The impact of temozolomide and lonafarnib on the stemness marker expression of glioblastoma cells in multicellular spheroids.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a poor prognosis. The GBM microenvironment is highly heterogeneous and is composed of many cell types including astrocytes and endothelial cells (ECs) along with tumor cells, which are responsible for heightened resistance to standard chemotherapeutic drugs such as Temozolomide (TMZ). Here, we investigated how drug treatments impact stemness marker expression of GBM cells in multicellular tumor spheroid (MCTS) models. Co- and tri-culture MCTS constructed using U87-MG GBM cells, astrocytes, and/or ECs were cultured for 7 days. At Day 7, 5 µM lonafarnib (LNF), 100 µM TMZ, or combination of 5 µM LNF + 100 µM TMZ was added and the MCTS were cultured for an additional 48 h. We assessed the spheroid sizes and expression of stemness markers- NESTIN, SOX2, CD133, NANOG, and OCT4- through qRT-PCR and immunostaining. Following 48 h treatment with LNF, TMZ or their combination (LNF + TMZ), the spheroid sizes decreased compared to the untreated control. We also observed that the expression of most of the stemness markers significantly increased in the LNF + TMZ treated condition as compared to the untreated condition. These results indicate that drug treatment can influence the stemness marker expression of GBM cells in MCTS models and these aspects must be considered while evaluating therapies. In future, by incorporating other relevant cell types, we can further our understanding of their crosstalk, eventually leading to the development of new therapeutic strategies.

Neural Stem Cells Secretome Increased Neurogenesis and Behavioral Performance and the Activation of Wnt/ß-Catenin Signaling Pathway in Mouse Model of Alzheimer's Disease.

Alzheimer's disease is a progressive and age-related neurodegenerative disorder that is manifested by neuropathological changes and clinical symptoms. Recently, cell-based therapeutic interventions have been considered as the promising and effective strategies in this field. Herein, we investigated therapeutic effects of neural stem cell secretome on Alzheimer's disease-like model by triggering of Wnt/ß-catenin signaling pathway. In this study, mice were randomly allocated into three different groups as follows: Control, AD + Vehicle, and AD + NSCs-CM groups. To induce mouse model of AD, Aß1-42 was injected into intracerebroventricular region. Following AD-like confirmation through thioflavin S staining and Passive avoidance test, about 5 µl mouse NSCs-CM was injected into the target areas 21 days after AD induction. For evaluation of endogenous proliferation rate (BrdU/Nestin+ cells), 50 µg/kbW BrdU was intraperitoneally injected for 5 consecutive days. To track NSC differentiation, percent of BrdU/NeuN+ cells were monitored via immunofluorescence staining. Histological Nissl staining was done to neurotoxicity and cell death in AD mice after NSCs-CM injection. Morris Water maze test was performed to assess learning and memory performance. Data showed that NSCs-CM could reverse the learning and memory deficits associated with Aß pathology. The reduced expression of Wnt/ß-catenin-related genes such as PI3K, Akt, MAPK, and ERK in AD mice was increased. Along with these changes, NSCs-CM suppressed overactivity of GSK3ß activity induced by Aß deposition. Besides, NSCs increased BrdU/Nestin+ and BrdU/NeuN+ cells in a paracrine manner, indicating proliferation and neural differentiation of NSCs. Moreover, neurotoxicity rate and cell loss were deceased after NSCs-CM injection. In summary, NSCs can regulate adult neurogenesis through modulating of Wnt/ß-catenin signaling pathway and enhance the behavioral performance in the AD mice. These data present the alternative and effective approach in the management of AD and other cognitive impairments.

Nestin Improves Preeclampsia-Like Symptoms by Inhibiting Activity of Cyclin-Dependent Kinase 5.

BACKGROUND/AIMS: Preeclampsia (PE) is a pregnancy-specific hypertensive disorder that is characterised by a high incidence of hypertension and proteinuria. Podocytes are involved in the formation of a split membrane, which is the last barrier preventing the leakage of protein into the urine. Nestin, a cytoskeleton protein, is expressed stably in podocytes. However, the association between the Nestin concentration in urine and the progression of PE and the role of Nestin in PE remains unclear. METHODS: In the present study, a mouse podocyte cell line, PE-like animal model and PE patients' urine samples were used. Eilsa kits were used to detect the levels of proteins expression in urine samples from patients and animal models. Western Blotting and immunofluorescence were used to detect proteins expression levels in cell samples and animal tissue samples. Flow cytometry was used to detect the level of apoptosis in cells. Tunel assay was used to detect the levels of apoptosis in animal tissue samples. RESULTS: Nestin levels were significantly increased in PE patients than in hypertensive patients and healthy subjects, and positively correlated with proteinuria and podocalyxin. Ang II treatment decreased the expression of Nestin and Podocin in a time- and dose- dependent manner in podocytes. Restoration of the Nestin levels could reverse Ang II-induced F-actin degradation and attenuate Ang II-mediated podocyte apoptosis, while knockdown of the Nestin level exhibited the opposite. Moreover, the protective role of Nestin on podocytes is mediated by inhibition of the kinase activity of CDK5. In PE-like animal model induced by L-NAME injection, restoration of Nestin lowered the pressure and proteinuria concentration, attenuated the loss of podocytes, and decreased the expression of p35, p53 and the activity of CDK5 kinase, as compared with the control. CONCLUSIONS: Our findings suggest that Nestin could improve preeclampsia-like symptoms by inhibiting the activity of CDK5, and Nestin may become a new prognostic factor and a potential therapy target for PE.