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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.
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Inflamação/etiologia , Inflamação/cirurgia , Transdução de Sinais/fisiologia , Traumatismos da Medula Espinal/complicações , Transplante de Células-Tronco/métodos , Fator de Transcrição RelA/metabolismo , Animais , Linhagem Celular Transformada , Citocinas/metabolismo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/cirurgia , Humanos , Masculino , Ratos , Ratos Wistar , Células-Tronco/fisiologia , Fatores de TempoRESUMO
Well known for its anti-oxidative and anti-inflammation properties, curcumin is a polyphenol found in the rhizome of Curcuma longa. In this study, we evaluated the effects of curcumin on behavioral recovery, glial scar formation, tissue preservation, axonal sprouting, and inflammation after spinal cord injury (SCI) in male Wistar rats. The rats were randomized into two groups following a balloon compression injury at the level of T9-T10 of the spinal cord, namely vehicle- or curcumin-treated. Curcumin was applied locally on the surface of the injured spinal cord immediately following injury and then given intraperitoneally daily; the control rats were treated with vehicle in the same manner. Curcumin treatment improved behavioral recovery within the first week following SCI as evidenced by improved Basso, Beattie, and Bresnahan (BBB) test and plantar scores, representing locomotor and sensory performance, respectively. Furthermore, curcumin treatment decreased glial scar formation by decreasing the levels of MIP1α, IL-2, and RANTES production and by decreasing NF-κB activity. These results, therefore, demonstrate that curcumin has a profound anti-inflammatory therapeutic potential in the treatment of spinal cord injury, especially when given immediately after the injury.
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Curcumina/farmacologia , Imunomodulação , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/fisiopatologia , Animais , Anti-Inflamatórios/farmacologia , Modelos Animais de Doenças , Masculino , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Glioblastoma (GBM) is the most common primary brain tumor in adults and demonstrates a 1-year median survival time. Codon-specific hotspot mutations of p53 result in constitutively active mutant p53, which promotes aberrant proliferation, anti-apoptosis, and cell cycle checkpoint failure in GBM. Recently identified CD133(+) cancer stem cell populations (CSC) within GBM also confer therapeutic resistance. We studied targeted therapy in a codon-specific p53 mutant (R273H) created by site-directed mutagenesis in U87MG. The effects of arsenic trioxide (ATO, 1 µM) and all-trans retinoic acid (ATRA, 10 µM), possible targeted treatments of CSCs, were investigated in U87MG neurospheres. The results showed that U87-p53(R273H) cells generated more rapid neurosphere growth than U87-p53(wt) but inhibition of neurosphere proliferation was seen with both ATO and ATRA. U87-p53(R273H) neurospheres showed resistance to differentiation into glial cells and neuronal cells with ATO and ATRA exposure. ATO was able to generate apoptosis at high doses and proliferation of U87-p53(wt) and U87-p53(R273H) cells was reduced with ATO and ATRA in a dose-dependent manner. Elevated pERK1/2 and p53 expression was seen in U87-p53(R273H) neurospheres, which could be reduced with ATO and ATRA treatment. Additionally, differential responses in pERK1/2 were seen with ATO treatment in neurospheres and non-neurosphere cells. In conclusion, codon-specific mutant p53 conferred a more aggressive phenotype to our CSC model. However, ATO and ATRA could potently suppress CSC properties in vitro and may support further clinical investigation of these agents.
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Antineoplásicos/farmacologia , Arsenicais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Códon , Genes p53 , Glioblastoma/tratamento farmacológico , Mutação , Óxidos/farmacologia , Tretinoína/farmacologia , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Neoplasias Encefálicas/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glioblastoma/genética , Humanos , Dados de Sequência Molecular , Células-Tronco Neoplásicas/patologiaRESUMO
Transplantation of mesenchymal stem cells (MSC) improves functional recovery in experimental models of spinal cord injury (SCI); however, the mechanisms underlying this effect are not completely understood. We investigated the effect of intrathecal implantation of human MSC on functional recovery, astrogliosis and levels of inflammatory cytokines in rats using balloon-induced spinal cord compression lesions. Transplanted cells did not survive at the lesion site of the spinal cord; however, functional recovery was enhanced in the MSC-treated group as was confirmed by the Basso, Beattie, and Bresnahan (BBB) and the flat beam test. Morphometric analysis showed a significantly higher amount of remaining white matter in the cranial part of the lesioned spinal cords. Immunohistochemical analysis of the lesions indicated the rearrangement of the glial scar in MSC-treated animals. Real-time PCR analysis revealed an increased expression of Irf5, Mrc1, Fgf2, Gap43 and Gfap. Transplantation of MSCs into a lesioned spinal cord reduced TNFα, IL-4, IL-1ß, IL-2, IL-6 and IL-12 and increased the levels of MIP-1α and RANTES when compared to saline-treated controls. Intrathecal implantation of MSCs reduces the inflammatory reaction and apoptosis, improves functional recovery and modulates glial scar formation after SCI, regardless of cell survival. Therefore, repeated applications may prolong the beneficial effects induced by MSC application.
