Prototype Salivary Assay for Quantification of Two Biomarkers for In Vitro Diagnosis of Endometriosis.

Endometriosis, a very common disease in women, is characterized by endometrial structures outside the uterine cavity. The lack of a reliable noninvasive diagnostic test and the often nonspecific symptoms of this pathology are responsible for the delay in definitive diagnosis of this disease. Recently, through a proteomics approach, our research group has identified two potential diagnostic markers for endometriosis in serum (Zn-alpha2-glycoprotein and complement C3 precursor). In this article, we describe the experimental conditions of a simple ELISA for rapid quantification of these two biomarkers in the saliva of patients with endometriosis. Finally, preliminary experiments on a small cohort of patients and controls have confirmed the potential diagnostic value of this assay.

Immunohistochemical expression of HMGB1 and related proteins in the skin as a possible tool for determining post-mortem interval: a preclinical study.

Determining the post-mortem interval (PMI) is one of forensic pathology's primary objectives and one of its most challenging tasks. Numerous studies have demonstrated the accuracy of histomorphology and immunohistochemical investigations in determining the time of death. Nevertheless, the skin, a robust and easy-to-remove tissue, has only been partially analyzed so far. By studying 20 adult male mice, we tried to determine whether post-mortem immunohistochemical detection in the skin of HMGB1 proteins and associated components (Beclin1 and RAGE) could be used for this purpose. We discovered that nuclear HMGB1 overexpression indicates that death occurred within the previous 12 h, nuclear HMGB1 negativization with high cytoplasmic HMGB1 intensity indicates that death occurred between 12 and 36 h earlier and cytoplasmic HMGB1 negativization indicates that more than 48 h have passed since death. RAGE and Beclin1 levels in the cytoplasm also decreased with time. The latter proteins' negativization might indicate that more than 24 and 36 h, respectively, have passed from the time of death. These indicators might potentially be helpful in forensic practice for determining the PMI using immunohistochemistry.

Adenogenesis Factors FGF7, FGF10, FGF23, IFN-τ and HGF in Endometriosis Tissue Respect to Eutopic Endometrium: An Immunohistochemical Study.

Endometriosis is a pathological condition defined by the occurrence of endometrial glandular and stromal structures in anatomical compartments different from the uterine cavity. Endometriosis is a genetic polymorphism, estrogen-dependent inflammatory disease. This very common pathological entity causes a high level of morbidity in patients; it is also considered one of the most important causes of infertility. We and others have proposed as a pathogenetic mechanism of endometriosis a modification in the fine tuning of the processes of organogenesis of the uterus. We have correlated the immunohistochemical expression in deep endometriotic lesions and in normal endometrial tissue of several molecular factors that are implicated in the embryonic development of the uterine glands. We noticed a significant higher expression both for epithelium and stroma in the controls respect to the endometriosis samples for FGF7, FGF-10 and HGF. Interestingly, regarding FGF-23 and IFN-τ, we observed a significant higher expression in the ectopic endometrial stroma compared to the eutopic endometrium, while thepithetlium expression did not display a significant differential expression in endometriosis tissues respect to normal endometrium. The data generated support the fact that endometriosis tissues, both the epithelial and stromal component, have a different phenotype respect to the eutopic endometrium and sustain the hypothesis that alterations in the molecular mechanisms in control for adenogenesis and survival of endometrial structures are linked to the genesis and survival of endometriosis lesions outside of the uterus.

Differential Expression in Endometriosis Tissue versus Endometrium of the Uterine Adenogenesis Factors PRL-R, GH, IGF1, and IGF2.

