Exosomes derived from human umbilical cord mesenchymal stem cells pretreated by monosialoteterahexosyl ganglioside alleviate intracerebral hemorrhage by down-regulating autophagy.

PURPOSE: Intracerebral hemorrhage (ICH) results in substantial morbidity, mortality, and disability. Depleting neural cells in advanced stages of ICH poses a significant challenge to recovery. The objective of our research is to investigate the potential advantages and underlying mechanism of exosomes obtained from human umbilical cord mesenchymal stem cells (hUMSCs) pretreated with monosialoteterahexosyl ganglioside (GM1) in the prevention of secondary brain injury (SBI) resulting from ICH. PATIENTS AND METHODS: In vitro, hUMSCs were cultured and induced to differentiate into neuron-like cells after they were pretreated with 150 µg/mL GM1. The exosomes extracted from the culture medium following a 6-h pretreatment with 150 µg/mL GM1 were used as the treatment group. Striatal infusion of collagenase and hemoglobin (Hemin) was used to establish in vivo and in vitro models of ICH. RESULTS: After being exposed to 150 µg/mL GM1 for 6 h, specific cells displayed typical neuron-like cell morphology and expressed neuron-specific enolase (NSE). The rate of differentiation into neuron-like cells was up to (15.9 ± 5.8) %, and the synthesis of N-Acetylgalactosaminyltransferase (GalNAcT), which is upstream of GM1, was detected by Western blot. This study presented an increase in the synthesis of GalNAcT. Compared with the ICH group, apoptosis in the treatment group was remarkably reduced, as detected by TUNEL, and mitochondrial membrane potential was restored by JC-1. Additionally, Western blot revealed the restoration of up-regulated autophagy markers Beclin-1 and LC3 and the down-regulation of autophagy marker p62 after ICH. CONCLUSION: These findings suggest that GM1 is an effective agent to induce the differentiation of hUMSCs into neuron-like cells. GM1 can potentially increase GalNAcT production through "positive feedback", which generates more GM1 and promotes the differentiation of hUMSCs. After pretreatment with GM1, exosomes derived from hUMSCs (hUMSCs-Exos) demonstrate a neuroprotective effect by inhibiting autophagy in the ICH model. This study reveals the potential mechanism by which GM1 induces differentiation of hUMSCs into neuron-like cells and confirms the therapeutic effect of hUMSCs-Exos pretreated by GM1 (GM1-Exos) on an ICH model, potentially offering a new direction for stem cell therapy in ICH.

SD-36 promotes growth inhibition and induces apoptosis via suppression of Mcl-1 in glioma.

Glioma is one of the most commonly observed tumours, representing approximately 75% of brain tumours in the adult population. Generally, glioma therapy includes surgical resection followed by radiotherapy and chemotherapy. The transcription factor STAT3 (signal transducer and activator of transcription 3) is a promising target for the treatment of cancer and several other diseases. At nanomolar concentrations, SD-36 induces rapid cellular degradation of STAT3 but cannot degrade other STAT proteins. The current study demonstrates the therapeutic efficacies of the STAT3 degraders SD-36 against glioma, as well as understanding the elucidating mechanisms and identifying molecular markers that determine cell sensitivity to STAT3 degraders. Glioma cell lines possessed similar response patterns to SD-36 but different responses to the STAT3 inhibitor Stattic. SD-36 potently induced apoptosis in glioma cells along with a reduction in Mcl-1 levels, which are critical for mediating the induction of apoptosis and enhancing TMZ-induced apoptosis. Accordingly, SD-36 sensitizes the antitumour effect of TMZ in patient-derived xenograft. In addition, the downregulation of Mcl-1 expression-mediated antitumour effect of SD-36 was analysed in cell-derived xenograft. These observations need to be validated clinically to confirm the efficacy of STAT3 degraders in glioma.

Lestaurtinib potentiates TRAIL-induced apoptosis in glioma via CHOP-dependent DR5 induction.

