Overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type‐dependent manner

Objectives

Human CAP10‐like protein 46 kDa (hCLP46), also known as Poglut1, has been shown to be an essential regulator of Notch signalling. hCLP46 is overexpressed in primary acute myelogenous leukaemia, T‐acute lymphoblastic leukaemia samples and other leukaemia cell lines. However, effects of hCLP46 overexpression, up to now, have remained unknown.

Materials and methods

In this study, we established stable 293TRex cell lines inducibly overexpressing hCLP46, and knocked down hCLP6 with a specific small interfering RNA to explore function of the protein in Notch signalling and cell proliferation.

Results

hCLP46 overexpression enhanced Notch1 activation in 293Trex cells in a ligand‐dependent manner, with increased Notch signalling enhancing Hes1 expression. We further verified that overexpression of hCLP46 inhibited proliferation of 293TRexs and was correlated with increases in cyclin dependent kinase inhibitors p21 and p27, whereas reduced hCLP46 expression moderately increased cell proliferation. In addition, p21 and p27 protein levels were higher when Notch signalling was activated by EDTA treatment.

Conclusions

Taken together, hCLP46 enhanced Notch activation and inhibited 293TRex cell proliferation through CDKI signalling.

     

Low‐intensity pulsed ultrasound induced enhanced adipogenesis of adipose‐derived stem cells

Objective

The aim of this study was to investigate effects of low‐intensity pulsed ultrasound (LIPUS) on differentiation of adipose‐derived stem cells (ASCs), in vitro.

Materials and methods

Murine ASCs were treated with LIPUS for either three or five days, immediately after adipogenic induction, or delayed for 2 days. Expression of adipogenic genes PPAR‐γ1, and APN, was examined by real‐time PCR. Immunofluorescence (IF) staining was performed to test for PPAR‐γ at the protein level.

Results

Our data revealed that specific patterns of LIPUS up‐regulated levels of both PPAR‐γ1 and APN mRNA, and PPAR‐γ protein.

Conclusions

In culture medium containing adipogenic reagents, LIPUS enhanced ASC adipogenesis.

     

Schisandrin B down‐regulated lncRNA BCYRN1 expression of airway smooth muscle cells by improving miR‐150 expression to inhibit the proliferation and migration of ASMC in asthmatic rats

Objective

The mechanism of Schisandrin B on the proliferation and migration of airway smooth muscle cells (ASMCs) in asthmatic rats was explored.

Methods

SD rats were divided into three groups: control (group 1), model (group 2) and model + Schisandrin B (group 3). miR‐150 and lncRNA BCYRN1 levels were measured by qRT‐PCR. The combination of BCYRN1 and miR‐150 was detected by RNA pull down. ASMCs’ viability/proliferation/migration were examined by WST‐1 assay and 24‐well Transwell system.

Results

Schisandrin B up‐regulated miR‐150 expression and down‐regulated BCYRN1 expression in sensitized rats. Schisandrin B reversed the expression of miR‐150 and BCYRN1 in MV‐treated ASMCs. In addition, Schisandrin B inhibited the viability, proliferation and migration of MV‐induced ASMCs. We also found miR‐150 inhibited BCYRN1 expression which was proved by experiments using ASMCs transfected with miR‐150 inhibitor.

Conclusion

Schisandrin B increased miR‐150 expression and decreased BCYRN1, and BCYRN1 expression was inhibited by miR‐150, which indicated that Schisandrin B could regulate BCYRN1 through miR‐150.

     

Effects of simvastatin and rosuvastatin on RAS protein,matrix metalloproteinases and NF‐κB in lung cancer and in normal pulmonary tissues

Objectives

In this study, we have evaluated effects of 24‐hour treatments with simvastatin or rosuvastatin on RAS protein, NF‐κB and MMP expression in LC tissues obtained from 12 patients undergoing thoracic surgery.

Materials and methods

Normal and lung tumour tissues obtained from each sample were exposed to simvastatin (2.5–30 μm ) or rosuvastatin (1.25–30 μm ) and western blot analysis was then performed.