Endometriosis is characterized by the presence of endometrial glandular and stromal structures outside the uterine cavity. It is an inflammatory estrogen dependent disease characterized by gene polymorphisms. This is a very frequent pathology and represents one of the most important causes of infertility, as well as having an important level of morbidity in patients. Recently, an alteration of the processes of organogenesis of the uterus has been proposed as a pathogenetic mechanism of endometriosis. In this article we have compared the expression in deep endometriotic lesions and in normal endometrial tissue of some of the molecular factors known to be involved in the embryonic development of the uterine glands. In detail, we found by immunohistochemistry a significant higher expression both for epithelium and stroma in the controls respect to the endometriosis samples for insulin growth factor 1 (IGF1) and IGF2, whereas for the prolactin receptor (PRL-R), this result was detected only for the epithelium. On the other hand, we found for growth hormone (GH) a significant higher expression in the epithelium of endometriosis samples respect to the controls. The correlation data generated can give indications on some of the molecular mechanisms responsible for the adenogenesis and survival of endometriosis structures outside of the uterus.

Electrochemotherapy in Translational Medicine: From Veterinary Experience to Human Oncology.

Electrochemotherapy (ECT) is a tumor treatment that, through the application of electric pulses with suitable amplitude and waveforms, favors the systemic or local delivery of chemotherapy agents. This procedure significantly increases the permeability of cancer cells to anticancer drugs, making them more effective and allowing their use at lower doses with less morbidity for patients. Its use in veterinary oncology is consolidated and it is currently adopted as first-line treatment for different cancers with successful results. In human oncology, ECT use is mainly in the treatment of cutaneous tumors and for the palliation of cutaneous metastases of malignant tumors. A standard operating procedure has been formulated. Currently, several preclinical and phase I and II studies are under way involving various cancers in humans to better define the efficacy and tolerability of this therapy. This review summarizes the state of the art of ECT in veterinary and human oncology, describing the most significant results achieved to date.

Novel insights into the BAP1-inactivated melanocytic tumor.

BAP1-inactivated melanocytic tumor (BIMT) is a group of melanocytic neoplasms with epithelioid cell morphology molecularly characterized by the loss of function of BAP1, a tumor suppressor gene located on chromosome 3p21, and a mutually exclusive mitogenic driver mutation, more commonly BRAF. BIMTs can occur as a sporadic lesion or, less commonly, in the setting of an autosomal dominant cancer susceptibility syndrome caused by a BAP1 germline inactivating mutation. Owing to the frequent identification of remnants of a conventional nevus, BIMTs are currently classified within the group of combined melanocytic nevi. "Pure" lesions can also be observed. We studied 50 BIMTs from 36 patients. Most lesions were composed of epithelioid melanocytes of varying size and shapes, resulting extreme cytomorphological heterogeneity. Several distinctive morphological variants of multinucleated/giant cells were identified. Some hitherto underrecognized microscopic features, especially regarding nuclear characteristics included nuclear blebbing, nuclear budding, micronuclei, shadow nuclei, peculiar cytoplasmic projections (ant-bear cells) often containing micronuclei and cell-in-cell structures (entosis). In addition, there were mixed nests of conventional and BAP1-inactivated melanocytes and squeezed remnants of the original nevus. Of the 26 lesions studied, 24 yielded a BRAF mutation, while in the remaining two cases there was a RAF1 fusion. BAP1 biallelic and singe allele mutations were found in 4/22 and 16/24 neoplasms, respectively. In five patients, there was a BAP1 germline mutation. Six novel, previously unreported BAP1 mutations have been identified. BAP1 heterozygous loss was detected in 11/22 lesions. Fluorescence in situ hybridization for copy number changes revealed a related amplification of both RREB1 and MYC genes in one tumor, whereas the remaining 20 lesions studied were negative; no TERT-p mutation was found in 14 studied neoplasms. Tetraploidy was identified in 5/21 BIMTs. Of the 21 patients with available follow-up, only one child had a locoregional lymph node metastasis. Our results support a progression of BIMTs from a conventional BRAF mutated in which the original nevus is gradually replaced by epithelioid BAP1-inactivated melanocytes. Some features suggest more complex underlying pathophysiological events that need to be elucidated.

The abundance of the long intergenic non-coding RNA 01087 differentiates between luminal and triple-negative breast cancers and predicts patient outcome.

