Sonic hedgehog, Wnt, and brain-derived neurotrophic factor cell signaling pathway crosstalk: potential therapy for depression.

There are various theories to explain the pathophysiology of depression and support its diagnosis and treatment. The roles of monoamines, brain-derived neurotrophic factor (BDNF), and Wnt signaling are well researched, but sonic hedgehog (Shh) signaling and its downstream transcription factor Gli1 are not well studied in depression. Shh signaling plays a fundamental role in embryonic development and adult hippocampal neurogenesis and also involved in the growth of cancer. In this article, we summarize the evidence for the Shh signaling pathway in depression and the potential crosstalk of Shh with Wnt and BDNF. Antidepressants are known to upregulate the adult hippocampal neurogenesis to treat depression. Shh plays an important role in adult hippocampal neurogenesis, and its downstream signaling components regulate the synthesis of Wnt proteins. Moreover, the expression of Gli1 and Smo is downregulated in depression. BDNF and Wnt signaling are also regulated by various available antidepressants, so there is the possibility that Shh may be involved in the pathophysiology of depression. Therefore, the crosstalk between the Shh, Wnt, and BDNF signaling pathways is being discussed to identify the potential targets. Specifically, the potential role of the Shh signaling pathway in depression is explored as a new target for better therapies for depression.

Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

Genome-wide methylation analysis in vestibular schwannomas shows putative mechanisms of gene expression modulation and global hypomethylation at the HOX gene cluster.

Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein-coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA-21, Met Proto-Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets.

Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples.

Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease.

Quantitative method for in vitro matrigel invasiveness measurement through image analysis software.

The determination of cell invasion by matrigel assay is usually evaluated by counting cells able to pass through a porous membrane and attach themselves to the other side, or by an indirect quantification of eluted specific cell staining dye by means of optical density measurement. This paper describes a quantitative analytical imaging approach for determining the invasiveness of tumor cells using a simple method, based on images processing with the public domain software, ImageJ. Images obtained by direct capture are split into the red channel, and the generated image is used to measure the area that cells cover in the picture. To overcome the several disadvantages that classical cell invasion determinations present, we propose this method because it generates more accurate and sensitive determinations, and it could be a reasonable option for improving the quality of the results. The cost-effective alternative method proposed is based on this simple and robust software that is worldwide affordable.

Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide-lomeguatrib combination.

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ-lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ-lomeguatrib (50 µM) was used in AA cell lines, IC50 values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib-TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.

Mesenchymal-Stem-Cell-Based Therapy against Gliomas.

Glioblastoma is the most aggressive, malignant, and lethal brain tumor of the central nervous system. Its poor prognosis lies in its inefficient response to currently available treatments that consist of surgical resection, radiotherapy, and chemotherapy. Recently, the use of mesenchymal stem cells (MSCs) as a possible kind of cell therapy against glioblastoma is gaining great interest due to their immunomodulatory properties, tumor tropism, and differentiation into other cell types. However, MSCs seem to present both antitumor and pro-tumor properties depending on the tissue from which they come. In this work, the possibility of using MSCs to deliver therapeutic genes, oncolytic viruses, and miRNA is presented, as well as strategies that can improve their therapeutic efficacy against glioblastoma, such as CAR-T cells, nanoparticles, and exosomes.

Codeletion of 1p and 19q determines distinct gene methylation and expression profiles in IDH-mutated oligodendroglial tumors.

Oligodendroglial tumors (OTs) are primary brain tumors that show variable clinical and biological behavior. The 1p/19q codeletion is frequent in these tumors, indicating a better prognosis and/or treatment response. Recently, the prognostically favorable CpG island methylator phenotype (CIMP) in gliomas (G-CIMP+) was associated with mutations in the isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 (IDH) genes, as opposed to G-CIMP- tumors, highlighting the relevance of epigenetic mechanisms. We performed a whole-genome methylation study in 46 OTs, and a gene expression study of 25 tumors, correlating the methylation and transcriptomic profiles with molecular and clinical variables. Here, we identified two different epigenetic patterns within the previously described main G-CIMP+ profile. Both IDH mutation-associated methylation profiles featured one group of OTs with 1p/19q loss (CD-CIMP+), most of which were pure oligodendrogliomas, and a second group with intact 1p/19q and frequent TP53 mutation (CIMP+), most of which exhibited a mixed histopathology. A third group of OTs lacking the CIMP profile (CIMP-), and with a wild-type IDH and an intact 1p/19q, similar to the G-CIMP- subgroup, was also observed. The three CIMP groups presented a significantly better (CD-CIMP+), intermediate (CIMP+) or worse (CIMP-) prognosis. Furthermore, transcriptomic analyses revealed CIMP-specific gene expression signatures, indicating the impact of genetic status (IDH mutation, 1p/19q codeletion, TP53 mutation) on gene expression, and pointing to candidate biomarkers. Therefore, the CIMP profiles contributed to the identification of subgroups of OTs characterized by different prognoses, histopathologies, molecular features and gene expression signatures, which may help in the classification of OTs.

