Edoxaban, a Factor Xa-Specific Direct Oral Anticoagulant, Significantly Suppresses Tumor Growth in Colorectal Cancer Colon26-Inoculated BALB/c Mice.

Introduction Certain low-molecular-weight heparins have been reported to reduce tumor growth and metastasis in tumor cell-inoculated mouse models and cancer patients. Recently, direct oral anticoagulants (DOACs) have been widely used in patients with thromboembolism. This study was aimed at investigating the effect of DOACs, which target thrombin or factor Xa, on tumor growth in a syngeneic mouse model comprising BALB/c mice inoculated with colon cancer Colon26 cells. Materials and Methods DOACs targeting thrombin (dabigatran etexilate [DABE]) or factor Xa (rivaroxaban [RVX] and edoxaban [EDX]) were orally administered daily to male BALB/c mice inoculated with Colon26 cells, followed by analyses of tumor growth and plasma levels of coagulation- and tumor-related factors such as tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), and matrix metalloproteinase-2 (MMP-2). Results Colon26 cells expressed significant amounts of functionally active TF. Tumor growth in Colon26-inoculated mice was significantly suppressed in DABE- or RVX-treated mice ( p <0.05) and was suppressed more significantly in EDX-treated mice ( p <0.01). Therefore, the antitumor mechanism of action of EDX was investigated next. Plasma levels of TF, PAI-1, IL-6, and MMP-2 were elevated in Colon26-inoculated mice but were significantly reduced in EDX-treated mice ( p <0.01). The expression of protease-activated receptor (PAR)1, PAR2, signal transducer and activator of transcription-3 (STAT3), cyclin D1, and Ki67 was increased in tumor tissue of Colon26-inoculated mice but (except for PAR1) was significantly decreased in tumor tissues of EDX-treated mice ( p <0.01). In addition, apoptotic cells and p53 protein levels were significantly increased in tumor tissues of EDX-treated mice. Conclusion The data suggest that among the tested DOACs, EDX significantly suppresses tumor cell proliferation via the factor Xa-PAR2 pathway, which is activated by coagulation and inflammation in Colon26-inoculated mice and induces tumor cell apoptosis.

Development of a novel automatic ascites filtration and concentration equipment with multi-ring-type roller pump units for cell-free and concentrated ascites reinfusion therapy.

Cell-free and concentrated ascites reinfusion therapy (CART) is an effective therapy for refractory ascites. However, CART is difficult to perform as ascites filtration and concentration is a complicated procedure. Moreover, the procedure requires the constant assistance of a clinical engineer or/and the use of an expensive equipment for the multi-purpose blood processing. Therefore, we developed a CART specialized equipment (mobility CART [M-CART]) that could be used safely with various safety measures and automatic functions such as automatic washing of clogged filtration filter and self-regulation of the concentration ratio. Downsizing, lightning of the weight, and automatic processing in M-CART required the use of newly developed multi-ring-type roller pump units. This equipment was approved under Japanese regulations in 2018. In performing 41 sessions of CART (for malignant ascites, 22 sessions; and hepatic ascites, 19 sessions) using this equipment in 17 patients, no serious adverse event occurred. An average of 4494 g of ascites was collected and the total amount of ascites was processed in all the sessions without any trouble. The mean weight of the processed ascites was 560 g and the mean concentration ratio was 8.0. The ascites were processed at a flow rate of 50 mL/min. The mean ascites processing time was 112.5 minutes and a 106.5-minutes (95.2%) ascites processing was performed automatically. The operator responded to alarms or support information 3.2 times on average (3.1 minutes, 2.1% of ascites processing time). Human errors related to ascites processing were detected by M-CART at 0.4 times per session on average and were appropriately addressed by the operator. The frequencies of automatic washing of clogged filtration filter and self-regulation of the concentration ratio were 31.7% and 53.7%, respectively. The mean recovery rates (recovery dose) of protein, albumin, and immunoglobulin G were 72.9%, 72.9%, and 71.2% (65.9 g, 34.9 g, and 13.2 g), respectively. Steroids were administered in 92.7% of the sessions to prevent fever and the mean increase in body temperature was 0.53°C. M-CART is a compact and lightweight automatic CART specialized equipment that can safely and easily process a large quantity of ascites without the constant assistance of an operator.

