Biomarkers of traumatic brain injury in vitreous humor: A pilot study.

BACKGROUND: Traumatic brain injury (TBI) is one of the major causes of morbidity and mortality worldwide. The patients' and injuries' heterogeneity associated with TBI, alongside with its variable clinical manifestations, make it challenging to make diagnosis and predict prognosis. Therefore, the identification of reliable prognostic markers would be relevant both to support clinical decision-making and forensic evaluation of polytraumatic deaths and cases of medical malpractice. This pilot study aimed to evaluate some of the main biomarkers specific for brain damage in sTBI and mmTBI deaths in samples of vitreous humor (VH) in order to verify whether predictors of prognosis in TBI can be found in this matrix. METHODS: VH were obtained from both eyes (right and left) of 30 cadavers (20 sTBI and 10 mmTBI) and analysed. These factors were evaluated: NSE (neuron-specific enolase), S100 calcium-binding protein (S100), glial fibrillary acidic protein (GFAP), Brain-derived neurotrophic factor (BDNF), Copeptin, Interleukin 6 (IL-6), Ferritin, Lactate dehydrogenase (LDH), C-Reactive Protein (CRP), Procalcitonin (PCT), Glucose and Neutrophil gelatinase-associated lipocalin (N-Gal). RESULTS: Four of the analysed proteins (LDH, ferritin, S100 and NSE) proved to be particularly promising. In particular, logistic regression analysis found a good discriminatory power. CONCLUSIONS: Given the peculiarity of the matrix and the poor standardization of the sampling, such promising results need to be furtherly investigated in serum before being implemented in the forensic practice.

A microRNA profile of pediatric glioblastoma: The role of NUCKS1 upregulation.

MicroRNAs (miRNAs/miRs) are a novel class of gene regulators that may be involved in tumor chemoresistance. Recently, specific miRNA expression profiles have been identified in adult glioblastoma (aGBM), but there are only limited data available on the role of miRNAs in pediatric GBM (pGBM). In the present study, the expression profile of miRNAs was examined in seven pGBMs and three human GBM cell lines (U87MG, A172 and T98G), compared with a non-tumoral pool of pediatric cerebral cortex samples by microarray analysis. A set of differentially expressed miRNAs was identified, including miR-490, miR-876-3p, miR-876-5p, miR-448 and miR-137 (downregulated), as well as miR-501-3p (upregulated). Through bioinformatics analysis, a series of target genes was predicted. In addition, similar gene expression patterns in pGBMs and cell lines was confirmed. Of note, drug resistant T98G cells had upregulated nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) expression, a protein overexpressed in many tumors that serves an important role in cell proliferation and progression. On the basis of the present preliminary report, it could be intriguing to further investigate the relationship between each of the identified differentially expressed miRNAs and NUCKS1, in order to clarify their involvement in the multi-drug resistance mechanism of pGBMs.

Combined Treatment with Doxorubicin and Rapamycin Is Effective against In Vitro and In Vivo Models of Human Glioblastoma.

Despite numerous clinical trials, glioblastoma (GBM) remains a tumor that is difficult to treat. The aim of this study was to investigate the potential of a new pharmacological approach, combining doxorubicin (Dox) and rapamycin (Rapa), in in vitro and in vivo GBM models. Cytotoxic and anti-proliferative effects of Rapa plus Dox treatments were analyzed in GBM cell lines. The in vivo effectiveness of these treatments was investigated in an orthotopic xenograft mice model of GBM. In vitro results demonstrated that prolonged exposure to Rapa sensitize GBM cells to Dox treatments. In vivo results demonstrated that Rapa (5 mg/kg) plus Dox (5 mg/kg) determined the major tumor growth inhibition (-97.29% vs. control) but results in greater toxicity. The combination Rapa plus Dox (2.5 mg/kg) showed a tumor inhibition like Rapa plus Dox (5 mg/kg) with a toxicity comparable to Rapa alone. Thus, this study demonstrated the efficacy of this pharmacological approach, providing the rationale for a clinical application of this combinational therapy in "poor-responder" GBM patients.

Aldoxorubicin and Temozolomide combination in a xenograft mice model of human glioblastoma.