Lestaurtinib, also called CEP-701, is an inhibitor of tyrosine kinase, causes haematological remission in patients with AML possessing FLT3-ITD (FLT3 gene) internal tandem duplication and strongly inhibits tyrosine kinase FLT3. Treatment with lestaurtinib modulates various signalling pathways and leads to cell growth arrest and programmed cell death in several tumour types. However, the effect of lestaurtinib on glioma remains unclear. In this study, we examined lestaurtinib and TRAIL interactions in glioma cells and observed their synergistic activity on glioma cell apoptosis. While U87 and U251 cells showed resistance to TRAIL single treatment, they were sensitized to apoptosis induced by TRAIL in the presence of lestaurtinib because of increased death receptor 5 (DR5) levels through CHOP-dependent manner. We also demonstrated using a xenograft model of mouse that the tumour growth was absolutely suppressed because of the combined treatment compared to TRAIL or lestaurtinib treatment carried out singly. Our findings reveal a potential new strategy to improve antitumour activity induced by TRAIL in glioma cells using lestaurtinib through a mechanism dependent on CHOP.

CDK4/6 inhibition suppresses tumour growth and enhances the effect of temozolomide in glioma cells.

In adults, glioma is the most commonly occurring and invasive brain tumour. For malignant gliomas, the current advanced chemotherapy includes TMZ (temozolomide). However, a sizeable number of gliomas are unyielding to TMZ, hence, giving rise to an urgent need for more efficient treatment choices. Here, we report that cyclin-dependent kinases 4 (CDK4) is expressed at significantly high levels in glioma cell lines and tissues. CDK4 overexpression enhances colony formation and proliferation of glioma cells and extends resistance to inhibition of TMZ-mediated cell proliferation and induction of apoptosis. However, CDK4 knockdown impedes colony formation and cell proliferation, and enhances sensitivity of glioma cells to TMZ. The selective inhibition of CDK4/6 impedes glioma cell proliferation and induces apoptotic induction. The selective inhibitors of CDK4/6 may enhance glioma cell sensitivity to TMZ. We further showed the possible role of RB phosphorylation mediated by CDK4 for its oncogenic function in glioma. The growth of glioma xenografts was inhibited in vivo, through combination treatment, and corresponded to enhanced p-RB levels, reduced staining of Ki-67 and enhanced activation of caspase 3. Therefore, CDK4 inhibition may be a favourable strategy for glioma treatment and overcomes TMZ resistance.

MiR-3116 sensitizes glioma cells to temozolomide by targeting FGFR1 and regulating the FGFR1/PI3K/AKT pathway.

Glioma is a brain tumour that is often diagnosed, and temozolomide (TMZ) is a common chemotherapeutic drug used in glioma. Yet, resistance to TMZ is a chief hurdle towards curing the malignancy. The current work explores the pathways and involvement of miR-3116 in the TMZ resistance. miR-3116 and FGFR1 mRNA were quantified by real-time PCR in malignant samples and cell lines. Appropriate assays were designed for apoptosis, viability, the ability to form colonies and reporter assays to study the effects of the miR-3116 or FGFR1. The involvement of PI3K/AKT signalling was assessed using Western blotting. Tumorigenesis was evaluated in an appropriate xenograft mouse model in vivo. This work revealed that the levels of miR-3116 dipped in samples resistant to TMZ, while increased miR-3116 caused an inhibition of the tumour features mentioned above to hence augment TMZ sensitivity. miR-3116 was found to target FGFR1. When FGFR1 was overexpressed, resistance to TMZ was augmented and reversed the sensitivity caused by miR-3116. Our findings further confirmed PI3K/AKT signalling pathway is involved in this action. In conclusion, miR-3116 sensitizes glioma cells to TMZ through FGFR1 downregulation and the PI3K/AKT pathway inactivation. Our results provide a strategy to overcome TMZ resistance in glioma treatment.

miR-152-5p suppresses glioma progression and tumorigenesis and potentiates temozolomide sensitivity by targeting FBXL7.