The molecular complexity of human breast cancer (BC) renders the clinical management of the disease challenging. Long non-coding RNAs (lncRNAs) are promising biomarkers for BC patient stratification, early detection, and disease monitoring. Here, we identified the involvement of the long intergenic non-coding RNA 01087 (LINC01087) in breast oncogenesis. LINC01087 appeared significantly downregulated in triple-negative BCs (TNBCs) and upregulated in the luminal BC subtypes in comparison to mammary samples from cancer-free women and matched normal cancer pairs. Interestingly, deregulation of LINC01087 allowed to accurately distinguish between luminal and TNBC specimens, independently of the clinicopathological parameters, and of the histological and TP53 or BRCA1/2 mutational status. Moreover, increased expression of LINC01087 predicted a better prognosis in luminal BCs, while TNBC tumors that harbored lower levels of LINC01087 were associated with reduced relapse-free survival. Furthermore, bioinformatics analyses were performed on TNBC and luminal BC samples and suggested that the putative tumor suppressor activity of LINC01087 may rely on interferences with pathways involved in cell survival, proliferation, adhesion, invasion, inflammation and drug sensitivity. Altogether, these data suggest that the assessment of LINC01087 deregulation could represent a novel, specific and promising biomarker not only for the diagnosis and prognosis of luminal BC subtypes and TNBCs, but also as a predictive biomarker of pharmacological interventions.

Curcumin as an Anticancer Agent in Malignant Mesothelioma: A Review.

Malignant mesothelioma is an infrequent tumor that initiates from the mesothelial cells lining of body cavities. The great majority of mesotheliomas originate in the pleural cavity, while the remaining cases initiate in the peritoneal cavity, in the pericardial cavity or on the tunica vaginalis. Usually, mesotheliomas grow in a diffuse pattern and tend to enclose and compress the organs in the various body cavities. Mesothelioma incidence is increasing worldwide and still today, the prognosis is very poor, with a reported median survival of approximately one year from presentation. Thus, the development of alternative and more effective therapies is currently an urgent requirement. The aim of this review article was to describe recent findings about the anti-cancer activity of curcumin and some of its derivatives on mesotheliomas. The potential clinical implications of these findings are discussed.

Definition of a Novel Plasmid-Based Gene Transfection Protocol of Mammalian Skeletal Muscles by Means of In Vivo Electroporation.

We describe an original electroporation protocol for in vivo plasmid DNA transfection. The right hind limbs of C57 mice are exposed to a specifically designed train of permeabilizing electric pulses by transcutaneous application of tailored needle electrodes, immediately after the injection of pEGFP-C1 plasmid encoding GFP (Green Fluorescente Protein). The electroporated rodents show a greater GFP expression than the controls at three different time points (4, 10, and 15 days). The electroporated muscles display only mild interstitial myositis, with a significant increase in inflammatory cell infiltrates. Finally, mild gait abnormalities are registered in electroporated mice only in the first 48 h after the treatment. This protocol has proven to be highly efficient in terms of expression levels of the construct, is easy to apply since it does not require surgical exposure of the muscle and is well tolerated by the animals because it does not cause evident morphological and functional damage to the electroporated muscle.

Helper-dependent adenovirus-mediated gene transfer of a secreted LDL receptor/transferrin chimeric protein reduces aortic atherosclerosis in LDL receptor-deficient mice.

Familial hypercholesterolemia (FH) is a genetic hyperlipidemia characterized by elevated concentrations of plasma LDL cholesterol. Statins are not always effective for the treatment of FH patients; unresponsive patients have poor prognosis and rely on LDL apheresis. In the past, we developed safe and effective gene therapy strategies for the expression of anti-atherogenic proteins using PEGylated helper-dependent adenoviral (HD-Ad) vectors. We recently developed a HD-Ad vector for the expression of the soluble form of the extracellular portion of the human LDL receptor (LDLR) fused with a rabbit transferrin dimer (LDLR-TF). We evaluated the efficacy of the LDLR-TF chimeric protein  in CHOLDLA7, a cell line lacking LDLR expression, restoring the ability to uptake LDL. Subsequently, we administered intravenously 1 × 10E13 vp/kg of this vector in LDLR-deficient mice and observed amelioration of lipid profile and reduction of aortic atherosclerosis. Finally, we studied LDL distribution after HD-Ad vector-mediated expression of LDLR-TF in LDLR-deficient mice and found LDL accumulation in liver, and in heart and intestine. These results support the possibility of lowering LDL-C levels and reducing aortic atherosclerosis using a secreted therapeutic transgene; the present strategy potentially can be modified and adapted to non-systemic gene transfer with expression of the secreted chimeric protein in muscle or other tissues. Intramuscular or local administration strategies could improve the safety profile of this strategy and facilitate applicability.

