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1.
Cancers (Basel) ; 16(18)2024 Sep 15.
Article in English | MEDLINE | ID: mdl-39335139

ABSTRACT

In non-small cell lung cancer (NSCLC) treatment, radiotherapy responses are not durable and toxicity limits therapy. We find that AM-101, a synthetic benzodiazepine activator of GABA(A) receptor, impairs the viability and clonogenicity of both primary and brain-metastatic NSCLC cells. Employing a human-relevant ex vivo 'chip', AM-101 is as efficacious as docetaxel, a chemotherapeutic used with radiotherapy for advanced-stage NSCLC. In vivo, AM-101 potentiates radiation, including conferring a significant survival benefit to mice bearing NSCLC intracranial tumors generated using a patient-derived metastatic line. GABA(A) receptor activation stimulates a selective-autophagic response via the multimerization of GABA(A) receptor-associated protein, GABARAP, the stabilization of mitochondrial receptor Nix, and the utilization of ubiquitin-binding protein p62. A high-affinity peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis that triggers autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity.

2.
bioRxiv ; 2023 Dec 01.
Article in English | MEDLINE | ID: mdl-38076805

ABSTRACT

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

3.
J Vis Exp ; (196)2023 06 16.
Article in English | MEDLINE | ID: mdl-37395566

ABSTRACT

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.


Subject(s)
Brain Neoplasms , Channelopathies , Humans , Early Detection of Cancer , Ion Channels/metabolism , Tumor Microenvironment
4.
Sci Adv ; 9(26): eadf2746, 2023 06 30.
Article in English | MEDLINE | ID: mdl-37390205

ABSTRACT

Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.


Subject(s)
Hydrogels , Triple Negative Breast Neoplasms , Humans , Animals , Mice , Immunogenic Cell Death , CD8-Positive T-Lymphocytes , Triple Negative Breast Neoplasms/drug therapy , Ceramides , Disease Models, Animal , Immunosuppressive Agents , Unfolded Protein Response , Tumor Microenvironment
5.
Basic Clin Pharmacol Toxicol ; 133(4): 353-363, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37337931

ABSTRACT

Brain-specific angiogenesis inhibitor 3 (ADGRB3/BAI3) belongs to the family of adhesion G protein-coupled receptors. It is most highly expressed in the brain where it plays a role in synaptogenesis and synapse maintenance. Genome-wide association studies have implicated ADGRB3 in disorders such as schizophrenia and epilepsy. Somatic mutations in ADGRB3 have also been identified in cancer. To better understand the in vivo physiological role of ADGRB3, we used CRISPR/Cas9 editing to generate a mouse line with a 7-base pair deletion in Adgrb3 exon 10. Western blot analysis confirmed that homozygous mutants (Adgrb3∆7/∆7 ) lack full-length ADGRB3 expression. The mutant mice were viable and reproduced in Mendelian ratios but demonstrated reduced brain and body weights and deficits in social interaction. Measurements of locomotor function, olfaction, anxiety levels and prepulse inhibition were comparable between heterozygous and homozygous mutants and wild-type littermates. Since ADGRB3 is also expressed in organs such as lung and pancreas, this new mouse model will facilitate elucidation of ADGRB3's role in non-central nervous system-related functions. Finally, since somatic mutations in ADGRB3 were identified in patients with several cancer types, these mice can be used to determine whether loss of ADGRB3 function contributes to tumour development.


Subject(s)
Epilepsy , Neoplasms , Humans , Mice , Animals , Genome-Wide Association Study , Brain/metabolism , Receptors, G-Protein-Coupled/metabolism , Neoplasms/metabolism
6.
Addict Sci Clin Pract ; 17(1): 60, 2022 11 05.
Article in English | MEDLINE | ID: mdl-36335381

