Bending of a lipid membrane edge by annexin A5 trimers.

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a "neck"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

[Effect of adenovirus-mediated shRNA down-regulates SHP2 expression on the apoptosis of human hepatic stellate cells LX-2].

Objective: To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro. Methods: The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group: (1) Control group: LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group: transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group: transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons. Results: shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells (P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) (P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) (P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Conclusions: SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.

Immunogenicity, antibody responses and vaccine efficacy of recombinant annexin B30 against Schistosoma mansoni.

AIMS: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model. METHODS AND RESULTS: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected. CONCLUSION: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development.

Human Embryonic Stem Cell-Derived Neural Lineages as In Vitro Models for Screening the Neuroprotective Properties of Lignosus rhinocerus (Cooke) Ryvarden.

In the biomedical field, there is growing interest in using human stem cell-derived neurons as in vitro models for pharmacological and toxicological screening of bioactive compounds extracted from natural products. Lignosus rhinocerus (Tiger Milk Mushroom) is used by indigenous communities in Malaysia as a traditional medicine to treat various diseases. The sclerotium of L. rhinocerus has been reported to have medicinal properties, including various bioactivities such as neuritogenic, anti-inflammatory, and anticancer effects. This study aims to investigate the neuroprotective activities of L. rhinocerus sclerotial extracts. Human embryonic stem cell (hESC)-derived neural lineages exposed to the synthetic glucocorticoid, dexamethasone (DEX), were used as the in vitro models. Excess glucocorticoids have been shown to adversely affect fetal brain development and impair differentiation of neural progenitor cells. Screening of different L. rhinocerus sclerotial extracts and DEX on the hESC-derived neural lineages was conducted using cell viability and neurite outgrowth assays. The neuroprotective effects of L. rhinocerus sclerotial extracts against DEX were further evaluated using apoptosis assays and Western blot analysis. Hot aqueous and methanol extracts of L. rhinocerus sclerotium promoted neurite outgrowth of hESC-derived neural stem cells (NSCs) with negligible cytotoxicity. Treatment with DEX decreased viability of NSCs by inducing apoptosis. Coincubation of L. rhinocerus methanol extract with DEX attenuated the DEX-induced apoptosis and reduction in phospho-Akt (pAkt) level in NSCs. These results suggest the involvement of Akt signaling in the neuroprotection of L. rhinocerus methanol extract against DEX-induced apoptosis in NSCs. Methanol extract of L. rhinocerus sclerotium exhibited potential neuroprotective activities against DEX-induced toxicity in hESC-derived NSCs. This study thus validates the use of human stem cell-derived neural lineages as potential in vitro models for screening of natural products with neuroprotective properties.

Precision medicine in lupus nephritis: can biomarkers get us there?

Patients with systemic lupus erythematosus frequently develop lupus nephritis (LN), a condition that can lead to end-stage kidney disease. Multiple serum and urine biomarkers for LN have been proposed in recent years, yet none have become incorporated into clinical use. The majority of studies have been single center with significant variability in cohorts, assays, and sample storage, leading to inconclusive results. It has become clear that no single biomarker is likely to be sufficient to diagnose LN, identify flares, and define the response to therapy and prognosis. A more likely scenario is a panel of urine, serum, tissue, and genetic biomarkers. In this review, we summarize traditional and novel biomarkers and discuss how they may be utilized in order to bring precision medicine to clinical practice in LN.

Annexins in Glaucoma.

Glaucoma is one of the leading causes of irreversible visual loss, which has been estimated to affect 3.5% of those over 40 years old and projected to affect a total of 112 million people by 2040. Such a dramatic increase in affected patients demonstrates the need for continual improvement in the way we diagnose and treat this condition. Annexin A5 is a 36 kDa protein that is ubiquitously expressed in humans and is studied as an indicator of apoptosis in several fields. This molecule has a high calcium-dependent affinity for phosphatidylserine, a cell membrane phospholipid externalized to the outer cell membrane in early apoptosis. The DARC (Detection of Apoptosing Retinal Cells) project uses fluorescently-labelled annexin A5 to assess glaucomatous degeneration, the inherent process of which is the apoptosis of retinal ganglion cells. Furthermore, this project has conducted investigation of the retinal apoptosis in the neurodegenerative conditions of the eye and brain. In this present study, we summarized the use of annexin A5 as a marker of apoptosis in the eye. We also relayed the progress of the DARC project, developing real-time imaging of retinal ganglion cell apoptosis in vivo from the experimental models of disease and identifying mechanisms underlying neurodegeneration and its treatments, which has been applied to the first human clinical trials. DARC has potential as a biomarker in neurodegeneration, especially in the research of novel treatments, and could be a useful tool for the diagnosis and monitoring of glaucoma.