Mushroom Sparassis crispa (Hanabiratake) Fermented with Lactic Acid Bacteria Significantly Enhances Innate Immunity of Mice.

Sparassis crispa (SC; Japanese name: Hanabiratake) is a mushroom with high ß(1-3)-glucan content. We here studied the effects of SC and lactic acid bacteria-fermented SC (SCL) on innate immunity. In in vivo studies using mice, oral administration of SC or SCL enhanced the accumulation of macrophages, neutrophils, natural killer (NK) cells, and C-C chemokine receptor type 2- or phospho-Syk-expressing cells in the jejunum epithelial villi and spleen, with significantly higher cell numbers in the SCL group than in the SC group. In addition, mRNA levels of genes encoding tissue factor (TF) and tumor necrosis factor (TNF)-α were increased in monocytes/macrophages from the peritoneal cavity of mice orally administered SCL. In in vitro studies using cultured human monocytes, SC and SCL enhanced the expression of gees involved in blood coagulation and inflammation, as well as those encoding various innate immune-related factors, such as TF, TNF-α, plasminogen activator inhibitor (PAI)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß, IL-8, IL-12ß, and IL-17, in a dose-dependent manner. In particular, the expression levels of all these factors in monocytes were significantly higher with SCL treatment than with SC treatment. SCL significantly enhanced the phagocytosis of pH-sensitive fluorescent dye-labeled Escherichia coli by human monocytes compared to SC. The effect of SCL on phagocytosis was significantly reduced to approximately 30% by pre-digestion of SCL with ß-glucanase, suggesting that ß(1-3)-glucan in SCL is a major contributor to the effect. These data suggest that oral administration of SCL significantly enhances innate immunity in mice and possibly humans.

Host protein C inhibitor inhibits tumor growth, but promotes tumor metastasis, which is closely correlated with hypercoagulability.

INTRODUCTION: Protein C inhibitor (PCI), a member of the serine protease inhibitor family, is expressed in various human tissues, including liver and kidneys. In the plasma, PCI physiologically inhibits an anticoagulant serine protease, activated protein C (APC). PCI expressed by cancer cells suppresses tumor invasion by inhibiting urokinase-type plasminogen activator, and inhibits tumor growth and metastasis, which are independent of its protease-inhibitory activity. In the present study, we clarified the effects of host PCI on growth and metastasis of B16 melanoma (B16) cells by comparing between wild-type mice and mice transgenic for human PCI gene (hPCI-TG), which have a tissue distribution of PCI similar to that observed in humans. MATERIALS AND METHODS: Growth of intracutaneously-injected B16 cells was evaluated by measuring the tumor volume, and metastatic behavior of intravenously-injected B16 cells by counting the number of metastatic lung nodules. RESULTS: Growth of intracutaneously injected B16 cells was significantly faster in wild-type mice than in hPCI-TG mice; however, hPCI-TG mice developed more metastatic nodules of B16 cells in the lungs. Immunohistochemical analysis using anti-mouse fibrinogen antibody revealed more fibrin deposition in the lung in hPCI-TG mice than in wild-type mice. Furthermore, the more invasive behavior observed in hPCI-TG mice was reduced by rabbit anti-human PCI IgG, APC, or soluble TM administration for 3 consecutive days including the day that B16 cells were injected. CONCLUSIONS: Our results suggest that like PCI expressed in tumor cells, host PCI also inhibits tumor growth, but host PCI promotes tumor metastasis via its procoagulant properties.

Novel p53 splicing site mutation in Li-Fraumeni-like syndrome with osteosarcoma.

We describe a 15-year-old girl with a novel germline p53 splice site mutation who developed an osteosarcoma. She received several cycles of chemotherapy with complete resection of the primary tumor without amputation, and has maintained remission for 18 months. Li-Fraumeni-like syndrome was suspected based on familial history. Sequence analysis revealed the presence of a novel germline p53 gene mutation resulting in a G to A transition at position +1 at the donor splice site of intron 6, creating a 6 amino acid insertion. This case provides interesting insight into the phenotype-genotype correlation in LFL syndrome with a TP53 splicing mutation.

