Human placenta mesenchymal stem cell protection in ischemic stroke is angiotensin converting enzyme-2 and masR receptor-dependent.

Thromboembolic stroke remains a major cause of neurological disability and death. Current stroke treatments (aspirin, tissue plasminogen activator) are significantly limited by timing and risks for hemorrhage which have driven researchers to explore other approaches. Stem cell-based therapy appears to be an effective option for ischemic stroke. Besides trans-differentiation into neural cells, stem cells also provide acute protection via paracrine signaling pathways through which releasing neuroprotective factors. We previously reported that intraperitoneal administration of human placenta mesenchymal stem cell (hPMSC) therapy upon reperfusion significantly protected the brain against middle cerebral artery occlusion (MCAO)-induced injury. In the present study, we specifically investigated the role of hPMSC-derived angiotensin converting enzyme-2 (ACE-2) in protection of MCAO-induced brain injury by measurement of brain tissue viability, cerebral blood flow, and neurological score. Here, we report for the first time that hPMSC expressing substantial amount of ACE-2, which mediates hPMSC protection in the MCAO model. Strikingly, we found that the protective effects of hPMSC in MCAO-induced brain injury could be attenuated by pretreatment of hPMSCs with MLN-4760, a specific inhibitor of ACE-2 activity, or by transfection of hPMSCs with ACE-2-shRNA-lentivirus. The hPMSC-derived ACE-2 specific protective mechanism was further demonstrated by administration of PD123319, an Angiotensin type-2 receptor antagonist, or A779, a MasR antagonist. Importantly, our study demonstrated that the protective effects of hPMSC in experimental stroke are ACE-2/MasR dependent and this signaling pathway represents an innovative and highly promising approach for targeted stroke therapy.

Three-Dimensional Printing of Cell Exclusion Spacers (CES) for Use in Motility Assays.

PURPOSE: Cell migration/invasion assays are widely used in commercial drug discovery screening. 3D printing enables the creation of diverse geometric restrictive barrier designs for use in cell motility studies, permitting on-demand assays. Here, the utility of 3D printed cell exclusion spacers (CES) was validated as a cell motility assay. METHODS: A novel CES fit was fabricated using 3D printing and customized to the size and contour of 12 cell culture plates including 6 well plates of basal human brain vascular endothelial (D3) cell migration cells compared with 6 well plates with D3 cells challenged with 1uM cytochalasin D (Cyto-D), an F-actin anti-motility drug. Control and Cyto-D treated cells were monitored over 3 days under optical microscopy. RESULTS: Day 3 cell migration distance for untreated D3 cells was 1515.943µm ± 10.346µm compared to 356.909µm ± 38.562µm for the Cyt-D treated D3 cells (p < 0.0001). By day 3, untreated D3 cells reached confluency and completely filled the original voided spacer regions, while the Cyt-D treated D3 cells remained significantly less motile. CONCLUSIONS: Cell migration distances were significantly reduced by Cyto-D, supporting the use of 3D printing for cell exclusion assays. 3D printed CES have great potential for studying cell motility, migration/invasion, and complex multi-cell interactions.

SKLB188 inhibits the growth of head and neck squamous cell carcinoma by suppressing EGFR signalling.

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. Several small molecule EGFR inhibitors have been tested in clinical trials for treatment of HNSCC, but none of them are more effective than the current chemotherapeutic drugs. Thus, it is urgently needed to develop novel EGFR inhibitors for HNSCC treatment. METHODS: By screening an in-house focused library containing approximately 650 000 known kinase inhibitors and kinase inhibitor-like compounds containing common kinase inhibitor core scaffolds, we identified SKLB188 as a lead compound for inhibition of EGFR. The anticancer effects of SKLB188 on HNSCC cells were investigated by in vitro cell growth, cell cycle and apoptosis assays, as well as in vivo FaDu xenograft mouse model. Molecular docking, in vitro kinase profiling and western blotting were performed to characterise EGFR as the molecular target. RESULTS: SKLB188 inhibited HNSCC cell proliferation by inducing G1 cell cycle arrest, which was associated with downregulating the expression of Cdc25A, cyclins D1/A and cyclin-dependent kinases (CDK2/4), and upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (p21Cip1 and p27Kip1), leading to decreased phosphorylation of Rb. SKLB188 also induced caspase-dependent apoptosis of HNSCC cells by downregulating the expression of Mcl-1 and survivin. Molecular docking revealed that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay showed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC50=5 nM). Western blot analysis demonstrated that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose-dependently inhibited FaDu xenograft growth in nude mice, and concurrently inhibited the phosphorylation of Erk1/2 and Akt in the tumours. CONCLUSIONS: SKLB188 potently inhibits the growth of HNSCC cells in vitro and in vivo by targeting EGFR signalling. The results provide a basis for further clinical investigation of SKLB188 as a targeted therapy for HNSCC. Our findings may open a new avenue for development of novel EGFR inhibitors for treatment of HNSCC and other cancers.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells.