Effect of lomeguatrib-temozolomide combination on MGMT promoter methylation and expression in primary glioblastoma tumor cells.

Temozolomide (TMZ) is commonly used in the treatment of glioblastoma (GBM). The MGMT repair enzyme (O (6)-methylguanine-DNA methyltransferase) is an important factor causing chemotherapeutic resistance. MGMT prevents the formation of toxic effects of alkyl adducts by removing them from the DNA. Therefore, MGMT inhibition is an interesting therapeutic approach to circumvent TMZ resistance. The aim of the study was to investigate the effect of the combination of lomeguatrib (an MGMT inactivator) with TMZ, on MGMT expression and methylation. Primary cell cultures were obtained from GBM tumor tissues. The sensitivity of primary GBM cell cultures and GBM cell lines to TMZ, and to the combination of TMZ and lomeguatrib, was determined by a cytotoxicity assay (MTT). MGMT and p53 expression, and MGMT methylation were investigated after drug application. In addition, the proportion of apoptotic cells and DNA fragmentation was analyzed. The combination of TMZ and lomeguatrib in primary GBM cell cultures and glioma cell lines decreased MGMT expression, increased p53 expression, and did not change MGMT methylation. Moreover, apoptosis was induced and DNA fragmentation was increased in cells. In addition, we also showed that lomeguatrib-TMZ combination did not have any effect on the cell cycle. Finally, we determined that the sensitivity of each primary GBM cells and glioma cell lines to the lomeguatrib-TMZ combination was different and significantly associated with the structure of MGMT methylation. Our study suggests that lomeguatrib can be used with TMZ for GBM treatment, although further clinical studies will be needed so as to determine the feasibility of this therapeutic approach.

DNA copy gains of tumor-related genes in vestibular schwannoma.

DNA copy gains are a common event in tumor growth. This study determines the gene dosage/amplification of seven tumor-related genes in patients undergoing vestibular schwannoma (VS) surgery and analyzes its clinical implications. Thirty-three patients undergoing surgery for VS were studied. Seven genes (EGFR, ERBB2, ERBB3, ERBB4, MDM2, MDM4, and NMYC) were analyzed by Quantitative real-time PCR. Copy gains were correlated with demographic, clinical and radiological data. Of the 33 samples, 48 % were positive for copy gains in at least one gene. There were no positive samples for gene amplification. A clinical correlation between tumor size and copy gains of ERBB2 was found. Patients with copy gains of this gene had larger tumors measured by diameter (p = 0.027) and volume (p = 0.005). Copy gains of EGFR, ERBB2, ERBB4, and MDM4 were associated with preoperative tinnitus. Contrary to other tumors of the central nervous system, development of VS does not appear to involve gene amplification. However, copy gains of certain tumor-related genes may play a role in the biological behavior of these neoplasms. Our findings support the role of ERBB2 in VS development and growth.

Glioblastoma Biology, Genetics and Possible Therapies.

Glioblastoma is the most aggressive intracranial tumor [...].

A Comprehensive Review of miRNAs and Their Epigenetic Effects in Glioblastoma.

Glioblastoma is the most aggressive form of brain tumor originating from glial cells with a maximum life expectancy of 14.6 months. Despite the establishment of multiple promising therapies, the clinical outcome of glioblastoma patients is abysmal. Drug resistance has been identified as a major factor contributing to the failure of current multimodal therapy. Epigenetic modification, especially DNA methylation has been identified as a major regulatory mechanism behind glioblastoma progression. In addition, miRNAs, a class of non-coding RNA, have been found to play a role in the regulation as well as in the diagnosis of glioblastoma. The relationship between epigenetics, drug resistance, and glioblastoma progression has been clearly demonstrated. MGMT hypermethylation, leading to a lack of MGMT expression, is associated with a cytotoxic effect of TMZ in GBM, while resistance to TMZ frequently appears in MGMT non-methylated GBM. In this review, we will elaborate on known miRNAs linked to glioblastoma; their distinctive oncogenic or tumor suppressor roles; and how epigenetic modification of miRNAs, particularly via methylation, leads to their upregulation or downregulation in glioblastoma. Moreover, we will try to identify those miRNAs that might be potential regulators of MGMT expression and their role as predictors of tumor response to temozolomide treatment. Although we do not impact clinical data and survival, we open possible experimental approaches to treat GBM, although they should be further validated with clinically oriented studies.

