Anti Protein Research Articles

A study of p53 expression in transitional cell carcinoma of urinary bladder in Erbil

Background and objectives: This study aimed to evaluate p53 protein expression in both normal bladder epithelium and in cases of transitional cell carcinoma (TCC) of urinary bladder by immunohistochemical study and to correlate p53 expression in urothelial cancer with other clinico-pathological parameters. Methods:This is a retrospective and prospective study for sample collection during the period from January 2006-May 2009. The samples studied included 105 formalin fixed, paraffin embedded urinary bladder tissue specimens; they consisted of the following diagnostic categories: chronic non specific cystitis (n=5) and urothelial cancer (n=100). In this study the nuclear p53 protein expression was detected in tissue samples by Dako Cytomation. LSAB + System-HRP staining protocol using monoclonal mouse anti human protein DO-7.  Results:None of the chronic non specific cystitis cases showed p53 nuclear immunostaining, while 93% of urothelial cancer specimens examined showed immunopositivity for p53 protein. In this study, a statistically significant correlation was observed between p53 overexpression rate with the tumor grade (p= <0.001) and histological architecture (p= 0.023), but not with other clinico-pathological parameters like age and gender. Conclusion: Results of the present study showed the validity and simplicity of application of immunohistochemistry in determining the status of p53 protein expression. The results suggest that p53 overexpression is strongly associated with the aggressiveness of urothelial cancer.

Fluorescent demonstration of neuronal degeneration in cyanide neurotoxicity through anti protein G-colloidal gold conjugate labeling

The study of the mechanism of neuronal cell death has been an important aspect of cell biology in the last decade. Considering the somewhat non-specific nature of the methods and the complexity of the neuronal cell cycle, a multi-technique approach has been suggested. This study is aimed at describing a method for demonstration of neuronal degeneration in the visual cortex (molecular layer) of adult wistar rats. It involves the use of colloidal gold (40 nm), conjugated with an antibody against protein G (transmembrane protein) to generate fluorescence; in essence to label the regions of cells with functional membrane, while the region of necrotic cells are empty. This method is complemented by histology to generate overlap images and to detect loss of membrane integrity in neurons at the early stages of progressive neuronal degeneration. An in vivo approach was used; 50 mg/Kg BW of potassium cyanide-Sigma (KCN) was administered orally to adult Wistar rats to induce neurotoxicity. The animals were sacrificed, and sections were labeled with anti Protein G-colloidal gold conjugate. The 40 nm gold particles generated fluorescence in the region of the cells with functional membrane properties, while empty spaces were found in regions of necrotic cells at a magnification of 400× (low magnification). Key words: Immunoglobin G (IgG), nanoparticles, cell death, bovine serum albumin (BSA).

Staphylococcal Extracellular Adherence Protein Induces Platelet Activation by Stimulation of Thiol Isomerases

Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Reduced Hepatic Injury in Toll-Like Receptor 4–Deficient Mice Following D-Galactosamine/Lipopolysaccharide-Induced Fulminant Hepatic Failure

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-ĸ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IĸB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-ĸB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.

Gene expression profiling in male genital lichen sclerosus

Male genital lichen sclerosus (MGLSc) has a bimodal distribution in boys and men. It is associated with squamous cell carcinoma (SCC). The pathogenesis of MGLSc is unknown. HPV and autoimmune mechanisms have been mooted. Anti extracellular matrix protein (ECM)1 antibodies have been identified in women with GLSc. The gene expression pattern of LSc is unknown. Using DNA microarrays we studied differences in gene expression in healthy and diseased prepuces obtained at circumcision in adult males with MGLSc (n = 4), paediatric LSc (n = 2) and normal healthy paediatric foreskin (n = 4). In adult samples 51 genes with significantly increased expression and 87 genes with significantly reduced expression were identified; paediatric samples revealed 190 genes with significantly increased expression and 148 genes with significantly reduced expression. Concordance of expression profiles between adult and paediatric samples indicates the same disease process. Functional analysis revealed increased expression in the adult and child MGSLc samples in the immune response/cellular defence gene ontology (GO) category and reduced expression in other categories including genes related to squamous cancer. No specific HPV, autoimmune or squamous carcinogenesis-associated gene expression patterns were found. ECM1 and CABLES1 expression were significantly reduced in paediatric and adult samples respectively.

