Deregulation of Bmi-1 is associated with enhanced migration,invasion and poor prognosis in salivary adenoid cystic carcinoma

Background

Bmi-1 had been found to involve in self renewal of stem cells and tumorigenesis in various malignancies. In this study, we investigated the role of Bmi-1 in the development of salivary adenoid cystic carcinoma (SACC).

Methods

At first, we confirmed that the deregulation of Bmi-1 was a frequent event in SACC; up-regulation of Bmi-1 was correlated with clinical stages, vital status and distant metastasis and associated with reduced overall survival and disease free survival. SACC-LM cells, higher migration and invasion abilities, elevated the expression of Bmi-1 protein, epithelial-mesenchymal transition (EMT) related proteins (Snail, Slug and Vimentin) and cancer stem cells (CSCs) related proteins (ABCG₂, Notch, ALDH-1, Oct-4, Nanog and Epcam) compared to the SACC-83 cells (lower migration and invasion abilities). The migration and invasion abilities were inhibited in SACC-LM cells upon Bmi-1 knockdown. Meanwhile, Bmi-1 knockdown resulted in simultaneous loss of stem cell markers and EMT markers in SACC-LM cells.

Conclusion

Our studies confirm that Bmi-1 deregulation plays an important role in the development of SACC and contributes to the migration and the invasion abilities of SACC, which is involved in EMT and CSCs.

General significance

To our knowledge, this is the first study revealing that Bmi-1 deregulation is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma.     

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells

Background

Methyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration.

Methods

MG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence.

Results

Treatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser⁸³ and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca^2 + and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor.

General significance

Our results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.     

Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Background

Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective

We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods

Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results

At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion

Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.     

Dicer knockdown induces fibronectin-1 expression in HEK293T cells via induction of Egr1

Background

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs); depletion of Dicer was found to impair the migration of endothelial cells.

Methods

siRNA transfection, cell migration assay, real-time RT–PCR, chromatin immunoprecipitation, Western blotting, ELISA, caspase-3 activity assay, and annexin-V–FITC assay were utilized.

Results

Knockdown of Dicer impairs the migratory capacity of HEK293T cells and induces fibronectin-1. The upregulation of fibronectin-1 is dependent on Egr1. Fibronectin-1/Dicer double-knockdown cells showed a marked increase in apoptosis compared with fibronectin-1 single knockdown cells.

Conclusions

Decreased Dicer expression induces fibronectin-1 expression via an Egr1-dependent manner.

General significance

Our data suggest that upregulation of fibronectin-1 protects Dicer knockdown HEK293T cells against apoptosis.     

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

Background

Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.

Results

By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.

Conclusion

The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

Cardiac stem cell therapy to modulate inflammation upon myocardial infarction

Background

After myocardial infarction (MI) a local inflammatory reaction clears the damaged myocardium from dead cells and matrix debris at the onset of scar formation. The intensity and duration of this inflammatory reaction are intimately linked to post-infarct remodeling and cardiac dysfunction. Strikingly, treatment with standard anti-inflammatory drugs worsens clinical outcome, suggesting a dual role of inflammation in the cardiac response to injury. Cardiac stem cell therapy with different stem or progenitor cells, e.g. mesenchymal stem cells (MSC), was recently found to have beneficial effects, mostly related to paracrine actions. One of the suggested paracrine effects of cell therapy is modulation of the immune system.

Scope of review

MSC are reported to interact with several cells of the immune system and could therefore be an excellent means to reduce detrimental inflammatory reactions and promote the switch to the healing phase upon cardiac injury. This review focuses on the potential use of MSC therapy for post-MI inflammation. To understand the effects MSC might have on the post-MI heart the cellular and molecular changes in the myocardium after MI need to be understood.

Major conclusions

By studying the general pathways involved in immunomodulation, and examining the interactions with cell types important for post-MI inflammation, it becomes clear that MSC treatment might provide a new therapeutic opportunity to improve cardiac outcome after acute injury.

General significance

Using stem cells to target the post-MI inflammation is a novel therapy which could have considerable clinical implications. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Extracellular matrix: A dynamic microenvironment for stem cell niche

Background

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche.

Scope of review

We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions.

Major conclusions

ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior.

General significance

ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Matrix regulators in neural stem cell functions

Background

Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism.

Scope of review

In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior.

Major conclusions

The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors.

General significance

The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

CCL2 increases αvβ3 integrin expression and subsequently promotes prostate cancer migration

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family which is associated with the disease status and outcomes of cancers. Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. However, the effect of CCL2 on human prostate cancer cells is largely unknown. The aim of this study was to examine the role of CCL2 in integrin expression and migratory activity in prostate cancers.

Methods

Prostate cancer migration was examined using Transwell, wound healing, and invasion assay. The PKCδ and c-Src phosphorylations were examined by using western blotting. The qPCR was used to examine the mRNA expression of integrins. A transient transfection protocol was used to examine AP-1 activity.