Results

We documented increased expression of proteins, MMP‐2, MMP‐9 and NF‐κB‐p65 in LC tissues, with respect to normal tissues (P < 0.01). In the malignant tissues, simvastatin and rosuvastatin significantly (P < 0.01) and dose‐dependently reduced RAS protein, MMP‐2/9 and NF‐κB‐p65 expression.

Conclusions

In conclusion, our results suggest that simvastatin and rosuvastatin could play a role in LC treatment by modulation of RAS protein, MMP‐2/9 and NF‐κB‐p65.

     

Mesenchymal‐like stem cells in canine ovary show high differentiation potential

Objectives

Recent studies have reported the existence of stem cells in ovarian tissue that show enhanced proliferative and differentiation potential compared to other adult tissues. Based on this evidence, we hypothesized that ovarian tissue contained mesenchymal‐like stem cells (MSC) that could be isolated using a novel rapid plastic adhesion technique.

Materials and methods

We established MSC lines derived from ovarian and adipose tissue based on their ability to rapidly adhere to plastic culture dishes in the first 3 hours after plating and studied their potentiality in terms of molecular markers and differentiation capacity.

Results

Morphological and kinetic properties of in vitro cultured ovarian MSC were similar to adipose‐derived MSC, and both reached senescence after similar passage numbers. Ovarian‐derived MSC expressed mesenchymal (CD90 and CD44) but not haematopoietic markers (CD34 and CD45), indicating similarity to adipose‐derived MSC. Moreover, ovarian‐derived MSC expressed NANOG, TERT, SOX2, OCT4 and showed extensive capacity to differentiate not only into adipogenic, osteogenic and chondrogenic tissue but also towards neurogenic and endodermal lineages and even precursors of primordial germ cells.

Conclusion

These results show for the first time the derivation of ovarian cells with the molecular properties of MSC as well as wide differentiation potential. Canine ovarian tissue is accessible, expandable, multipotent and has high plasticity, holding promise for applications in regenerative medicine.

     

Critical role of inflammatory mast cell in fibrosis: Potential therapeutic effect of IL‐37

Background

Fibrosis involves the activation of inflammatory cells, leading to a decrease in physiological function of the affected organ or tissue.

Aims

To update and synthesize relevant information concerning fibrosis into a new hypothesis to explain the pathogenesis of fibrosis and propose potential novel therapeutic approaches.

Materials and Methods

Literature was reviewed and relevant information is discussed in the context of the pathogenesis of fibrosis.

Results

A number of cytokines and their mRNA are involved in the circulatory system and in organs of patients with fibrotic tissues. The profibrotic cytokines are generated by several activated immune cells, including fibroblasts and mast cells (MCs), which are important for tissue inflammatory responses to different types of injury. MC‐derived TNF, IL‐1, and IL‐33 contribute crucially to the initiation of a cascade of the host defence mechanism(s), leading to the fibrosis process. Inhibition of TNF and inflammatory cytokines may slow the progression of fibrosis and improve the pathological status of the affected subject. IL‐37 is generated by various types of immune cells and is an IL‐1 family member protein. IL‐37 is not a receptor antagonist; it binds IL‐18 receptor alpha (IL‐18Rα) and delivers the inhibitory signal by using TIR8. It has been shown that IL‐37 can be protective in inflammation and injury, and inhibits both innate and adaptive immunity.

Discussion

IL‐37 may be useful for suppression of inflammatory diseases induced by inhibiting MyD88‐dependent TLR signalling. In addition, IL‐37 downregulates NF‐κB induced by TLR2 or TLR4 through a mechanism dependent on IL‐18Rα.

Conclusion

This review summarizes current knowledge on the role of MC in inflammation and tissue/organ fibrosis, with a focus on the therapeutic potential of IL‐37‐targeting cytokines.

