mTOR pathway inhibition alters proliferation as well as differentiation of neural stem cells.

Introduction: Neural stem cells (NSCs) are essential for both embryonic development and adult neurogenesis, and their dysregulation causes a number of neurodevelopmental disorders, such as epilepsy and autism spectrum disorders. NSC proliferation and differentiation in the developing brain is a complex process controlled by various intrinsic and extrinsic stimuli. The mammalian target of rapamycin (mTOR) regulates proliferation and differentiation, among other cellular functions, and disruption in the mTOR pathway can lead to severe nervous system development deficits. In this study, we investigated the effect of inhibition of the mTOR pathway by rapamycin (Rapa) on NSC proliferation and differentiation. Methods: The NSC cultures were treated with Rapa for 1, 2, 6, 24, and 48 h. The effect on cellular functions was assessed by immunofluorescence staining, western blotting, and proliferation/metabolic assays. Results: mTOR inhibition suppressed NSC proliferation/metabolic activity as well as S-Phase entry by as early as 1 h of Rapa treatment and this effect persisted up to 48 h of Rapa treatment. In a separate experiment, NSCs were differentiated for 2 weeks after treatment with Rapa for 24 or 48 h. Regarding the effect on neuronal and glial differentiation (2 weeks post-treatment), this was suppressed in NSCs deficient in mTOR signaling, as evidenced by downregulated expression of NeuN, MAP2, and GFAP. We assume that the prolonged effect of mTOR inhibition is realized due to the effect on cytoskeletal proteins. Discussion: Here, we demonstrate for the first time that the mTOR pathway not only regulates NSC proliferation but also plays an important role in NSC differentiation into both neuronal and glial lineages.

Discrete Mechanistic Target of Rapamycin Signaling Pathways, Stem Cells, and Therapeutic Targets.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions via its discrete binding partners to form two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin-sensitive mTORC1, which regulates protein synthesis and cell growth, is tightly controlled by PI3K/Akt and is nutrient-/growth factor-sensitive. In the brain, mTORC1 is also sensitive to neurotransmitter signaling. mTORC2, which is modulated by growth factor signaling, is associated with ribosomes and is insensitive to rapamycin. mTOR regulates stem cell and cancer stem cell characteristics. Aberrant Akt/mTOR activation is involved in multistep tumorigenesis in a variety of cancers, thereby suggesting that the inhibition of mTOR may have therapeutic potential. Rapamycin and its analogues, known as rapalogues, suppress mTOR activity through an allosteric mechanism that only suppresses mTORC1, albeit incompletely. ATP-catalytic binding site inhibitors are designed to inhibit both complexes. This review describes the regulation of mTOR and the targeting of its complexes in the treatment of cancers, such as glioblastoma, and their stem cells.

Brain Metastases Are Regulated by Immuno-inflammatory Signaling Pathways Governed by STAT3, MAPK and Tumor Suppressor p53 Status: Possible Therapeutic Targets.

BACKGROUND/AIM: Brain metastasis (BM) is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1), an immunotherapy target. Tumor suppressor p53, dysregulated in cancers, regulates STAT3 and ERK1/2 signaling. This study examined the roles of STAT3, MAPK and p53 status in BM initiation and maintenance. MATERIALS AND METHODS: Twenty-six BM, with various primary malignancies, were used (IRB-approved) to determine mutant p53 (p53mt), pSTAT3Tyr705, pERK1/2Thr202/Tyr204, and PD-L1 expression using immunohistochemistry. cDNA microarray was used for gene expression analysis. Brain-metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 (NSC74859) or MAPK/ERK1/2 (U0126) inhibitors in regular or astrocytic media. ERK1/2 pathway was assessed using western blotting, and cell proliferation and migration were determined using MTT and scratch-wound assays, respectively. RESULTS: pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed at tumor margins, whereas p53mt and PD-L1 were uniformly expressed, with significant overlap between expression of these proteins. Gene expression analysis identified alterations in 18 p53- and 32 STAT3- or MAPK-associated genes contributing to dysregulated immune responses and cell cycle regulation. U0126 and NSC74859 reduced pERK1/2Thr202/Tyr204 expression. Cell proliferation decreased following each treatment (p≤0.01). Migration stagnated following U0126 treatment in astrocytic media (p≤0.01). CONCLUSION: Activation of STAT3 and ERK1/2 promotes BM and provides compelling evidence for use of STAT3, ERK1/2 and p53 status as potential immunotherapeutic targets in BM.

