AMPA receptors play an important role in the biological consequences of spinal cord injury: Implications for AMPA receptor modulators for therapeutic benefit.

Spinal cord injury (SCI) afflicts millions of individuals globally. There are few therapies available to patients. Ascending and descending excitatory glutamatergic neural circuits in the central nervous system are disrupted by SCI, making α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) a potential therapeutic drug target. Emerging research in preclinical models highlights the involvement of AMPARs in vital processes following SCI including breathing, pain, inflammation, bladder control, and motor function. However, there are no clinical trial data reported in this patient population to date. No work on the role of AMPA receptors in sexual dysfunction after SCI has been disclosed. Compounds with selective antagonist and potentiating effects on AMPA receptors have benefit in animal models of SCI, with antagonists generally showing protective effects early after injury and potentiators (ampakines) producing improved breathing and bladder function. The role of AMPARs in pathophysiology and recovery after SCI depends upon the time post injury, and the timing of AMPAR augmentation or antagonism. The roles of inflammation, synaptic plasticity, sensitization, neurotrophic factors, and neuroprotection are considered in this context. The data summarized and discussed in this paper document proof of principle and strongly encourage additional studies on AMPARs as novel gateways to therapeutic benefit for patients suffering from SCI. The availability of both AMPAR antagonists such as perampanel and AMPAR allosteric modulators (i.e., ampakines) such as CX1739, that have been safely administered to humans, provides an expedited means of clinical inquiry for possible therapeutic advances.

Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo.

Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment. SIGNIFICANCE STATEMENT: We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings.

From protein-protein interactions to immune modulation: Therapeutic prospects of targeting Neuropilin-1 in high-grade glioma.

In the past several years there has been a marked increase in our understanding of the pathophysiological hallmarks of glioblastoma development and progression, with specific respect to the contribution of the glioma tumor microenvironment to the rapid progression and treatment resistance of high-grade gliomas. Despite these strides, standard of care therapy still only targets rapidly dividing tumor cells in the glioma, and does little to curb the pro-tumorigenic functions of non-cancerous cells entrenched in the glioma microenvironment. This tumor promoting environment as well as the heterogeneity of high-grade gliomas contribute to the poor prognosis of this malignancy. The interaction of non-malignant cells in the microenvironment with the tumor cells accentuate phenotypes such as rapid proliferation or immunosuppression, so therapeutically modulating one target expressed on one cell type may be insufficient to restrain these rapidly developing neoplasias. With this in mind, identifying a target expressed on multiple cell types and understanding how it governs tumor-promoting functions in each cell type may have great utility in better managing this disease. Herein, we review the physiology and pathological effects of Neuropilin-1, a transmembrane co-receptor which mediates signal transduction pathways when associated with multiple other receptors. We discuss its effects on the properties of endothelial cells and on immune cell types within gliomas including glioma-associated macrophages, microglia, cytotoxic T cells and T regulatory cells. We also consider its effects when elaborated on the surface of tumor cells with respect to proliferation, stemness and treatment resistance, and review attempts to target Neuroplin-1 in the clinical setting.

Lucanthone Targets Lysosomes to Perturb Glioma Proliferation, Chemoresistance and Stemness, and Slows Tumor Growth In Vivo.