Annexin A9 promotes invasion and metastasis of colorectal cancer and predicts poor prognosis.

Annexin A9 (ANXA9), a member of annexin family, has been reported be associated with colorectal cancer (CRC) carcinogenesis. However, the clinical significance of ANXA9 in CRC, particularly its correlation to invasion and metastasis remains ambiguous. The aim of the present study was to investigate the significance of ANXA9 in CRC and understand the molecular mechanism of ANXA9 in CRC invasion and metastasis. Expression levels of the ANXA9 protein in CRC tissues were detected using immunohistochemistry (IHC), and the clinical and prognostic value of ANXA9 was investigated. ANXA9­siRNA was utilized to investigate the effect and molecular mechanism of ANXA9 in HCT116 cells. The IHC result demonstrated that the positivity rate of the ANXA9 protein in CRC tissue was significantly higher than that in adjacent mucosa (P<0.05), which was consistent with the western blot results. ANXA9 protein expression levels are associated with invasion depth and lymphatic metastasis. Furthermore, patients with ANXA9­positive expression demonstrated a poor prognosis and ANXA9 was an independent risk factor for survival (P<0.05). After inhibiting ANXA9 in HCT116 cells, the activity and metastatic and invasion capacity of cells decreased significantly, and expression levels of ADAM metallopeptidase domain 17 and matrix metallopeptidase 9 were significantly downregulated, while the expression levels of tissue inhibitors of metalloproteinases­1 and E­cadherin were upregulated (P<0.05). Thus, positive ANXA9 expression may present as a novel marker for predicting poor prognosis in CRC patients, and ANXA9 may promote the invasion and metastasis of CRC by regulating invasion and metastasis­associated genes.

Annexin A10 optimally differentiates between intrahepatic cholangiocarcinoma and hepatic metastases of pancreatic ductal adenocarcinoma: a comparative study of immunohistochemical markers and panels.

Discriminating intrahepatic cholangiocarcinoma (ICC) from hepatic metastases of pancreatic ductal adenocarcinoma (mPDAC) can be challenging. While pathologists might depend on clinical information regarding a primary tumor, their diagnosis will lead the patient either to potentially curative surgery (for ICC) or to palliation (for mPDAC). Beyond the validation of recently published potential biomarkers for PDAC (primary or metastatic) in a large cohort, we assessed diagnostic performance of the most promising candidates in the challenging task of discriminating metastatic PDAC (mPDAC) from ICC. In a training set of 87 ICC and 88 pPDAC, our previously identified biomarkers Annexin A1 (ANXA1), ANXA10, and ANXA13 were tested and compared with 11 published biomarkers or panels (MUCIN 1, Agrin, S100P, MUC5 AC, Laminin, VHL, CK 17, N-Cadherin, ELAC2, PODXL and HSPG2). Biomarkers with best results were further tested in an independent series of biopsies of 27 ICC and 36 mPDAC. Highest AUC values (between 0.72 and 0.84) for the discrimination between ICC and pPDAC were found in the training set for Annexin A1, Annexin A10, MUC5 AC, CK17, and N-Cadherin. These markers were further tested on an independent series of liver biopsies containing ICC or mPDAC. Diagnostic characteristics were evaluated for individual markers as well as for 3× panels. ANXA 10 showed the highest diagnostic potential of all single markers, correctly classifying 75% of mPDAC and 85% of ICC. Our results suggest that ANXA10 may be useful to differentiate between ICC and mPDAC, when only a tissue specimen is available.

Factors controlling permeability of the blood-brain barrier.