     

Effect of substrate stiffness on proliferation and differentiation of periodontal ligament stem cells

Objectives

The aim of this study was to understand the effect of substrate stiffness (a mechanical factor of the extracellular matrix) on periodontal ligament stem cells (PDLSCs) and its underlying mechanism.

Materials and methods

Elastic substrates were fabricated by mixing 2 components, a base and curing agent in proportions of 10:1, 20:1, 30:1 or 40:1. PDLSC morphology was observed using scanning electron microscopy (SEM). Cell proliferation and differentiation were assessed after PDLSCs was cultured on various elastic substrates. Data were analysed using one‐way ANOVA.

Results

SEM revealed variations in the morphology of PDLSCs cultured on elastic substrates. PDLSC proliferation increased with substrate stiffness (P < .05). Osteogenic differentiation of PDLSCs was higher on stiff substrates. Notch pathway markers were up‐regulated in PDLSCs cultured on stiff substrates.

Conclusions

Results suggested that the osteogenic differentiation of PDLSCs might be promoted by culturing them in a stiffness‐dependent manner, which regulates the Notch pathway. This might provide a new method of enhancing osteogenesis in PDLSCs.

     

Epigenetically induced changes in nuclear textural patterns and gelatinase expression in human fibrosarcoma cells

Objective

Chromatin texture patterns of tumour cell nuclei can serve as cancer biomarkers, either to define diagnostic classifications or to obtain relevant prognostic information, in a large number of human tumours. Epigenetic mechanisms, mainly DNA methylation and histone post‐translational modification, have been shown to influence chromatin packing states, and therefore nuclear texture. The aim of this study was to analyse effects of these two mechanisms on chromatin texture, and also on correlation with gelatinase expression, in human fibrosarcoma tumour cells.

Materials and methods

We investigated effects of DNA hypomethylating agent 5‐aza‐2′‐deoxycytidine (5‐azadC) and histone deacetylase inhibitor trichostatin A (TSA) on nuclear textural characteristics of human HT1080 fibrosarcoma cells, evaluated by image cytometry, and expression of gelatinases MMP‐2 and MMP‐9, two metalloproteinases implicated in cancer progression and metastasis.

Results

5‐azadC induced significant variation in chromatin higher order organization, particularly chromatin decondensation, associated with reduction in global DNA methylation, concomitantly with increase in MMP‐9, and to a lesser extent, MMP‐2 expression. TSA alone did not have any effect on HT1080 cells, but exhibited differential activity when added to cells treated with 5‐azadC. When treated with both drugs, nuclei had higher texture abnormalities. In this setting, reduction in MMP‐9 expression was observed, whereas MMP‐2 expression remained unaffected.

Conclusions

These data show that hypomethylating drug 5‐azadC and histone deacetylase inhibitor TSA were able to induce modulation of higher order chromatin organization and gelatinase expression in human HT1080 fibrosarcoma cells.

     

SNRPA enhances tumour cell growth in gastric cancer through modulating NGF expression

Objectives

SNRPA is a protein component of U1 small nuclear ribonucleoprotein (U1 snRNP) complex, which takes part in the splicing of pre‐mRNAs. Its expression and function in tumour remain unknown. Herein, we elucidated the functional contribution of SNRPA to the progression of gastric cancer (GC).

Materials and methods

SNRPA expression was investigated in a GC tissue microarray by immunohistochemical staining. Cell proliferation was evaluated by CCK‐8, colony formation and EdU incorporation assays. A mouse xenograft model was used to detect the tumourigenicity. Gene expression profiling was performed and then the potential target genes were verified by quantitative real‐time PCR and western blot analyses. The functional relevance between SNRPA and its target gene was examined by cell growth assays.

Results

SNRPA expression was higher in tumour tissues than in matched normal gastric mucosa tissues, and it was positively correlated with the tumour size and progression. High SNRPA expression indicated poor prognosis of GC patients. Silencing SNRPA in GC cells markedly inhibited cell proliferation in vitro and tumour growth in a xenograft model, while overexpressing SNRPA exhibited opposite results. Moreover, we identified NGF (Nerve growth factor) as a downstream effector of SNRPA and further proved that NGF was crucial for SNRPA‐mediated GC cell growth.