Glioblastoma is the most common and aggressive primary brain tumor in adults. Median survival time remains at 16-20 months despite multimodal treatment with surgical resection, radiation, temozolomide and tumor-treating fields therapy. After genotoxic stress glioma cells initiate cytoprotective autophagy, which contributes to treatment resistance, limiting the efficacy of these therapies and providing an avenue for glioma recurrence. Antagonism of autophagy steps has recently gained attention as it may enhance the efficacy of classical chemotherapies and newer immune-stimulating therapies. The modulation of autophagy in the clinic is limited by the low potency of common autophagy inhibitors and the inability of newer ones to cross the blood-brain barrier. Herein, we leverage lucanthone, an anti-schistosomal agent which crosses the blood-brain barrier and was recently reported to act as an autophagy inhibitor in breast cancer cells. Our studies show that lucanthone was toxic to glioma cells by inhibiting autophagy. It enhanced anti-glioma temozolomide (TMZ) efficacy at sub-cytotoxic concentrations, and suppressed the growth of stem-like glioma cells and temozolomide-resistant glioma stem cells. In vivo lucanthone slowed tumor growth: reduced numbers of Olig2+ glioma cells, normalized tumor vasculature, and reduced tumor hypoxia. We propose that lucanthone may serve to perturb a mechanism of temozolomide resistance and allow for successful treatment of TMZ-resistant glioblastoma.

Deciphering the dual role and prognostic potential of PINK1 across cancer types.

Metabolic rewiring and deregulation of the cell cycle are hallmarks shared by many cancers. Concerted mutations in key tumor suppressor genes, such as PTEN, and oncogenes predispose cancer cells for marked utilization of resources to fuel accelerated cell proliferation and chemotherapeutic resistance. Mounting research has demonstrated that PTEN-induced putative kinase 1 (PINK1) acts as a pivotal regulator of mitochondrial homeostasis in several cancer types, a function that also extends to the regulation of tumor cell proliferative capacity. In addition, involvement of PINK1 in modulating inflammatory responses has been highlighted by recent studies, further expounding PINK1's multifunctional nature. This review discusses the oncogenic roles of PINK1 in multiple tumor cell types, with an emphasis on maintenance of mitochondrial homeostasis, while also evaluating literature suggesting a dual oncolytic mechanism based on PINK1's modulation of the Warburg effect. From a clinical standpoint, its expression may also dictate the response to genotoxic stressors commonly used to treat multiple malignancies. By detailing the evidence suggesting that PINK1 possesses distinct prognostic value in the clinical setting and reviewing the duality of PINK1 function in a context-dependent manner, we present avenues for future studies of this dynamic protein.

Interactions between Tumor Cells, Neurons, and Microglia in the Glioma Microenvironment.

Despite significant strides made in understanding the pathophysiology of high-grade gliomas over the past two decades, most patients succumb to these neoplasias within two years of diagnosis. Furthermore, there are various co-morbidities associated with glioma and standard of care treatments. Emerging evidence suggests that aberrant glutamate secretion in the glioma microenvironment promotes tumor progression and contributes to the development of co-morbidities, such as cognitive defects, epilepsy, and widespread neurodegeneration. Recent data clearly illustrate that neurons directly synapse onto glioma cells and drive their proliferation and spread via glutamatergic action. Microglia are central nervous system-resident myeloid cells, modulate glioma growth, and possess the capacity to prune synapses and encourage synapse formation. However, current literature has yet to investigate the potential role of microglia in shaping synapse formation between neurons and glioma cells. Herein, we present the literature concerning glutamate's role in glioma progression, involving hyperexcitability and excitotoxic cell death of peritumoral neurons and stimulation of glioma proliferation and invasion. Furthermore, we discuss instances in which microglia are more likely to sculpt or encourage synapse formation during glioma treatment and propose studies to delineate the role of microglia in synapse formation between neurons and glioma cells. The sex-dependent oncogenic or oncolytic actions of microglia and myeloid cells, in general, are considered in addition to the functional differences between microglia and macrophages in tumor progression. We also put forth tractable methods to safely perturb aberrant glutamatergic action in the tumor microenvironment without significantly increasing the toxicities of the standard of care therapies for glioma therapy.

PINK1 depletion sensitizes non-small cell lung cancer to glycolytic inhibitor 3-bromopyruvate: Involvement of ROS and mitophagy.

