Premarin Reduces Neurodegeneration and Promotes Improvement of Function in an Animal Model of Spinal Cord Injury.

Spinal cord injury (SCI) causes significant mortality and morbidity. Currently, no FDA-approved pharmacotherapy is available for treating SCI. Previously, low doses of estrogen (17ß-estradiol, E2) were shown to improve the post-injury outcome in a rat SCI model. However, the range of associated side effects makes advocating its therapeutic use difficult. Therefore, this study aimed at investigating the therapeutic efficacy of Premarin (PRM) in SCI. PRM is an FDA-approved E2 (10%) formulation, which is used for hormone replacement therapy with minimal risk of serious side effects. The effects of PRM on SCI were examined by magnetic resonance imaging, immunofluorescent staining, and western blot analysis in a rat model. SCI animals treated with vehicle alone, PRM, E2 receptor antagonist (ICI), or PRM + ICI were graded in a blinded way for locomotor function by using the Basso-Beattie-Bresnahan (BBB) locomotor scale. PRM treatment for 7 days decreased post-SCI lesion volume and attenuated neuronal cell death, inflammation, and axonal damage. PRM also altered the balance of pro- and anti-apoptotic proteins in favor of cell survival and improved angiogenesis and microvascular growth. Increased expression of estrogen receptors (ERs) ERα and ERß following PRM treatment and their inhibition by ER inhibitor indicated that the neuroprotection associated with PRM treatment might be E2-receptor mediated. The attenuation of glial activation with decreased inflammation and cell death, and increased angiogenesis by PRM led to improved functional outcome as determined by the BBB locomotor scale. These results suggest that PRM treatment has significant therapeutic implications for the improvement of post-SCI outcome.

Regulation of autophagy as a therapeutic option in glioblastoma.

Around three out of one hundred thousand people are diagnosed with glioblastoma multiforme, simply called glioblastoma, which is the most common primary brain tumor in adults. With a dismal prognosis of a little over a year, receiving a glioblastoma diagnosis is oftentimes fatal. A major advancement in its treatment was made almost two decades ago when the alkylating chemotherapeutic agent temozolomide (TMZ) was combined with radiotherapy (RT). Little progress has been made since then. Therapies that focus on the modulation of autophagy, a key process that regulates cellular homeostasis, have been developed to curb the progression of glioblastoma. The dual role of autophagy (cell survival or cell death) in glioblastoma has led to the development of autophagy inhibitors and promoters that either work as monotherapies or as part of a combination therapy to induce cell death, cellular senescence, and counteract the ability of glioblastoma stem cells (GSCs) for initiating tumor recurrence. The myriad of cellular pathways that act upon the modulation of autophagy have created contention between two groups: those who use autophagy inhibition versus those who use promotion of autophagy to control glioblastoma growth. We discuss rationale for using current major therapeutics, their molecular mechanisms for modulation of autophagy in glioblastoma and GSCs, their potentials for making strides in combating glioblastoma progression, and their possible shortcomings. These shortcomings may fuel the innovation of novel delivery systems and therapies involving TMZ in conjunction with another agent to pave the way towards a new gold standard of glioblastoma treatment.

Synergism of 4HPR and SAHA increases anti-tumor actions in glioblastoma cells.

Glioblastoma is the most malignant and prevalent brain tumor in adults. It can grow and spread quickly causing harm to the brain health. One of the major challenges in treatment of glioblastoma is drug resistance. Use of synergistic combination of two drugs with different anti-tumor effects is nowadays highly considered in the development of effective therapeutic strategies for many malignancies. In the present study, we showed synergistic therapeutic efficacies of two chemical compounds, N-(4-hydroxyphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA), for significant reduction in cell viability of rat C6 and human T98G glioblastoma cells. These compounds (4HPR and SAHA) were used alone or in synergistic combination for evaluating their various anti-tumor effects. The results showed that combination of 4HPR and SAHA significantly induced morphological and molecular features of astrocytic differentiation in C6 and T98G glioblastoma cells. Combination of 4HPR and SAHA proved to be an important therapeutic strategy for inhibiting cell growth and inducing differentiation in glioblastoma cells. Furthermore, combination of the two drugs showed more efficacies than either dug alone in reducing in vitro cell invasion (transwell assay), cell migration (wound healing assay), and angiogenesis (tube formation assay) due to down regulation of the molecules involved in these processes. The ultimate of goal of using this combination of drugs was induction of apoptosis. The results showed that these drugs in synergistic combination contributed highly to increases in morphological and molecular features of apoptotic death in the tumor cells. The results from molecular studies indicated that cell death occurred via activation of the extrinsic and intrinsic pathways of apoptosis in both C6 and T98G cells. The drugs in combination also contributed to dramatic inhibition of histone deacetylase 1, an important epigenetic player in promoting growth in glioblastoma cells. This novel combination of drugs should also be considered as a promising therapeutic strategy for the treatment of glioblastoma in vivo.