ABSTRACT

BACKGROUND: Medication for opioid use disorder (MOUD) reduces mortality, but few patients access MOUD. At a Federally Qualified Health Center (FQHC), we implemented a low barrier model of MOUD, including same-day MOUD initiation and a harm reduction philosophy. OBJECTIVE: To investigate whether low barrier MOUD improved retention in care compared to traditional treatment. DESIGN AND PARTICIPANTS: Retrospective cohort study of patients with at least one visit seeking MOUD at the FQHC during a historical control period (3/1/2018-3/31/2019) and a low barrier intervention period (11/1/2019-7/31/2020). MAIN MEASURES: Primary outcomes were any MOUD prescription within 6 months of the index visit and 3- and 6-month retention in treatment without care gap, with care gap defined as 60 consecutive days without a visit or prescription. Secondary outcomes were all-cause hospitalization and emergency department visit within 6 months of the index visit. KEY RESULTS: Baseline characteristics were similar between the intervention (n = 113) and control (n = 90) groups, except the intervention group had higher rates of uninsured, public insurance and diabetes. Any MOUD prescription within 6 months of index visit was higher in the intervention group (97.3% vs 70%), with higher adjusted odds of MOUD prescription (OR = 4.01, 95% CI 2.08-7.71). Retention in care was similar between groups at 3 months (61.9% vs 60%, aOR = 1.06, 95% CI 0.78-1.44). At 6 months, a higher proportion of the intervention group was retained in care, but the difference was not statistically significant (53.1% vs 45.6%, aOR 1.27, 95% CI 0.93-1.73). There was no significant difference in adjusted odds of 6-month hospitalization or ED visit between groups. CONCLUSIONS: Low barrier MOUD engaged a higher risk population and did not result in any statistically significant difference in retention in care compared with a historical control. Future research should determine what interventions improve retention of patients engaged through low barrier care. Primary care clinics can implement low barrier treatment to make MOUD accessible to a broader population.


Subject(s)
Buprenorphine , Opioid-Related Disorders , Humans , Buprenorphine/therapeutic use , Retrospective Studies , Opioid-Related Disorders/drug therapy , Opioid-Related Disorders/epidemiology , Harm Reduction , Emergency Service, Hospital , Analgesics, Opioid/therapeutic use , Opiate Substitution Treatment
7.
Cancers (Basel) ; 14(7)2022 Mar 23.
Article in English | MEDLINE | ID: mdl-35406398

ABSTRACT

Glioblastoma, or glioblastoma multiforme (GBM, WHO Grade IV), is a highly aggressive adult glioma. Despite extensive efforts to improve treatment, the current standard-of-care (SOC) regimen, which consists of maximal resection, radiotherapy, and temozolomide (TMZ), achieves only a 12-15 month survival. The clinical improvements achieved through immunotherapy in several extracranial solid tumors, including non-small-cell lung cancer, melanoma, and non-Hodgkin lymphoma, inspired investigations to pursue various immunotherapeutic interventions in adult glioblastoma patients. Despite some encouraging reports from preclinical and early-stage clinical trials, none of the tested agents have been convincing in Phase III clinical trials. One, but not the only, factor that is accountable for the slow progress is the blood-brain barrier, which prevents most antitumor drugs from reaching the target in appreciable amounts. Herein, we review the current state of immunotherapy in glioblastoma and discuss the significant challenges that prevent advancement. We also provide thoughts on steps that may be taken to remediate these challenges, including the application of ultrasound technologies.

8.
Front Physiol ; 13: 839437, 2022.
Article in English | MEDLINE | ID: mdl-35350689

ABSTRACT

Ligand-gated ion channels are an ionotropic receptor subtype characterized by the binding of an extracellular ligand, followed by the transient passage of ions through a transmembrane pore. Ligand-gated ion channels are commonly subcategorized into three superfamilies: purinoreceptors, glutamate receptors, and Cys-loop receptors. This classification is based on the differing topographical morphology of the receptors, which in turn confers functional differences. Ligand-gated ion channels have a diverse spatial and temporal expression which implicate them in key cellular processes. Given that the transcellular electrochemical gradient is finely tuned in eukaryotic cells, any disruption in this homeostasis can contribute to aberrancies, including altering the activity of pro-tumorigenic molecular pathways, such as the MAPK/ERK, RAS, and mTOR pathways. Ligand-gated ion channels therefore serve as a potential targetable system for cancer therapeutics. In this review, we analyze the role that each of the three ligand-gated ion channel superfamilies has concerning tumor proliferation and as a target for the treatment of cancer symptomatology.

9.
Exp Biol Med (Maywood) ; 246(19): 2128-2135, 2021 10.
Article in English | MEDLINE | ID: mdl-34649481

ABSTRACT

γ-aminobutyric acid or GABA is an amino acid that functionally acts as a neurotransmitter and is critical to neurotransmission. GABA is also a metabolite in the Krebs cycle. It is therefore unsurprising that GABA and its receptors are also present outside of the central nervous system, including in immune cells. This observation suggests that GABAergic signaling impacts events beyond brain function and possibly human health beyond neurological disorders. Indeed, GABA receptor subunits are expressed in pathological disease states, including in disparate cancers. The role that GABA and its receptors may play in cancer development and progression remains unclear. If, however, those cancers have functional GABA receptors that participate in GABAergic signaling, it raises an important question whether these signaling pathways might be targetable for therapeutic benefit. Herein we summarize the effects of modulating Type-A GABA receptor signaling in various cancers and highlight how Type-A GABA receptors could emerge as a novel therapeutic target in cancer.


