A Perspective on the Characterization of Early Neural Progenitor Cell-Derived Extracellular Vesicles for Targeted Delivery to Neuroblastoma Cells.

As an element of the cellular signaling systems, extracellular vesicles (EVs) exhibit many desirable traits for usage as targeted delivery vehicles. When administered, EVs cause little to no toxic or immune response, stay in circulation for longer periods compared to synthetic carriers, preferentially accumulate in tissues that are the same or similar to their cell-of-origin and can pass through the blood-brain barrier. Combined, these traits make neural EVs a particularly promising tool for delivering drugs to the brain. This study aims to combine tissue and EVs engineering to prepare neural differentiated cells derived EVs that exhibit neural properties, to develop an effective, tissue-homing drug and gene delivery platform for the brain. Early neural differentiated cell-derived EVs were produced with neural characteristics from neural differentiated human neonatal dermal fibroblasts. The EVs carried key neural proteins such as Nestin, Sox2 and Doublecortin. The cellular uptake of early neural differentiated cell-derived EVs was higher compared to non-neural EVs during in vitro uptake assays on neuroblastoma cells. Moreover, eND-EVs were significantly decreased the viability of neuroblastoma cells. In conclusion, this study revealed that early neural differentiated cell-derived EVs have potential as a promising drug carrier for the treatment of various neural disorders.

Synergistic anti-proliferative and apoptotic effect of NVP-BEZ235 and curcumin on human SH-SY5Y neuroblastoma cells.

Neuroblastoma, a tumor of the sympathetic nervous system, is one of the most common tumors found in children. Most patients develop resistance to therapy and show poor prognosis, thus there is a need of novel therapeutic agents for the treatment of neuroblastoma. NVP-BEZ235 is a dual Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) kinase inhibitor that induces apoptosis and suppresses the growth of cancer. Curcumin acts as an anticancer agent in certain cancers. This study investigated the synergetic effect of NVP-BEZ235 and curcumin against neuroblastoma SH-SY5Y cell line. In the current study, the synergic effect of NVP-BEZ235 and curcumin in SH-SY5Y was examined in terms of the cell growth by cell viability and colony forming assay, cell cycle and apoptotic cell death by flow cytometry and mRNA expression levels by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). Curcumin, NVP-BEZ235 or a combination of both, showed cytotoxicity in a dose and time dependent manner in SH-SY5Y cells. 10 µM curcumin and 200 nM NVP-BEZ235 were chosen as combination therapy, as the combination index showed synergism. Colony forming assay showed decrease in cell growth in combination group. The cell cycle distribution for combination group demonstrated a decrease in G0/G1 phase at 48 h. Annexin V showed an anticancer effect in combination group when compared to control group. Moreover, qRT-PCR results showed a significant increase in caspase 3, caspase 7, Bax and p53 genes, while a decrease in Bcl-2 gene expression levels. These findings suggest that combination therapy of NVP-BEZ235 and curcumin may be a promising therapeutic candidate for treatment of neuroblastoma.

The emerging role of breast cancer derived extracellular vesicles-mediated intercellular communication in ovarian cancer progression and metastasis.

Breast cancer is one of the most occurring cancer types in women worldwide and metastasizes to several organs such as bone, lungs, liver, brain, and ovaries. Extracellular vesicles (EVs) mediate intercellular signaling which has a profound effect on tumor development and metastasis. Recent developments in the field of EVs provide an opportunity to investigate the roles of EVs released from tumor cells in metastasis. In this study, we compared the effects of metastatic breast cancer-derived EVs on both nonluteinized granulosa HGrC1 and ovarian cancer OVCAR-3 cells in terms of proliferation, invasion, apoptosis, and gene expression levels. EVs were isolated from the culture medium of metastatic breast cancer cell line MDA-MB-231 by ultracentrifugation. Cell proliferation, apoptosis, cell cycle, invasion, and cellular uptake analysis were performed to clarify the roles of tumor-derived EVs in both cells. 6.85 × 108 nanoparticles of BCD-EVs were markedly increased cell proliferation as well as invasion capacity. Exposing the cells with BCD-EVs for 24 h, resulted in an accumulation of both cells in G2/M phase as determined by flow cytometry. The apoptosis assay results were consistent with cell proliferation and cell cycle results. The uptake of the BCD-EVs was efficiently internalized by both cells. In addition, marked variations in fatty acid composition between cells were observed. BCD-EVs appeared new fatty acids in HGrC1. Besides, BCD-EVs upregulated epithelial-mesenchymal transition (EMT) and proliferation-related genes. In conclusion, an environment of tumor-derived EVs changes the cellular phenotype of cancer and noncancerous cells and may lead to tumor progression and metastasis.