Diosgenin as a Novel Alternative Therapy for Inhibition of Growth, Invasion, and Angiogenesis Abilities of Different Glioblastoma Cell Lines.

Fenugreek (Trigonella foenum-graecum) seeds and roots of wild yam (Dioscorea villosa) possess nutritional and medicinal properties and have been used for centuries in traditional medicine to treat different diseases and inflammatory responses. Diosgenin is a natural steroidal sapogenin extracted from fenugreek and wild yam and it is one of the major bioactive compounds used in the treatment of diabetes, hypercholesterolemia, and inflammation. Recent studies have shown a promising effect of diosgenin as an anti-tumor agent for inhibition of cell proliferation and induction of apoptosis in many cancers such as colon cancer, leukemia, breast cancer, and liver cancer. We examined the effects of different concentrations (5, 10, 15, 20, and 25 µM) of diosgenin on proliferation of rat C6 and human T98G glioblastoma cell lines. We noticed that diosgenin had a high inhibitory effect on the growth of both C6 and T98G cell lines. Diosgenin induced the differentiation of glioblastoma cells, as determined by the increase in the expression of the differentiation marker glial fibrillary acidic protein (GFAP); and decreased the dedifferentiation of the cells, as shown by the decrease in the abundance of the dedifferentiation marker proteins Id2, N-Myc, telomerase reverse transcriptase (TERT), and Notch-1. It also induced apoptosis in C6 and T98G cell lines and the molecular mechanisms involved in the induction of apoptosis included increase in pro-apoptotic Bax protein and decrease in anti-apoptotic Bcl-2 protein. Further, the diosgenin-induced suppression of cell migration was correlated with the decrease in expression of matrix metalloproteinase 2 (MMP2) and MMP9; and the inhibition of angiogenesis, as determined by the tube formation assay, was correlated with a decrease in the protein levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). In conclusion, diosgenin showed anti-tumor effects in glioblastoma cells by induction of differentiation and apoptosis and inhibition of migration, invasion, and angiogenesis.

Quercetin and Sodium Butyrate Synergistically Increase Apoptosis in Rat C6 and Human T98G Glioblastoma Cells Through Inhibition of Autophagy.

This study investigated the efficacy of quercetin (QCT) in combination with sodium butyrate (NaB) in enhancing apoptosis in rat C6 and human T98G glioblastoma cells though blockage of autophagy under nutrient-starvation. The most synergistic doses of the drugs were determined to be 25 µM QCT and 1 mM NaB in both cell lines. After QCT and QCT + NaB treatments, autophagy quantification with acridine orange staining showed a drastic decrease in protective autophagy in the cells under nutrient-starvation. Decrease in autophagy was correlated with decreases in expression of Beclin-1 and LC3B II. Combination treatment increased the morphological signs of apoptosis including membrane blebbing, nuclear fragmentation, and chromatin condensation. Annexin V staining was also performed for detection and quantification of increases in apoptosis. Western blotting results showed that combination of QCT and NaB increased apoptosis by decreasing anti-apoptotic Bcl-2 and increasing pro-apoptotic Bax, decreasing survivin, activating caspase-3, and degrading poly (ADP-ribose) polymerase (PARP). This study demonstrated the therapeutic potentials of a novel combination therapy in inhibiting protective autophagy to enhance apoptosis in rat C6 and human T98G glioblastoma cells.