The antagonistic effects of temozolomide and trichostatin a combination on MGMT and DNA mismatch repair pathways in Glioblastoma.

Glioblastoma is the most aggressive and fatal form of brain cancer. Despite new advancements in treatment, the desired outcomes have not been achieved. Temozolomide (TMZ) is the first-choice treatment for the last two decades and has improved survival rates. Emerging studies have shown that targeting epigenetics in glioblastoma can be beneficial when combined with clinically used treatments. Trichostatin A (TSA), a histone deacetylase inhibitor, has anti-cancer properties in various cancers. No data concerning the TMZ and TSA relationship was shown previously in glioblastoma therefore, we aimed to determine the likely therapeutic effect of the TMZ and TSA combination in glioblastoma. The T98G and U-373 MG, glioblastoma cell lines, were used in this study. TMZ and TSA cytotoxicity and combination index were performed by MTT assay. The expression of DNA repair genes (MGMT, MLH-1, PMS2, MSH2 and MSH6) was detected using RT-PCR. One-way analysis of variance (ANOVA) was used for statistical analysis. Combination index calculations revealed antagonistic effects of TMZ and TSA in terms of cytotoxicity. Antagonistic effects were more apparent in the T98G cell line, which is expressing MGMT relatively higher. MGMT and DNA Mismatch Repair (MMR) genes were upregulated in the T98G cell line, whereas downregulated in the U373-MG cell lines under TMZ and TSA combination treatment. It is concluded that MGMT might be playing a more active part than MMR genes in TMZ resistance to TMZ and TSA antagonism. This is the first study elucidating the TMZ and TSA relationship in cancer cell lines.

Role of Circular RNA in Brain Tumor Development.

Central nervous system tumors are a leading cause of cancer-related death in children and adults, with medulloblastoma (MB) and glioblastoma (GBM) being the most prevalent malignant brain tumors, respectively. Despite tremendous breakthroughs in neurosurgery, radiation, and chemotherapeutic techniques, cell heterogeneity and various genetic mutations impacting cell cycle control, cell proliferation, apoptosis, and cell invasion result in unwanted resistance to treatment approaches, with a 5-year survival rate of 70-80% for medulloblastoma, and the median survival time for patients with glioblastoma is only 15 months. Developing new medicines and utilizing combination medications may be viewed as excellent techniques for battling MB and GBM. Circular RNAs (circRNAs) can affect cancer-developing processes such as cell proliferation, cell apoptosis, invasion, and chemoresistance in this regard. As a result, several compounds have been introduced as prospective therapeutic targets in the fight against MB and GBM. The current study aims to elucidate the fundamental molecular and cellular mechanisms underlying the pathogenesis of GBM in conjunction with circRNAs. Several mechanisms were examined in detail, including PI3K/Akt/mTOR signaling, Wnt/-catenin signaling, angiogenic processes, and metastatic pathways, in order to provide a comprehensive knowledge of the involvement of circRNAs in the pathophysiology of MB and GBM.

ABCG2 is required to control the sonic hedgehog pathway in side population cells with stem-like properties.

The Sonic Hedgehog (Hh) pathway has been implicated in the maintenance of stem or progenitor cells in many adult tissues. Importantly, abnormal Hh pathway activation is also associated with initiation of neoplasia, but its role in tumor growth is still unclear. Here, we demonstrate that cyclopamine, a plant-derived alkaloid product used to inhibit the Hh signaling pathway, reduces the Side Population (SP) obtained by Hoechst 33342 (Ho342) dye measurements. In addition, cyclopamine is able to modulate, along with oxysterols and other products, the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Therefore, if the SP is solely measured as a Ho342 dye extruding fraction, this may be significantly modulated by the inhibition of ABCG2 transport fraction, independently from the action of cyclopamine on the Hh pathway. Our results indicate that ABCG2 may act in the upstream regulation of the Hh signaling pathway to protect the stemness of the SP compartment, giving support to the cancer stem cell hypothesis and suggesting that ABCG2 is not only critical for increased resistance to anticancer agents.

Expression and epigenetic modulation of sonic hedgehog-GLI1 pathway genes in neuroblastoma cell lines and tumors.