As the primary protective barrier for neurons in the brain, the blood-brain barrier (BBB) exists between the blood microcirculation system and the brain parenchyma. The normal BBB integrity is essential in protecting the brain from systemic toxins and maintaining the necessary level of nutrients and ions for neuronal function. This integrity is mediated by structural BBB components, such as tight junction proteins, integrins, annexins, and agrin, of a multicellular system including endothelial cells, astrocytes, pericytes, etc. BBB dysfunction is a significant contributor to the pathogeneses of a variety of brain disorders. Many signaling factors have been identified to be able to control BBB permeability through regulating the structural components. Among those signaling factors are inflammatory mediators, free radicals, vascular endothelial growth factor, matrix metalloproteinases, microRNAs, etc. In this review, we provide a summary of recent progress regarding these structural components and signaling factors, relating to their roles in various brain disorders. Attention is also devoted to recent research regarding impact of pharmacological agents such as isoflurane on BBB permeability and how iron ion passes across BBB. Hopefully, a better understanding of the factors controlling BBB permeability helps develop novel pharmacological interventions of BBB hyperpermeability under pathological conditions.

Annexin A10 expression in colorectal cancers with emphasis on the serrated neoplasia pathway.

AIM: To validate the utility of Annexin A10 as a surrogate marker of the serrated neoplasia pathway in invasive colorectal cancers (CRCs). METHODS: A total of 1133 primary CRC patients who underwent surgical resection at Seoul National University Hospital between January 2004 and December 2007 were enrolled. Expression of Annexin A10 was evaluated by immunohistochemistry using tissue microarray and paired to our findings on clinicopathologic and molecular characteristics of each individual. CpG island methylator phenotype was determined by MethyLight assay and microsatellite instability was determined by high performance liquid chromatography. KRAS and BRAF mutation status was evaluated by direct sequencing and allele-specific PCR. Univariate and stage-specific survival analyses were performed to reveal the prognostic value of Annexin A10 expression. RESULTS: Annexin A10 expression was observed in 66 (5.8%) of the 1133 patients. Annexin A10 expression was more commonly found in females and was associated with proximal location, ulcerative gross type, advanced T category, N category and TNM stage. CRCs with Annexin A10 expression showed an absence of luminal necrosis, luminal serration and mucin production. CRCs with Annexin A10 expression were associated with CpG island methylator phenotype, microsatellite instability and BRAF mutation. In survival analysis, Annexin A10 expression was associated with poor overall survival and progression-free survival, especially in stage IV CRCs. CONCLUSION: Annexin A10 expression is associated with poor clinical behavior and can be used a supportive surrogate marker of the serrated neoplasia pathway in invasive CRCs.

Effect of high-fat diet on hepatic proteomics of hamsters.

A high-fat diet contributes to the etiology of metabolic diseases. As the liver plays a crucial role in metabolism, an insight into the hepatic proteomics will help to illustrate the physiological effect of a high-fat diet. Fourteen nine-week old male Syrian hamsters were maintained on either control (C) or high-fat (HF) diets (0.2% cholesterol +22% fat) for 8 weeks. Hamsters were chosen because they show close similarity to human lipid metabolism. At the end of study, blood and livers were collected for analysis. Liver proteins were fractionated by electrophoresis, digested by trypsin, and then separated by label-free nano-LC/MS/MS. The TurboSequest algorithm was used to identify the peptide sequences against the hamster database in Universal Proteins Resource Knowledgebase (UniProt). The results indicate that 1191 hepatic proteins were identified and 135 of them were expressed differentially in the high-fat group (p < 0.05). Some of these 135 proteins that involve in metabolic diseases were further validated by Western blotting. The animals maintained on the high-fat diet had significantly (p < 0.05) higher serum triglyceride, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and uric acid. Animals consuming a high-fat diet also had significantly (p < 0.05) more accumulation of triglyceride and cholesterol in livers. Xanthine dehydrogenase (XDH), which plays an important role in uric acid synthesis, was up-regulated by the high-fat diet (p < 0.05). The α-subunit of hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (HADHA), which catalyzes the second and third reactions of ß-oxidation, was down-regulated by the high-fat diet (p < 0.05). Aconitate hydratase 2 (ACO2), which catalyzes the conversion of citrate to isocitrate in TCA cycle, was down-regulated in animals of the high-fat group (p < 0.05). Inflammatory markers annexin A3 (ANXA3) and annexin A5 (ANXA5) were up-regulated by the high-fat diet (p < 0.05). Moreover, enzymes involved in the urea cycle were suppressed by high-fat diet, including carbamoyl phosphate synthase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase 1 (ARG 1). Post-translational modifications (PTM) of ANXA3, ANXA5, and XDH were also analyzed. A set of differentially expressed proteins were identified as molecular markers for elucidating the pathological mechanism of high-fat diet.