BACKGROUND: Despite significant strides in understanding the pathophysiology of non-small cell lung cancer (NSCLC), these neoplasms typically present with intrinsic chemo- and radiotherapeutic resistance. Transcriptomic analyses of patient NSCLC tumors stratified by survival times have identified the PTEN-induced putative kinase 1 (PINK1) as a molecular governor of tumor aggressiveness and patient survival time. PINK1 has been shown to confer neuroprotection in models of Parkinson Disease by ensuring proper mitochondrial turnover (mitophagy), the upkeep of ATP production and sequestering of reactive oxygen species (ROS). METHODS: We utilized an shRNA against PINK1 and the glycolytic inhibitor 3-BP to assess effects on NSCLC viability via MTS cell viability assay. ATP levels, caspase-9 activation, mitophagy and ROS production were determined with standardly available kits. Cytochrome c cellular localization and phosphorylated parkin levels were determined using an ELISA. RESULTS: Our results demonstrate that PINK1 depletion in the NSCLC cell line A549 via shRNA, reduced cancer cell proliferation, increased cell death, reduced ATP production, inhibited mitophagy and increased ROS and caspase-9-dependent apoptosis. PINK1 depleted cells were more susceptible to the glycolytic inhibitor 3-bromopyruvate (3-BP), which further perturbed ATP production. PINK1 depletion and 3-BP synergistically increased ROS production, caspase-9-dependent apoptosis and additively repressed mitophagy. CONCLUSIONS: These results suggest that PINK1 depletion alters energetic metabolism and confers sensitivity to agents that inhibit glycolysis. Targeting accelerated tumor cell metabolism may prove useful in the clinical setting while sparing non-malignant tissue.

Effects of chronic systemic low-impact ampakine treatment on neurotrophin expression in rat brain.

Neurotrophin dysregulation has been implicated in a large number of neurodegenerative and neuropsychiatric diseases. Unfortunately, neurotrophins cannot cross the blood brain barrier thus, novel means of up regulating their expression are greatly needed. It has been demonstrated previously that neurotrophins are up regulated in response to increases in brain activity. Therefore, molecules that act as cognitive enhancers may provide a clinical means of up regulating neurotrophin expression. Ampakines are a class of molecules that act as positive allosteric modulators of AMPA-type glutamate receptors. Currently, they are being developed to prevent opioid-induced respiratory depression without sacrificing the analgesic properties of the opioids. In addition, these molecules increase neuronal activity and have been shown to restore age-related deficits in LTP in aged rats. In the current study, we examined whether two different ampakines could increase levels of BDNF and NGF at doses that are active in behavioral measures of cognition. Results demonstrate that ampakines CX516 and CX691 induce differential increases in neurotrophins across several brain regions. Notable increases in NGF were observed in the dentate gyrus and piriform cortex while notable BDNF increases were observed in basolateral and lateral nuclei of the amygdala. Taken together, our data demonstrates that chronic administration of clinically relevant doses of ampakines have the ability to elevate neurotrophin expression in different brain regions, and may have therapeutic benefit in multiple neurodegenerative and/or neuropsychiatric disorders.

Ampakines Attenuate Staurosporine-induced Cell Death in Primary Cortical Neurons: Implications in the 'Chemo-Brain' Phenomenon.

BACKGROUND/AIM: Mounting evidence suggests that trophic cell signaling can be mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activation. It has been demonstrated that exogenous application of brain-derived neurotrophic factor (BDNF) is highly neuroprotective in vitro against neurotoxic insults such as standard chemotherapies. MATERIALS AND METHODS: Because positive allosteric modulation of AMPARs with ampakines can increase both BDNF mRNA and protein in vitro and in vivo, we examined whether application of the ampakines CX614 and CX729 promoted neuroprotection against staurosporine-induced cell death in rat primary cortical neurons using propidium iodide to stain for dead cells. RESULTS: A transient 2-h pretreatment with CX614 or CX729 performed 24 h prior to staurosporine produced significant, time-dependent neuroprotection that was resistant to the AMPAR antagonists NBQX or GYKI 52466, suggesting that this effect may be independent of ion flow. Furthermore, the pretreatment time requirements of CX729 matched the time course for increased BDNF expression previously reported to occur in hippocampal slices, suggesting that increased neurotrophin expression might be associated with the neuroprotective effects conferred by ampakines. CONCLUSION: Our data demonstrate that ampakines may be able to perturb neuronal toxicity and peripheral neuropathy of front-line chemotherapies.