Subject(s)
Neoplasms/metabolism , Receptors, GABA-A/metabolism , Animals , Humans , Signal Transduction/physiology , gamma-Aminobutyric Acid/metabolism
10.
Cancer Invest ; 39(10): 854-870, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34569407

ABSTRACT

The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.


Subject(s)
Brain Neoplasms/drug therapy , CD58 Antigens/pharmacology , Glioma/drug therapy , Proto-Oncogene Proteins c-akt/metabolism , Animals , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , CD58 Antigens/therapeutic use , Cell Cycle Checkpoints/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Glioma/metabolism , Glioma/pathology , Male , PTEN Phosphohydrolase/analysis , Phosphorylation , Proto-Oncogene Proteins c-mdm2/analysis , Rats , Rats, Wistar , Tumor Microenvironment , Tumor Suppressor Protein p53/analysis , bcl-Associated Death Protein/metabolism
11.
Oncogene ; 40(33): 5182-5191, 2021 08.
Article in English | MEDLINE | ID: mdl-34218269

ABSTRACT

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive <1 year, highlighting the urgent need for new therapies. TCGA has recently revealed that a hypoxia gene signature is associated with poor UM patient prognosis. Here we show that expression of hypoxia-regulated collagen prolyl-4-hydroxylase genes P4HA1 and P4HA2 is significantly upregulated in UM patients with metastatic disease and correlates with poor prognosis, suggesting these enzymes might be key tumor drivers. We targeted hypoxia-induced expression of P4HA1/2 in UM with KCN1, a hypoxia inducible factor-1 (HIF-1) pathway inhibitor and found potent inhibition of primary and metastatic disease and extension of animal survival, without overt side effects. At the molecular level, KCN1 antagonized hypoxia-induced expression of P4HA1 and P4HA2, which regulate collagen maturation and deposition in the extracellular matrix. The treatment decreased prolyl hydroxylation, induced proteolytic cleavage and rendered a disordered structure to collagen VI, the main collagen produced by UM, and reduced UM cell invasion. Together, these data demonstrate that extracellular collagen matrix formation can be targeted in UM by inhibiting hypoxia-induced P4HA1 and P4HA2 expression, warranting further development of this strategy in patients with uveal melanoma.


Subject(s)
Hypoxia-Inducible Factor-Proline Dioxygenases , Extracellular Matrix , Humans , Hydroxylation , Melanoma , Transcriptional Activation , Up-Regulation , Uveal Neoplasms
12.
Pharmaceuticals (Basel) ; 14(2)2021 Jan 28.
Article in English | MEDLINE | ID: mdl-33525329

ABSTRACT

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. The current standard of care for GBM is the Stupp protocol which includes surgical resection, followed by radiotherapy concomitant with the DNA alkylator temozolomide; however, survival under this treatment regimen is an abysmal 12-18 months. New and emerging treatments include the application of a physical device, non-invasive 'tumor treating fields' (TTFs), including its concomitant use with standard of care; and varied vaccines and immunotherapeutics being trialed. Some of these approaches have extended life by a few months over standard of care, but in some cases are only available for a minority of GBM patients. Extensive activity is also underway to repurpose and reposition therapeutics for GBM, either alone or in combination with the standard of care. In this review, we present select molecules that target different pathways and are at various stages of clinical translation as case studies to illustrate the rationale for their repurposing-repositioning and potential clinical use.

13.
Int J Radiat Oncol Biol Phys ; 109(4): 1040-1053, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33289666

ABSTRACT

PURPOSE: Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. METHODS AND MATERIALS: We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. RESULTS: Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. CONCLUSIONS: This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.


Subject(s)
Immune Checkpoint Inhibitors/therapeutic use , Melanoma/therapy , Receptors, GABA-A/physiology , T-Lymphocytes/immunology , Animals , Benzodiazepines/pharmacology , Benzodiazepines/therapeutic use , Cell Proliferation/drug effects , Combined Modality Therapy , Female , Humans , Melanoma/pathology , Membrane Proteins/analysis , Mice , Mice, Inbred C57BL , Radiation-Sensitizing Agents/pharmacology , Receptors, GABA-A/analysis
14.
J Neurooncol ; 150(1): 77-83, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32451770