Ceramide and Sphingosine Regulation of Myelinogenesis: Targeting Serine Palmitoyltransferase Using microRNA in Multiple Sclerosis.

Ceramide and sphingosine display a unique profile during brain development, indicating their critical role in myelinogenesis. Employing advanced technology such as gas chromatography-mass spectrometry, high performance liquid chromatography, and immunocytochemistry, along with cell culture and molecular biology, we have found an accumulation of sphingosine in brain tissues of patients with multiple sclerosis (MS) and in the spinal cord of rats induced with experimental autoimmune encephalomyelitis. The elevated sphingosine leads to oligodendrocyte death and fosters demyelination. Ceramide elevation by serine palmitoyltransferse (SPT) activation was the primary source of the sphingosine elevation as myriocin, an inhibitor of SPT, prevented sphingosine elevation and protected oligodendrocytes. Supporting this view, fingolimod, a drug used for MS therapy, reduced ceramide generation, thus offering partial protection to oligodendrocytes. Sphingolipid synthesis and degradation in normal development is regulated by a series of microRNAs (miRNAs), and hence, accumulation of sphingosine in MS may be prevented by employing miRNA technology. This review will discuss the current knowledge of ceramide and sphingosine metabolism (synthesis and breakdown), and how their biosynthesis can be regulated by miRNA, which can be used as a therapeutic approach for MS.

Targeting autophagy for combating chemoresistance and radioresistance in glioblastoma.

Autophagy is an evolutionarily conserved catabolic process that plays an essential role in maintaining cellular homeostasis by degrading unneeded cell components. When exposed to hostile environments, such as hypoxia or nutrient starvation, cells hyperactivate autophagy in an effort to maintain their longevity. In densely packed solid tumors, such as glioblastoma, autophagy has been found to run rampant due to a lack of oxygen and nutrients. In recent years, targeting autophagy as a way to strengthen current glioblastoma treatment has shown promising results. However, that protective autophagy inhibition or autophagy overactivation is more beneficial, is still being debated. Protective autophagy inhibition would lower a cell's previously activated defense mechanism, thereby increasing its sensitivity to treatment. Autophagy overactivation would cause cell death through lysosomal overactivation, thus introducing another cell death pathway in addition to apoptosis. Both methods have been proven effective in the treatment of solid tumors. This systematic review article highlights scenarios where both autophagy inhibition and activation have proven effective in combating chemoresistance and radioresistance in glioblastoma, and how autophagy may be best utilized for glioblastoma therapy in clinical settings.

Sphingosine Toxicity in EAE and MS: Evidence for Ceramide Generation via Serine-Palmitoyltransferase Activation.

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.

Broad targeting of resistance to apoptosis in cancer.

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Calpain inhibition reduces structural and functional impairment of retinal ganglion cells in experimental optic neuritis.

Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis. ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells, and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrificing (Day 26). Visual function was determined by electroretinogram recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias toward unilateral OKR impairment among EAE-ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern electroretinogram amplitudes, and paralysis, with attenuation of retinal ganglion cell death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE-ON parallels the clinical presentation of ON exacerbations associated with multiple sclerosis. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON. As in multiple sclerosis (MS) patients, optic neuritis (ON) and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE, experimental autoimmune encephalomyelitis). Daily oral treatment with the calpain inhibitor SNJ 1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a, brain-specific homeobox/POU domain protein 3A) and their axons (MOSP, myelin oligodendrocyte-specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in ON.

Administration of low dose estrogen attenuates gliosis and protects neurons in acute spinal cord injury in rats.