Endothelial cell-derived extracellular vesicles induce pro-angiogenic responses in mesenchymal stem cells.

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin-1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)-particularly EVs of vascular origin-could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)-derived small EVs (<200 nm; HU-sEVs) were investigated as a potential pro-angiogenic agent. Treating mesenchymal stem cells (MSCs) and mature HUVEC cells with HU-sEVs induced their tube formation under in vitro conditions and significantly increased the expression of angiogenesis-related genes, such as Ang1, VEGF, Flk-1 (VEGF receptor 2), Flt-1 (VEGF receptor 1), and vWF (von Willebrand Factor), in a dose-dependent manner. These results indicate that HU-sEVs take part in angiogenesis activities in physiological systems, and suggest endothelial EVs as a potential therapeutic candidate for the treatment of angiogenesis-related diseases.

Antineoplastic effects of erufosine on small cell and non-small cell lung cancer cells through induction of apoptosis and cell cycle arrest.

BACKGROUND: Lung cancer (LC) is the most common types of cancer worldwide and is marked by high mortality rate. LC is classified into two major types due to their molecular and histological properties; non-small cell lung cancer (NSCLC) A549 and small cell lung cancer (SCLC). Currently, surgery, chemotherapy and radiation therapy are the most common treatment options of LC. However, the survival rate of LC is still very poor. Therefore, new treatment strategies are urgently needed. Erufosine (ErPC3) is a novel alkylphosphocholine and inhibits the translocation of Akt to the plasma membrane. METHODS AND RESULTS: In the current study, the effects of ErPC3 in NSCLC cell line A549 and SCLC cell line DMS 114 in terms of cell viability, induction of apoptosis, cell cycle phase distribution, gene and protein expression levels, and migration capacity were investigated. 25 µM ErPC3 exhibited dose-dependent cytotoxicity against in both cancer cells. However, DMS 114 was more sensitive to ErPC3 than A549. Similarly, ErPC3 induced apoptotic cell ratio in DMS114 was significantly greater than A549. 25 µM ErPC3 caused the accumulation of both cell in G2/M phase. The levels of BCL-2 were downregulated and CASPASE 3-7 and BAX were upregulated while p-Akt levels were reduced in A549 and DMS 114 cells treated with 25 µM ErPC3. Besides, ErPC3 displayed anti-migratory effect on A549 and DMS 114. CONCLUSION: These findings suggest that ErPC3 may be a promising novel therapeutic candidate for treatment of LC. ErPC3 treatment merits further investigation as potential agent against LC.

Dual Akt and Bcl-2 inhibition induces cell-type specific modulation of apoptotic and autophagic signaling in castration resistant prostate cancer cell lines.

BACKGROUND: Cancer cell survival depends on the cross-regulation between apoptosis and autophagy which share common signaling pathways including PI3K/Akt/mTOR and Bcl-2. The aim of this study was to elucidate the modulation patterns between apoptosis and autophagy following dual inhibition by Akt inhibitor erufosine and Bcl-2 inhibitor ABT-737 in castration-resistant prostate cancer (CRPC) cell lines, PC-3 (Bax+) and DU-145 (Bax-). METHODS AND RESULTS: Cell cycle progression, apoptotic and autophagic signaling were examined by flow cytometry, multi-caspase assay, Hoechst staining, acridine orange staining of acidic vesicular organelles (AVOs), qRT-PCR and Western Blot. Dual inhibition increased G2/M arrest in PC-3 and DU-145, but not in the healthy prostate epithelium cells, PNT-1A. Only in PC-3, dual inhibition induced synergistic apoptotic and additive autophagic effects. In DU-145 and PNT-1A cells, ABT-737 did not display any remarkable effect on multicaspase activity and erufosine and ABT-737, neither alone nor in combination induced AVOs. By dual inhibition, AKT, BCL-2 and NF-κB gene expressions were downregulated in PC-3, both ATG-5 and BECLIN-1 gene expressions were upregulated in DU-145 but Beclin-1 protein expression was substantially reduced in both CRPC cells. Dual inhibition-induced synergistic multicaspase activation in PC-3 degrades and disrupts autophagic activity of Beclin-1, enhancing caspase-dependent apoptosis. However, in DU-145, following dual inhibition, rate of multicaspase induction and apoptosis are lower but autophagy is completely abolished despite markedly increased BECLIN-1 gene expression. CONCLUSION: In conclusion, antineoplastic drug combinations may display cell-type specific modulation of apoptotic and autophagic signaling and lack of protective autophagy may not necessarily indicate increased chemotherapeutic sensitivity in heterogenous tumor subpopulations.

Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing.

Due to the prevalence of individuals suffering from chronic wounds, developing safe and effective wound care agents are one of the more prominent fields of research in biology. However, wound healing is a complex, multi-stage biological process, involving multiple sequences of biological responses from different types of cells, secreted mediators, and extracellular matrix elements. Plants have a long history of use in the treatment of wounds. Plant-derived extracellular vesicles, which are secreted nano vesicle messengers responsible for intercellular communications, show promise as a new, biotechnological wound-care agent. In this study, we assessed the wound healing potential of extracellular vesicles isolated from grapefruits - a plant with well-known anti-inflammatory and wound healing properties. Grapefruit extracellular vesicles (GEVs) increased cell viability and cell migration while reducing intracellular ROS production in a dose-dependent manner in HaCaT cells. Expression of proliferation and migration-related genes were raised by GEV treatment in a dose dependent manner. Additionally, GEV treatment increased the tube formation capabilities of treated HUVEC cells. These findings suggest that GEVs can be used as plant-derived wound healing agents, and have shown potential as a biotechnological agent for wound healing. Further development and study of plant-derived extracellular vesicles may lead to the realization of their full potential.

Sodium Pentaborate Pentahydrate ameliorates lipid accumulation and pathological damage caused by high fat diet induced obesity in BALB/c mice.

BACKGROUND: Obesity is one of the most popular topic in the field of research. In order to defeat this highly widespread disease, the mechanism of fat accumulation at the molecular level and its elimination are crucial. The use of boron has been showing promising results during the recent years. METHODS: In this study, anti-obesity potential of Sodium Pentaborate Pentahydrate (SPP) used as a dietary supplement on BALB/c mice fed with a high-fat diet was evaluated. Mice were divided into four groups with different diets, consisting of a normal diet, a high-fat diet (HFD) (containing 60 % fat), a HFD-supplemented with 0.5 mg/g body weight (BW) of SPP and a HFD-supplemented with 1.5 mg/g body weight (BW) of SPP. The animals were then observed for 10 weeks and physically monitored, and were sacrificed at the end of the experiment for physical and physicochemical evaluation. RESULTS: According to the physical parameters measured -body weight, food and water intake ratios-, the results indicate that SPP decreased weight gain in a dose dependent manner. Measurement of the hormone levels in the blood and fat accumulation in organs of mice also supported the anti-obesity effects of SPP. Expressions of adipogenesis related genes were also negatively regulated by SPP administration in white adipose tissue (WAT) tissue. CONCLUSION: These findings promise a treatment approach and drug development that can be used against obesity when SPP is used in the right doses. As a future aspect, clinical studies with SPP will reveal the effect of boron derivatives on obesity.

Evaluation of the neuroprotective potential of caffeic acid phenethyl ester in a cellular model of Parkinson's disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, and oxidative stress and mitochondrial dysfunction play a major role in the pathogenesis of PD. Since conventional therapeutics are not sufficient for the treatment of PD, the development of new agents with anti-oxidant potential is crucial. Caffeic Acid Phenethyl Ester (CAPE), a biologically active flavonoid of propolis, possesses several biological properties such as immunomodulatory, anti-inflammatory and anti-oxidative. In the present study, we investigated the neuroprotective effects of CAPE against 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y cells. The neuroprotective effects were detected by using cell viability, Annexin V, Hoechst staining, total caspase activity, cell cycle, as well as western blotting. Besides, the anti-oxidative activity was measured by the production of reactive oxygen species and mitochondrial function was determined by measurement of mitochondrial membrane potential (ΔΨm). We found that CAPE significantly increased cell viability and decreased apoptotic cell death (~20%) after 150 µM 6-OHDA exposure following 24 h. 1.25 µM CAPE also prevented 6-OHDA-induced changes in condensed nuclear morphology. Furthermore, treatment with 1.25 µM CAPE increased mitochondrial membrane potential in 6-OHDA-exposed cells. CAPE inhibited 6-OHDA-induced caspase activity (~2 fold) and production of reactive oxygen species. In addition, 150 µM 6-OHDA-induced down-regulation of Bcl-2 and Akt levels and up-regulation of Bax and cleaved caspase-9/caspase-9 levels were partially restored by 1.25 µM CAPE treatment. These results revealed a neuroprotective potential of CAPE against 6-OHDA-induced apoptosis in an in vitro PD model and may be a potential therapeutic candidate for the prevention of neurodegeneration in Parkinson's Disease.