Immune response of cattle immunized with a conjugate of the glycolipid glucose monomycolate and protein

Strong anti glycolipid IgG responses can occur in humans and animals, but contrary to anti protein responses and anti glycoprotein responses, the exact mechanism of induction is unknown. We have previously shown that experimental immunization with the glycolipid glucose monomycolate (GMM) causes the development of specific T cell responses, but not of anti GMM antibodies. However, cattle naturally infected with Mycobacterium avium ssp. paratuberculosis produce high levels of anti GMM IgG. In the present study, we tested whether vaccination with GMM conjugated to a protein mimics natural infection in its capacity to induce the production of antibodies against GMM. Cattle were immunized ( n = 5 per group) with GMM conjugated to a protein, or GMM and protein non-conjugated and administered at contralateral locations, or carrier only. Although immunization with the GMM–protein conjugate vaccine and the non-conjugated vaccine induced protein specific antibody responses, GMM specific antibodies were not detected in either of the groups. In conclusion, the generation of isotype-switched anti lipid antibodies appears to require more than providing peptide epitopes for T helper cells to support glycolipid specific B cells in antibody production.

An endodontic sealer induces a pathological condition when associated with persistent tissue toxicity and presence of myofibroblasts

The aims of this study were to evaluate the ratio between inflammatory reactions induced by four endodontic sealers and the occurrence of fibrosis and the number of myofibroblasts with positivity to α-smooth-actin muscle (α-SMA). Polyethylene tubes were filled with a root canal sealer (Endofill, AH Plus, Acroseal and Epiphany) and inserted into 4 site at the dorsal region of 24 Wistar rats; 2 empty tubes (control) were grafted in 6 rats. After 7, 21, and 45 days, 8 animals were euthanized, providing 6 specimens per test group and 2 specimens from the control group. The fragments were subjected to histological processing and immunohistochemical analysis for anti α-SMA protein. All specimens, except those from the control group, presented severe inflammatory reaction on the 7th postoperative day, which also coincided with a large number of myofibroblasts. On the 21st and 45th days post-surgery, the inflammatory reaction induced by Endofill, AH Plus and Acroseal decreased significantly, which coincided with reduced presence of myofibroblasts and usual collagen deposition. In contrast, in the group filled with Epiphany, significant inflammatory cell infiltrate was present in all analyzed periods. The persistence of an inflammatory reaction induced by endodontic sealer may also induce the development of fibrosis in combination with presence of myofibroblasts.

Novel nanoplatform for oral delivery of anti-cancer biomacromolecules

Oral administration of bio–macromolecules is an uphill task and the challenges from varying pH and enzymatic activity are difficult to overcome. In this regard, nanotechnology promises the new hope and offers advantages such as controlled release, target specific delivery, combinatorial therapy and many more. In this study, we demonstrate the formulation of a novel alginate enclosed, chitosan coated ceramic, anti cancer nano carrier (ACSC NC). These NC were loaded with multi functional anti cancer bovine lactoferrin (Lf), a natural milk based protein, for improvement of intestinal absorption, in order to develop a novel platform to carry anti cancer protein and/or peptides for oral therapy. Here we demonstrate the size, morphology, internalisation and release profiles of the nanoparticles (NC) under varying pH as perceived in human digestive system. We further determine the uptake of these particles by colon cancer cell lines by measuring the endocytosis and transcytosis of the NC. These NC can be used for future targeted protein/peptide or nucleic acid based drug delivery to treat difficult diseases including cancer.

