Fluoxetine induces autophagic cell death via eEF2K‐AMPK‐mTOR‐ULK complex axis in triple negative breast cancer

Objectives

Triple negative breast cancer (TNBC) is a complex and intrinsically aggressive tumour with poor prognosis, and the discovery of targeted small‐molecule drugs for TNBC treatment still remains in its infancy. In this study, we aimed to discover a small‐molecule agent for TNBC treatment and illuminate its potential mechanisms.

Materials and methods

Cell viability was detected by using methylthiazoltetrazolium (MTT) assay. Electron microscopy, GFP‐LC3 transfection, monodansylcadaverine staining and apoptosis assay were performed to determine Fluoxetine‐induced autophagy and apoptosis. Western blotting and siRNA transfection were carried out to investigate the mechanisms of Fluoxetine‐induced autophagy. iTRAQ‐based proteomics analysis was used to explore the underlying mechanisms.

Results

We have demonstrated that Fluoxetine had remarkable anti‐proliferative activities and induced autophagic cell death in MDA‐MB‐231 and MDA‐MB‐436 cells. The mechanism for Fluoxetine‐induced autophagic cell death was associated with inhibition of eEF2K and activation of AMPK‐mTOR‐ULK complex axis. Further iTRAQ‐based proteomics and network analyses revealed that Fluoxetine‐induced mechanism was involved in BIRC6, BNIP1, SNAP29 and Bif‐1.

Conclusions

These results demonstrate that Fluoxetine induces apoptosis and autophagic cell death in TNBC, which will hold a promise for the future TNBC therapy.

     

Identification of novel caspase/autophagy‐related gene switch to cell fate decisions in breast cancers

Objectives

Caspases, a family of cysteine proteases with unique substrate specificities, contribute to apoptosis, whereas autophagy‐related genes (ATGs) regulate cytoprotective autophagy or autophagic cell death in cancer. Accumulating evidence has recently revealed underlying mechanisms of apoptosis and autophagy; however, their intricate relationships still remain to be clarified. Identification of caspase/ATG switches between apoptosis and autophagy may address this problem.

Materials and methods

Identification of caspase/ATG switches was carried out using a series of elegant systems biology & bioinformatics approaches, such as network construction, hub protein identification, microarray analyses, targeted microRNA prediction and molecular docking.

Results

We computationally constructed the global human network from several online databases and further modified it into the basic caspase/ATG network. On the basis of apoptotic or autophagic gene differential expressions, we identified three molecular switches [including androgen receptor, serine/threonine‐protein kinase PAK‐1 (PAK‐1) and mitogen‐activated protein kinase‐3 (MAPK‐3)] between certain caspases and ATGs in human breast carcinoma MCF‐7 cells. Subsequently, we identified microRNAs (miRNAs) able to target androgen receptor, PAK‐1 and MAPK‐3, respectively. Ultimately, we screened a range of small molecule compounds from DrugBank, able to target the three above‐mentioned molecular switches in breast cancer cells.

Conclusions

We have systematically identified novel caspase/ATG switches involved in miRNA regulation, and predicted targeted anti‐cancer drugs. These findings may uncover intricate relationships between apoptosis and autophagy and thus provide further new clues towards possible cancer drug discovery.

     

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells

Objectives

Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti‐cancer agent. However, the molecular mechanisms involved remain poorly understood.

Materials and methods

Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA‐β‐gal assay, western blotting and immunofluorescence were performed to determine CGN‐induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN‐induced senescence and autophagy. The anti‐tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models.

Results

We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell‐cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN‐mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti‐tumour effects in vivo.

Conclusions

Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN‐mediated anti‐cancer therapy.

     

Galantamine inhibits β‐amyloid‐induced cytostatic autophagy in PC12 cells through decreasing ROS production

Objectives

Alzheimer's disease (AD) is one of the most prevalent brain diseases among the elderly, majority of which is caused by abnormal deposition of amyloid beta‐peptide (Aβ). Galantamine, currently the first‐line drug in treatment of AD, has been shown to diminish Aβ‐induced neurotoxicity and exert favourable neuroprotective effects, but the detail mechanisms remain unclear.

Materials and methods

Effects of galantamine on Aβ‐induced cytotoxicity were checked by MTT, clone formation and apoptosis assays. The protein variations and reactive oxygen species (ROS) production were measured by western blotting analysis and dichloro‐dihydro‐fluorescein diacetate assay, respectively.

Results

Galantamine reversed Aβ‐induced cell growth inhibition and apoptosis in neuron cells PC12. Aβ activated the entire autophagy flux and accumulation of autophagosomes, and the inhibition of autophagy decreased the protein level of cleaved‐caspase‐3 and Aβ‐induced cytotoxicity. Meanwhile, galantamine suppressed Aβ‐mediated autophagy flux and accumulation of autophagosomes. Moreover, Aβ upregulated ROS accumulation, while ROS scavengers N‐acetyl‐l ‐cysteine impaired Aβ‐mediated autophagy. Further investigation showed that galantamine downregulated NOX4 expression to inhibit Aβ‐mediated ROS accumulation and autophagy.