Tarps differentially affect the pharmacology of ampakines.

Transmembrane AMPA receptor regulatory proteins (TARPs) govern AMPA receptor cell surface expression and distinct physiological properties including agonist affinity, desensitization and deactivation kinetics. The prototypical TARP, STG or γ2 and TARPs γ3, γ4, γ7 and γ8 are all expressed to varying degrees in the mammalian brain and differentially regulate AMPAR gating parameters. Positive allosteric AMPA receptor modulators or ampakines alter receptor rates of agonist binding/unbinding, channel opening and can offset receptor desensitization and deactivation. The effects of the two ampakines, CX614 and cyclothiazide (CTZ) were evaluated on homomeric GluR1-flip receptors and GluR2-flop receptors expressed on HEK293 cells by transient transfection with or without different TARPs γ2, γ3, γ4 or γ8 genes. γ4 was the most robust TARP in increasing the affinities of CX614 and CTZ on GluR1-flip receptors, but had no such effect on GluR2-flop receptors. However, γ8 gave the most significant increases in affinities of CX614 and CTZ on GluR2-flop. These data show that TARPs differentially affect the surface expression and kinetics of the AMPA receptor, as well as the pharmacology of ampakines for the AMPA receptor. The modulatory effects of TARPs on ampakine pharmacology are complex, being dependent on both the TARP subtype and the AMPA receptor subtypes/isoforms.

Stargazin differentially modulates ampakine gating kinetics and pharmacology.

It was previously reported that Stargazin (STG) enhances the surface expression of AMPA receptors, controls receptor gating and slows channel desensitization as an auxiliary subunit of the receptors. Ampakines are a class of AMPA receptor positive allosteric modulators that modify rates of transmitter binding, channel activity and desensitization parameters. As such, they have shown efficacy in animal models of neurodegenerative diseases, where excitatory synaptic transmission is compromised. Given the functional similarities between STG and ampakines, the current study sought to probe interactions between STG and ampakine gating properties. The effects of the high impact ampakines, CX614 and cyclothiazide (CTZ), were compared with homomeric GluR1-flip (Glur1i) and GluR2-flop (Glur2o) receptors expressed in HEK293 cells by transient transfection with or without STG gene. STG dramatically enhanced the surface expression of AMPA receptors and increased glutamate-induced steady-state currents during desensitization. STG also increased ratios of 500 µM kainate and 500 µM glutamate activated steady-state currents. STG reduced association rates of ampakines and differentially affected the dissociation rates for both CX614 and CTZ on desensitized receptors. The estimated Kd value for CX614 was lowered from 340 µM to 70 µM, whereas that for CTZ was lowered from 170 µM to 6 µM by STG. The data suggest that Stargazin can dramatically alter the conformation of the receptor dimer interface where CX614 and CTZ are known to bind. This work also demonstrates the importance of considering STG interactions when developing ampakines to treat neurodegenerative diseases in which AMPAergic signaling is compromised.

Our ACE in the HOLE: Justifying the Use of Angiotensin-converting Enzyme Inhibitors as Adjuvants to Standard Chemotherapy.