Spinal cord injury (SCI) is a debilitating condition with neurological deficits and loss of motor function that, depending on the severity, may lead to paralysis. The only treatment currently available is methylprednisolone, which is widely used and renders limited efficacy in SCI. Therefore, other therapeutic agents must be developed. The neuroprotective efficacy of estrogen in SCI was studied with a pre-clinical and pro-translational perspective. Acute SCI was induced in rats that were treated with low doses of estrogen (1, 5, 10, or 100 µg/kg) and compared with vehicle-treated injured rats or laminectomy control (sham) rats at 48 h post-SCI. Changes in gliosis and other pro-inflammatory responses, expression and activity of proteolytic enzymes (e.g., calpain, caspase-3), apoptosis of neurons in SCI, and cell death were monitored via Western blotting and immunohistochemistry. Negligible pro-inflammatory responses or proteolytic events and very low levels of neuronal death were found in sham rats. In contrast, vehicle-treated SCI rats showed profound pro-inflammatory responses with reactive gliosis, elevated expression and activity of calpain and caspase-3, elevated Bax:Bcl-2 ratio, and high levels of neuronal death in lesion and caudal regions of the injured spinal cord. Estrogen treatment at each dose reduced pro-inflammatory and proteolytic activities and protected neurons in the caudal penumbra in acute SCI. Estrogen treatment at 10 µg was found to be as effective as 100 µg in ameliorating the above parameters in injured animals. Results from this investigation indicated that estrogen at a low dose could be a promising therapeutic agent for treating acute SCI. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript.

Administration of low dose estrogen attenuates persistent inflammation, promotes angiogenesis, and improves locomotor function following chronic spinal cord injury in rats.

Spinal cord injury (SCI) causes loss of neurological function and, depending upon the severity of injury, may lead to paralysis. Currently, no FDA-approved pharmacotherapy is available for SCI. High-dose methylprednisolone is widely used, but this treatment is controversial. We have previously shown that low doses of estrogen reduces inflammation, attenuates cell death, and protects axon and myelin in SCI rats, but its effectiveness in recovery of function is not known. Therefore, the goal of this study was to investigate whether low doses of estrogen in post-SCI would reduce inflammation, protect cells and axons, and improve locomotor function during the chronic phase of injury. Injury (40 g.cm force) was induced at thoracic 10 in young adult male rats. Rats were treated with 10 or 100 µg 17ß-estradiol (estrogen) for 7 days following SCI and compared with vehicle-treated injury and laminectomy (sham) controls. Histology (H&E staining), immunohistofluorescence, Doppler laser technique, and Western blotting were used to monitor tissue integrity, gliosis, blood flow, angiogenesis, the expression of angiogenic factors, axonal degeneration, and locomotor function (Basso, Beattie, and Bresnahan rating) following injury. To assess the progression of recovery, rats were sacrificed at 7, 14, or 42 days post injury. A reduction in glial reactivity, attenuation of axonal and myelin damage, protection of cells, increased expression of angiogenic factors and microvessel growth, and improved locomotor function were found following estrogen treatment compared with vehicle-treated SCI rats. These results suggest that treatment with a very low dose of estrogen has significant therapeutic implications for the improvement of locomotor function in chronic SCI. Experimental studies with low dose estrogen therapy in chronic spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes that could ameliorate the degenerative pathways in chronic SCI as shown in (a). Furthermore, the alterations in local spinal blood flow could be significantly alleviated with low dose estrogen therapy. This therapy led to the preservation of the structural integrity of the spinal cord (b), which in turn led to the improved functional recovery as shown (c).

Neuron-microglia interaction induced bi-directional cytotoxicity associated with calpain activation.

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Anti-tumor activities of luteolin and silibinin in glioblastoma cells: overexpression of miR-7-1-3p augmented luteolin and silibinin to inhibit autophagy and induce apoptosis in glioblastoma in vivo.

