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Background: In the fight against GBM, drug repurposing emerges as a viable and time-saving approach to explore new treatment options. Chlorpromazine, an old antipsychotic medication, has recently arisen as a promising candidate for repositioning in GBM therapy in addition to temozolomide, the first-line standard of care. We previously demonstrated the antitumor efficacy of chlorpromazine and its synergistic effects with temozolomide in suppressing GBM cell malignant features in vitro. This prompted us to accomplish a Phase II clinical trial to evaluate the efficacy and safety of adding chlorpromazine to temozolomide in GBM patients with unmethylated MGMT gene promoter. In this in vitro study, we investigate the potential role of chlorpromazine in overcoming temozolomide resistance. Methods: In our experimental set, we analyzed Connexin-43 expression at both the transcriptional and protein levels in control- and chlorpromazine-treated GBM cells. DNA damage and subsequent repair were assessed by immunofluorescence of γ-H2AX and Reverse-Phase Protein microArrays in chlorpromazine treated GBM cell lines. To elucidate the relationship between DNA repair systems and chemoresistance, we analyzed a signature of DNA repair genes in GBM cells after treatment with chlorpromazine, temozolomide and Connexin-43 downregulation. Results: Chlorpromazine treatment significantly downregulated connexin-43 expression in GBM cells, consequently compromising connexin-dependent cellular resilience, and ultimately contributing to cell death. In line with this, we observed concordant post-translational modifications of molecular determinants involved in DNA damage and repair pathways. Our evaluation of DNA repair genes revealed that temozolomide elicited an increase, while chlorpromazine, as well as connexin-43 silencing, a decrease in DNA repair gene expression in GBM cells. Conclusions: Chlorpromazine potentiates the cytotoxic effects of the alkylating agent temozolomide through a mechanism involving downregulation of Cx43 expression and disruption of the cell cycle arrest essential for DNA repair processes. This finding suggests that chlorpromazine may be a potential therapeutic strategy to overcome TMZ resistance in GBM cells by inhibiting their DNA repair mechanisms. [ABSTRACT FROM AUTHOR]

MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211–RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis.

In recent years many articles have underlined the key role of nanovesicles, i.e., exosomes, as information carriers among biological systems including cancer. Tumor-derived exosomes (TEXs) are key players in the dynamic crosstalk between cancer cells and the microenvironment while promote immune system control evasion. In fact, tumors are undoubtedly capable of silencing the immune response through multiple mechanisms, including the release of exosomes. TEXs have been shown to boost tumor growth and promote progression and metastatic spreading via suppression or stimulation of the immune response towards cancer cells. The advantage of immunotherapeutic treatment alone over combining immuno- and conventional therapy is currently debated. Understanding the role of tumor exosome-cargo is of crucial importance for our full comprehension of neoplastic immonosuppression and for the construction of novel therapies and vaccines based on (nano-) vesicles. Furthermore, to devise new anti-cancer approaches, diverse groups investigated the possibility of engineering TEXs by conditioning cancer cells' own cargo. In this review, we summarize the state of art of TEX-based immunomodulation with a particular focus on the molecular function of non-coding family genes, microRNAs. Finally, we will report on recent efforts in the study of potential applications of engineered exosomes in cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma Multiforme is the deadliest type of brain tumor and is characterized by very poor prognosis with a limited overall survival. Current optimal therapeutic approach has essentially remained unchanged for more than a decade, consisting in maximal surgical resection followed by radiotherapy plus temozolomide. Main body: Such a dismal patient outcome represents a compelling need for innovative and effective therapeutic approaches. Given the development of new drugs is a process presently characterized by an immense increase in costs and development time, drug repositioning, finding new uses for existing approved drugs or drug repurposing, re-use of old drugs when novel molecular findings make them attractive again, are gaining significance in clinical pharmacology, since it allows faster and less expensive delivery of potentially useful drugs from the bench to the bedside. This is quite evident in glioblastoma, where a number of old drugs is now considered for clinical use, often in association with the first-line therapeutic intervention. Interestingly, most of these medications are, or have been, widely employed for decades in non-neoplastic pathologies without relevant side effects. Now, the refinement of their molecular mechanism(s) of action through up-to-date technologies is paving the way for their use in the therapeutic approach of glioblastoma as well as other cancer types. Short conclusion: The spiraling costs of new antineoplastic drugs and the long time required for them to reach the market demands a profoundly different approach to keep lifesaving therapies affordable for cancer patients. In this context, repurposing can represent a relatively inexpensive, safe and fast approach to glioblastoma treatment. To this end, pros and cons must be accurately considered. [ABSTRACT FROM AUTHOR]

We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer. [ABSTRACT FROM AUTHOR]

The article focuses on Galectin-3 (Gal-3) and its role in muscle-to-bone crosstalk, demonstrating that Gal-3 is released from myoblast cells during differentiation into myotubes and interferes with early bone formation processes, including signaling pathways and extracellular matrix interactions.

The article offers information on effects of dynamic shear stress on the biological properties of pericyte-like cells from human dental pulp in an inflammatory micro environment. Topics include information on low immunogenicity and immunomodulatory/anti-inflammatory properties; how they regulate numerous functions including vessel growth; and pericytes localized in the head, dental pulp, thymus and the outflow tract of the aorta.