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

MicroRNA 17-92 expressed by a transposone-based vector changes expression level of cell-cycle-related genes.

The miR-17-92 cluster is composed of seven miRNAs (microRNAs; miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1). Previous studies have indicated that this cluster is involved in cell proliferation and their overexpression has been seen in several types of cancer. We have assessed the overexpression effects of miR-17-92 on the expression of several genes associated with cell-cycle regulation. The human miR-17-92 gene was cloned into a transposone-based vector, piggyBac and transfected into HEK-293T [HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)] cell line. Gene expression analysis indicated that up-regulation of this cluster causes significant changes in the expression of several cell-cycle related genes, including CDK2 (cyclin-dependent kinase 2), cyclin-D2, c-Myc and CREB (cAMP-response-element-binding protein). Other methods of transcripts assessment confirmed miR-17-92 overexpression enhances cell proliferation.

Applications of acellular human amniotic membrane in regenerative medicine.

The human amniotic membrane (hAM) surrounds and protects the fetus. It mainly consists of a thick basement membrane and an avascular stromal matrix. The structural, physical, and biological properties of AM make it a wonderful choice to use in regenerative medicine applications. This review discusses recent applications of hAM in skin transplantation and wound healing, ophthalmology, orthopedics, cell culture matrix, cell delivery system, cardiac injury, male and female reproductive systems, and liver. Moreover, an overview is presented on the advantages and disadvantages of using hAM and some methods to systematically refine and standardize hAM preparation.

The Role of the ACE2/MasR Axis in Ischemic Stroke: New Insights for Therapy.

Ischemic stroke remains the leading cause of neurologically based morbidity and mortality. Current stroke treatment is limited to two classes of FDA-approved drugs: thrombolytic agents (tissue plasminogen activator (tPA)) and antithrombotic agents (aspirin and heparin), which have a narrow time-window (<4.5 h) for administration after onset of stroke symptoms. While thrombolytic agents restore perfusion, they carry serious risks for hemorrhage, and do not influence damage responses during reperfusion. Consequently, stroke therapies that can suppress deleterious effects of ischemic injury are desperately needed. Angiotensin converting enzyme-2 (ACE2) has been recently suggested to beneficially influence experimental stroke outcomes by converting the vasoconstrictor Ang II into the vasodilator Ang 1-7. In this review, we extensively discuss the protective functions of ACE2-Ang (1-7)-MasR axis of renin angiotensin system (RAS) in ischemic stroke.

Human placental mesenchymal stem cells improve stroke outcomes via extracellular vesicles-mediated preservation of cerebral blood flow.

BACKGROUND: Besides long-term trans-differentiation into neural cells, benefits of stem cell therapy (SCT) in ischemic stroke may include secretion of protective factors, which partly reflects extracellular vesicle (EVs) released by stem cell. However, the mechanism(s) by which stem cells/EVs limit stroke injury have yet to be fully defined. METHODS: We evaluated the protection effect of human placenta mesenchymal stem cells (hPMSC) as a potential form of SCT in experimental ischemic stroke 'transient middle cerebral artery occusion (MCAO)/reperfusion' mice model. FINDINGS: We found for the first time that intraperitoneal administration of hPMSCs or intravenous hPMSC-derived EVs, given at the time of reperfusion, significantly protected the ipsilateral hemisphere from ischemic injury. This protection was associated with significant restoration of normal blood flow to the post-MCAO brain. More importantly, EVs derived from hPMSC promote paracrine-based protection of SCT in the MCAO model in a cholesterol/lipid-dependent manner. INTERPRETATION: Together, our results demonstrated beneficial effects of hPMSC/EVs in experimental stroke models which could permit the rapid "translation" of these cells into clinical trials in the near-term.