Glioblastoma is the deadliest brain tumor in humans. High systemic toxicity of conventional chemotherapies prompted the search for natural compounds for controlling glioblastoma. The natural flavonoids luteolin (LUT) and silibinin (SIL) have anti-tumor activities. LUT inhibits autophagy, cell proliferation, metastasis, and angiogenesis and induces apoptosis; while SIL activates caspase-8 cascades to induce apoptosis. However, synergistic anti-tumor effects of LUT and SIL in glioblastoma remain unknown. Overexpression of tumor suppressor microRNA (miR) could enhance the anti-tumor effects of LUT and SIL. Here, we showed that 20 µM LUT and 50 µM SIL worked synergistically for inhibiting growth of two different human glioblastoma U87MG (wild-type p53) and T98G (mutant p53) cell lines and natural combination therapy was more effective than conventional chemotherapy (10 µM BCNU or 100 µM TMZ). Combination of LUT and SIL caused inhibition of growth of glioblastoma cells due to induction of significant amounts of apoptosis and complete inhibition of invasion and migration. Further, combination of LUT and SIL inhibited rapamycin (RAPA)-induced autophagy, a survival mechanism, with suppression of PKCα and promotion of apoptosis through down regulation of iNOS and significant increase in expression of the tumor suppressor miR-7-1-3p in glioblastoma cells. Our in vivo studies confirmed that overexpression of miR-7-1-3p augmented anti-tumor activities of LUT and SIL in RAPA pre-treated both U87MG and T98G tumors. In conclusion, our results clearly demonstrated that overexpression of miR-7-1-3p augmented the anti-tumor activities of LUT and SIL to inhibit autophagy and induce apoptosis for controlling growth of different human glioblastomas in vivo.

Neuron specific enolase: a promising therapeutic target in acute spinal cord injury.

Enolase is a multifunctional protein, which is expressed abundantly in the cytosol. Upon stimulatory signals, enolase can traffic to cell surface and contribute to different pathologies including injury, autoimmunity, infection, inflammation, and cancer. Cell-surface expression of enolase is often detected on activated macrophages, microglia/macrophages, microglia, and astrocytes, promoting extracellular matrix degradation, production of pro-inflammatory cytokines/chemokines, and invasion of inflammatory cells in the sites of injury and inflammation. Inflammatory stimulation also induces translocation of enolase from the cytosolic pool to the cell surface where it can act as a plasminogen receptor and promote extracellular matrix degradation and tissue damage. Spinal cord injury (SCI) is a devastating debilitating condition characterized by progressive pathological changes including complex and evolving molecular cascades, and insights into the role of enolase in multiple inflammatory events have not yet been fully elucidated. Neuronal damage following SCI is associated with an elevation of neuron specific enolase (NSE), which is also known to play a role in the pathogenesis of hypoxic-ischemic brain injury. Thus, NSE is now considered as a biomarker in ischemic brain damage, and it has recently been suggested to be a biomarker in traumatic brain injury (TBI), stroke and anoxic encephalopathy after cardiac arrest and acute SCI as well. This review article gives an overview of the current basic research and clinical studies on the role of multifunctional enolase in neurotrauma, with a special emphasis on NSE in acute SCI.

Carbon Nanomaterials for Drug Delivery and Cancer Therapy.

Nanotechnology is one of the most exciting disciplines and it incorporates physics, chemistry, materials science, and biology. It can be applied to design cancer medicines with improved therapeutic indices. At the basic level, carbon nanotubes (CNTs) and graphene are sp2 carbon nanomaterials. Their unique physical and chemical properties make them interesting candidates of research in a wide range of areas including biological systems and different diseases. Recent research has been focused on exploring the potential of the CNTs as a carrier or vehicle for intracellular transport of drugs, proteins, and targeted genes in vitro and in vivo. Several research groups are actively involved to find out a functional CNT carrier capable of transporting targeted drug molecules in animal models with least toxicity. Current investigations are also focused on graphene, an allotrope of carbon, which appears to be a promising agent for successful delivery of biomolecules in various animal models. But potential clinical implementations of CNTs are still hampered by distinctive barriers such as poor bioavailability and intrinsic toxicity, which pose difficulties in tumor targeting and penetration as well as in improving therapeutic outcome. This article presents recent progresses in the design and evaluation of closely related CNTs for experimental cancer therapy and explores their implications in bringing nanomedicines into the clinics.

Retinoic acid signaling pathways in development and diseases.