The Role of Green Tea Catechin Epigallocatechin Gallate (EGCG) and Mammalian Target of Rapamycin (mTOR) Inhibitor PP242 (Torkinib) in the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition that has physical and psychological consequences for patients. SCI is accompanied by scar formation and systemic inflammatory response leading to an intense degree of functional loss. The catechin, epigallocatechin gallate (EGCG), an active compound found in green tea, holds neuroprotective features and is known for its anti-inflammatory potential. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two functionally distinct complexes termed mTOR complex 1 and 2 (mTORC1; mTORC2). Inhibition of mTORC1 by rapamycin causes neuroprotection, leading to partial recovery from SCI. In this study the effects of EGCG, PP242 (an inhibitor of both complexes of mTOR), and a combination of EGCG and PP242 in SCI have been examined. It has been found that both EGCG and PP242 significantly improved sensory/motor functions following SCI. However, EGCG appeared to be more effective (BBB motor test, from 2 to 8 weeks after SCI, p = 0.019, p = 0.007, p = 0.006, p = 0.006, p = 0.05, p = 0.006, and p = 0.003, respectively). The only exception was the Von Frey test, where EGCG was ineffective, while mTOR inhibition by PP242, as well as PP242 in combination with EGCG, significantly reduced withdrawal latency starting from week three (combinatorial therapy (EGCG + PP242) vs. control at 3, 5, and 7 weeks, p = 0.011, p = 0.007, and p = 0.05, respectively). It has been found that EGCG was as effective as PP242 in suppressing mTOR signaling pathways, as evidenced by a reduction in phosphorylated S6 expression (PP242 (t-test, p < 0.0001) or EGCG (t-test, p = 0.0002)). These results demonstrate that EGCG and PP242 effectively suppress mTOR pathways, resulting in recovery from SCI in rats, and that EGCG acts via suppressing mTOR pathways.

Defining the role of mTOR pathway in the regulation of stem cells of glioblastoma.

The mechanistic target of rapamycin (mTOR), a serine/threonine kinase, functions by forming two multiprotein complexes termed mTORC1 and mTORC2. Glioblastoma (GBM) is a uniformly fatal brain tumor that remains incurable partly due to the existence of untreatable cancer stem cells (CSC). The pathogenesis of GBM is largely due to the loss of the tumor suppressor gene PTEN, which is implicated in the aberrant activation of the mTOR pathway. The major cause of tumor recurrence, growth, and invasion is the presence of the unique population of CSC. Resistance to conventional therapies appears to be caused by both extensive genetic abnormalities and dysregulation of the transcription landscape. Consequently, CSCs have emerged as targets of interest in new treatment paradigms. Evidence suggests that inhibition of the mTOR pathway can also be applied to target CSCs. Here we explored the role of the mTOR pathway in the regulation of stem cells of GBM by treating them with inhibitors of canonical PI3K/AKT/mTOR pathways such as rapamycin (mTORC1 inhibitor), PP242 (ATP binding mTORC1/2 inhibitor), LY294002 (PI3K inhibitor), and MAPK inhibitor, U0126. A significant number of GBM tumors expressed stem cell marker nestin and activated mTOR (pmTORSer2448), with most tumor cells co-expressing both markers. The expression of stem cell marker NANOG was suppressed following rapamycin treatment. The neurospheres were disrupted following rapamycin and LY294002 treatments. Rapamycin or PP242 along with differentiating agent All-trans-retinoic acid reduced stem cell proliferation. Treatment with novel small molecule inhibitors of mTORC1/2 demonstrated that Torin1 and Torin2 suppressed the proliferation of GBM CSC, while XL388 was less effective. Torin1 and XL388 delay the process of self-renewal as compared to controls, whereas Torin2 halted self-renewal. Torin2 was able to eradicate tumor cells. In conclusion, Torin2 effectively targeted CSCs of GBM by halting self-renewal and inhibiting cell proliferation, underscoring the use of Torin2 in the treatment of GBM.