Glioblastoma Multiforme (GBM) is still an incurable disease. The front-line Temozolomide (TMZ)-based therapy suffers from poor efficacy, underlining the need of new therapies. Preclinically, Aldoxorubicin (Aldox), a novel prodrug of Doxorubicin (Dox), has been successfully tested against GBM, encouraging the study of its association with other agents. For the first time, we evaluated the effectiveness of Aldox combined to TMZ in preclinical models of GBM. Our in vitro results demonstrated that the anti-glioma effect of Aldox was more marked than TMZ and their combination increased the killing effect of the anthracycline in TMZ-resistant GBM cells. Moreover, unlike Dox, Aldox was able to accumulate in P-glycoprotein (P-gp)-overexpressed cells due to a negative regulation of the P-gp function. We also compared efficacy and safety of weekly administrations of Aldox (16 mg/kg), with or without TMZ (0.9 mg/kg, daily injections), in the U87 xenograft mouse model. Aldox therapy induced a moderate tumor volume inhibition (TVI) and an increased survival rate (+12.5% vs vehicle). On the other hand, when combined to TMZ, Aldox caused a significant TVI (P=0.0175 vs vehicle) and delayed the mortality during the experimental period, although TVI and endpoint survival percentage (+37.5% vs vehicle) were not significantly different from TMZ alone. Our preliminary data showed that Aldox exerts anti-glioma effects in vitro and in vivo. It also enhances its antitumor activity when combined with TMZ, resulting in a superior efficacy compared to the single agents, without adverse side effects.

Glioblastoma Chemoresistance: The Double Play by Microenvironment and Blood-Brain Barrier.

For glioblastoma, the tumor microenvironment (TME) is pivotal to support tumor progression and therapeutic resistance. TME consists of several types of stromal, endothelial and immune cells, which are recruited by cancer stem cells (CSCs) to influence CSC phenotype and behavior. TME also promotes the establishment of specific conditions such as hypoxia and acidosis, which play a critical role in glioblastoma chemoresistance, interfering with angiogenesis, apoptosis, DNA repair, oxidative stress, immune escape, expression and activity of multi-drug resistance (MDR)-related genes. Finally, the blood brain barrier (BBB), which insulates the brain microenvironment from the blood, is strongly linked to the drug-resistant phenotype of glioblastoma, being a major physical and physiological hurdle for the delivery of chemotherapy agents into the brain. Here, we review the features of the glioblastoma microenvironment, focusing on their involvement in the phenomenon of chemoresistance; we also summarize recent advances in generating systems to modulate or bypass the BBB for drug delivery into the brain. Genetic aspects associated with glioblastoma chemoresistance and current immune-based strategies, such as checkpoint inhibitor therapy, are described too.

Tumor response of temozolomide in combination with morphine in a xenograft model of human glioblastoma.

Despite multimodal treatments comprising, radiation therapy (RT) and chemotherapy with temozolomide (TMZ), the prognosis of glioblastoma multiforme (GBM) remains dismal and consolidated therapy yields a median survival of 14.6 months. Blood Brain Barrier (BBB) mediated chemoresistance and high dose related toxicity make necessary the development of new therapeutic approach to sensitize GBM to TMZ. The aim of the present study was to investigate the potential of the treatment morphine plus TMZ metronmic doses (1,77 and 0,9 mg/kg) in GBM therapy. The effect of morphine, on tumor cell growth and P-glycoprothein (P-gp) activity, was investigate in in vitro models. The results demonstrated that GBM cells growth is not influenced by morphine treatment and, for the first time, we show that morphine is an inhibitor of the activity of P-gp efflux transporter who is markedly expressed on BBB. In vivo, response to the treatments TMZ plus morphine was investigated in an orthotopic nude mice model of GBM. Animals treated with TMZ metronomic doses showed a significant tumor growth inhibition compared to untreated mice and association with morphine appears to improve TMZ efficacy. Moreover, the combination of morphine with lower dose of TMZ result in a cytostatic effect on tumor growth over the period of the pharmacological treatments. In conclusion this novel approach could be a successful strategy to overcome chemoresistance and side effects TMZ mediated, reducing drug dosage and improving long term response, in GBM therapy.