Conclusions

These findings suggested that SNRPA may contribute to GC progression via NGF and could be a prognostic biomarker for GC.

     

Downregulation of heat shock protein B8 decreases osteogenic differentiation potential of dental pulp stem cells during in vitro proliferation

Objectives

Tissue‐derived stem cells, such as dental pulp stem cells (DPSCs), reduce differentiation capability during in vitro culture. We found that cultured DPSCs reduce expression of heat shock protein B8 (HspB8) and GIPC PDZ domain containing family member 2 (Gipc2). Our objectives were to evaluate the changes in DPSC composition during in vitro proliferation and to determine whether HspB8 and Gipc2 have function in differentiation potential of DPSCs.

Materials and Methods

Different passages of rat DPSCs were evaluated for changes in CD90+ and/or CD271+ stem cells and changes in osteogenic potential. Real‐time RT‐PCR and immunostaining were conducted to determine expression of HspB8 and Gipc2. Expression of the genes in DPSCs was knocked down by siRNA, followed by osteogenic induction to evaluate the function of the genes.

Results

About 90% of cells in the DPSC cultures were CD90+ and/or CD271+ cells without dramatic change during in vitro proliferation. The DPSCs at passages 3 to 5 (P3 to P5) possess strong osteogenic potential, but such potential was greatly reduced at later passages. Expression of HspB8 and Gipc2 was significantly reduced at P11 versus P3. Knock‐down of HspB8 expression abolished osteogenic potential of the DPSCs, but knock‐down of Gipc2 had no effect.

Conclusions

CD90+ and CD271+ cells are the major components of DPSCs in in vitro culture. High‐level expression of HspB8 was critical for maintaining differentiation potential of DPSCs.

     

2‐(2‐nitrobenzylidene) indolin‐3‐one compound inhibits transmembrane prostate androgen‐induced protein (TMEPAI) expression and cancer cell proliferation

Objectives

The transmembrane prostate androgen‐induced protein (TMEPAI) is aberrantly expressed in many cancer and plays a crucial role in tumourigenesis, which makes it a potential cancer therapeutic target for drug discovery.

Materials and methods

Here, we employed a firefly luciferase reporter driven by the TMEPAI gene promoter to screen for compound capable of inhibiting the expression of TMEPAI, and the effects of TMEPAI inhibitor on cancer cell proliferation were evaluated using the colony formation assay, cell cycle analysis, Ki‐67 immunofluorescence assay and EdU incorporation assay.

Results

2‐(2‐nitrobenzylidene) indolin‐3‐one (JHY‐A007‐50) was identified and shown to effectively inhibit the TMEPAI promoter activity. Further studies revealed that JHY‐A007‐50 specifically inhibited the expression of TMEPAI at both the mRNA and protein levels. Moreover, we found that JHY‐A007‐50 could inhibit cell proliferation and induce cell cycle arrest at the G1 phase. Our results showed that overexpression of TMEPAI decreased the inhibitory effects of JHY‐A007‐50 on cancer cell proliferation, and JHY‐A007‐50 did not affect the cell viability of HeLa cells knocked down of TMEPAI.

Conclusions

Taken together, these results suggest that compound JHY‐A007‐50 mediates the downregulation of TMEPAI expression and inhibits cell proliferation in cancer cells.

     

Sustained release of stem cell factor in a double network hydrogel for ex vivo culture of cord blood‐derived CD34+ cells

Objectives

Stem cell factor (SCF) is considered as a commonly indispensable cytokine for proliferation of haematopoietic stem cells (HSCs), which is used in large dosages during ex vivo culture. The work presented here aimed to reduce the consumption of SCF by sustained release but still support cells proliferation and maintain the multipotency of HSCs.