Functional studies of a novel germline p53 splicing mutation identified in a patient with Li-Fraumeni-like syndrome.

Most p53 mutations identified in Li-Fraumeni syndrome (LFS) are missense mutations; splicing mutations have rarely been reported. A novel splicing p53 mutation was identified in a patient with Li-Fraumeni-like syndrome (LFL). Usually, p53 missense mutants identified in LFS and cancer cells function as dominant negative mutations interfering with wild-type p53 function. However, the mechanism by which p53 haploinsufficiency causes carcinogenesis is not well characterized. In this study, we describe a novel splicing mutation that results in the loss-of-function of p53. These findings suggest a linkage between the loss-of-function type p53 mutation and a LFL phenotype.

[Usefulness of survivin/glyceraldehyde-3-phosphate dehydrogenase ratio in urine exfoliated cells for the detection of bladder tumor].

OBJECTIVE: Survivin is one of the apoptosis inhibitor proteins and is rarely expressed in adult normal tissues. However, survivin expression has been detected in various tumors. In this study, we evaluated the usefulness of urinary survivin/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio as a marker for bladder tumor. PATIENTS AND METHODS: Urine samples were obtained from 72 patients with bladder tumor, 36 with urinary tract inflammation as controls. Survivin and GAPDH mRNA expression was measured by quantitative real-time PCR assay in urine cells. The GAPDH housekeeping gene was used for normalization of survivin expression. We also analyzed survivin protein levels using urine samples and recombinant protein by western blotting. RESULTS: High expression of survivin was confirmed on the protein level using urine samples of bladder tumor by western blotting. Survivin/GAPDH mRNA ratios of bladder tumor quantified by real-time PCR was significantly higher than those of controls (p=0.001). In pathological stage of bladder tumor, survivin/ GAPDH mRNA ratio of pTis was significantly high compared with pTa and pT1 (p < 0.001, p=0.001, respectively). Grade3 tumors expressed high level of survivin/GAPDH mRNA ratio compared with Grade 1 and Grade 2 tumors (p=0.03). The sensitivity, the specificity and AUC(area under the curve) of survivin/ GAPDH mRNA ratio was 83.3%, 86.1% and 0.898, respectively. CONCLUSION: Measuring survivin/GAPDH mRNA ratio in urine is non-invasive and high sensitive examination. Therefore, survivin/GAPDH mRNA ratio is useful marker for the detection of bladder tumor, especially to detect carcinoma in situ.

Personalized treatment in the eradication therapy for Helicobacter pylori.

Helicobacter pylori (HP) is known to be a causative bacterium of gastritis and peptic ulcers. The combination treatment consisting of a proton pump inhibitor (PPI), amoxicillin and clarithromycin (CAM) is widely used in eradication therapy, but the eradication fails in some patients. The main causes are CAM resistance of HP and individual variability in PPI metabolism related to the activity of the cytochrome P450 2C19 (CYP2C19) enzyme. In this study, we examined the usefulness of the prediction of the pharmacotherapeutic efficacy using a newly developed analysis system for HP CAM resistance and CYP2C19 genotypes. After obtaining the informed consent from 45 subjects with HP-positive peptic ulcers, biopsy specimens of the gastric mucosa were obtained by endoscopy. HP DNA extracted from the gastric mucosa was examined by the SELMAP-PCR method, the direct sequencing method or the single-nucleotide primer extension (SNuPE) method. HP detection rates by culture and the SELMAP-PCR method were 71% and 100%, respectively. Among 32 cultured HP, CAM resistance was confirmed in 6 samples by the in vitro drug susceptibility test. CAM-resistant gene mutations were also examined by the SELMAP-PCR method using 32 DNAs from cultured HP and the results were consistent with the drug susceptibility test. Among 22 patients, the eradication rate was 77%. Among 4 patients with CAM resistance determined by both the in vitro drug susceptibility test and the SNuPE method, eradication was successful in one intermediate metabolizer (IM), but not in three extensive metabolizers (EMs). Patients were divided into three groups according to their CYP2C19 phenotype: EMs, IMs and poor metabolizers (PMs). The eradication rates for 6 EMs, 12 IMs and 4 PMs were 33.3%, 91.7% and 100%, respectively. Based on these results, the information on CAM resistance in HP and CYP2C19 phenotypes in carriers could predict the pharmacotherapeutic efficacy and probability of eradication. It can then be possible to vary the dosing or to select another drug by the prediction of the pharmacotherapeutic efficacy.