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Quimiocina CCL5/metabolismo , Interleucinas/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Traumatismos da Medula Espinal/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Quimiocina CCL5/genética , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interleucinas/genética , Locomoção , Masculino , Ratos , Ratos Wistar , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Traumatismos da Medula Espinal/terapia , Fator de Necrose Tumoral alfa/genéticaRESUMO
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.
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Glioblastoma , Células-Tronco Neoplásicas , Sirolimo , Humanos , Glioblastoma/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
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.
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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.
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Antígeno B7-H1 , Neoplasias Encefálicas , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular TumoralRESUMO
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.
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Glioblastoma , Sirolimo , Humanos , Sirolimo/farmacologia , Transdução de Sinais , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proliferação de Células , Células-Tronco/metabolismo , Linhagem Celular TumoralRESUMO
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.
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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.
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Glioblastoma , Proliferação de Células , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
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.
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Neoplasias Cerebelares , Meduloblastoma , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/terapia , Criança , Aberrações Cromossômicas , Perfilação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/terapia , MutaçãoRESUMO
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.
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Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de MTOR/farmacologia , Naftiridinas/farmacologia , Sulfonas/farmacologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidoresRESUMO
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.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Esferoides Celulares/fisiologia , Antígeno AC133/análise , Animais , Neoplasias Encefálicas/irrigação sanguínea , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Glioblastoma/irrigação sanguínea , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/fisiologia , Neovascularização Patológica/etiologiaRESUMO
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.
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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.
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Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Nanopartículas de Magnetita/uso terapêutico , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Ensaios Clínicos como Assunto , Meios de Contraste/química , Sistemas de Liberação de Medicamentos , Glioblastoma/diagnóstico por imagem , HumanosRESUMO
OBJECT: One mechanism that contributes to cerebral vasospasm is the impairment of potassium channels in vascular smooth muscles. Adenosine triphosphate-sensitive potassium channel openers (PCOs) appear to be particularly effective for dilating cerebral arteries in experimental models of subarachnoid hemorrhage (SAH). A mode of safe administration that provides timed release of PCO drugs is still a subject of investigation. The authors tested the efficacy of locally delivered intrathecal cromakalim, a PCO, incorporated into a controlled-release system to prevent cerebral vasospasm in a rat model of SAH. METHODS: Cromakalim was coupled to a viscous carrier, hyaluronan, 15% by weight. In vitro release kinetics studies showed a steady release of cromakalim over days. Fifty adult male Sprague-Dawley rats weighing 350-400 g each were divided into 10 groups and treated with various doses of cromakalim or cromakalim/hyaluronan in a rat double SAH model. Treatment was started 30 minutes after the second SAH induction. Animals were killed 3 days after treatment, and the basilar arteries were processed for morphometric measurements and histological analysis. RESULTS: Controlled release of cromakalim from the cromakalim/hyaluronan implant at a dose of 0.055 mg/kg significantly increased lumen patency in a dose-dependent manner up to 94 +/- 8% (mean +/- standard error of the mean) of the basilar arteries of the sham group compared with the empty polymer group (p = 0.006). Results in the empty polymer group were not different from those in the SAH-only group, with a lumen patency of 65 +/- 12%. Lumen patencies of the cromakalim-only groups did not differ in statistical significance at low (64 +/- 9%) or high (66 +/- 7%) doses compared to the SAH-only group. CONCLUSIONS: Treatment of SAH with a controlled-release cromakalim/hyaluronan implant prevented experimental cerebral vasospasm in this rat double hemorrhage model; this inhibition was dose-dependent. The authors' results confirm that sustained delivery of cromakalim perivascularly to cerebral vessels could be an effective therapeutic strategy in the treatment of cerebral vasospasm after SAH.