Retinoids comprise a group of compounds each composed of three basic parts: a trimethylated cyclohexene ring that is a bulky hydrophobic group, a conjugated tetraene side chain that functions as a linker unit, and a polar carbon-oxygen functional group. Biochemical conversion of carotenoid or other retinoids to retinoic acid (RA) is essential for normal regulation of a wide range of biological processes including development, differentiation, proliferation, and apoptosis. Retinoids regulate various physiological outputs by binding to nuclear receptors called retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which themselves are DNA-binding transcriptional regulators. The functional response of RA and their receptors are modulated by a host of coactivators and corepressors. Retinoids are essential in the development and function of several organ systems; however, deregulated retinoid signaling can contribute to serious diseases. Several natural and synthetic retinoids are in clinical use or undergoing trials for treating specific diseases including cancer. In this review, we provide a broad overview on the importance of retinoids in development and various diseases, highlighting various retinoids in the drug discovery process, ranging all the way from retinoid chemistry to clinical uses and imaging.

miR-138 overexpression is more powerful than hTERT knockdown to potentiate apigenin for apoptosis in neuroblastoma in vitro and in vivo.

Decrease in expression of the tumor suppressor microRNA-138 (miR-138) correlates well with an increase in telomerase activity in many human cancers. The ability of almost all human cancer cells to grow indefinitely is dependent on presence of telomerase activity. The catalytic component of human telomerase reverse transcriptase (hTERT) regulates telomerase activity in most of the human cancers including malignant neuroblastoma. We observed an indirect increase in the expression of miR-138 after the transfection with hTERT short hairpin RNA (shRNA) plasmid in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines. Transfection with hTERT shRNA plasmid followed by treatment with the flavonoid apigenin (APG) further increased expression of miR-138. Direct transfection with miR-138 mimic was more powerful than transfection with hTERT shRNA plasmid in potentiating efficacy of APG for decreasing cell viability and colony formation capability of both cell lines. Upregulation of miR-138 was also more effective than down regulation of hTERT in enhancing efficacy of APG for induction of apoptosis in malignant neuroblastoma cells in vitro and in vivo. We delineated that apoptosis occurred with induction of molecular components of the extrinsic and intrinsic pathways in SK-N-DZ and SK-N-BE2 cells both in vitro and in vivo. In conclusion, these results demonstrate that direct miR-138 overexpression is more powerful than hTERT down regulation in enhancing pro-apoptotic effect of APG for controlling growth of human malignant neuroblastoma in cell culture and animal models.

TUNEL-n-DIFL Method for Detection and Estimation of Apoptosis Specifically in Neurons and Glial Cells in Mixed Culture and Animal Models of Central Nervous System Diseases and Injuries.

Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in a specific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS cell type in the mixed culture and animal models of the CNS diseases and injuries.

Epigallocatechin-3-Gallate and Genistein for Decreasing Gut Dysbiosis, Inhibiting Inflammasomes, and Aiding Autophagy in Alzheimer's Disease.

There are approximately 24 million cases of Alzheimer's disease (AD) worldwide, and the number of cases is expected to increase four-fold by 2050. AD is a neurodegenerative disease that leads to severe dementia in most patients. There are several neuropathological signs of AD, such as deposition of amyloid beta (Aß) plaques, formation of neurofibrillary tangles (NFTs), neuronal loss, activation of inflammasomes, and declining autophagy. Several of these hallmarks are linked to the gut microbiome. The gastrointestinal (GI) tract contains microbial diversity, which is important in regulating several functions in the brain via the gut-brain axis (GBA). The disruption of the balance in the gut microbiota is known as gut dysbiosis. Recent studies strongly support that targeting gut dysbiosis with selective bioflavonoids is a highly plausible solution to attenuate activation of inflammasomes (contributing to neuroinflammation) and resume autophagy (a cellular mechanism for lysosomal degradation of the damaged components and recycling of building blocks) to stop AD pathogenesis. This review is focused on two bioflavonoids, specifically epigallocatechin-3-gallate (EGCG) and genistein (GS), as a possible new paradigm of treatment for maintaining healthy gut microbiota in AD due to their implications in modulating crucial AD signaling pathways. The combination of EGCG and GS has a higher potential than either agent alone to attenuate the signaling pathways implicated in AD pathogenesis. The effects of EGCG and GS on altering gut microbiota and GBA were also explored, along with conclusions from various delivery methods to increase the bioavailability of these bioflavonoids in the body.