Molecular Stratification of Medulloblastoma: Clinical Outcomes and Therapeutic Interventions.

Medulloblastoma (MB) is the most common malignant pediatric posterior fossa tumor. Recent genetic, epigenetic, and transcriptomic analyses have classified MB into three subgroups, Wingless Type (WNT), Sonic Hedgehog (SHH), and non-WNT/non-SHH (originally termed Group 3 and Group 4), with discrete patient profiles and prognoses. WNT is the least common subgroup with the best prognosis, characterized by nuclear ß-catenin expression, mutations in Catenin beta-1 (CTNNB1), and chromosome 6 monosomy. SHH tumors contain mutations and alterations in GLI1, GLI2, SUFU, and PTCH1 genes, which constitutively activate the SHH pathway. Originally, the presence of TP53 gene alterations and/or MYC amplifications was considered the most reliable prognostic factor. However, recent molecular analyses have subdivided SHH MB into several subtypes with distinct characteristics such as age, TP53 mutation, MYC amplification, presence of metastases, TERT promoter alterations, PTEN loss, and other chromosomal alterations as well as SHH pathway-related gene mutations. The third non-WNT/non-SHH MB (Group3/4) subgroup is genetically highly heterogeneous and displays several molecular patterns, including MYC and OTX2 amplification, GFI1B activation, KBTBD4 mutation, GFI1 rearrangement, PRDM6 enhancer hijacking, KDM6A mutation, LCA histology, chromosome 10 loss, isochromosome 17q, SNCAIP duplication, and CDK6 amplification. However, based on molecular profiling and methylation patterns, additional non-WNT/non-SHH MB subtypes have been described. Recent WHO (2021) guidelines stratified MB into four molecular subgroups with four and eight further subgroups for SHH and non-WNT/non-SHH MB, respectively. In this review, we discuss advancements in genetics, epigenetics, and transcriptomics for better characterization, prognostication, and treatment of MB using precision medicine.

Disentangling the signaling pathways of mTOR complexes, mTORC1 and mTORC2, as a therapeutic target in glioblastoma.

Aberrant signaling of mechanistic target of rapamycin (mTOR aka mammalian target of rapamycin) is shown to be linked to tumorigenesis of numerous malignancies including glioblastoma (GB). mTOR is a serine threonine kinase that functions by forming two multiprotein complexes. These complexes are named mTORC1 and mTORC2 and activate downstream substrates that execute cellular and metabolic functions. This signaling cascade of PI3K/AKT/mTOR is often upregulated due to frequent loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. Since rapamycin and its analogue are less effective in treatment of GB, we used novel ATP-competitive dual inhibitors of mTORC1 and mTORC2, namely, Torin1, Torin2, and XL388. Torin2 caused a concentration dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6KSer235/236 and 4E-BP1Thr37/46 as well as the mTORC2 substrate AKTSer473 resulting in suppression of tumor cell proliferation and migration. Torin1 showed similar effects only at higher doses. Another small molecule compound, XL388 suppressed cell proliferation at a higher dose but failed to inhibit cell migration. Torin1 suppressed phosphorylation of PRAS40Thr246, however, Torin2 completely abolished it. XL388 treatment inhibited the phosphorylation of PRAS40Thr246 at higher doses only. These findings underscore the use of novel compounds in treatment of cancer. In addition, formulation of third generation mTOR inhibitor "Rapalink-1" may provide new aspects to target mTOR pathways. Numerous inhibitors are currently being used in clinical trials that are aimed to target activated mTOR pathways.

Targeting the mTOR pathway using novel ATP­competitive inhibitors, Torin1, Torin2 and XL388, in the treatment of glioblastoma.