Morphine modulates doxorubicin uptake and improves efficacy of chemotherapy in an intracranial xenograft model of human glioblastoma.

Morphine may alter the permeability of Blood-Brain Barrier (BBB), enhancing the access of molecules normally unable to cross it, as Doxorubicin (Dox). In addition, morphine seems to mediate the uptake of Dox into the brain by its reduced efflux mediated by P-glycoprotein (P-gp). We evaluated the antitumor efficacy of Dox plus morphine treatment by an orthotopic glioblastoma xenograft model. Foxn1 mice were injected with U87MG-luc cells in the left lobe of the brain and treated with Dox (5 mg/kg and 2.5 mg/kg, weekly) with or without morphine pretreatment (10 mg/kg, weekly). Bioluminescence imaging (BLI) was used to monitoring tumor growth and response to therapy. Additionally, we investigated the role of morphine on the uptake of Dox by MDCKII cells transfected with human MDR1 gene encoding for P-gp. The data demonstrate that only Dox 5 mg/kg determined a significant tumor regression while the lower dose (2.5 mg/kg) was not effective. However, if combined with morphine, the group treated with Dox 2.5 mg/kg showed a decreasing tumor growth. The average BLI for Dox 2.5 mg/kg plus morphine was 5 fold lower than Dox 2.5 mg/kg alone (P=0.0053) and 8 fold lower than vehicle (P=0.0004). Additionally, Dox increased in MDCKII-P-gp transfected cells only in the presence of morphine with a significantly higher level comparing control group (3.84) vs Dox plus morphine group (12.29, P<0.05). Our results indicate that Dox alone and in combination with morphine appear to be effective in controlling the growth of glioblastoma in a xenograft mouse model.

The Use of Anthracyclines for Therapy of CNS Tumors.

Despite being long lived, anthracyclines remain the "evergreen" drugs in clinical practice of oncology, showing a potent effect in inhibiting cell growth in many types of tumors, including brain neoplasms. Unfortunately, they suffer from a poor penetration into the brain when intravenously administered due to multidrug resistance mechanism, which hampers their delivery across the blood brain barrier. In this paper, we summarize the current literature on the role of anthracyclines in cancer therapy and highlight recent efforts on 1) development of tumor cell resistance to anthracyclines and 2) the new approaches to brain drug delivery across the blood brain barrier.

Anti-miR21 oligonucleotide enhances chemosensitivity of T98G cell line to doxorubicin by inducing apoptosis.

Various signal transduction pathways seem to be involved in chemoresistance mechanism of glioblastomas (GBMs). miR-21 is an important oncogenic miRNA which modulates drug resistance of tumor cells. We analyzed the expression of 5 miRNAs, previously found to be dysregulated in high grade gliomas, in 9 pediatric (pGBM) and in 5 adult (aGBM) GBMs. miR-21 was over-expressed, with a significant difference between pGBMs and aGBMs represented by a 4 times lower degree of expression in the pediatric compared to the adult series (p = 0.001). Doxorubicin (Dox) seems to be an effective anti-glioma agent with high antitumor activity also against glioblastoma stem cells. We therefore evaluated the chemosensitivity to Dox in 3 GBM cell lines (A172, U87MG and T98G). Dox had a cytotoxic effect after 48 h of treatment in A172 and U87MG, while T98G cells were resistant. TUNEL assay verified that Dox induced apoptosis in A172 and U87MG but not in T98G. miR-21 showed a low basal expression in treated cells and was over-expressed in untreated cells. To validate the possible association of miR-21 with drug resistance of T98G cells, we transfected anti-miR-21 inhibitor into the cells. The expression level of miR-21 was significantly lower in T98G transfected cells (than in the parental control cells). Transfected cells showed a high apoptotic rate compared to control after Dox treatment by TUNEL assay, suggesting that combined Dox and miR-21 inhibitor therapy can sensitize GBM resistant cells to anthracyclines by enhancing apoptosis.

Delivery of doxorubicin across the blood-brain barrier by ondansetron pretreatment: a study in vitro and in vivo.