Obstructive sleep apnea intensifies stroke severity following middle cerebral artery occlusion.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is a sleep disorder caused by transient obstruction of the upper airway and results in intermittent hypoxia, sleep fragmentation, sympathetic nervous system activation, and arousal which can have an adverse effect on cardiovascular disease. It is theorized that OSA might intensify stroke injury. Our goal here was to develop a new model of experimental OSA and test its ability to aggravate behavioral and morphological outcomes following transient brain ischemia/reperfusion. METHODS: We used a 3D printed OSA device to expose C57BL6 mice to 3 h of OSA (obstructive apnea index of 20 events per hour) for three days. These mice were then subjected to ischemia/reperfusion using the middle cerebral artery occlusion model (MCAO) stroke and examined for overall survival, infarct size and neurological scoring. RESULTS: We found that OSA transiently decreased respiration and reduced oxygen saturation with bradycardia and tachycardia typical of human responses during apneic events. Brain injury from MCAO was significantly increased by OSA as measured by infarct size and location as well as by intensification of neurological deficits; mortality following MCAO was also increased in OSA animals. CONCLUSIONS: Our findings suggest that our new model of OSA alters respiratory and cardiovascular physiological functions and is associated with enhanced ischemia/reperfusion mediated injury in our non-invasive, OSA intensified model of stroke.

Brain Endothelial Cells Release Apical and Basolateral Microparticles in Response to Inflammatory Cytokine Stimulation: Relevance to Neuroinflammatory Stress?

Microparticles (MP) are regarded both as biomarkers and mediators of many forms of pathology, including neurovascular inflammation. Here, we characterized vectorial release of apical and basolateral MPs (AMPs and BMPs) from control and TNF-α/IFN-γ treated human brain endothelial monolayers, studied molecular composition of AMPs and BMPs and characterized molecular pathways regulating AMP and BMP release. The effects of AMPs and BMPs on blood-brain barrier properties and human brain microvascular smooth muscle tonic contractility in vitro were also evaluated. We report that human brain microvascular endothelial cells release MPs both apically and basolaterally with both AMP and BMP release significantly increased following inflammatory cytokine challenge (3.5-fold and 3.9-fold vs. control, respectively). AMPs and BMPs both carry proteins derived from parent cells including those in BBB junctions (Claudin-1, -3, -5, occludin, VE-cadherin). AMPs and BMPs represent distinct populations whose release appears to be regulated by distinctly separate molecular pathways, which depend on signaling from Rho-associated, coiled-coil containing protein kinase (ROCK), calpain as well as cholesterol depletion. AMPs and BMPs modulate functions of neighboring cells including BBB endothelial solute permeability and brain vascular smooth muscle contractility. While control AMPs enhanced brain endothelial barrier, cytokine-induced AMPs impaired BBB. Cytokine-induced but not control BMPs significantly impaired human brain smooth muscle contractility as early as day 1. Taken together these results indicate that AMPs and BMPs may contribute to neurovascular inflammatory disease progression both within the circulation (AMP) and in the brain parenchyma (BMP).

High-throughput scaffold-free microtissues through 3D printing.

BACKGROUND: Three-dimensional (3D) cell cultures and 3D bioprinting have recently gained attention based on their multiple advantages over two-dimensional (2D) cell cultures, which have less translational potential to recapitulate human physiology. 3D scaffold supports, cell aggregate systems and hydrogels have been shown to accurately mimic native tissues and support more relevant cell-cell interactions for studying effects of drugs and bioactive agents on cells in 3D. The development of cost-effective, high-throughput and scaffold-free microtissue assays remains challenging. In the present study, consumer grade 3D printing was examined as a fabrication method for creation of high-throughput scaffold-free 3D spheroidal microtissues. RESULTS: Consumer grade 3D printing was capable of forming 96-well cell culture inserts to create scaffold-free microtissues in liquid suspensions. The inserts were seeded with human glioblastoma, placental-derived mesenchymal stem cells, and intestinal smooth muscle cells. These inserts allowed for consistent formation of cell density-controllable microtissues that permit screening of bioactive agents. CONCLUSION: A variety of different cell types, co-cultures, and drugs may be evaluated with this 3D printed microtissue insert. It is suggested that the microtissue inserts may benefit 3D cell culture researchers as an economical assay solution with applications in pharmaceuticals, disease modeling, and tissue-engineering.