Background Spinocerebellar ataxia type 1 (SCA1) is a genetic disorder characterized by severe ataxia associated with progressive loss of cerebellar Purkinje cells. The mGlu1 metabotropic glutamate receptor plays a key role in mechanisms of activity-dependent synaptic plasticity in the cerebellum, and its dysfunction is linked to the pathophysiology of motor symptoms associated with SCA1. We used SCA1 heterozygous transgenic mice (Q154/Q2) as a model for testing the hypothesis that drugs that enhance mGlu1 receptor function may be good candidates for the medical treatment of SCA1. Results Symptomatic 30-week old SCA1 mice showed reduced mGlu1 receptor mRNA and protein levels in the cerebellum. Interestingly, these mice also showed an intense expression of mGlu5 receptors in cerebellar Purkinje cells, which normally lack these receptors. Systemic treatment of SCA1 mice with the mGlu1 receptor positive allosteric modulator (PAM), Ro0711401 (10 mg/kg, s.c.), caused a prolonged improvement of motor performance on the rotarod and the paw-print tests. A single injection of Ro0711401 improved motor symptoms for several days, and no tolerance developed to the drug. In contrast, the mGlu5 receptor PAM, VU0360172 (10 mg/kg, s.c.), caused only a short-lasting improvement of motor symptoms, whereas the mGlu1 receptor antagonist, JNJ16259685 (2.5 mg/kg, i.p.), further impaired motor performance in SCA1 mice. The prolonged symptomatic benefit caused by Ro0711401 outlasted the time of drug clearance from the cerebellum, and was associated with neuroadaptive changes in the cerebellum, such as a striking reduction of the ectopically expressed mGlu5 receptors in Purkinje cells, increases in levels of total and Ser880 phosphorylated GluA2 subunit of AMPA receptors, and changes in the length of spines in the distal dendrites of Purkinje cells. Conclusions These data demonstrate that pharmacological enhancement of mGlu1 receptors causes a robust and sustained motor improvement in SCA1 mice, and lay the groundwork for the development of mGlu1 receptor PAMs as novel "cerebellum-specific", effective, and safe symptomatic drugs for the treatment of SCA1 in humans. [ABSTRACT FROM AUTHOR]

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway. [ABSTRACT FROM AUTHOR]

Background: Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome. Methodology/Principal Findings: We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as "tumor spheres" and as "monolayers" in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells. Conclusions/Significance: EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients. [ABSTRACT FROM AUTHOR]

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146+ MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions. J. Cell. Physiol. 227: 3291-3300, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Tumor-initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor-initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo-like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer-initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133+ colon cancer cells leading to complete growth arrest of colon cancer stem cell-derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133+ cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor-initiating cells. Quiescent CD133+ cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor-initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer. S tem C ells 2012;30:1819-1830 [ABSTRACT FROM AUTHOR]

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Kinases are important drug targets, as such kinase network information could become the basis for development of therapeutic strategies for improving treatment outcome. An urgent clinical goal is to identify functionally important molecular networks associated with subpopulations of patients that may not respond to conventional combination chemotherapy. Reverse-phase protein microarrays are a technology platform designed for quantitative, multiplexed analysis of specific phosphorylated, cleaved, or total (phosphorylated and nonphosphorylated) forms of cellular proteins from a limited amount of sample. This class of microarray can be used to interrogate cellular samples, serum or body fluids. This review focuses on the application of reverse-phase protein microarrays for translational research and therapeutic drug target discovery. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining 'classic' gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays. [ABSTRACT FROM AUTHOR]

Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer.We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1-/- mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses. [ABSTRACT FROM AUTHOR]

: Study objectiveTo evaluate the potential of compounds commonly used in anesthesia practice to affect the intracellular oxidant-antioxidant homeostasis of peripheral blood lymphocytes at clinically relevant concentrations; and to study the changes in reactive oxygen species production and measure the mitochondrial glutathione content.: DesignProspective, in vitro study.: SettingExperimental medical research laboratory at a University Hospital.: MeasurementsLymphocytes were isolated from the peripheral blood of 15 healthy donors and incubated for 12 hours at 37°C with the following drug concentrations: thiopental sodium 20 mmoL/mL, droperidol 130 μmol/mL, propofol 60 mmoL/mL, and succinylcholine 17 mmoL/mL. Reactive oxygen species (ROS) generation was determined by hydroethidine and 2′,7′-dichlorofluorescein diacetate methods. Mitochondrial glutathione level was assessed using monobromobimane staining.: Measurements and main resultsThiopental-treated lymphocytes exhibited an overgeneration of ROS, but no change was detected in mitochondrial glutathione quantity. Propofol and droperidol could not induce any perturbative effect on the oxidative state of T cells, whereas succinylcholine was found to markedly affect lymphocyte oxidative state both by impairing glutathione content and promoting exaggerated production of ROS.: ConclusionDrugs commonly used in anesthesia practice may significantly alter the oxidative state of peripheral T cells. This mechanism could contribute to the immune suppression that occurs transiently in the early postoperative period. [Copyright &y& Elsevier]

The scenario of proteogenomics is rapidly evolving and novel technologies are enabling comprehensive molecular exploration down to single cells. Likewise, digital (immuno-)assays are revolutionizing the field of biomarker detection and have reached the grade for population-level screenings with single-molecule sensitivity. Nonetheless, cost- and time-effective, high-throughput targeted phospho-proteomics at a preclinical stage still relies on ad hoc microarray platforms, such as the Reverse-Phase Protein microArrays (RPPA). Although this technique requires specific knowledge and equipment and different laboratories worldwide have implemented alternative methodological strategies, the application of RPPA to biomarker discovery has proven successful on diverse types of samples, including tissues and biological fluids as well as nanovesicles and in vitro cultured lines. Among these, cancer stem(-like) cells (CSC) represent an ideal experimental model system for preclinical discovery and definition of novel drug targets. The present methodological article provides the basic knowledge and steps on how to deploy an RPPA analysis with specific reference to an ideal experimental setup of drug testing on CSC., Competing Interests: Disclosures Authors have no conflicts of interest to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target., (Copyright © 2017 Elsevier Inc. All rights reserved.)