Zooming in across the Skin: A Macro-to-Molecular Panorama.

Maintaining integrity of the skin and its appendages still preserves its top-ranking in priorities of survival for the modern human as it probably once did for the ancient individual, -not only- because it is the primary barrier to external assaults, but also because of social and psychological impact of healthy skin during their life-span. Healing wounds in order to shield off the internal organs from infections and damage, restoring its ability to adapt to various environmental stimuli, and slowing-down and reversing aging of the skin in the quest for an everlasting youth can be named as a few of the main drivers behind the multi-million investments dedicated to the advancement of our understanding of skin's physiology. Over the years, these tremendous efforts culminated in the breakthrough discovery of skin stem cells the regenerative capacity of which accounted for the resilience of the skin through their unique capacity as a special cell type that can both self-renew and differentiate into various lineages. In this review, first we summarize the current knowledge on this amazing organ both at a structural and functional level. Next, we provide a comprehensive -in depth- discussion on epidermal as well as dermal stem cells in terms of the key regulatory pathways as well as the main genetic factors that have been implicated in the orchestration of the skin stem cell biology in regards to the shifts between quiescence and entry into distinct differentiation programs.

A Novel Approach to Septal Perforation Repair: Septal Cartilage Cells Induce Chondrogenesis of hASCs In Vitro.

The aim of this study was to investigate the effect of medium harvested from septal cartilage cells on chondrogenic differentiation of adipose stem cells (hASCs) and to compare/contrast its properties to those of a commonly used standard medium formulation in terms of induction and maintenance of chondrogenic hASCs. Differentiation was carried out under three different conditions: septal cartilage medium-SCM, chondrogenic differentiation medium-CM, and 50:50 mixture of CM/SCM. Mesenchymal stem cells (MSCs) markers were determined by flow cytometry. The cytotoxic and apoptotic effects were determined by MTS and Annexin V assay, respectively. The differentiation status of the cells was confirmed by Alcian blue staining, and quantitative real-time flow cytometry showed that hASCs were positive for MSCs, negative for hematopoietic stem cells and endothelial cell surface markers. According to MTS analysis, the first condition was not toxic at any concentration tested. Annexin V assay revealed that the application of different concentrations of SCM did not result in any cell death. The Alcian blue and gene expression analyses showed that the cells in the SCM group underwent the highest cartilage cell formation. The observed increase in chondrogenesis may offer better treatment options for the cartilage defects seen in nasal septum perforation.

A Novel Virtue in Stem Cell Research: Exosomes and Their Role in Differentiation.

In the past decade a number of different stem cell types have entered the clinical applications increasingly as a therapeutic option, due to their tissue maintenance capacity at the site where they localize. Although it was initially thought that conferral of resilience to damaged tissue largely depends on the stem cells themselves through orchestration of signaling among the local epithelial and immune systems at the injury site, recent findings point out that the remarkable regenerative capacity of stem cells is rather due to their nanovesicular products that emerge as the new active players of tissue repair processes. Among these extracellular vesicles exosomes generated particularly by stem cells have been receiving a substantial interest both in the fields of stem cell biology and extracellular vesicles. In this chapter fundamental facts about stem cell biology, biogenesis of extracellular vesicles and exosomes, their structure, and function are summarized. Moreover, properties of both tumor-derived exosomes as well as those derived from stem cells are discussed relatively in-depth in terms of their influence on proximal and distal tissue physiology. Last but not the least, among countless studies in an exploding field, we summarize those that attempt to unravel the complex signaling networks through which stem cell-derived exosomes alter the fate of differentiating stem cells as well as the molecular make-up of exosomes released from differentiating stem cells by conducting thorough proteomic and genomic analyses with the ultimate goal of identifying effector gene products mediating exosomal cues in stem cell biology.