Aberrant differentiation of urothelial cells in patients with ureteropelvic junction obstruction.

AIM: To investigate the urothelial changes in the pathogenesis of ureteropelvic junction obstruction (UPJ-O). METHODS: A total of 12 patients of UPJ-O were respectively studied. The expression of Annexin A7, Annexin A11, EGFR, Keratin 5, uroplakin III, and SMA in the urothelium of obstructed UPJ segment and of the normal ureter below the obstructed segment were determined by immunofluorescence. Transmission electron microscopy was used to determine the morphological changes in UPJ epithelium in compared to normal ureteral epithelium. RESULTS: We found that Annexin A7, Annexin A11, EGFR, Keratin 5, and SMA were upregulated, while uroplakin III was downregulated in the urothelium of UPJ-O patients. Furthermore, ultrastructural analyses showed that intercellular spaces between urothelial cells were dilated and the number of microvilli on superficial cells was increased in UPJ-O patients. CONCLUSIONS: We propose that a disrupted urothelial barrier in UPJ-O may results in urothelial inflammatory response and truncated differentiated urothelial cells, which may play an important role in the development and pathogenesis of UPJO.

Morphologic and molecular characterization of traditional serrated adenomas of the distal colon and rectum.

Of the serrated polyps, the origin, morphologic features, molecular alterations, and natural history of traditional serrated adenomas (TSAs) are the least understood. Recent studies suggest that these polyps may arise from precursor lesions. The frequencies of KRAS and BRAF mutations vary between these studies, and only 1 small study has measured CpG island methylation using current markers of methylation. Mutations in GNAS, a gene commonly mutated in colorectal villous adenomas, have not been fully evaluated in TSAs. Finally, the expression of annexin A10 (ANXA10), a recently discovered marker of sessile serrated adenomas/polyps, has not been studied in these polyps. To further characterize these polyps, 5 gastrointestinal pathologists reviewed 55 left-sided polyps diagnosed as TSA at a single institution. Pathologists assessed various histologic features including cytoplasmic eosinophilia, ectopic crypt foci, presence of conventional dysplasia, and presence of precursor serrated lesions. KRAS, BRAF, and GNAS mutational analysis was performed, as well as CpG island methylation and ANXA10 immunohistochemistry. Ectopic crypt foci were seen in 62% of TSAs. Precursor lesions were seen in 24% of the study polyps, most of which were hyperplastic polyps. KRAS and BRAF mutations were common and were present in 42% and 48% of polyps, respectively. GNAS mutations occurred in 8% of polyps, often in conjunction with a BRAF mutation. Unlike sessile serrated adenomas/polyps, TSAs rarely had diffuse expression of ANXA10. Importantly, BRAF-mutated TSAs had more widespread methylation of a 5-marker CpG island panel compared with KRAS-mutated polyps. However, ectopic crypt foci, a proposed defining feature of TSA, were not associated with any specific molecular alteration.

Immunohistochemistry for annexin A10 can distinguish sporadic from Lynch syndrome-associated microsatellite-unstable colorectal carcinoma.

Differentiating sporadic microsatellite-unstable colorectal carcinoma due to MLH1 promoter hypermethylation from Lynch syndrome (LS)-associated tumors due to mutations in mismatch-repair proteins is time consuming, cost intensive, and requires advanced laboratory testing. A mutation in BRAF has been shown to be highly specific for sporadic tumors; however, a significant proportion of sporadic microsatellite-unstable tumors lack BRAF mutations. MLH1 promoter methylation analysis is subsequently used to differentiate LS and sporadic tumors, but both tests require specialized laboratories and are costly. Through previous gene expression profiling of serrated polyps, we identified annexin A10 as a protein highly expressed in sessile serrated adenomas/polyps. As these polyps give rise to the majority of sporadic microsatellite-unstable tumors, we evaluated the ability of annexin A10 expression to discriminate between LS and sporadic tumors. A marked increase in annexin A10 mRNA was observed in sporadic microsatellite-unstable tumors compared with LS tumors (378-fold increase, P<0.001). Using immunohistochemistry, annexin A10 was expressed in 23/53 (43%) BRAF-mutated and 9/22 (41%) BRAF wild-type sporadic tumors. In contrast, only 3/56 (5%) LS tumors were positive for annexin A10 (P<0.0001). One patient had a deleterious MSH2 mutation, and another had a variant of uncertain significance in MSH6. These 2 tumors could be easily distinguished from sporadic tumors using mismatch-repair protein immunohistochemistry. Only 1/28 (4%) LS tumors with loss of MLH1 was positive for annexin A10. This patient did not have a deleterious MLH1 mutation but rather germline promoter hypermethylation of MLH1. On the basis of these results, immunohistochemistry for annexin A10 may be a useful marker to distinguish sporadic from LS-associated microsatellite-unstable colon cancer.