Results

Stimulation of prostate cancer cell lines (PC3, DU145, and LNCaP) induced migration and expression of integrin αvβ3. Treatment of cells with αvβ3 antibody or siRNA abolished CCL2-increased cell migration. CCL2-increased migration and integrin expression were diminished by CCR2 but not by CCR4 inhibitors, suggesting that the CCR2 receptor is involved in CCL2-promoted prostate cancer migration. CCL2 activated a signal transduction pathway that includes PKCδ, c-Src, and AP-1. Reagents that inhibit specific components of this pathway each diminished the ability of CCL2 to effect cell migration and integrin expression.

Conclusions

Interaction between CCL2 and CCR2 enhances migration of prostate cancer cells through an increase in αvβ3 integrin production.

General significance

CCL2 is a critical factor of prostate cancer metastasis.     

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

Studying the enhancement of programmed cell death by combined AG1024 and paclitaxel in a model of chronic myelogenous leukemia

Aims

Chronic myelogenous leukemia is a clonal malignancy of the pluripotent hematopoietic stem cells that is characterized by the uncontrolled proliferation and expansion of myeloid progenitors. Myeloid progenitors express the fusion oncogene BCR–ABL, which has uncontrollable activity in malignant cells and prevents the cell apoptosis caused by some antineoplastic agents, such as paclitaxel. Targeting these abnormalities by blocking the tyrosine kinase enzymes of BCR–ABL is a promising approach for chronic myelogenous leukemia therapy.

Main methods

Conventional Liu's staining is an auxiliary technique used in microscopy to enhance the contrast in microscopic images, aiding the observation of cell morphology. The MTT assay, flow cytometry of the sub-G1 analysis and the TUNEL assay were applied to estimate the apoptosis levels. RT-PCR and western blot methods were used to evaluate the key molecules conferring anti-cell-death properties.

Key findings

The effects of the tyrosine kinase inhibitor AG1024 were evaluated with regard to the regulation of BCR–ABL expression, inhibition of cell proliferation, and enhanced paclitaxel-induced apoptosis in BCR–ABL-expressing K562 cell lines. AG1024 downregulated the expression of BCR–ABL and anti-apoptosis factors, such as Bcl-2 and Bcl-xL, which were present in K562 cells. Moreover, the combination of AG1024 with paclitaxel inhibited cell proliferation and enhanced paclitaxel-induced apoptosis within 24 h.

Significance

In summary, the present study shows that the combination of AG1024 with paclitaxel inhibited model cancer cell proliferation, suggesting a new use of paclitaxel-based chemotherapy for cancer control.     

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

Background

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.

Methods

EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.

Results

The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm^− 1, 1083 cm^− 1, 937 cm^− 1, 858 cm^− 1, 577 cm^− 1 and 482 cm^− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.

Conclusions

The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.

General significance

As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.     

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

Background and aims

The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.

Methods

Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.

Results

We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.

Conclusions

The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.     

Major signaling pathways in intestinal stem cells

Background

The discovery of markers to identify the intestinal stem cell population and the generation of powerful transgenic mouse models to study stem cell physiology have led to seminal discoveries in stem cell biology.

Scope of review

In this review we give an overview of the current knowledge in the field of intestinal stem cells (ISCs) highlighting the most recent progress on markers defining the ISC population and pathways governing intestinal stem cell maintenance and differentiation. Furthermore we review their interaction with other stem cell related pathways. Finally we give an overview of alteration of these pathways in human inflammatory gastrointestinal diseases.

Major conclusions

We highlight the complex network of interactions occurring among different pathways and put in perspective the many layers of regulation that occur in maintaining the intestinal homeostasis.

General significance

Understanding the involvement of ISCs in inflammatory diseases can potentially lead to new therapeutic approaches to treat inflammatory GI pathologies such as IBD and celiac disease and could reveal the molecular mechanisms leading to the pathogenesis of dysplasia and cancer in inflammatory chronic conditions. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Biochemistry and biology: Heart-to-heart to investigate cardiac progenitor cells

Background

Cardiac regenerative medicine is a rapidly evolving field, with promising future developments for effective personalized treatments. Several stem/progenitor cells are candidates for cardiac cell therapy, and emerging evidence suggests how multiple metabolic and biochemical pathways strictly regulate their fate and renewal.

Scope of review

In this review, we will explore a selection of areas of common interest for biology and biochemistry concerning stem/progenitor cells, and in particular cardiac progenitor cells. Numerous regulatory mechanisms have been identified that link stem cell signaling and functions to the modulation of metabolic pathways, and vice versa. Pharmacological treatments and culture requirements may be exploited to modulate stem cell pluripotency and self-renewal, possibly boosting their regenerative potential for cell therapy.

Major conclusions

Mitochondria and their many related metabolites and messengers, such as oxygen, ROS, calcium and glucose, have a crucial role in regulating stem cell fate and the balance of their functions, together with many metabolic enzymes. Furthermore, protein biochemistry and proteomics can provide precious clues on the definition of different progenitor cell populations, their physiology and their autocrine/paracrine regulatory/signaling networks.

General significance

Interdisciplinary approaches between biology and biochemistry can provide productive insights on stem/progenitor cells, allowing the development of novel strategies and protocols for effective cardiac cell therapy clinical translation. This article is part of a Special Issue entitled Biochemistry of Stem Cells.