Monitoring peripheral blood CD4+ adenosine triphosphate activity after living donor liver transplantation: impact of combination assays of immune function and CYP3A5 genotype.

BACKGROUND: The immune response after living donor liver transplantation (LDLT) was evaluated, with specific attention focused on the inter-relationship among clinical conditions, pharmacokinetics of tacrolimus, and cytochrome P450 3A5 (CYP3A5) genotypes. METHODS: Forty 40 adult patients who underwent LDLT in the period 2002-2009 were enrolled in the study. Peripheral blood was collected from these patients between June 2009 and December 2009 and analyzed for CD4+ adenosine triphosphate (ATP) activity (ImmuKnow assay), tacrolimus concentration, and CYP3A5 genotype. Based on the results of the ImmuKnow assay (i.e., strength of immune response), each patient was categorized into one of the three established zones of immune response: Group A (low response, n = 13), Group B (moderate response, n = 24), and Group C (strong response, n = 3). RESULTS: There was no correlation between the concentration of tacrolimus and ATP levels in the blood. The patients in Group A required much higher tacrolimus doses to maintain the blood concentration, as evidenced by the tacrolimus concentration/dose (C/D) ratios in Group A being significantly lower than those in Group B. Almost half of the patients in Group A suffered from infectious complications. The C/D ratios were significantly lower in the six patients with the CYP3A5 1 allele than in the 14 patients with the CYP3A5 3 allele; one-half of the patients with the CYP3A5 1 allele belonged to Group A. CONCLUSIONS: Our results demonstrate that the immunKnow assay of immune function is an excellent tool for monitoring immune response, especially in the patients with CYP3A5 1 allele (expressors).

Multicenter evaluation of prototype real-time PCR assays for Epstein-Barr virus and cytomegalovirus DNA in whole blood samples from transplant recipients.

Quantitative PCR is becoming widespread for diagnosing and monitoring post-transplantation diseases associated with EBV and CMV. These assays need to be standardized to manage patients in different facilities. Five independent laboratories in Japan compared home-brew assays and a prototype assay system to establish a standard quantitative procedure for measuring EBV and CMV. Reference standards and a total of 816 (642 EBV and 174 CMV) whole blood samples from post-transplantation recipients were used for this multicenter evaluation. The prototype reference standard for EBV was compared to a panel of samples, with a theoretical expected value made using EBV-positive cells containing two virus genome copies per cell. The mean ratio of the reference standard at each site to the standard of the prototype assay was ≤ 4.15 for EBV among three different sites and ≤ 3.0 for CMV between two laboratories. The mean of the theoretical expected number of the EBV genome: prototype reference was close to 1.0. The correlation coefficients between the viral copy numbers determined using the prototype assay and those using each home-brew assay were high (EBV, 0.73-0.83, median = 0.78; CMV, 0.54-0.60, median = 0.57). The dynamics of the EBV and CMV loads in transplant recipients were similar between the assay types. There was an inter-laboratory difference among the quantification results, indicating that a unified protocol and kit are favorable for standardizing the quantification of EBV and CMV. Such standardization will help to standardize the diagnosis and monitoring of diseases associated with EBV and CMV.

The association of protein S Tokushima-K196E with a risk of deep vein thrombosis.