Mechanistic target of rapamycin (mTOR), which functions via two multiprotein complexes termed mTORC1 and mTORC2, is positioned in the canonical phosphoinositide 3­kinase­related kinase (PI3K)/AKT (PI3K/AKT) pathways. These complexes exert their actions by regulating other important kinases, such as 40S ribosomal S6 kinases (S6K), eukaryotic translation initiation factor 4E (elF4E)­binding protein 1 (4E­BP1) and AKT, to control cell growth, proliferation, migration and survival in response to nutrients and growth factors. Glioblastoma (GB) is a devastating form of brain cancer, where the mTOR pathway is deregulated due to frequent upregulation of the Receptor Tyrosine Kinase/PI3K pathways and loss of the tumor suppressor phosphatase and tensin homologue (PTEN). Rapamycin and its analogs were less successful in clinical trials for patients with GB due to their incomplete inhibition of mTORC1 and the activation of mitogenic pathways via negative feedback loops. Here, the effects of selective ATP­competitive dual inhibitors of mTORC1 and mTORC2, Torin1, Torin2 and XL388, are reported. Torin2 exhibited concentration­dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6KSer235/236 and 4E­BP1Thr37/46 as well as the mTORC2 substrate AKTSer473 resulting in suppression of tumor cell migration, proliferation and S­phase entry. Torin1 demonstrated similar effects, but only at higher doses. XL388 suppressed cell proliferation at a higher dose, but failed to inhibit cell migration. Treatment with Torin1 suppressed phosphorylation of proline rich AKT substrate of 40 kDa (PRAS40) at Threonine 246 (PRAS40Thr246) whereas Torin2 completely abolished it. XL388 treatment suppressed the phosphorylation of PRAS40Thr246 only at higher doses. Drug resistance analysis revealed that treatment of GB cells with XL388 rendered partial drug resistance, which was also seen to a lesser extent with rapamycin and Torin1 treatments. However, treatment with Torin2 completely eradicated the tumor cell population. These results strongly suggest that Torin2, compared to Torin1 or XL388, is more effective in suppressing mTORC1 and mTORC2, and therefore in the inhibition of the GB cell proliferation, dissemination and in overcoming resistance to therapy. These findings underscore the significance of Torin2 in the treatment of GB.

Involvement of mTOR Pathways in Recovery from Spinal Cord Injury by Modulation of Autophagy and Immune Response.

Traumatic spinal cord injury (SCI) is untreatable and remains the leading cause of disability. Neuroprotection and recovery after SCI can be partially achieved by rapamycin (RAPA) treatment, an inhibitor of mTORC1, complex 1 of the mammalian target of rapamycin (mTOR) pathway. However, mechanisms regulated by the mTOR pathway are not only controlled by mTORC1, but also by a second mTOR complex (mTORC2). Second-generation inhibitor, pp242, inhibits both mTORC1 and mtORC2, which led us to explore its therapeutic potential after SCI and compare it to RAPA treatment. In a rat balloon-compression model of SCI, the effect of daily RAPA (5 mg/kg; IP) and pp242 (5 mg/kg; IP) treatment on inflammatory responses and autophagy was observed. We demonstrated inhibition of the mTOR pathway after SCI through analysis of p-S6, p-Akt, and p-4E-BP1 levels. Several proinflammatory cytokines were elevated in pp242-treated rats, while RAPA treatment led to a decrease in proinflammatory cytokines. Both RAPA and pp242 treatments caused an upregulation of LC3B and led to improved functional and structural recovery in acute SCI compared to the controls, however, a greater axonal sprouting was seen following RAPA treatment. These results suggest that dual mTOR inhibition by pp242 after SCI induces distinct mechanisms and leads to recovery somewhat inferior to that following RAPA treatment.

Understanding the Biological Basis of Glioblastoma Patient-derived Spheroids.

