Mesenchymal stem cell-derived rapid drug screening system for Alzheimer's disease for the identification of novel drugs.

A reliable and efficient in vitro model is needed to screen drugs for Alzheimer's disease (AD), as many drugs are currently in the developmental stage. To address this, we developed an in vitro model using amniotic membrane-derived mesenchymal stem cells (AM-MSCs) to screen novel drugs for AD. We differentiated AM-MSCs into neurons and degenerated them using beta amyloid1-42 (Aß). We then tested AD drugs, which are commercially available such as donepezil, rivastigmine, memantine, citicoline, and two novel drugs, that is, probucol, an anti-hyperlipidaemic drug, and NMJ-2, a cinnamic acid analogue for their potential to protect the cells against neurodegeneration. We used gene expression and immunofluorescence staining to assess the neuroprotective ability of these drugs. We also measured the ability of these drugs to reduce lactate dehydrogenase, reactive oxygen species, and nitric oxide levels, as well as their ability to stabilize the mitochondrial membrane potential and increase acetylcholine (ACh) levels. The AD drugs and novel drugs reduced cytotoxicity and oxidative stress, stabilized mitochondrial membrane potential, and restored ACh levels. Furthermore, they reduced BACE1 expression, with a concomitant increase in the expression of cholinergic markers. This AM-MSCs-based AD-like model has immense potential to be an accurate human model and an alternative to animal models for testing a large number of lead compounds in a short time. Our results also suggest that the novel drugs probucol and NMJ-2 may protect against Aß-induced neurodegeneration.

Want of Wnt in Parkinson's disease: Could sFRP disrupt interplay between Nurr1 and Wnt signaling?

Nuclear receptor related 1 (Nurr1) is a transcription factor known to regulate the development and maintenance of midbrain dopaminergic (mDA) neurons. Reports have confirmed that defect or obliteration of Nurr1 results in neurodegeneration and motor function impairment leading to Parkinson's disease (PD). Studies have also indicated that Nurr1 regulates the expression of alpha-synuclein (α-SYN) and mutations in Nurr1 cause α-SYN overexpression, thereby increasing the risk of PD. Nurr1 is modulated via various pathways including Wnt signaling pathway which is known to play an important role in neurogenesis, and deregulation of it contributes to PD pathogenesis. Both Wnt/ß-catenin dependent and independent pathways are implicated in the activation of Nurr1 and subsequent downregulation of α-SYN. This review highlights the interaction between Nurr1 and Wnt signaling pathways in mDA neuronal development. We further hypothesize how modulation of Wnt signaling pathway by its antagonist, secreted frizzled related proteins (sFRPs) could be a potential route to treat PD.

Identification of a Novel Wnt Antagonist Based Therapeutic and Diagnostic Target for Alzheimer's Disease Using a Stem Cell-Derived Model.

Currently, all the existing treatments for Alzheimer's disease (AD) fail to stall progression due to longer duration of time between onset of the symptoms and diagnosis of the disease, raising the necessity of effective diagnostics and novel treatment. Specific molecular regulation of the onset and progression of disease is not yet elucidated. This warranted investigation of the role of Wnt signaling regulators which are thought to be involved in neurogenesis. The AD model was established using amyloid beta (Aß) in human mesenchymal stem cells derived from amniotic membranes which were differentiated into neuronal cell types. In vivo studies were carried out with Aß or a Wnt antagonist, AD201, belonging to the sFRP family. We further created an AD201-knockdown in vitro model to determine the role of Wnt antagonism. BACE1 upregulation, ChAT and α7nAChR downregulation with synapse and functionality loss with increases in ROS confirmed the neurodegeneration. Reduced ß-catenin and increased AD201 expression indicated Wnt/canonical pathway inhibition. Similar results were exhibited in the in vivo study along with AD-associated behavioural and molecular changes. AD201-knockdown rescued neurons from Aß-induced toxicity. We demonstrated for the first time a role of AD201 in Alzheimer's disease manifestation, which indicates a promising disease target and biomarker.

An Overview of Ovarian Cancer: The Role of Cancer Stem Cells in Chemoresistance and a Precision Medicine Approach Targeting the Wnt Pathway with the Antagonist sFRP4.

Ovarian cancer is one of the most prevalent gynecological cancers, having a relatively high fatality rate with a low five-year chance of survival when detected in late stages. The early detection, treatment and prevention of metastasis is pertinent and a pressing research priority as many patients are diagnosed only in stage three of ovarian cancer. Despite surgical interventions, targeted immunotherapy and adjuvant chemotherapy, relapses are significantly higher than other cancers, suggesting the dire need to identify the root cause of metastasis and relapse and present more precise therapeutic options. In this review, we first describe types of ovarian cancers, the existing markers and treatment modalities. As ovarian cancer is driven and sustained by an elusive and highly chemoresistant population of cancer stem cells (CSCs), their role and the associated signature markers are exhaustively discussed. Non-invasive diagnostic markers, which can be identified early in the disease using circulating tumor cells (CTCs), are also described. The mechanism of the self-renewal, chemoresistance and metastasis of ovarian CSCs is regulated by the Wnt signaling pathway. Thus, its role in ovarian cancer in promoting stemness and metastasis is delineated. Based on our findings, we propose a novel strategy of Wnt inhibition using a well-known Wnt antagonist, secreted frizzled related protein 4 (sFRP4), wherein short micropeptides derived from the whole protein can be used as powerful inhibitors. The latest approaches to early diagnosis and novel treatment strategies emphasized in this review will help design precision medicine approaches for an effective capture and destruction of highly aggressive ovarian cancer.

An efficient human stem cells derived cardiotoxicity testing platform for testing oncotherapeutic analogues of quercetin and cinnamic acid.