A generally used chemotherapeutic drug for glioma, a frequently diagnosed brain tumour, is temozolomide (TMZ). Our study investigated the activity of FBXL7 and miR-152-5p in glioma. Levels of microRNA-152-5p (miR-152-5p) and the transcript and protein of FBXL7 were assessed by real-time PCR and Western blotting, respectively. The migratory and invasive properties of cells were measured by Transwell migration and invasion assay and their viability were examined using CCK-8 assay. Further, the putative interaction between FBXL7 and miR-152-5p were analysed bioinformatically and by luciferase assay. The activities of FBXL7, TMZ and miR-152-5p were analysed in vivo singly or in combination, on mouse xenografts, in glioma tumorigenesis. The expression of FBXL7 in glioma tissue is significantly up-regulated, which is related to the poor prognosis and the grade of glioma. TMZ-induced cytotoxicity, proliferation, migration and invasion in glioma cells were impeded by the knock-down of FBXL7 or overexpressed miR-152-5p. Furthermore, the expression of miR-152-5p reduced remarkably in glioma cells and it exerted its activity through targeted FBXL7. Overexpression of miR-152-5p and knock-down of FBXL7 in glioma xenograft models enhanced TMZ-mediated anti-tumour effect and impeded tumour growth. Thus, the miR-152-5p suppressed the progression of glioma and associated tumorigenesis, targeted FBXL7 and increased the effect of TMZ-induced cytotoxicity in glioma cells, further enhancing our knowledge of FBXL7 activity in glioma.

miR-1254 inhibits progression of glioma in vivo and in vitro by targeting CSF-1.

The role of miRNAs (microRNAs) has been implicated in glioma initiation and progression, although the inherent biochemical mechanisms still remain to be unravelled. This study strived to evaluate the association between CSF-1 and miR-1254 and their effect on advancement of glioma cells. The levels of miR-1254 in glioma cells and tissues were determined by real-time RT-PCR. Proliferation, apoptosis and cell cycle arrest, invasion and migration, were assessed by CCK-8 assay, colony formation assay, flow cytometry, transwell assay and wound-healing assay, respectively. The targeted relationship between miR-1254 and CSF-1 was confirmed by dual-luciferase reporter assay. The effects of CSF-1 on cellular functions were also assessed. The in vivo effect of miR-1254 on the formation of a tumour was explored by using the mouse xenograft model. We found in both glioma tissues and glioma cells, the down-regulated expressions of miR-1254 while that of CSF-1 was abnormally higher than normal level. The target relationship between CSF-1 and miR-1254 was validated by dual-luciferase reporter assay. The CSF-1 down-regulation or miR-1254 overexpression impeded the invasion, proliferation and migratory ability of U251 and U87 glioma cells, concurrently occluded the cell cycle and induced cell apoptosis. Moreover, in vivo tumour development was repressed due to miR-1254 overexpression. Thus, CSF-1 is targeted directly by miR-1254, and the miR-1254/CSF-1 axis may be a potential diagnostic target for malignant glioma.

Effects of IGFBP-3 and GalNAc-T14 on proliferation and cell cycle of glioblastoma cells and its mechanism.

OBJECTIVE: The purpose of this study was to determine the effects of IGFBP-3 and GalNAc-T14 on the proliferation and cell cycle of glioblastoma cells and to explore the mechanisms of action. METHODS: U87MG and U251MG glioblastoma cells were treated with recombinant human IGFBP-3 (rhIGFBP-3). Furthermore, IGFBP-3-overexpressed cells and cells co-overexpressing IGFBP-3 and GalNAc-T14 were constructed by transfection. Cell viability, cell colony formation ability, cell cycle and protein expression were determined by MTT assay, colony formation assay, flow cytometry and Western blotting, respectively. KEY FINDINGS: Both rhIGFBP-3 treatment and overexpression of IGFBP-3 induced the proliferation, colony formation, and G1/S phase transformation of U87MG and U251MG cells. In addition, the expression of cyclinE, CDK2 and p-ERK1/2 proteins was up-regulated in the cells. In cells co-overexpressing, IGFBP-3 and GalNAc-T14, cell proliferation, colony formation and G1/S phase transformation were inhibited, and the expression of CyclinE, CDK2 and p-ERK1/2 was significantly down-regulated, when compared with IGFBP-3-overexpressed cells. CONCLUSIONS: IGFBP-3 can promote the proliferation, colony formation and G1/S phase transformation of U87MG and U251MG cells, which may be related to the activation of ERK signalling pathway and the up-regulation of cyclinE and CDK2 proteins. Furthermore, our study demonstrated that GalNAc-T14 can inhibit the functions of IGFBP-3.