Deep vein thrombosis (DVT) is a multifactorial disease caused by acquired risk factors such as a bed rest, surgery and malignancies. Although the factor V Leiden and the prothrombin-20210G>A mutation do not exist in Japanese populations, a mutation in protein S (PS) Tokushima (K196E) has been attracting attention in Japan. In this study, the genetic contribution of PS Tokushima (K196E) was evaluated in 60 Japanese patients with thrombosis in comparison to 234 healthy volunteers and 88 patients without thrombosis. Genes associated with the response to warfarin, cytochrome P450 2C9 (CYP2C9), vitamin K epoxide reductase complex subunit 1 (VKORC1), and γ-glutamyl carboxylase (GGCX) were also investigated simultaneously. PS Tokushima (K196E) was detected in 6 patients with thrombosis, in 3 without thrombosis and in 3 healthy volunteers, indicating that there is a high frequency of the PS Tokushima (K196E). There were no significant differences of CYP2C9, VKORC1 or GGCX between the patients with and without DVT. Therefore, PS Tokushima (K196E) is an important genetic risk factor for DVT in the Japanese population.

Neurotrophic receptor tyrosine kinase B induces c-fos-associated cell survival.

The neurotrophic receptor tyrosine kinase B (TrkB) is a cell surface receptor for brain-derived neurotrophic factor (BDNF) with kinase activity. This protein is expressed in various tumors and thought to participate in various cellular processes. In this study, we established 293T cells stably expressing human TrkB to elucidate its intracellular functions. Using this cell system, we examined the biological roles of TrkB and its downstream target molecules. The TrkB expressing cells showed an increased survival rate through increased c-fos mRNA expression by BDNF, which were completely suppressed by TrkB inhibitor. Moreover, the combination of inhibitors of mitogen-activated protein kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) partially reduced both the cell survival rate and c-fos mRNA expression, whereas monotreatment of these reagents could not affect cell survival nor c-fos mRNA expression. These results suggested that TrkB could play a role in c-fos-associated cell survival through both MEK and PI3K pathway. It is conceivable that activation of TrkB has a significant impact on tumorigenesis and metastasis.

Neurotrophic receptor, tropomyosin-related kinase B as an independent prognostic marker in gastric cancer patients.

BACKGROUND AND OBJECTIVES: Neurotrophic receptor tyrosine kinase TrkB has been associated with clinical outcome and chemotherapy resistance in neuroblastoma and certain human malignancies. Recent studies have focused on the association between metastatic potential and TrkB expression in tumor cells. METHODS: To determine the role of TrkB in gastric cancer, we analyzed TrkB mRNA levels by real-time reverse transcription polymerase chain reaction in 90 patients with gastric cancer. TrkB levels were correlated with clinicopathological variables. The association between TrkB and overall survival was evaluated by univariate and multivariate analyses. RESULTS: The mean TrkB level in gastric cancer tissue was 2.96 (range, 0-27.1). Thirty-eight (42%) of 90 patients showed detectable TrkB levels, whereas the remainder had no detectable TrkB. There was no significant association between clinicopathological variables and TrkB positivity. TrkB level was significantly associated with extent of lymph node metastasis in node positive patients (P = 0.03). TrkB positivity (n = 38) was significantly correlated with worse patient survival (P = 0.03). Multivariate analysis showed TrkB to be an independent prognostic parameter for overall survival (P = 0.04). CONCLUSIONS: TrkB is an independent prognostic marker in patients with gastric cancer and appears to be associated with metastatic potential.

The effectiveness of measuring for fragmented red cells using an automated hematology analyzer in patients with thrombotic microangiopathy.

Thrombotic microangiopathy (TMA) or thrombotic thrombocytopenic purpura (TTP) is a life-threatening syndrome characterized by increased number of fragmented red cells (FRCs) and thrombocytopenia. FRCs can be measured using the recently developed automated hematology analyzer XE-2100. The normal range for FRCs is 0% to 0.205%, as determined by the automated hematology analyzer XE-2100. The FRC count is significantly elevated in patients with TMA associated with liver transplantation, bone marrow transplantation, or TTP. In patients with TMA after liver transplantation, the FRC count is significantly higher than in those without TMA. In receiver operating characteristic analysis for the diagnosis of TMA, the area under the curve is 0.986, suggesting that FRC is a useful marker for the diagnosis of TMA. When the cutoff value of FRC for TMA is 1.2%, the sensitivity is 90% and the specificity is 96%, indicating that FRC is the most useful screening test for the diagnosis of TMA.