Materials and methods

Stem cell factor was physically encapsulated within a hyaluronic acid/gelatin double network (HGDN) hydrogel to achieve a slow release rate. CD34⁺ cells were cultured within the SCF‐loaded HGDN hydrogel for 14 days. The cell number, phenotype and functional capacity were investigated after culture.

Results

The HGDN hydrogels had desirable properties and encapsulated SCF kept being released for more than 6 days. SCF remained the native bioactivity, and the proliferation of HSCs within the SCF‐loaded HGDN hydrogel was not affected, although the consumption of SCF was only a quarter in comparison with the conventional culture. Moreover, CD34⁺ cells harvested from the SCF‐loaded HGDN hydrogels generated more multipotent colony‐forming units (CFU‐GEMM).

Conclusion

The data suggested that the SCF‐loaded HGDN hydrogel could support ex vivo culture of HSCs, thus providing a cost‐effective culture protocol for HSCs.

     

Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration

Objectives

Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration.

Materials and methods

Assays were performed in HuH‐7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen‐induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures.

Results

In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle‐related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen‐induced restrictions to restore cycling.

Conclusions

Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure.

     

The role of Sprouty1 in the proliferation,differentiation and apoptosis of epidermal keratinocytes

Objectives

Sprouty (SPRY) 1 is one of the SPRY proteins that inhibits signalling from various growth factors pathways and has also been known as a tumour suppressor in various malignancies. However, no study elucidates the role of SPRY1 in the skin. Our study was conducted to determine the function of SPRY1 in human keratinocytes and the epidermis.

Materials and methods

In vitro primary cultured epidermal keratinocytes were used to investigate the proliferation, differentiation and apoptosis of these cells. We also established overexpression of SPRY1 in vitro and K14‐SPRY1 transgenic mice.

Results

SPRY1 was mainly located in the cytoplasm of the epidermal keratinocytes from the granular epidermal layer of the skin and cultured cells. Overexpressed SPRY1 in keratinocytes resulted in up‐regulation of P21, P27 and down‐regulation of cyclin B1; decrease in MMP3 and integrin α6. SPRY1‐overexpressed primary keratinocytes exhibited a lower proliferation and migration capability and higher rates of apoptosis. Epidermis of SPRY1‐TG mice represented delayed wound healing. Proteomics analysis and GO enrichment showed DEPs of SPRY1 TG mice epidermis is significantly enriched in immune‐ and inflammatory‐associated biological process.

Conclusions

In summary, SPRY1 expression was inversely correlated with cell proliferation, migration and promote cell apoptosis of keratinocytes. SPRY1 maybe a negative feedback regulator in normal human epidermal keratinocytes and cutaneous inflammatory responses. Our study raised the possibility that enhancing expression of SPRY1 may have the potential to promote anti‐inflammatory effects.

     

Low level laser therapy induces increased viability and proliferation in isolated cancer cells

Objectives

Low level laser therapy (LLLT), which stimulates natural biological processes in the application region, is frequently used in dental treatments. The aim of our study was to evaluate the effects of LLLT which could activate precancerous cells or increase existing cancerous tissue in case of clinically undetectable situations.

Materials and methods

Saos‐2 osteoblast‐like osteosarcoma cells and A549 human lung carcinoma cells were used. Twenty‐four hours after preparation of cell culture plates, laser irradiation was performed 1, 2 and 3 times according to the test groups using Nd:YAG laser with the power output 0.5, 1, 2 and 3 W. Cell proliferation analysis was performed by MTT assay at the 24th hour following the last laser applications.

Results

Generally, it was observed that the proliferation rates increased as the number of applications increased, when compared to the controls, especially in those cases in which the irradiation was performed 2 or 3 times more.

Conclusion

The findings of this study have led to the conclusion that LLLT increases cancer cell proliferation, depending on the power output level of the laser and the number of applications. In addition to the proliferation and mitotic activity of the cancer tissue cells, we concluded that LLLT, which is frequently used in dental practice, could activate precancerous cells or increase existing cancerous tissue.