by anticoagulant glycosaminoglycans in dogs is associated with the inhibition of thrombin-induced platelet aggregation.

INTRODUCTION: The clinical use of unfractionated heparin (UFH) is complicated by hemorrhage. This has led to a search for safer alternatives, one of which, the recently identified depolymerized holothurian glycosaminoglycan (DHG), causes less bleeding and exhibits a better antithrombotic-hemorrhagic ratio in rats and dogs than UFH and low-molecular-weight heparin (LMWH). In contrast to UFH and LMWH, which exert their anticoagulant effects by inhibiting thrombin in the presence of antithrombin III (AT), DHG exerts its anticoagulant effect by inhibiting the intrinsic factor Xase complex and thrombin in the presence of heparin cofactor II (HCII). MATERIALS AND METHODS: The hemorrhagic effect of DHG was compared with those of UFH and LMWH in healthy dogs, and the mechanism responsible for prolonging bleeding time was examined both in dogs and with human platelets. RESULTS: DHG prolonged template-bleeding time in dogs less than UFH and LMWH do. Although the maximum noneffective concentrations of each glycosaminoglycan (GAG) that prolong the bleeding time are almost the same as the concentrations that inhibit thrombin-induced platelet aggregation, they are not related to those that inhibit ADP-induced platelet aggregation. Results of experiments on gel-filtered platelets from humans indicate that the inhibition of thrombin-induced platelet aggregation caused by UFH and LMWH in the presence of AT is more prominent than that caused by DHG with HCII. CONCLUSIONS: These results suggest that the prolongation of bleeding time caused by GAGs are associated with the inhibition of thrombin-induced platelet aggregation, and DHG may cause less bleeding than UFH and LMWH because of its different thrombin inhibition mechanism in platelet-rich plasma (PRP).

Expression of UDP-N-acetyl-alpha-D-galactosamine-polypeptide galNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma.

BACKGROUND: Tumor development usually is accompanied by alterations of O-glycosylation. Initial glycosylation of mucin-type, O-linked proteins is catalyzed by one of the UDP-GalNAc-polypeptide N-acetyl-galactosaminyl transferases, such as GalNAc-T3, which is expressed in adenocarcinoma cells. The authors investigated whether such expression influenced tumor differentiation or prognosis in patients with colorectal carcinoma. METHODS: The expression of GalNAc-T3 was evaluated immunohistochemically in 106 paraffin embedded samples from surgically resected colorectal carcinomas and was related to patient and tumor characteristics. Western blot analysis was performed on seven samples of frozen tissue. RESULTS: Strong tumor expression of GalNAc-T3 predicted 5-year survival in patients with colorectal carcinoma (67.2% vs. 43.6% for weak expression; P = 0.017). GalNAc-T3 expression was not associated with age, gender, tumor size, tumor location, or disease stage but was related to histologic differentiation (P = 0.049) and depth of invasion (P = 0.031). Univariate analysis showed that strong GalNAc-T3 expression significantly enhanced the likelihood of survival. Multivariate Cox survival analysis identified enzyme expression as an independent prognostic factor that was second only to TNM stage. CONCLUSIONS: GalNAc-T3 expression is a novel and useful indicator of tumor differentiation, disease aggressiveness, and prognosis in patients with colorectal carcinoma.