Doxorubicin (Dox) has got a limited efficacy in the treatment of central nervous system tumors because of its poor penetration through blood-brain barrier mediated by MDR efflux transporters. We investigated the possibility that ondansetron (Ond) enhances Dox cytotoxicity in cell lines interfering with P-glycoprotein and increases Dox concentration in rat brain tissues. The MDR phenotype was studied using human hepatocellular carcinoma cell line PLC/PRF/5 (P5 and P1(0.5) clones), two subclones of NIH 3T3 cells (PSI-2 and PN1A) and two glioblastoma cell lines (A172, U87MG). Rats were pretreated with Ond before injection of Dox. Quantitative analysis of Dox was performed by mass spectrometry. Our in vitro experiments demonstrated that Ond at 10 µg/ml is not toxic to all cell lines. However, Ond reverses the MDR phenotype in P1(0.5) and PN1A cell lines. In addition, we showed that pretreatment with Ond increases Dox concentration in rat brain tissues, without increasing acute heart and renal toxicity.

Familial hemophagocytic lymphohistiocytosis: when rare diseases shed light on immune system functioning.

The human immune system depends on the activity of cytotoxic T lymphocytes (CTL), natural killer (NK) cells, and NKT cells in order to fight off a viral infection. Understanding the molecular mechanisms during this process and the role of individual proteins was greatly improved by the study of familial hemophagocytic lymphohistiocytosis (FHL). Since 1999, genetic sequencing is the gold standard to classify patients into different subgroups of FHL. The diagnosis, once based on a clinical constellation of abnormalities, is now strongly supported by the results of a functional flow-cytometry screening, which directs the genetic study. A few additional congenital immune deficiencies can also cause a resembling or even identical clinical picture to FHL. As in many other rare human disorders, the collection and analysis of a relatively large number of cases in registries is crucial to draw a complete picture of the disease. The conduction of prospective therapeutic trials allows investigators to increase the awareness of the disease and to speed up the diagnostic process, but also provides important functional and genetic confirmations. Children with confirmed diagnosis may undergo hematopoietic stem cell transplantation, which is the only cure known to date. Moreover, detailed characterization of these rare patients helped to understand the function of individual proteins within the exocytic machinery of CTL, NK, and NKT cells. Moreover, identification of these genotypes also provides valuable information on variant phenotypes, other than FHL, associated with biallelic and monoallelic mutations in the FHL-related genes. In this review, we describe how detailed characterization of patients with genetic hemophagocytic lymphohistiocytosis has resulted in improvement in knowledge regarding contribution of individual proteins to the functional machinery of cytotoxic T- and NK-cells. The review also details how identification of these genotypes has provided valuable information on variant phenotypes.

Deregulation of ion channel and transporter encoding genes in pediatric gliomas.

Brain tumors, including the majority gliomas, are the leading cause of cancer-related death in children. World Health Organization has divided pediatric brain tumors into different grades and, based upon cDNA microarray data identifying gene expression profiles (GEPs), it has become evident in the last decade that the various grades involve different types of genetic alterations. However, it is not known whether ion channel and transporter genes, intimately involved in brain functioning, are associated with such GEPs. We determined the GEPs in an available cohort of 10 pediatric brain tumors initially by comparing the data obtained from four primary tumor samples and corresponding short-term cultures. The correspondence between the two types of samples was statistically significant. We then performed bioinformatic analyses on those samples (a total of nine) which corresponded to tumors of glial origin, either tissues or cell cultures, depending on the best "RNA integrity number." We used R software to evaluate the genes which were differentially expressed (DE) in gliomas compared with normal brain. Applying a p-value below 0.01 and fold change ≥4, led to identification of 2284 DE genes. Through a Functional Annotation Analysis (FAA) using the NIH-DAVID software, the DE genes turned out to be associated mainly with: immune/inflammatory response, cell proliferation and survival, cell adhesion and motility, neuronal phenotype, and ion transport. We have shown that GEPs of pediatric brain tumors can be studied using either primary tumor samples or short-term cultures with similar results. From FAA, we concluded that, among DE genes, pediatric gliomas show a strong deregulation of genes related to ion channels and transporters.