Ciclopirox inhibits cancer cell proliferation by suppression of Cdc25A.

Ciclopirox olamine (CPX), an off-patent fungicide, has recently been identified as a novel anticancer agent. However, the molecular mechanism underlying its anticancer action remains to be elucidated. Here we show that CPX inhibits cell proliferation in part by downregulating the protein level of Cdc25A in tumor cells. Our studies revealed that CPX did not significantly reduce Cdc25A mRNA level or Cdc25A protein synthesis, but remarkably promoted Cdc25A protein degradation. This resulted in inhibition of G1-cyclin dependent kinases (CDKs), as evidenced by increased inhibitory phosphorylation of G1-CDKs. Since Cdc25A degradation is tightly related to its phosphorylation status, we further examined whether CPX alters Cdc25A phosphorylation. The results showed that CPX treatment increased the phosphorylation of Cdc25A (S76 and S82), but only Cdc25A-S82A mutant was resistant to CPX-induced degradation. Furthermore, ectopic expression of Cdc25A-S82A partially conferred resistance to CPX inhibition of cell proliferation. Therefore, our findings indicate that CPX inhibits cell proliferation at least in part by promoting Cdc25A degradation.

Fusarochromanone-induced reactive oxygen species results in activation of JNK cascade and cell death by inhibiting protein phosphatases 2A and 5.

Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death.

Both mTORC1 and mTORC2 are involved in the regulation of cell adhesion.

mTOR is a central controller for cell growth/proliferation and survival. Recent studies have shown that mTOR also regulates cell adhesion, yet the underlying mechanism is not known. Here we found that inhibition of mTOR by rapamycin reduced the basal or type I insulin-like growth factor (IGF-1)-stimulated adhesion of cancer cells. Further research revealed that both mTORC1 and mTORC2 were involved in the regulation of cell adhesion, as silencing expression of raptor or rictor inhibited cell adhesion. Also, PP242, an mTORC1/2 kinase inhibitor, inhibited cell adhesion more potently than rapamycin (mTORC1 inhibitor). Of interest, ectopic expression of constitutively active and rapamycin-resistant mutant of p70 kinase 1 (S6K1) or downregulation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) conferred resistance to rapamycin inhibition of cell adhesion, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or downregulation of S6K1 suppressed cell adhesion. In contrast, neither genetic manipulation of Akt activity nor pharmacological inhibition of Akt affected cell adhesion. The results suggest that both mTORC1 and mTORC2 are involved in the regulation of cell adhesion; and mTORC1 regulates cell adhesion through S6K1 and 4E-BP1 pathways, but mTORC2 regulates cell adhesion via Akt-independent mechanism.

Maduramicin inhibits proliferation and induces apoptosis in myoblast cells.

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.

Fusarochromanone induces G1 cell cycle arrest and apoptosis in COS7 and HEK293 cells.

Fusarochromanone (FC101), a mycotoxin produced by the fungus Fusarium equiseti, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G1 cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death.

Biological activities of fusarochromanone: a potent anti-cancer agent.

BACKGROUND: Fusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity. RESULTS: Herein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 µM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells. CONCLUSIONS: Our data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101's molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery.

Study of Anti Cancer Property of Scrophularia striata Extract on the Human Astrocytoma Cell Line (1321).

There are considerable efforts to identify naturally occurring substances as new drugs in cancer therapy. Many components of medicinal plants have been identified that possess substantial anticancerous properties. This prompted us to investigate the effect of Scrophularia striata (an Iranian species belonging to the Scrophulariace family) extract on the growth of astrocyte cancer cell line (1321). The 1321 cell line were seeded in 96-well culture plates in the presence and absence of various concentrations of either leaf and seed filtered and unfiltered extract of Scrophularia striata to determine their probable anticancer effects in comparison with etoposide (chemical anticancer reagent). filtered leaf extract of S. Striata showed strong anticancer effect on 1321cell line as compared to control group (cells not exposed to extracts), and even the group (adenocarcinoma gastric cell line) exposed to etoposide. Unlike the leaf extract, the seed extract activated cell proliferation in all experiments. Flow cytometry findings indicated that apoptosis is the mechanism by which the leaf extract inhibits cell proliferation. Our findings indicate that both leaves and seeds of S. Striata contain both anti cancer and cell growth enhancing agents.