BACKGROUND/AIM: Resistance to glioblastoma (GB) therapy is attributed to the presence of glioblastoma stem cells (GSC). Here, we defined the behavior of GSC as it pertains to proliferation, migration, and angiogenesis. MATERIALS AND METHODS: Human-derived GSC were isolated and cultured from GB patient tumors. Xenograft GSC were extracted from the xenograft tumors, and spheroids were created and compared with human GSC spheroids by flow cytometry, migration, proliferation, and angiogenesis assays. Oct3/4 and Sox2, GFAP, and Ku80 expression was assessed by immunoanalysis. RESULTS: The xenograft model showed the formation of two different tumors with distinct characteristics. Tumors formed at 2 weeks were less aggressive with well-defined margins, whereas tumors formed in 5 months were diffuse and aggressive. Expression of Oct3/4 and Sox2 was positive in both human and xenograft GSC. Positive Ku80 expression in xenograft GSC confirmed their human origin. Human and xenograft GSC migrated vigorously in collagen and Matrigel, respectively. Xenograft GSC displayed a higher rate of migration and invasion than human GSC. CONCLUSION: Human GSC were more aggressive in growth and proliferation than xenograft GSC, while xenograft GSC had increased invasion and migration compared to human GSC. A simple in vitro spheroid system for GSC provides a superior platform for the development of precision medicine in the treatment of GB.

Potential for Treatment of Glioblastoma: New Aspects of Superparamagnetic Iron Oxide Nanoparticles.

Glioblastoma (GB) is a highly aggressive and infiltrative brain tumor characterized by poor outcomes and a high rate of recurrence despite maximal safe resection, chemotherapy, and radiation. Superparamagnetic iron oxide nanoparticles (SPIONs) are a novel tool that can be used for many applications including magnetic targeting, drug delivery, gene delivery, hyperthermia treatment, cell tracking, or multiple simultaneous functions. SPIONs are studied as a magnetic resonance imaging tumor contrast agent by targeting tumor cell proteins or tumor vasculature. Drug delivery to GB tumor has been targeted with SPIONs in murine models. In addition to targeting tumor cells for imaging or drug-delivery, SPION has also been shown to be effective at targeting for hyperthermia. Along with animal models, human trials have been conducted for a number of different modes of SPION utilization, with important findings and lessons for further preclinical and clinical experiments. SPIONs are opening up several new avenues for monitoring and treatment of GB tumors; here, we review the current research and a variety of possible clinical applications.

The effect of 808 nm and 905 nm wavelength light on recovery after spinal cord injury.

We investigated the effect of a Multiwave Locked System laser (with a simultaneous 808 nm continuous emission and 905 nm pulse emission) on the spinal cord after spinal cord injury (SCI) in rats. The functional recovery was measured by locomotor tests (BBB, Beam walking, MotoRater) and a sensitivity test (Plantar test). The locomotor tests showed a significant improvement of the locomotor functions of the rats after laser treatment from the first week following lesioning, compared to the controls. The laser treatment significantly diminished thermal hyperalgesia after SCI as measured by the Plantar test. The atrophy of the soleus muscle was reduced in the laser treated rats. The histopathological investigation showed a positive effect of the laser therapy on white and gray matter sparing. Our data suggests an upregulation of M2 macrophages in laser treated animals by the increasing number of double labeled CD68+/CD206+ cells in the cranial and central parts of the lesion, compared to the control animals. A shift in microglial/macrophage polarization was confirmed by gene expression analysis by significant mRNA downregulation of Cd86 (marker of inflammatory M1), and non-significant upregulation of Arg1 (marker of M2). These results demonstrated that the combination of 808 nm and 905 nm wavelength light is a promising non-invasive therapy for improving functional recovery and tissue sparing after SCI.

Diverse signaling mechanisms of mTOR complexes: mTORC1 and mTORC2 in forming a formidable relationship.