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Implantes Absorvíveis , Cromakalim/administração & dosagem , Sistemas de Liberação de Medicamentos , Canais de Potássio/efeitos dos fármacos , Hemorragia Subaracnóidea/complicações , Vasodilatadores/administração & dosagem , Vasoespasmo Intracraniano/prevenção & controle , Animais , Ácido Hialurônico , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) remains the most aggressive and frequently occurring brain neoplasm. Members of the Rho family of small GTP-binding proteins, including Rho, Rac, and Cdc42, have been shown to participate in cell growth differentiation and motility. The mitogen-activated protein kinase (MAPK) pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), has been shown to regulate cell growth, differentiation and motility. Here, the involvement of the Rho and Rho-associated protein kinase (ROCK) pathway, along with MAPK, was investigated to determine their roles in GBM cell migration and proliferation. MATERIALS AND METHODS: In vitro studies utilized the human malignant glioblastoma cell line LN-18. The cells were treated with Y-27632, a ROCK inhibitor, and U0126, an upstream MAPK kinase inhibitor (MEK), alone or in combination with one another. Immunoblotting analysis established the levels of phosphorylated ERK1/2. Cell migration was determined by radial migration assay and cell proliferation by MTT. RESULTS: Y-27632 reduced phosphorylation of ERK1/2 at 0.5 and 2 h. U0126 in combination with Y-27632 led to a more pronounced repression of platelet-derived growth factor (PDGF)- or fibronectin (FN)-induced ERK1/2 activation than U0126 treatment alone. Y-27632 treatment for 24 h suppressed GBM cell migration and resulted in a reduction in LN-18 cell proliferation. Furthermore, PDGF and FN-induced cell proliferation was suppressed by pre-treatment with Y-27632 or U0126, with the greatest reduction achieved by a combination of the two inhibitors. CONCLUSION: Rho/ROCK signaling is involved in GBM cell migration and proliferation, and this pathway may be linked to ERK signaling.
Assuntos
Movimento Celular/fisiologia , Glioblastoma/enzimologia , Glioblastoma/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismo , Amidas/farmacologia , Butadienos/farmacologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibronectinas/farmacologia , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
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.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Transdução de Sinais , Animais , Proliferação de Células , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
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.
Assuntos
Terapia com Luz de Baixa Intensidade/métodos , Traumatismos da Medula Espinal/terapia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Locomoção , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Ratos , Ratos Wistar , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Regeneração da Medula EspinalRESUMO
PURPOSE: It has been hypothesized that brain tumors are derived from stem cell or transiently dividing precursor transformation. Furthermore, c-Jun NH(2)-terminal kinases (JNKs) have been involved in gliomagenesis. This study analyzes stem cell marker nestin and JNK expression in glioblastoma multiforme (GBM) and peritumor tissue and assesses their possible prognostic implications. EXPERIMENTAL DESIGN: Nestin and both total JNK (tJNK) and phosphorylated JNK (pJNK) expression was investigated by immunohistochemistry in 20 GBMs. Samples were derived from tumors (first area), from tissues at a distance <1 cm (second area), and between 1 and 3.5 cm (third area) from the macroscopic tumor border. The relationships between patients' age, Karnofsky performance status, gender, protein expression, and survival were analyzed. RESULTS: Nestin cytoplasmic immunoreactivity was observed in the majority of cells in tumor but infrequently in peritumor areas. tJNK, observed in the nucleus and cytoplasm, was widely expressed in the three areas; pJNK, mostly located in the nuclei, was found in a variable percentage of cells in the tumor and peritumor tissue. Nestin and JNK expression in peritumor areas was independent of the presence of neoplastic cells. Univariate analysis indicated that survival was longer (19 versus 12 months; P = 0.01) for patients whose pJNK/nestin and (pJNK/tJNK)/nestin ratios in the second area were > or =2.619 and > or =0.026, respectively. The same variables showed an independent prognostic value in multivariate analysis. CONCLUSIONS: Nestin and JNK expression indicates that peritumor tissue, independently of the presence of neoplastic cells, may present signs of transformation. Moreover, pJNK/nestin and (pJNK/tJNK)/nestin ratios in that tissue seem to have some prognostic implications in GBM patients.