Angiotensin-I-converting enzyme (ACE) inhibitors have been very effective in treating cardiac hypertension since their clinical inception over four decades ago. Since then, it has been established that angiotensin II, the product of ACE, has oncogenic and pro-proliferative qualities, which begs the question as to whether ACE inhibitors may have oncolytic characteristics. In fact, scattered reports suggest that ACE inhibitors are oncolytic and oncopreventive, but the available literature has yet to be thoroughly examined. In the present review, we examine the available literature and determine that ACE inhibitors would have great utility in the prevention and treatment of cancer. At the same time, they would augment the efficacy of chemo- and radiotherapy as well as mitigating damage to healthy tissue by standard chemotherapeutic regimens. We review some of the mounting clinical evidence and show that ACE inhibitors have oncolytic activity in multiple types of cancer and discuss the ability of ACE inhibitors to prevent cardiotoxicity of multiple chemotherapies. Our analysis demonstrates that the actions of ACE inhibitors converge on vascular endolthelial growth factor to reduce its levels in tumors and prevent construction of blood vessels to masses, leaving them nutrient-depleted and subsequently hindering their growth. Given that ACE inhibitors are approved by the Federal Drug Administration and the therapeutic dose for hypertension treatment also slows the growth of multiple cancers types, ACE inhibitors are in a perfect position to be repurposed as oncolytic agents, that would widely increase their utility in the clinic.

Oncolytic Properties of Ampakines In Vitro.

BACKGROUND/AIM: The 5-year survival rate of glioblastoma (GBM) is ~10%, demonstrating that a new therapeutic modality for this cancer is desperately needed. Complicating the search for such a modality is that most large molecules cannot pass through the blood brain barrier, so molecules demonstrating efficacy in vitro may not be useful in vivo because they never reach the brain. Recently, the selective serotonin reuptake inhibitor (SSRI) fluoxetine (FLX) was identified as an effective agent in targeting GBM in vitro and in vivo by agonizing AMPA-glutamate receptors (AMPARs), eliciting massive calcium influx and mitochondrial calcium overload and apoptosis. MATERIALS AND METHODS: In the current study, we used a colorimetric cell viability assay to determine if we could enhance the oncolytic effect of FLX in vitro by pre-treating cells with an AMPAR-positive allosteric modulator (Ampakine). RESULTS: Our results demonstrated for the first time that concentrations of the Class I ampakine CX614, which increase AMPAR agonist binding affinity, possess oncolytic activity as a sole agent and synergistically reduce GBM viability when paired with FLX. FLX also demonstrates a dose-dependent induction of apoptosis in cancer cells originating outside the CNS that overexpress calcium-permeable AMPARs. Likewise, CX614 inhibits cancer cell viability in a dose-dependent fashion and its combination with FLX synergistically reduces cell viability. These effects of CX614 were not seen with the Class II ampakines, CX717 and CX1739. CONCLUSION: CX614 inhibits the growth of multiple cancers in vitro and bolsters the oncolytic activity of FLX in several cancers.

BDNF: An Oncogene or Tumor Suppressor?

Neurotrophins are a family of growth factors that are vital to the proper development of the central nervous system. Their effects on cells are governed by the expression and activation of the tyrosine kinase receptors TrkA, TrkB and TrkC. TrkB has been immensely implicated in mediating neuronal migration, development and differentiation. It has also been shown to protect several neuronal cell types from an array of cytotoxic stressors after activation by its conjugate ligand brain-derived neurotrophic factor (BDNF). Over the past two decades, it has been shown that TrkB and BDNF are up-regulated in many types of cancers, conferring aggressive phenotypes underpinned by their resistance to several standard chemotherapeutic agents. This resistance to chemotherapy is modulated by the downstream targets of the TrkB receptor which include the well-characterized PI3K /Akt growth pathway, a hallmark of uncontrolled cancer cell growth and proliferation. Pre-clinical efforts to develop inhibitors of this receptor are promising, and such inhibitors also seem to sensitize cancer cells to standard chemotherapies. However, new evidence suggests that BDNF overexpression in the hypothalamus has immunoaugmenting properties, eliciting an increased anti-tumor immune response and reducing the activity of several proteins that would normally confer resistance to chemotherapeutic agents. In the current work, we provide a global analysis of the physiological consequences of TrkB receptor activation in vitro and discuss the dynamic consequences of TrkB activation in vivo. Finally, we propose a clinically-feasible option for increasing BDNF expression in the hypothalamus to more readily utilize the oncolytic effects of BDNF.