Phenotypic effects of chronic and acute use of methiopropamine in a mouse model.

Methiopropamine (MPA) is a structural analogue of methamphetamine and belongs to the category of the novel psychoactive substances. To the best of our knowledge, no experimental study has been performed to evaluate the organ damage evoked by MPA administration in an animal model. Therefore, the main purpose of the present study was to investigate the histological changes in CD-1 male mice following the chronic administration of MPA. MPA-chronically treated mice showed myocardial damage with features consistent with repeated episodes of ischemia and a pattern of kidney damage and gastrointestinal ischemia, with ischemic-necrotic lesions of variable extent. In agreement with the analogies between MPA and methamphetamine, we link organ damage secondary to MPA administration to the vasoconstrictive effect exhibited by both compounds. Chronically MPA-treated mice did not show changes in body weight, food intake, thermoregulation, muscular strength and motor coordination in the accelerod test. However, acute MPA administration significantly increased their heart rate and promoted vasoconstriction, which were associated with the sudden death of a subset of animals (40% of all chronically treated mice). In conclusion, the present study demonstrates that MPA consumption could induce health hazards, highlighting the risk of sudden catastrophic events; therefore, clinicians should be aware of these data and consider MPA screening when no other drug is identified by a urine drug screen.

Enzymatic and Biological Characterization of Novel Sirtuin Modulators against Cancer.

Sirtuins, a family of nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylases, are promising targets for anticancer treatment. Recently, we characterized a novel pan-sirtuin (SIRT) inhibitor, MC2494, displaying antiproliferative effects and able to induce death pathways in several human cancer cell lines and decrease tumor growth in vivo. Based on the chemical scaffold of MC2494, and by applying a structure-activity relationship approach, we developed a small library of derivative compounds and extensively analyzed their enzymatic action at cellular level as well as their ability to induce cell death. We also investigated the effect of MC2494 on regulation of cell cycle progression in different cancer cell lines. Our investigations indicated that chemical substitutions applied to MC2494 scaffold did not confer higher efficacy in terms of biological activity and SIRT1 inhibition, but carbethoxy-containing derivatives showed higher SIRT2 specificity. The carbethoxy derivative of MC2494 and its 2-methyl analog displayed the strongest enzymatic activity. Applied chemical modifications improved the enzymatic selectivity of these SIRT inhibitors. Additionally, the observed activity of MC2494 via cell cycle and apoptotic regulation and inhibition of cell migration supports the potential role of SIRTs as targets in tumorigenesis and makes SIRT-targeting molecules good candidates for novel pharmacological approaches in personalized medicine.

Novel dietary supplement association reduces symptoms in endometriosis patients.

Endometriosis is characterized by disabling symptoms that afflict young women with severe physical discomfort, difficulty in relationship life, and infertility; however, the currently available therapeutic strategies are unsatisfactory. Goal of this research was to identify a new combination of natural active ingredients that, administered as dietary supplements, could have the effect of reducing inflammatory response in endometriosis patients, decreasing the symptoms the disease produces and its harmful effects on affected organs. A cohort of endometriosis patient was treated for 3 months with a composition including quercitin, curcumin, parthenium, nicotinamide, 5-methyltetrahydrofolate, and omega 3/6. Using a VAS scale, we demonstrated a significant reduction of the symptoms in endometriosis patients treated with the dietary composition respect to the controls. Moreover, we demonstrated also a significant reduction in the serum levels of PGE2 and CA-125. Further study are required to compare the effect of this combination of molecules with standard therapies and to evaluate if the use of these dietary supplements in combination with standard therapies may lead to the improvement of the regular medical treatment for endometriosis.