Significance of PGP9.5 Expression in Cancer-Associated Fibroblasts for Prognosis of Colorectal Carcinoma

To assess the expression of a cancer-associated fibroblasts (CAFs) marker as an indicator of prognosis, we raised anti-protein gene product 9.5 (PGP9.5) monoclonal antibody against cultured fibroblasts. PGP9.5 expression in cultured normal fibroblasts was increased by transforming growth factor beta stimulation, indicating the phenotypic alteration to activated fibroblast. We immunohistochemically evaluated PGP9.5 expression with the CAFs of 110 colorectal cancer cases under T3 stage. PGP9.5 immunoreactivity in 30% or more of CAFs was defined as high PGP9.5 expression, and the other cases were considered as having low PGP9.5 expression. Patients with high PGP9.5 expression (42.7%) had significantly shorter survival and a higher incidence of recurrence than the low PGP9.5 expression group (P = .002 and P < .001, respectively). Multivariate analysis indicated PGP9.5 expression as an independent prognostic factor for overall and recurrence-free survival partly as well as lymph node metastasis. These results indicate that PGP9.5 expression in CAFs is a helpful finding to represent the overall biologic behavior of advanced colorectal cancer.

Elongin B/C Recruitment Regulates Substrate Binding by CIS

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.

Effect of transferrin on hypomyelination induced by iron deficiency

We have used a model of iron deficiency in the rat to analyze the effects of a disruption in iron availability on oligodendroglial cell (OLGc) maturation and myelinogenesis and to explore the possible beneficial influence of an intracranial injection (ICI) of apotransferrin (aTf) at 3 days of age on this process. Studies carried out on postnatal days 17 and 24 showed that iron deficiency produced a decrease in myelin proteins and lipids at 24 days of age. Immunohistochemistry showed that in untreated iron-deficient (ID) rats, the immunoreactivity of anti-adenomatous polyposis coli (APC) and anti-MBP antibodies decreased markedly with reference to normal controls, whereas in ID rats treated with an ICI of aTf, the immunoreactivity of these markers increased. A similar situation occurred with the immunoreactivity of H-ferritin. In primary OLGc cultures from ID rats, there was a high number of cells positive to the antibody against the polysialylated form of the cell surface glycoprotein NCAM (PSA-NCAM) compared with in OLGc cultures prepared from normal controls or from ID animals treated with aTf. The number of MBP+ cells in cultures from ID rats increased after treatment with aTf. The presence of lipid rafts evaluated with a specific anti-protein prion cellular (PrPc) antibody showed a smaller number of PrPc-positive structures in ID rat cultures. Treatment of the ID animals with a single ICI of aTf stimulated myelination, producing a significant correction in the different biochemical parameters affected by ID.

STUDY OF TENASCIN-C (TN-C) PROTEIN ROLE IN ORAL MALIGNANCY PROGRESSIVITY PROCESS BY MOLECULAR BIOLOGICAL APPROACH

The progressiveness of malignant tumors influenced by various complex factors. One of the important factors is Tenascin-C (Tn-C) protein, which can interact with fibrinectin as an anti adhesive or anti modulation protein. Tenascin-C is an extra cellular matrix glycoprotein (EMG), which can be found in the oral tissue also as an up regulator. They can be associated with EMG, and strongly influenced promotion of the stromal cell as cell growth, migration, differentiation, angiogenesis, and apoptosis in cancer. Alternative splicing of fibronectin-like type III (FN III) repeats of Tn-C generates a number of splice variants, and influences tumor progressiveness. The conclusion of Tn-C role in tumor progressiveness depends on the molecular weight and alternative splicing of FN III.

Anti C-Reactive Protein Immobilization Onto Poly(glycidyl Methacrylate) Microspheres and its Use for Diagnosis

A method for a polymeric carrier based test was developed for the diagnosis of C-reactive protein (CRP) as an alternative to commercial kits. Poly(glycidyl methacrylate) (PGMA) particles were synthesized by dispersion polymerization method from the glycidyl methacrylate (GMA) monomer. The monomer transformation value was 88.67% by weight. In the first step the epoxide rings were opened to form amine groups on the particle surfaces. The optimized conditions were found for the antigen to be immobilized onto the surface-modified microspheres. Agglutination tests were carried out under in vitro conditions with the anti-human CRP immobilized microspheres with samples from patient serum. Relatively high sensitivity was obtained with a short reaction time between doing the test after receiving patient serum compared with the selected commercial kit now in use. By referring to the agglutination intensity scores, using only 10 mg/L samples of plasma, the CRP concentration was detectable with these new kits.