Activation of Mechanistic target of rapamycin (mTOR) signaling plays a crucial role in tumorigenesis of numerous malignancies including glioblastoma (GB). The Canonical PI3K/Akt/mTOR signaling cascade is commonly upregulated due to loss of the tumor suppressorm PTEN, a phosphatase that acts antagonistically to the kinase (PI3K) in conversion of PIP2 to PIP3. mTOR forms two multiprotein complexes, mTORC1 and mTORC2 which are composed of discrete protein binding partners to regulate cell growth, motility, and metabolism. These complexes are sensitive to distinct stimuli, as mTORC1 is sensitive to nutrients while mTORC2 is regulated via PI3K and growth factor signaling. The main function of mTORC1 is to regulate protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. On the other hand, mTORC2 is responsive to growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases like Akt and SGK and it also plays a crucial role in maintenance of normal and cancer cells through its association with ribosomes, and is involved in cellular metabolic regulation. mTORC1 and mTORC2 regulate each other, as shown by the fact that Akt regulates PRAS40 phosphorylation, which disinhibits mTORC1 activity, while S6K regulates Sin1 to modulate mTORC2 activity. Allosteric inhibitors of mTOR, rapamycin and rapalogs, remained ineffective in clinical trials of Glioblastoma (GB) patients, in part due to their incomplete inhibition of mTORC1 as well as unexpected activation of mTOR via the loss of negative feedback loops. In recent years, novel ATP binding inhibitors of mTORC1 and mTORC2 suppress mTORC1 activity completely by total dephosphorylation of its downstream substrate pS6KSer235/236, while effectively suppressing mTORC2 activity, as demonstrated by complete dephosphorylation of pAKTSer473. Furthermore by these novel combined mTORC1/mTORC2 inhibitors reduced the proliferation and self-renewal of GB cancer stem cells. However, a search of more effective way to target mTOR has generated a third generation inhibitor of mTOR, "Rapalink", that bivalently combines rapamycin with an ATP-binding inhibitor, which effectively abolishes the mTORC1 activity. All in all, the effectiveness of inhibitors of mTOR complexes can be judged by their ability to suppress both mTORC1/mTORC2 and their ability to impede both cell proliferation and migration along with aberrant metabolic pathways.

Transplantation of neural precursors generated from spinal progenitor cells reduces inflammation in spinal cord injury via NF-κB pathway inhibition.

BACKGROUND: Traumatic spinal cord injury (SCI) triggers a chain of events that is accompanied by an inflammatory reaction leading to necrotic cell death at the core of the injury site, which is restricted by astrogliosis and apoptotic cell death in the surrounding areas. Activation of nuclear factor-κB (NF-κB) signaling pathway has been shown to be associated with inflammatory response induced by SCI. Here, we elucidate the pattern of activation of NF-κB in the pathology of SCI in rats and investigate the effect of transplantation of spinal neural precursors (SPC-01) on its activity and related astrogliosis. METHODS: Using a rat compression model of SCI, we transplanted SPC-01 cells or injected saline into the lesion 7 days after SCI induction. Paraffin-embedded sections were used to assess p65 NF-κB nuclear translocation at days 1, 3, 7, 10, 14, and 28 and to determine levels of glial scaring, white and gray matter preservation, and cavity size at day 28 after SCI. Additionally, levels of p65 phosphorylated at Serine536 were determined 10, 14, and 28 days after SCI as well as levels of locally secreted TNF-α. RESULTS: We determined a bimodal activation pattern of canonical p65 NF-κB signaling pathway in the pathology of SCI with peaks at 3 and 28 days after injury induction. Transplantation of SCI-01 cells resulted in significant downregulation of TNF-α production at 10 and 14 days after SCI and in strong inhibition of p65 NF-κB activity at 28 days after SCI, mainly in the gray matter. Moreover, reduced formation of glial scar was found in SPC-01-transplanted rats along with enhanced gray matter preservation and reduced cavity size. CONCLUSIONS: The results of this study demonstrate strong immunomodulatory properties of SPC-01 cells based on inhibition of a major signaling pathway. Canonical NF-κB pathway activation underlines much of the immune response after SCI including cytokine, chemokine, and apoptosis-related factor production as well as immune cell activation and infiltration. Reduced inflammation may have led to observed tissue sparing. Additionally, such immune response modulation could have impacted astrocyte activation resulting in a reduced glial scar.

Molecular Sequence of Events and Signaling Pathways in Cerebral Metastases.