It is well known that sonic hedgehog signaling pathway plays a vital role during early embryonic development. It is also responsible for stem cell renewal and development of several cancers like colorectal and breast carcinoma and major brain tumors as medulloblastoma and glioblastoma. The role of sonic hedgehog signaling in the development of neuroblastoma has not been thoroughly investigated. In this study, we attempted to determine the expression of Bmi-1 stem cell marker and of Shh pathway downstream target genes glioma-associated oncogene homolog 1 (GLI1), protein patched homolog 1 (PTCH1), Cyclin D2, plakoglobin (γ catenin), NK2 homeobox 2 (NKX2.2), paired box gene 6 (PAX6), secreted frizzled-related protein 1 (SFRP1), and hedgehog interacting protein (HHIP) in 11 neuroblastoma cell lines and 41 neuroblastoma samples. Also, inhibition of the pathway was performed genetically by GLI1 knockdown siRNA or chemically by cyclopamine. After inhibition, low transcript expression was detected in downstream target genes like PTCH1, in the cell lines. We further preformed promoter methylation studies of Cyclin D2, PTCH1, HHIP, and SFRP1 genes by melting curve analysis-based methylation assay (MCA-Meth) and methylation-specific PCR (MSP). Results revealed no methylation in Cyclin D2 gene promoter in neuroblastoma samples or in cell lines; one cell line (MHH-NB-11) showed PTCH1 methylation; 3/11 (27%) cell lines and 9/41 (22%) neuroblastoma samples showed HHIP methylation; and 3/11 (27%) cell lines and 11/41 (27%) samples showed SFRP1 methylation. Taken together, our results suggest the possibility of two levels of control of the sonic hedgehog signaling pathway: transcriptional and epigenetic, which might offer new therapeutic possibilities to modulate the pathway and try to suppress tumor growth.

Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples.

Medulloblastoma is the most common pediatric brain tumor and its development is affected by genetic and epigenetic factors. In this study we found there is low or no expression of the hedgehog interacting protein (HHIP), a negative regulator of the sonic hedgehog pathway, in most medulloblastoma cell lines and primary samples explored. We proceeded to promoter methylation assays of this gene by MCA-Meth, and found that HHIP was hypermethylated in all medulloblastoma cell lines, but only in 2 out of 14 (14%) primary tumor samples. Methylation correlated with low or unexpressed HHIP in cell lines but not in primary tumor samples. These results suggest the possibility of epigenetic regulation of HHIP in medulloblastoma, similarly to gastric, hepatic and pancreatic cancer. However, HHIP seems to be not only under regulation of promoter methylation, but under other factors involved in the control of its low levels of expression in medulloblastoma.

KIT expression and methylation in medulloblastoma and PNET cell lines and tumors.

The stem cell factor/kit tyrosine kinase receptor pathway is related to tumor growth and progression in several cancers including Ewing sarcoma, a peripheral PNET (pPNET). Identifying additional groups of tumors that may use the pathway is important as they might be responsive to imatinib mesylate treatment. MB and central PNET (cPNET) are embryonal tumors of the CNS that share similar undifferentiated morphology with Ewing sarcomas and display aggressive clinical behavior. cPNET outcome is significantly lower than MB outcome, even for localized tumors treated with high-risk MB therapy. The elucidation of signaling pathways involved in MB and cPNET pathogenesis, and the discovery of new therapeutic targets is necessary to improve the treatment of these neoplasms. We analyzed KIT expression in 2 MB, one pPNET, one cPNET and 2 rhabdomyosarcoma (RMS) cell lines. Also, in 13 tumor samples (12 MB and one cPNET), we found KIT overexpression in the most aggressive cell lines (metastatic MB and pPNET). Hypermethylation of KIT was clear in the RMS non-expressing cell lines. Among MB tumors, we could see variable levels of KIT expression; a subset of them (25%) might be related in its growth pattern to KIT up-regulation. No methylated KIT was detected in the tumors expressing the lowest levels of KIT. Our results point to methylation as an epigenetic regulatory mechanism for KIT inhibition only in the KIT non-expressing RMS cell lines, and neither in the rest of the cell lines nor in the tumor samples.

Molecular and Cellular Mechanisms of Glioblastoma.

Glioblastoma is the most malignant primary brain tumor [...].

Glioblastoma and MiRNAs.

Glioblastoma (GB) is one of the most common types of lethal brain tumors. Although several treatment options are available including surgery, along with adjuvant chemo and radiotherapy, the disease has a poor prognosis and patients generally die within 14 months of diagnosis. GB is chemo and radio resistant. Thus, there is a critical need for new insights into GB treatment to increase the chance of therapeutic success. This is why microRNA (miRNA) is being potentially considered in the diagnosis and treatment of glioblastoma. The objective of our review is to provide a holistic picture of GB up-regulated and down-regulated miRNA, in relationship with the expression of other genes, cell signaling pathways, and their role in GB diagnosis and treatment. MiRNA treatment is being considered to be used against GB together with radiotherapy and chemotherapy. Moreover, the use of miRNA as a diagnostic tool has also begun. Knowing that miRNAs are isolated in almost all human body fluids and that there are more than 3000 miRNAs in the human genome, plus the fact that each miRNA controls hundreds of different mRNAs, there is still much study needed to explore how miRNAs relate to GB for its proliferation, progression, and inhibition.