ABSTRACT

INTRODUCTION: Brain tumors make up over a quarter of pediatric malignancies. Depending on the age of presentation and treatment, pediatric brain tumor survivors experience varying degrees of treatment induced morbidity and sequelae. Epigenetic mechanisms play a critical role in silencing of tumor suppressor genes and activation of driver genes involved in oncogenesis in different types of brain tumors. Epigenetic modifications in pediatric brain tumor patients may influence long-term survival and may refine the molecular response to treatment induced morbidity and sequelae. However, there is a dearth of studies on how epigenetics of pediatric brain tumors is connected with neurocognition and other treatment related sequelae in survivors. METHODS/RESULTS: In this review we explore epigenetic factors that may contribute to the survivorship and treatment of pediatric brain tumor patients. We focus on glioblastoma, medulloblastoma, and the neurocutaneous syndrome neurofibromatosis type-1 to highlight epigenetic biomarkers that can potentially serve not only as prognostic indicators of overall patient survival, but hopefully as indicators to the response to treatment neurocognitively and otherwise. CONCLUSIONS: Future studies will hopefully soon bridge the gap in our knowledge on how epigenetic modifications are linked to treatment related sequelae in pediatric brain tumor patients.


Subject(s)
Brain Neoplasms , Cerebellar Neoplasms , Medulloblastoma , Brain Neoplasms/genetics , Cerebellar Neoplasms/genetics , Child , Epigenesis, Genetic , Humans , Medulloblastoma/genetics , Survivorship
16.
Sci Rep ; 9(1): 4437, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30872606

ABSTRACT

CRISPR gene editing creates indels in targeted genes that are detected by genotyping. Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we developed a simple genotyping method that exploits the differential electrophoretic mobility of homoduplex versus heteroduplex DNA hybrids in high concentration agarose gels. First, the CRISPR target region is PCR amplified and homo- and hetero-duplexed amplicons formed during the last annealing cycle are separated by 4-6% agarose gel electrophoresis. WT/mutant heteroduplexes migrate more slowly and are distinguished from WT or mutant homoduplexes. Heterozygous alleles are immediately identified as they produce two distinct bands, while homozygous wild-type or mutant alleles yield a single band. To discriminate the latter, equal amounts of PCR products of homozygous samples are mixed with wild-type control samples, subjected to one denaturation/renaturation cycle and products are electrophoresed again. Samples from homozygous mutant alleles now produce two bands, while those from wild-type alleles yield single bands. This method is simple, fast and inexpensive and can identify indels >2 bp. in size in founder pups and genotype offspring in established transgenic mice colonies.


Subject(s)
CRISPR-Cas Systems , Electrophoresis, Agar Gel/methods , Gene Editing , INDEL Mutation , Membrane Proteins/genetics , NAV1.6 Voltage-Gated Sodium Channel/genetics , Nerve Tissue Proteins/genetics , Animals , Female , Genotype , Genotyping Techniques , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Mice , Mice, Transgenic , NAV1.6 Voltage-Gated Sodium Channel/chemistry , NAV1.6 Voltage-Gated Sodium Channel/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Polymerase Chain Reaction
17.
Rev Neurosci ; 30(5): 527-541, 2019 07 26.
Article in English | MEDLINE | ID: mdl-30645197

ABSTRACT

Melatonin (N-acetyl-5-methoxytryptamine) is a naturally synthesized hormone secreted from the pineal gland in a variety of animals and is primarily involved in the regulation of the circadian rhythm, which is the natural cycle controlling sleep in organisms. Melatonin acts on specific receptors and has an important role in overall energy metabolism. This review encompasses several aspects of melatonin activity, such as synthesis, source, structure, distribution, function, signaling and its role in normal physiology. The review highlights the cellular signaling and messenger systems involved in melatonin's action on the body and their wider implications, the distribution and diverse action of different melatonin receptors in specific areas of the brain, and the pharmacological agonists and antagonists that have specific action on these melatonin receptors. This review also incorporates the antitumor effects of melatonin in considerable detail, emphasizing on melatonin's role as an adjuvant therapeutic agent in glioma treatment. We conclude that the diminishing levels of melatonin have significant debilitating effects on normal physiology and can also be associated with malignant conditions such as glioma. Based on the review of the available evidence, our study provides a broad platform for a better understanding of the specific roles of melatonin and serves as a starting point for further investigation into the therapeutic effect of melatonin in glioma as an adjuvant therapeutic agent.


Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , Melatonin/metabolism , Signal Transduction , Animals , Antineoplastic Agents/therapeutic use , Antioxidants/therapeutic use , Brain Neoplasms/drug therapy , Glioma/drug therapy , Humans , Melatonin/therapeutic use
18.
Cancer Cell ; 33(6): 1004-1016.e5, 2018 06 11.
Article in English | MEDLINE | ID: mdl-29894688

ABSTRACT

Adhesion G protein-coupled receptors (ADGRs) encompass 33 human transmembrane proteins with long N termini involved in cell-cell and cell-matrix interactions. We show the ADGRB1 gene, which encodes Brain-specific angiogenesis inhibitor 1 (BAI1), is epigenetically silenced in medulloblastomas (MBs) through a methyl-CpG binding protein MBD2-dependent mechanism. Knockout of Adgrb1 in mice augments proliferation of cerebellar granule neuron precursors, and leads to accelerated tumor growth in the Ptch1+/- transgenic MB mouse model. BAI1 prevents Mdm2-mediated p53 polyubiquitination, and its loss substantially reduces p53 levels. Reactivation of BAI1/p53 signaling axis by a brain-permeable MBD2 pathway inhibitor suppresses MB growth in vivo. Altogether, our data define BAI1's physiological role in tumorigenesis and directly couple an ADGR to cancer formation.


Subject(s)
Angiogenic Proteins/metabolism , Cerebellar Neoplasms/metabolism , Medulloblastoma/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Angiogenic Proteins/genetics , Animals , Cell Line, Tumor , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/genetics , HCT116 Cells , Humans , Kaplan-Meier Estimate , Medulloblastoma/drug therapy , Medulloblastoma/genetics , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Proto-Oncogene Proteins c-mdm2/genetics , RNA Interference , Receptors, G-Protein-Coupled , Small Molecule Libraries/pharmacology , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
19.
J Cell Physiol ; 233(2): 759-770, 2018 Feb.
Article in English | MEDLINE | ID: mdl-28608562

ABSTRACT

Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K, and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes.


Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , CD58 Antigens/pharmacology , Glioma/drug therapy , Immunotherapy/methods , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , T-Lymphocytes/drug effects , Tumor Escape/drug effects , 3-Phosphoinositide-Dependent Protein Kinases/metabolism , Active Transport, Cell Nucleus , Animals , Brain Neoplasms/enzymology , Brain Neoplasms/immunology , Brain Neoplasms/pathology , CD28 Antigens/immunology , CD28 Antigens/metabolism , Cell Survival , Ethylnitrosourea , Female , Glioma/enzymology , Glioma/immunology , Glioma/pathology , Male , NF-kappa B/metabolism , PTEN Phosphohydrolase/metabolism , Phosphorylation , Rats , Signal Transduction/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/immunology
20.
J Cell Physiol ; 232(3): 526-539, 2017 Mar.
Article in English | MEDLINE | ID: mdl-27255231

ABSTRACT

Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor-associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma-associated brain endothelial cells. In the present study, we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma-associated brain endothelial cells. Annexin-V/PI staining showed that T11TS treatment in glioma-induced rats increases apoptosis of glioma-associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC-1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma-associated brain endothelial cells. Flowcytometry, immunoblotting, and in situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment-upmodulated expression of p53, Bax, Fas, FasL, and FADD in glioma associated endothelial cells and downregulated Bcl-2 protein. T11TS therapy induced cytochrome-c release into cytosol, activated caspase -9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma-associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas-dependent extrinsic pathway. The pro-apoptotic action of T11TS on glioma-associated endothelial cells provides crucial insight into how T11TS exerts its anti-angiogenic function in glioma. J. Cell. Physiol. 232: 526-539, 2017. © 2016 Wiley Periodicals, Inc.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Apoptosis/drug effects , Brain Neoplasms/drug therapy , Endothelial Cells/pathology , Glioma/pathology , Glycopeptides/pharmacology , Glycopeptides/therapeutic use , Membrane Glycoproteins/pharmacology , Membrane Glycoproteins/therapeutic use , Neovascularization, Pathologic/drug therapy , Angiogenesis Inhibitors/pharmacology , Animals , BH3 Interacting Domain Death Agonist Protein/metabolism , Brain Neoplasms/pathology , Caspases/metabolism , Cytochromes c/metabolism , Cytosol/metabolism , Endothelial Cells/drug effects , Enzyme Activation , Fas Ligand Protein/metabolism , Fas-Associated Death Domain Protein/metabolism , Female , Glycopeptides/administration & dosage , Male , Membrane Glycoproteins/administration & dosage , Membrane Potential, Mitochondrial/drug effects , Models, Biological , Neovascularization, Pathologic/pathology , Rats , Sheep , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/metabolism , fas Receptor/metabolism
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