Brain metastases are the leading cause of morbidity and mortality among cancer patients, and are reported to occur in about 40% of cancer patients with metastatic disease in the United States of America. Primary tumor cells appear to detach from the parent tumor site, migrate, survive and pass through the blood brain barrier in order to establish cerebral metastases. This complex process involves distinct molecular and genetic mechanisms that mediate metastasis from these primary organs to the brain. Furthermore, an interaction between the invading cells and cerebral milieu is shown to promote this process as well. Here, we review the mechanisms by which primary cancer cells metastasize to the brain via a mechanism called epithelial-to-mesenchymal transition, as well as the involvement of certain microRNA and genetic aberrations implicated in cerebral metastases from the lung, breast, skin, kidney and colon. While the mechanisms governing the development of brain metastases remain a major hindrance in treatment, understanding and identification of the aforementioned molecular pathways may allow for improved management and discovery of novel therapeutic targets.

Does combined therapy of curcumin and epigallocatechin gallate have a synergistic neuroprotective effect against spinal cord injury?

Systematic inflammatory response after spinal cord injury (SCI) is one of the factors leading to lesion development and a profound degree of functional loss. Anti-inflammatory compounds, such as curcumin and epigallocatechin gallate (EGCG) are known for their neuroprotective effects. In this study, we investigated the effect of combined therapy of curcumin and EGCG in a rat model of acute SCI induced by balloon compression. Immediately after SCI, rats received curcumin, EGCG, curcumin + EGCG or saline [daily intraperitoneal doses (curcumin, 6 mg/kg; EGCG 17 mg/kg)] and weekly intramuscular doses (curcumin, 60 mg/kg; EGCG 17 mg/kg)] for 28 days. Rats were evaluated using behavioral tests (the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test, flat beam test). Spinal cord tissue was analyzed using histological methods (Luxol Blue-cresyl violet staining) and immunohistochemistry (anti-glial fibrillary acidic protein, anti-growth associated protein 43). Cytokine levels (interleukin-1ß, interleukin-4, interleukin-2, interleukin-6, macrophage inflammatory protein 1-alpha, and RANTES) were measured using Luminex assay. Quantitative polymerase chain reaction was performed to determine the relative expression of genes (Sort1, Fgf2, Irf5, Mrc1, Olig2, Casp3, Gap43, Gfap, Vegf, NfκB, Cntf) related to regenerative processes in injured spinal cord. We found that all treatments displayed significant behavioral recovery, with no obvious synergistic effect after combined therapy of curcumin and ECGC. Curcumin and EGCG alone or in combination increased axonal sprouting, decreased glial scar formation, and altered the levels of macrophage inflammatory protein 1-alpha, interleukin-1ß, interleukin-4 and interleukin-6 cytokines. These results imply that although the expected synergistic response of this combined therapy was less obvious, aspects of tissue regeneration and immune responses in severe SCI were evident.

Anti-inflammatory compound curcumin and mesenchymal stem cells in the treatment of spinal cord injury in rats.

Spinal cord injury leads to a robust inflammatory response that is an unfavorable environment for stem cell implantation. In this study, we evaluated the effect of combined therapy of curcumin and mesenchymal stem cells (MSC) on behavioral recovery and tissue sparing, glial scar formation, axonal sprouting and inflammatory responses in a rat experimental model of spinal cord injury (SCI). Balloon-induced compression lesion was performed at thoracic (Th8-9) spinal level. Out of the four groups studied, two groups received curcumin on the surface of the spinal cord immediately after SCI and then once a week for 3 weeks together with an intraperitoneal daily curcumin injection for 28 days. The other two groups received saline. Seven days after SCI, human MSC were intrathecally implanted in one curcumin and one saline group. Both curcumin and curcumin combined with MSC treatment improved locomotor ability in comparison to the saline treated animals. The combined treatment group showed additional improvement in advanced locomotor performance. The combined therapy facilitated axonal sprouting, and modulated expression of pro-regenerative factors and inflammatory responses, when compared to saline and single treatments. These results demonstrate that preconditioning with curcumin, prior to the MSC implantation could have a synergic effect in the treatment of experimental SCI.

A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines.

Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. Behavior recovery was monitored by BBB, plantar, rotarod and flat-beam tests. The levels of inflammatory cytokines were determined on days 1, 3, 7, 10 and 14 after SCI. Additionally, NF-κB pathway activity was evaluated. The results demonstrated that EGCG-treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL-6, IL2, MIP1α and RANTES levels on days 1 and 3, and an upregulation of IL-4, IL-12p70 and TNFα 1 day following SCI in EGCG-treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF-κB dimer, and therefore canonical NF-κB pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG-treated rats. Further, EGCG influenced expression of M1 and M2 macrophage markers. Our results have demonstrated a therapeutic value of EGCG in SCI, as observed by better behavioral performance measured by flat beam test, modulation of inflammatory cytokines and induction of higher axonal sprouting.

Discrete signaling mechanisms of mTORC1 and mTORC2: Connected yet apart in cellular and molecular aspects.

Activation of PI3K/Akt/mTOR (mechanistic target of rapamycin) signaling cascade has been shown in tumorigenesis of numerous malignancies including glioblastoma (GB). This signaling cascade is frequently upregulated due to loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is responsive to growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases like Akt and SGK. mTORC2 plays a crucial role in maintenance of normal and cancer cells through its association with ribosomes, and is involved in cellular metabolic regulation. Both complexes control each other as Akt regulates PRAS40 phosphorylation, which disinhibits mTORC1 activity, while S6K regulates Sin1 to modulate mTORC2 activity. Another significant component of mTORC2 is Sin1, which is crucial for mTORC2 complex formation and function. Allosteric inhibitors of mTOR, rapamycin and rapalogs, have essentially been ineffective in clinical trials of patients with GB due to their incomplete inhibition of mTORC1 or unexpected activation of mTOR via the loss of negative feedback loops. Novel ATP binding inhibitors of mTORC1 and mTORC2 suppress mTORC1 activity completely by total dephosphorylation of its downstream substrate pS6KSer235/236, while effectively suppressing mTORC2 activity, as demonstrated by complete dephosphorylation of pAKTSer473. Furthermore, proliferation and self-renewal of GB cancer stem cells are effectively targetable by these novel mTORC1 and mTORC2 inhibitors. Therefore, the effectiveness of inhibitors of mTOR complexes can be estimated by their ability to suppress both mTORC1 and 2 and their ability to impede both cell proliferation and migration.

A Comparative Study of Three Different Types of Stem Cells for Treatment of Rat Spinal Cord Injury.

Three different sources of human stem cells-bone marrow-derived mesenchymal stem cells (BM-MSCs), neural progenitors (NPs) derived from immortalized spinal fetal cell line (SPC-01), and induced pluripotent stem cells (iPSCs)-were compared in the treatment of a balloon-induced spinal cord compression lesion in rats. One week after lesioning, the rats received either BM-MSCs (intrathecally) or NPs (SPC-01 cells or iPSC-NPs, both intraspinally), or saline. The rats were assessed for their locomotor skills (BBB, flat beam test, and rotarod). Morphometric analyses of spared white and gray matter, axonal sprouting, and glial scar formation, as well as qPCR and Luminex assay, were conducted to detect endogenous gene expression, while inflammatory cytokine levels were performed to evaluate the host tissue response to stem cell therapy. The highest locomotor recovery was observed in iPSC-NP-grafted animals, which also displayed the highest amount of preserved white and gray matter. Grafted iPSC-NPs and SPC-01 cells significantly increased the number of growth-associated protein 43 (GAP43+) axons, reduced astrogliosis, downregulated Casp3 expression, and increased IL-6 and IL-12 levels. hMSCs transiently decreased levels of inflammatory IL-2 and TNF-α. These findings correlate with the short survival of hMSCs, while NPs survived for 2 months and matured slowly into glia- and tissue-specific neuronal precursors. SPC-01 cells differentiated more in astroglial phenotypes with a dense structure of the implant, whereas iPSC-NPs displayed a more neuronal phenotype with a loose structure of the graft. We concluded that the BBB scores of iPSC-NP- and hMSC-injected rats were superior to the SPC-01-treated group. The iPSC-NP treatment of spinal cord injury (SCI) provided the highest recovery of locomotor function due to robust graft survival and its effect on tissue sparing, reduction of glial scarring, and increased axonal sprouting.