Immunohistochemical diagnosis of methylthioadenosine phosphorylase (MTAP) deficiency in non-small cell lung carcinoma.

Methylthioadenosine phosphorylase (MTAP) is involved in the metabolism of purines and converts methylthioadenosine (MTA) to adenine. It is abundant in all normal tissues but is deficient in various tumors. Here, we investigated MTAP deficiency in clinical samples of lung cancer using immunohistochemistry (IHC), and compared these results with those obtained by real-time PCR. Seventy-five samples were obtained from patients who underwent operations for non-small cell lung cancer (NSCLC). MTAP genetic analysis, using real-time PCR, and IHC were carried out on the samples. Methylation-specific primers were used to analyze methylation of the MTAP promoter, using DNA treated with sodium bisulfite. Sixty-nine of 75 samples were compared using both IHC and real-time PCR. The IHC results were consistent with those of real-time PCR in 56 samples. Of 62 positive samples tested by real-time PCR, only 49 (79%) were MTAP-positive by IHC. Seven samples were MTAP-negative by real-time PCR and IHC. In 13 samples of PCR (+) and IHC (-), six samples showed that the promoter region of MTAP was methylated. IHC is an accurate and useful diagnostic method for detecting MTAP deficiency in NSCLC, and the frequency of MTAP deficiency was found to be relatively high. The metabolic alterations diagnosed by IHC could be exploited for selective chemotherapy.

[Analysis of platelet production following liver transplantation using the immature platelet fraction (IPF) parameter].

Although liver transplantation (LT) is frequently associated with thrombocytopenia, thrombopoiesis following LT remains to be evaluated. We analyzed platelet counts (PLT), immature Platelet Fraction (IPF), and thrombopoietin (TPO) in 8 patients with LT. PLT and IPF were measured using Sysmex XE-2100. TPO was measured with Human TPO ELISA Kit. In 7 of 8 patients with LT, IPF increased prior to the elevation of platelet counts. In 5 of 7 patients with increased IPF, TPO levels increased simultaneously with IPF, suggesting that IPF may reflect the production of platelets in patients with LT. IPF and TPO might be useful to monitor the platelet production in patients following LT.

Methylthioadenosine phosphorylase deficiency in Japanese osteosarcoma patients.

Methylthioadenosine phosphorylase (MTAP) is an important enzyme in the salvage pathway of adenosine and methionine synthesis. MTAP is ubiquitously present in all normal cells and tissues, but deficient in a variety of malignant tumors. The enzyme deficiency is caused by either MTAP gene deletion or promoter hypermethylation. We investigated MTAP expression, MTAP gene deletion and promoter abnormality in 40 primary tumor samples from Japanese osteosarcoma patients and determined the frequency of the enzyme deficiency. We also tested whether or not the enzyme deficiency can be exploited for tumor-specific chemotherapy using osteosarcoma cell lines. For MTAP expression, immunohistochemistry (IHC) and Western blotting were used. Real-time quantitative PCR assay was used for the analysis of MTAP gene deletion in fifteen osteosarcoma samples. MTAP promoter abnormality was analyzed by methylation-specific PCR. Then, the relationship between MTAP expression and sensitivity to the inhibitors of de novo AMP synthesis was confirmed in an MTAP-negative and -positive osteosarcoma cell line. The MTAP protein was negative in 11 of 40 samples (27.5%) by IHC and in 4 of 6 osteosarcoma cell lines (66.7%) by Western blot analysis. Among 40 samples, 15 were subjected to quantitative real-time PCR and promoter methylation analysis. Of 6 samples that were negative by IHC, the MTAP gene was deleted in 3 and the MTAP promoter was methylated in 2. These results indicated that MTAP deficiency was caused by MTAP gene deletion or promoter methylation in all MTAP-negative samples except one that was negative with IHC although no deletion or promoter methylation was detected. In in vitro experiments using transfectoma along with the MTAP-negative parental cell line, the MTAP-negative parental cell line was more chemosensitive to the inhibitors of de novo AMP synthesis than MTAP-positive transfectoma. MTAP deficiency frequently found in osteosarcoma can be exploited for selective chemotherapy in MTAP-negative osteosarcoma patients with the inhibitors of de novo purine synthesis.