The difference of anti phenolic glycolipid-1 (PGL-1) immunoglobulin-M (IgM) level and nutritional intake in subclinical leprosy patients who reside at home and in the orphanage

Leprosy patients can be found in the orphanage, and ordinary house holds. The different living conditions in different places might cause differences in the environments, nutritional intake, and immunity, which in the end would affect the transmission of M. leprae. The aim of this study was to find out the difference in anti PGL-1 IgM level and nutritional intake between sub-clinical leprosy patients residing at home with their families and those living in the orphanage. This cross-section observational study was done in Semarang, Central Java, Indonesia. Thirty one sub-clinical leprosy patients living in an orphanage and 30 sub-clinical leprosy patients living at home were included in this study. Venous blood was taken from all of the subjects, the level of anti PGL-1 IgM was measured, and daily nutrient intake was analyzed. Differential test between the 2 groups was performed using independent t-test or Mann Whitney test, when the distribution was not normal. There was significant differences (P &lt; 0.05) in the level of anti PGL-1 IgM, protein, zinc, and vitamin C intake between the 2 groups. (Med J Indones 2007;16:233-6)

Hepatitis G Virus Exposure in Dialysis Staff

Hepatitis G virus (HGV) is a blood-borne virus. Some present data demonstrate an occupational risk of HGV infection in medical staff of dialysis units. The aim of this investigation was to assess the prevalence of HGV exposure in dialysis staff. This study was performed in a main dialysis unit in Iran. In 27 dialysis staff, HGV exposure was detected serologically by the presence of anti HGV envelope protein E2 (anti-E2) by an enzyme-linked immunosorbent assay, and compared with 77 hemodialysis (HD) and 13 continuous ambulatory peritoneal dialysis (CAPD) patients. All of them were also screened for hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs), and hepatitis C antibody (anti-HCV). A low prevalence of HGV exposure was found in the dialysis staff (0%), which nearly corresponded to the prevalence of the dialysis patients (HD 3.89%, CAPD 0%). The prevalence of anti-HCV and anti-HBs in staff was 37.03% and 33.33%, respectively, which was higher than HGV anti-E2. The prevalence of HGV exposure was low in dialysis staff in our study, and was near to the prevalence of HGV exposure in dialysis patients. Therefore, it can be concluded that the occupational risk for HGV exposure in our investigation was minimal.

Mutant Presenilin 1 Alters Synaptic Transmission in Cultured Hippocampal Neurons

Mutations in presenilins are the major cause of familial Alzheimer disease, but the precise pathogenic mechanism by which presenilin (PS) mutations cause synaptic dysfunction leading to memory loss and neurodegeneration remains unclear. Using autaptic hippocampal cultures from transgenic mice expressing human PS1 with the A246E mutation, we demonstrate that mutant PS1 significantly depressed the amplitude of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated synaptic currents. Analysis of the spontaneous miniature synaptic activity revealed a lower frequency of miniature currents but normal miniature amplitude. Both alterations could be rescued by the application of a gamma-secretase blocker. On the other hand, the application of synthetic soluble Abeta42 in wild-type neurons induced the PS1 mutant phenotype on synaptic strength. Together, these findings strongly suggest that the expression of mutant PS1 in cultured neurons depresses synaptic transmission by causing a physical reduction in the number of synapses. This hypothesis is consistent with morphometic and semiquantitative immunohistochemical analysis, revealing a decrease in synaptophysin-positive puncta in PS1 mutant hippocampal neurons.

An N-Linked Glycan Modulates the Interaction between the CD1d Heavy Chain and β2-Microglobulin

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.