Cathodal transcranial direct current stimulation affects seizures and cognition in fully amygdala-kindled rats.

This study evaluated the effects of weak transcranial direct current stimulation (tDCS), a new non-invasive brain stimulation technique, on amygdala-kindled rats. The seizure severity, i.e. seizure stage, afterdischarge duration (ADD), and AD threshold (ADT) in the animals were measured one day after the last cathodal tDCS session, comparing with those of pre-treatment controls. Furthermore, the effects of cathodal tDCS on cognitive function were also studied by a water maze test (WMT) two days after the last tDCS session. Cathodal tDCS treatment significantly improved the seizure stage and decreased ADD together with elevated ADT one day after the last tDCS session. The treatment also showed significant improvement in the performance of WMT. The findings suggest that cathodal tDCS has anticonvulsive after-effects last at least for one day on the amygdala-kindled rats and positively affects cognitive performance.

Targeting c-Myc on cell growth and vascular endothelial growth factor expression in IN500 glioblastoma cells.

BACKGROUND: The level of c-Myc is closely associated with high pathological grade and the poor prognosis of gliomas. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor that potently stimulates the proliferation and migration of vascular endothelial cells. This study aimed to address the biological importance of c-Myc in the development of gliomas, we downregulated the expression of c-Myc in the human glioblastoma cell line IN500 and studied the in vitro effect on cellular growth, proliferation, and apoptosis and the expression of VEGF and the in vivo effect on tumor formation in a xenograft mouse model. METHODS: IN500Δ cells were stably transfected with shRNA-expressing plasmids for either c-Myc (pCMYC-shRNA) or as a control (pCtrl-shRNA). Following establishment of stable cells, the mRNA expressions of c-Myc and VEGF were examined by reverse transcription (RT)-PCR, and c-Myc and VEGF proteins by Western blotting and immunohistochemistry. Cell-cycle progression and apoptosis were determined by flow cytometry. The in vivo effect of targeting c-Myc was determined by subcutaneous injection of stable cells into immunodeficient nude mice. RESULTS: The stable transfection of pCMYC-shRNA successfully knocked down the steady-state mRNA and protein levels of c-Myc in IN500, which positively correlated with the downregulation of VEGF. Downregulating c-Myc in vitro also led to G1-S arrest and enhanced apoptosis. In vivo, targeting c-Myc reduced xenograft tumor formation and resulted in significantly smaller tumors. CONCLUSIONS: c-Myc has multiple functions in glioblastoma development that include regulating cell-cycle, apoptosis, and VEGF expression. Targeting c-Myc expression may be a promising therapy for malignant glioma.

Transcranial direct current stimulation decreases convulsions and spatial memory deficits following pilocarpine-induced status epilepticus in immature rats.

PURPOSE: Transcranial direct current stimulation (tDCS) is a recently available, noninvasive brain stimulation technique. The effects of cathodal tDCS on convulsions and spatial memory after status epilepticus (SE) in immature animals were investigated. METHODS: Rats underwent lithium-pilocarpine-induced SE at postnatal day (P) 20-21 and received daily 30-min cathodal tDCS for 2 weeks at P23-36 through a unilateral epicranial electrode at 200µA. After tDCS, convulsions over 2 weeks were estimated by 20-h/day video monitoring. The rats were tested in a water maze for spatial learning at P50-53 and the brains were examined for cell loss and mossy fiber sprouting. RESULTS: Long-term treatment with weak cathodal tDCS reduced SE-induced hippocampal cell loss, supragranular and CA3 mossy fiber sprouting, and convulsions (reduction of 21%) in immature rats. The tDCS treatment also rescued cognitive impairment following SE. CONCLUSIONS: These findings suggested that cathodal tDCS has neuroprotective effects on the immature rat hippocampus after pilocarpine-induced SE, including reduced sprouting and subsequent improvements in cognitive performance. Such treatment might also have an antiepileptic effect.