Novel Instruments for the Implementation of Electrochemotherapy Protocols: From Bench Side to Veterinary Clinic.

Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). In the past 10 years, the clinical efficacy of this therapeutic approach in several spontaneous models of tumors in animals has been shown. Moreover, some of the molecular and cellular mechanisms responsible for this phenomenon have been elucidated. Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols, and their translation to humans. J. Cell. Physiol. 232: 490-495, 2017. © 2016 Wiley Periodicals, Inc.

Human equilibrative nucleoside transporter 1 gene expression is associated with gemcitabine efficacy in advanced leiomyosarcoma and angiosarcoma.

BACKGROUND: The expression of human equilibrative nucleoside transporter 1 (hENT1), the major gemcitabine transporter into cells, has been thoroughly investigated as a predictive marker of response to gemcitabine in pancreatic cancer and biliary tract cancers. Since gemcitabine is widely used in the treatment of leiomyosarcoma and angiosarcoma, we investigated the correlation between hENT1 expression and gemcitabine efficacy in these sarcoma subtypes. METHODS: We retrospectively identified 71 patients affected by advanced angiosarcoma (26) or leiomyosarcoma (45) treated within five Italian referral centres for sarcoma; among them, 49 patients (15 angiosarcoma, 34 leiomyosarcoma) were treated with gemcitabine. All tumour samples were analysed for hENT1 expression by real-time PCR. Median 2-ΔCt value was used as the cutoff to dichotomise patients into 'high' expression and 'low' expression groups. Kaplan-Meier analysis was performed to estimate progression-free survival (PFS) and overall survival (OS). RESULTS: We found a significant association between high hENT1 expression levels and favourable outcome in terms of PFS and OS compared to cases with low hENT1 expression in leiomyosarcoma treated with gemcitabine (PFS: 6.8 vs 3.2 months, P=0.004; OS: 14.9 vs 8.5 months, P=0.007). In addition, hENT1 overexpression correlated with a significant improvement in PFS (9.3 vs 4.5 months; P=0.02) and OS (20.6 vs 10.8 months; P=0.001) in angiosarcoma patients treated with gemcitabine. CONCLUSIONS: Our study suggests that higher hENT1 expression are associated to gemcitabine efficacy both in patients with advanced leiomyosarcoma and angiosarcoma.

Prototype of Multiplex Bead Assay for Quantification of Three Serum Biomarkers for In Vitro Diagnosis of Endometriosis.

Endometriosis is a very common disease, affecting 10% of women in the reproductive age. To date, a significant delay between onset of the symptoms and definitive diagnosis is caused by the lack of a reliable non-invasive diagnostic test. Recently, the potential value as diagnostic markers for endometriosis of three proteins (Zn-alpha2-glycoprotein, serum albumin, and complement C3 precursor), has been showed. In this article, we have defined the experimental conditions for the development of a multiplex bead array assay for rapid and simultaneous quantification of these three biomarkers in the serum of patients with endometriosis. Finally, pivotal experiments on a small cohort of patients have confirmed the diagnostic value of this assay. J. Cell. Physiol. 231: 2622-2627, 2016. © 2016 Wiley Periodicals, Inc.

Epigenetic silencing of serine protease HTRA1 drives polyploidy.

BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.

Non-metastatic 2 (NME2)-mediated suppression of lung cancer metastasis involves transcriptional regulation of key cell adhesion factor vinculin.

Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far, two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n=382), survival data (n=530) and lymph node metastases (n=100) in lung cancer patients identified non-metastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling. Importantly, we detected binding of NME2 in promoter of focal adhesion factor, vinculin. Reduced expression of NME2 led to enhanced transcription of vinculin. In comparison, NME1, a close homolog of NME2, did not bind to vinculin promoter nor regulate its expression. In line, enhanced metastasis of NME2-depleted lung cancer cells was found in zebrafish and nude mice tumor models. The metastatic potential of NME2-depleted cells was remarkably diminished upon selective RNA-i-mediated silencing of vinculin. Together, we demonstrate that reduced NME2 levels lead to transcriptional de-repression of vinculin and regulate lung cancer metastasis.