Role of transcription factor activator protein 1 (AP1) in epidermal growth factor‐mediated protection against apoptosis induced by a DNA‐damaging agent

We investigated the survival signals of epidermal growth factor (EGF) in human gastric adenocarcinoma cell line TMK-1. Treatment of TMK-1 cells with adriamycin (ADR) caused apoptosis and apoptosis-related reactions such as the release of cytochrome c from mitochondria and the activation of caspase 9. However, EGF treatment greatly reduced the ADR-induced apoptosis as well as these reactions. We previously reported that hepatocyte growth factor transmitted protective signals against ADR-induced apoptosis by causing activation of the phosphatidylinositol-3'-OH kinase (PtdIns3-K)/Akt signaling pathway in human epithelial cell line MKN74 [Takeuchi K & Ito F (2004) J Biol Chem279, 892-900]. However, PtdIns3-K/Akt signaling did not mediate the antiapoptotic action of EGF in TMK-1 cells. EGF increased the expression of the Bcl-X(L) protein, an antiapoptotic member of the Bcl-2 family, but not that of other anti (Bcl-2) or proapoptotic (Bad and Bax) protein members. Expression of the c-Fos and c-Jun, components of activator protein 1 (AP1), which are known to regulate bcl-X(L) gene transcription, were increased in response to EGF. Pretreatment of the cells with PD98059, an inhibitor of MAP kinase kinase, inhibited the EGF-induced c-Fos and c-Jun expression, AP1 DNA binding, Bcl-X(L) expression, and the resistance against ADR-induced apoptosis, suggesting that EGF transmitted the antiapoptotic signal in such a way that it activated AP1 via a MAP kinase signaling pathway. TMK-1 cells stably transfected with TAM67, c-Jun dominant-negative mutant, did not display EGF-induced Bcl-X(L) expression or resistance against ADR-induced apoptosis. These results indicate that AP1-mediated upregulation of Bcl-X(L) expression is critical for protection of TMK-1 cells against ADR-induced apoptosis.

The αPROX assay: fluorescence screening of the inhibitory effects of hydrophilic antioxidants on protein oxidation

We report on a new and convenient high-throughput fluorescence technique for determining antioxidant capacities of hydrophilic food samples. The new method is called alphaPROX (anti protein oxidation) and is based on an equimolar complex of diphenylhexatriene propionic acid (DPHPA) and bovine serum albumin (BSA) in aqueous buffer at pH 7.4. DPHPA is a reporter fluorophore that becomes nonfluorescent upon free radical-induced oxidation. In a typical assay, the DPHPA/BSA complex is challenged with peroxyl radicals and shows almost the same susceptibility to oxidation as unlabeled BSA. The progress of protein oxidation and its inhibition by antioxidants at physiological pH is determined from the time-dependent decrease in DPHPA fluorescence intensity. The alphaPROX method was compared to other techniques frequently used to measure antioxidant capacities. In this article, representative results are provided for the inhibitory effects of pure food components, fruit juices, wines, and various polar plant extracts on protein oxidation.

Restoration by aspirin of impaired plasma maspin level in human breast cancer

Maspin, an anti breast cancer protein, is produced in the normal mammary cells but not in malignant cells in breast cancer. We investigated the effect of aspirin induced increase of plasma nitric oxide (NO) on plasma maspin production in breast cancer patients. Fifteen breast cancer patients (35–65 years), who had not yet undergone any cancer therapy, and an equal number of age matched normal female volunteers participated in the study. They were asked not to take any medication for two weeks. All participants then ingested 150 mg of aspirin. Plasma NO and maspin levels were determined before and at 60 min after the ingestion of aspirin. It was found that the maspin level in plasma increased to 4.63±0.02 nM from the basal 0.95±0.012 nM (p < 0.001) with increase of plasma NO from 0.60±0.03 µM to 2.08±0.030 µM (p < 0.001) in breast cancer patients. In normal volunteers the basal maspin increased from 4.76±0.041 to 9.36±0.036 nM (p < 0.001) with increase of NO from 2.15±0.08 to 3.36±0.04 µM (p < 0.001) at the same period. These results indicated that the ingestion of aspirin might be beneficial for breast cancer through increased maspin production.