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor alpha-positive and -negative human hematopoietic progenitors.

Interleukin (IL)-6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL-6 receptor-alpha subunit (IL-6Ralpha)(+) but also IL-6Ralpha(-) hematopoietic progenitors via soluble IL-6R. The Notch ligand Delta-1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL-6 are influenced by Delta-1. When CD34(+)CD38(-) hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL-3, Delta-1, in combination with the IL-6R/IL-6 fusion protein FP6, increased the generation of glycophorin A(+) erythroid cells but counteracted the effects of IL-6 and FP6 on the generation of CD14(+) monocytic and CD15(+) granulocytic cells. Although freshly isolated CD34(+)CD38(-) cells expressed no or only low levels of IL-6Ralpha, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta-1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL-6Ralpha(-) erythroid progenitors. In contrast, Delta-1 antagonized the effects of IL-6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14(-)CD1a(+) dendritic cells, from the IL-6Ralpha(+) myeloid progenitors. These results indicate that Delta-1 interacts differentially with gp130 activation in IL-6Ralpha(-) erythroid and IL-6Ralpha(+) myeloid progenitors. The present data suggest a divergent interaction between Delta-1 and gp130 activation in human hematopoiesis.

Protein C inhibitor inhibits breast cancer cell growth, metastasis and angiogenesis independently of its protease inhibitory activity.

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway and also inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. In the present study, we evaluated the effect of human PCI and its inactive derivatives on tumor growth and metastasis of human breast cancer (MDA-231) cells, and on angiogenesis in vivo. The invasiveness of MDA-231 cells was inhibited by recombinant intact PCI, but not by reactive site-modified PCI (R354APCI) or by the N-terminal fragment of protease-cleaved PCI (NTPCI). The in vitro invasiveness of MDA-231 cells expressing intact PCI (MDA-PCI) was significantly decreased as compared to MDA-231 cells expressing R354APCI (MDA-R354APCI) or NTPCI (MDA-NTPCI). Further, in vivo growth and metastatic potential of MDA-PCI, MDA-R354APCI and MDA-NTPCI cells in severe combined immunodeficient (SCID) mice were significantly decreased as compared to MDA-Mock cells. Angiogenesis was also significantly decreased in Matrigel implant containing MDA-PCI, MDA-R354APCI or MDA-NTPCI cells as compared to that containing MDA-Mock cells. In vivo angiogenesis in rat cornea and in vitro tube formation were also inhibited by recombinant intact PCI, R354APCI and NTPCI. Furthermore, the anti-angiogenic activity of PCI was strong as cleaved antithrombin (AT), and slightly stronger than that of plasminogen activator inhibitor (PAI)-1 and pigment epithelium-derived factor (PEDF). Overall, this study showed that, in addition to a reactive site-dependent mechanism, PCI may also regulate tumor growth and metastasis independently of its protease inhibitory activity by inhibiting angiogenesis.

The interaction among protein C inhibitor, prostate-specific antigen, and the semenogelin system.

The kallikrein-like serine protease, prostate-specific antigen (PSA), is mixed in human seminal plasma with its protein substrates semenogelin (Sg) -I, Sg-II, and protein C inhibitor (PCI), which are produced in seminal vesicles. In the seminal plasma, PSA degrades Sg-I, and Sg-II, which are major components in insoluble coagula, and PCI inhibits PSA by forming a PSA-PCI complex. Digestion of seminal coagula with PSA releases PCI and PSA-PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI within the coagula. PCI forms a ternary complex with PSA and Sg-II in the seminal plasma. The binding of Sg-II to PSA and PCI is influenced by pH, ionic strength, heparin, negatively charged dextran sulfate, divalent cations, and particularly by Zn 2 +. These observations suggest that binding of PCI to Sg in seminal vesicles regulates the PSA-catalyzed degradation of Sg in seminal plasma; the complex formation among PCI, PSA, and Sg is modulated by several factors in seminal plasma.