Suppressive Role of Boron on Adipogenic Differentiation and Fat Deposition in Human Mesenchymal Stem Cells.

Over the past years, adipose tissue has become an invaluable source of mesenchymal stem cells (MSCs) due to development of improved isolation methodologies. In a recent work, our group established a primary culture of human adipose-derived stem cells (hADSCs), which were characterized for their stem cell characteristics in detail and studied their myogenic differentiation potential in presence of boron. In the current study, we focused on the effects of a boron-containing compound, sodium pentaborate pentahydrate (NaB), on the adipogenic differentiation of hADSCs. Incorporation of boron in various chemical derivates has been a novel interest in drug-discovery attempts due to increasing number of reports on their anticancer, antibacterial, antiviral, and antifungal activities. In this report, a striking suppressive activity of boron on adipogenic differentiation of hADSCs is observed in a dose-dependent manner. Higher concentrations of NaB (20, 50, and 100 µg/mL (68, 170 and 340 µM)) resulted in a progressive decrease of lipid deposition, suppressed master regulators of adipogenesis transcriptional programming at the mRNA and protein levels, while having no evident cytotoxicity on the cells. The findings of this study are encouraging to undertake further investigations on potential beneficial effects boron in terms of its impact on normal and dysfunctional adipose biology. In that respect, these results pave the path to evaluate boron-based compounds in prevention and treatment of obesity which is a modern age pandemic that is predominant worldwide and found in strong association with comorbidities, including type 2 diabetes, hypertension, cardiovascular disease, cancers, and others."

ABT-737 and erufosine combination against castration-resistant prostate cancer: a promising but cell-type specific response associated with the modulation of anti-apoptotic signaling.

A deeper understanding of the molecular basis of castration-resistant prostate cancer (CRPC) paved the way for the rational design and development of targeted therapies, which yielded promising preclinical results. However, translation of these potentially promising agents into clinics has usually failed, partly because of tumor heterogeneity. In this study, anticancer activities of the Bcl-2 inhibitor ABT-737 and the Akt-inhibitor erufosine (ErPC3) alone and in combination were compared between CRPC (PC-3 and DU-145) and healthy (PNT-1A) cell lines. The combination of ABT-737 and ErPC3 showed synergistic antiproliferative, antimigratory, and apoptotic effects in PC-3 cells. In DU-145 cells, ErPC3 showed a resistant profile, with half-maximal inhibitory concentration (IC50) values more than two-fold of PC-3, and combining ErPC3 with ABT-737 yielded no added benefit for all the incubation periods compared with ErPC3 alone. In PNT-1A cells, ABT-737 and ErPC3 alone and in combination reduced cell survival slightly and only at the highest concentrations. Apoptosis analysis showed that ABT-737 induced increased Akt expression and ErPC3 induced increased Mcl-1 expression in DU-145 cells. In conclusion, the ABT-737 and ErPC3 combination seems to be promising against CRPC, with a favorable safety profile in healthy cells. However, CRPC cell-type-specific resistance may be induced by enhancement of antiapoptotic signaling.

The effects of bisphosphonates on osteonecrosis of jaw bone: a stem cell perspective.

Bisphosphonate-induced osteonecrosis of the jaw (BIONJ) is a commonly encountered side effect of Bisphosphonates (BPs). Although certain aspects of BIONJ have been studied, the effects of BPs on the proliferation, differentiation, and maintenance of dental stem cells (DSC) in way that might account for development of BIONJ have not been evaluated. In the current study, Dental Pulp Stem Cells (DPSCs), Periodontal Stem Cells (PDLSCs), and human Tooth Germ Stem Cells (hTGSCs) were characterized and then each stem cell type were treated with selected BPs: Zoledronate (ZOL), Alendronate (ALE), and Risedronate (RIS). Negative effect on osteogenesis capacity of DSCs has not been observed after differentiation experiments in vitro. BPs exerted inhibitory effect on the migratory capacities of stem cells confirmed by in vitro scratch assay analysis. Angiogenesis of endothelial cells was blocked by BPs treatment in tube formation analysis. In conclusion, inhibitory effects of BPs on migration capacity of DSCs localized in close proximity to the jaw bone might be the primary reason for the side effects of BPs in the development of BIONJ process. Therefore, further in vivo evidence is required to investigate DSC properties in BP treated animals which might elucidate the importance of DSCs in BIONJ formation.