Oncotherapeutics research is progressing at a rapid pace, however, not many drugs complete the successful clinical trial because of severe off-target toxicity to cardiomyocytes which ultimately leads to cardiac dysfunction. It is thus important to emphasize the need for early testing for possible cardiotoxicity of emerging oncotherapeutics. In this study, we assessed a novel stem cell-derived cardiac model for testing for cardiotoxicity of novel oncotherapeutics. We evaluated the cardiotoxic effect of synthesized derivatives of oncotherapeutics, quercetin (QMJ-2, -5, and -6) and cinnamic acid (NMJ-1, -2, and -3) using human Wharton's jelly mesenchymal stem cells-derived cardiomyocytes (WJCM) against known cardiotoxic oncologic drugs, doxorubicin, 5-fluorouracil, cisplatin. QMJ-6, NMJ-2, and NMJ-3 were not cardiotoxic and had minimum cardiac side effects. They did not show any effect on cardiomyocyte viability, caused low LDH release, and intracellular ROS production kept the calcium flux minimal and protected the active mitochondrial status in cardiomyocytes. They persevered cardiac-specific gene expression as well. However, compounds QMJ-2, QMJ-5, and NMJ-1 were cardiotoxic and the concentration needs to be reduced to prevent toxic effects on cardiomyocytes. Significantly, we were able to demonstrate that WJCM is an efficient cardiac testing model to analyze the cardiotoxicity of drugs in a human context.

Inhibition of breast cancer stem-like cells by a triterpenoid, ursolic acid, via activation of Wnt antagonist, sFRP4 and suppression of miRNA-499a-5p.

Breast cancer, one of the leading causes of death in the world, has been largely considered to be drug resistant because of a small population of drug refractory cells, the cancer stem cells (CSCs). The CSCs are tightly regulated by self-renewal pathways such as the Wnt pathway, which is further regulated by a gamut of microRNAs. In this study, we investigated the effect of ursolic acid (UA), a natural triterpene, on breast CSCs enriched from breast cancer cell lines, MCF7, MDA-MB-231 and T47D and analysed the interplay of the Wnt inhibitor, sFRP4 and an miRNA, miR-499a-5p, in mediating the effect of UA. By using caspase 3/7, ROS, migration, TCF/LEF and CAM assays, overexpressing and inhibiting miR-499a-5p and NanoString PanCancer analysis, we observed that UA had significant anti-CSC ability. There was a link between UA and Wnt/ß-catenin pathway wherein, Wnt was suppressed by upregulation of the antagonist, sFRP4. Furthermore, expression of the oncogenic miR-499a-5p was substantially diminished in CSCs after UA treatment. Notably, the axis by which miR-499a-5p acts is via the TCF/LEF machinery of the Wnt/ß-catenin pathway. Our findings indicate for the first time that UA can target breast CSCs via Wnt by suppressing miR-499a-5p and upregulating the Wnt antagonist, sFRP4.

Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma.

BACKGROUND: Chemotherapeutic resistance of glioblastoma has been attributed to a self-renewing subpopulation, the glioma stem cells (GSCs), which is known to be maintained by the Wnt ß-catenin pathway. Our previous findings demonstrated that exogeneous addition of the Wnt antagonist, secreted fizzled-related protein 4 (sFRP4) hampered stem cell properties in GSCs. METHODS: To understand the molecular mechanism of sFRP4, we overexpressed sFRP4 (sFRP4 OE) in three human glioblastoma cell lines U87MG, U138MG, and U373MG. We also performed chromatin immunoprecipitation (ChIP) sequencing of sFRP4 OE and RNA sequencing of sFRP4 OE and sFRP4 knocked down U87 cells. RESULTS: We observed nuclear localization of sFRP4, suggesting an unknown nuclear role. ChIP-sequencing of sFRP4 pulldown DNA revealed a homeobox Cphx1, related to the senescence regulator ETS proto-oncogene 2 (ETS2). Furthermore, miRNA885, a p53-mediated apoptosis inducer, was upregulated in sFRP4 OE cells. RNA sequencing analysis suggested that sFRP4-mediated apoptosis is via the Fas-p53 pathway by activating the Wnt calcium and reactive oxygen species pathways. Interestingly, sFRP4 OE cells had decreased stemness, but when knocked down in multipotent mesenchymal stem cells, pluripotentiality was induced and the Wnt ß-catenin pathway was upregulated. CONCLUSIONS: This study unveils a novel nuclear role for sFRP4 to promote apoptosis by a possible activation of DNA damage machinery in glioblastoma.

Amelioration of methylmercury induced neural damage by essential oil of Selinum vaginatum (Edgew) C. B. Clarke.

Methylmercury (MeHg), an organometallic contaminant is a well spotted cause for a series of disorders, especially in the central nervous system. As there is no proper treatment, Selinum vaginatum (Edgew) C. B. Clarke, a traditional medicinal plant, is taken in the present study for assessing its neuroprotective effect against MeHg induced toxicity using rat brain mitochondrial fractions. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) assay indicated that there was a reduction in the mitochondrial viability in MeHg treated sample and IC50 value recorded was 2.5µg/ml. Biochemical analysis showed that there was a significant inhibition of glutathione levels (GSH) and catalase activity and an elevation of thiobarbituric acid reactive substances (TBARS) levels in MeHg treated sample. These changes were prevented by co-incubation with essential oil extracted from Selinum vaginatum. The GSH reduction caused by MeHg is restored by essential oil, endorsing its chelating effect, an important molecular mechanism of defense against oxidative injury. Some of the major compounds are detected in Gas chromatography-mass spectrometry (GC-MS) analysis of essential oil, which could be accountable for its neuroprotection against MeHg.