The tumor suppressor annexin A10 is a novel component of nuclear paraspeckles.

Annexin A10 is the latest identified member of the annexin family of Ca(2+)- and phospholipid-binding proteins. In previous studies, downregulation of annexin A10 was correlated with dedifferentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role. However, the biochemical characteristics and functions of annexin A10 remain unknown. We show that annexin A10 displays biochemical characteristics atypical for an annexin, indicating a Ca(2+)- and membrane-binding-independent function. Annexin A10 co-localizes with the mRNA-binding proteins SFPQ and PSPC1 at paraspeckles, an only recently discovered nuclear body, and decreases paraspeckle numbers when overexpressed in HeLa cells. In addition, annexin A10 relocates to dark perinucleolar caps upon transcriptional inhibition of RNA polymerase II. We mapped the cap-binding function of annexin A10 to the proximal part of the core domain, which is missing in the short isoform of annexin A10, and show its independence from the remaining functional type II Ca(2+)-binding site. In contrast to this, paraspeckle recruitment required additional core regions and was negatively affected by the mutation of the last type II Ca(2+)-binding site. Additionally, we show that overexpression of annexin A10 in HeLa cells increases their sensitivity to apoptosis and reduces colony formation. The identification of unique nuclear and biochemical characteristics of annexin A10 points towards its membrane-independent role in paraspeckle-associated mRNA regulation or processing.

Proteomic profiling of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy reveals a drastic decrease in laminin, nidogen and annexin.

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large-scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X-linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age-related alterations in the proteome of dystrophin-deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC-MS/MS. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild-type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy.

Gene expression profiling of serrated polyps identifies annexin A10 as a marker of a sessile serrated adenoma/polyp.

Sessile serrated adenomas/polyps (SSA/Ps) are precursors of colon cancer, particularly those that exhibit microsatellite instability. Distinguishing SSA/Ps from the related, but innocuous, microvesicular hyperplastic polyp (MVHP) can be challenging. In this study seven gastrointestinal pathologists reviewed 109 serrated polyps and identified 60 polyps with histological consensus. Microarray analysis was performed on six distal consensus MVHPs < 9 mm, six proximal consensus SSA/Ps > 9 mm, and six normal colon biopsies (three proximal, three distal). Comparative gene expression analysis confirmed the close relationship between SSA/Ps and MVHPs as there was overlapping expression of many genes. However, the gene expression profile in SSA/Ps had stronger and more numerous associations with cancer-related genes compared with MVHPs. Three genes (TFF2, FABP6, and ANXA10) were identified as candidates whose expression can differentiate SSA/Ps from MVHPs, and the differences in expression were confirmed by quantitative RT-PCR. As ANXA10 showed the most promise in differentiating these polyps, the expression of ANXA10 was evaluated by immunohistochemistry in consensus SSA/Ps (n = 26), MVHPs (n = 21), and normal colon (n = 9). Immunohistochemical expression of ANXA10 was not identified in separate samples of normal colon or in the normal colonic epithelium adjacent to the serrated polyps. Consistent with the microarray and quantitative RT-PCR experiments, immunohistochemical expression of ANXA10 was markedly increased in SSA/Ps compared to MVHPs (p < 0.0001). An ANXA10 score ≥ 3 has a sensitivity of 73% and a specificity of 95% in the diagnosis of an SSA/P. In conclusion, we show that SSA/Ps and MVHPs have significant overlap in gene expression, but also important differences, particularly in cancer-related pathways. Expression of ANXA10 may be a potential marker of the serrated pathway to colon cancer.

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters. METHODS: HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay. RESULTS: In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids. CONCLUSIONS: MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.