A current perspective on the oncopreventive and oncolytic properties of selective serotonin reuptake inhibitors.

Current cancer research strongly focuses on identifying novel pathways that can be selectively exploited in the clinic and identifying drugs capable of exploiting cancer vulnerabilities. Occasionally, drugs identified to exploit a cancer-specific vulnerability are on the market for clinical indications in another disease area. Rebranding them as anti-cancer drugs is a process commonly referred to as drug repurposing and is typically a faster method than bringing a novel drug to market. Selective serotonin reuptake inhibitors (SSRIs) are primarily used for treating several types of depression, but over the past two decades mounting evidence suggests that drugs in this class have oncolytic properties and reduce the risk of certain cancers. In the current work, we discuss how the secondary mechanisms of action associated with these drugs mediate their oncolytic effect. In particular, sertraline limits tumor growth by abrogating the PI3K/akt signaling pathway, a growth pathway shown to be constitutively active in multiple cancers. Fluoxetine has been shown to activate the AMPA-type glutamate receptor, induce massive calcium influx and mitochondrial calcium overload and induce caspase-9-dependent apoptosis. This receptor being highly overexpressed in cancer stem cells may explain why SSRIs lower the risk of multiple types of cancer. Fluoxetine has also been shown to inhibit multidrug resistance pumps, increasing the efficacy of several standard chemotherapies. Given the vast potential of SSRIs in treating cancer, these drugs should be more heavily used not only in treating cancer-related depression, but in combating cancer and increasing the efficacy of standard of care chemotherapies.

Acute ampakine treatment ameliorates age-related deficits in long-term potentiation.

Memory loss observed as a consequence of aging is paralleled by a down-regulation of AMPA-type glutamate receptors (AMPARs) that mediate fast excitatory synaptic transmission. Activation of these receptors enhances long-term potentiation (LTP), a neuronal process demonstrated to be crucial for memory storage and thought to be a cellular substrate of learning and memory. In the present studies, we determined that LTP was reduced in aged rats when compared to young rats and that acute treatment with CX1846, a novel AMPAR positive allosteric modulator, fifteen minutes prior to tetanic stimulation completely reversed the significant deficit in LTP observed in aged rats. These results suggest that CX1846 might be useful for the treatment of age-related memory impairments.

Delineating the molecular mechanisms of tamoxifen's oncolytic actions in estrogen receptor-negative cancers.

Since its clinical inception, tamoxifen (TAM) has proved to be a powerful tool in treating estrogen receptor-positive breast cancers while exhibiting manageable side effects. Although TAM was synthesized as an estrogen receptor antagonist, reports have found that a significant fraction of women with estrogen receptor-negative cancers have benefitted from TAM treatment, suggesting the possibility of an alternate anti-cancer mechanism. In this paper, we present a review of recent and past literature in an attempt to clarify how TAM inhibits cell proliferation and induces apoptosis in cells lacking the estrogen receptor. Our analysis indicates that micromolar concentrations of TAM selectively elevate intracellular calcium concentrations in malignant cells, possibly by inversely agonizing cannabinoid receptors, producing considerable mitochondrial distress followed by the rapid production of reactive oxygen species. In response, cytoplasmic proteins such as JNK1 are activated, which mediates the activation of caspase-8. Fyn kinase auto phosphorylates in response to increased reactive oxygen species and directs the ubiquitin ligase c-Cbl to tag growth factor receptors for ubiquitination, potentially abrogating constitutively active survival pathways that are hallmarks of cancer progression. We attempt to differentiate the effect that TAM has on purified Protein Kinase C (PKC) compared to that in an intact cell, suggesting that low micromolar concentrations of TAM indirectly inhibit PKC by inducing EGFR destruction and high micromolar concentrations of TAM inhibits PKC through a direct binding mechanism.