Conclusions

Galantamine inhibits Aβ‐induced cytostatic autophagy through decreasing ROS accumulation, providing new insights into deep understanding of AD progression and molecular basis of galantamine in neuroprotection.

     

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.

     

miR‐34c works downstream of p53 leading to dairy goat male germline stem‐cell (mGSCs) apoptosis

Objectives

Recent lines of evidence have indicated that miR‐34c can play important roles in regulation of the cell cycle, cell senescence and apoptosis of mouse and human tumour cells, spermatogenesis, and male germ‐cell apoptosis. However, there is little information on the effects of miR‐34c on proliferation and apoptosis of livestock male germ cells. The dairy goat is a convenient domestic species for biological investigation and application. The purpose of this study was to investigate the effects of miR‐34c on apoptosis and proliferation of dairy goat male germline stem cells (mGSCs), as well as to determine the relationship between p53 and miR‐34c in this species.

Materials and methods

Morphological observation, miRNA in situ hybridisation (ISH), bromodeoxyuridine staining, flow cytometry, quantitative‐RT‐PCR (Q‐RT‐PCR) and western blotting were utilized to ascertain apoptosis and proliferation of mGSCs, through transfection of miR‐34c mimics (miR‐34c), miR‐34c inhibitor (anti‐miR‐34c), miR‐34c mimics and inhibitors co‐transfected (mixture) compared to control groups.

Results

Results manifested that miR‐34c over‐expression promoted mGSCs apoptosis and suppressed their proliferation. Simultaneously, a variety of apoptosis‐related gene expression was increased while some proliferation‐related genes were downregulated. Accordingly, miR‐34c promoted apoptosis in mGSCs and reduced their proliferation; moreover, expression of miR‐34c was p53‐dependent.

Conclusions

This study is the first to provide a model for study of miRNAs and mechanisms of proliferation and apoptosis in male dairy goat germ cells.

     

TrkB expression level correlates with metastatic properties of L1 mouse sarcoma cells cultured in non‐adhesive conditions

Objectives

Ability of a cell to survive without adhesion, and to overcome anoikis, is indispensable for malignant cell invasion and metastasis formation. It has previously been shown that TrkB ‐neutrophin growth factor receptor might be involved in suppression of apoptosis, induced by the lack of adhesion. The aim of our study was to analyse changes in expression of genes and proteins as well as in biological properties of cancer cells cultured without adhesion. A mouse sarcoma, stable, adherent L1 cell line, derived from a spontaneously arisen Balb/c mouse lung tumour, was established in vitro.

Materials and methods

L1 cells resistant to anoikis were established by culture of L1 cells without adhesion, followed by selection of clones with elevated expression levels of TrkB protein. Biological characteristics of the cells were studied by migration/invasion tests and colony forming assay. Gene expression analysis was performed by with the aid of cDNA Gene Expression Array and Real‐Time PCR. In vivo experiments were conducted in syngeneic Balb/c mice.

Results

Significant changes in gene expression, including higher expression level of TrkB, were found in cells that were able to survive without adhesion. Selected TrkB‐expressing clones were found to have higher clonogenicity and invasive potential, formed more colonies in mouse lungs, and induced larger tumours, when injected subcutaneously into Balb/c mice.

Conclusion

Lack of adhesion induced significant changes in the cancer cells’ behaviour, which may result from alterations in gene and protein expression levels, including changes in anoikis‐connected protein – TrkB.

     

Protective effect of GDNF‐engineered amniotic fluid‐derived stem cells on the renal ischaemia reperfusion injury in vitro

Objectives

Amniotic fluid‐derived stem cells (AFSCs) possessing multilineage differentiation potential are proposed as a novel and accessible source for cell‐based therapy and tissue regeneration. Glial‐derived neurotrophic factor (GDNF) has been hypothesized to promote the therapeutic effect of AFSCs on markedly ameliorating renal dysfunction. The aim of this study was to investigate whether AFSCs equipped with GDNF (GDNF‐AFSCs) had capabilities of attenuating mouse renal tubular epithelial cells (mRTECs) apoptosis and evaluate its potential mechanisms.

Materials and methods

A hypoxia‐reoxygenation (H/R) model of the mRTECs was established. Injured mRTECs were co‐cultured with GDNF‐AFSCs in a transwell system. The mRNA expressions of hepatocytes growth factor (HGF) and fibroblast growth factor (bFGF) were detected by qRT‐PCR. Changes of intracelluar reactive oxygen species (ROS) and the level of superoxide dismutase (SOD) and malondialdehyde (MDA) were examined. The expressions of nitrotyrosine, Gp91‐phox, Bax, and Bcl‐2 were determined by Western blotting. Cell apoptosis was assayed by flow cytometry, and caspase‐3 activity was monitored by caspase‐3 activity assay kit.

Results

Our study revealed that expression of growth factors was increased and oxidative stress was dramatically counteracted in GDNF‐AFSCs‐treated group. Furthermore, apoptosis induced by H/R injury was inhibited in mRTECs by GDNF‐AFSCs.

Conclusions

These data indicated that GDNF‐AFSCs are beneficial to repairing damaged mRTECs significantly in vitro, which suggests GDNF‐AFSCs provide new hopes of innovative interventions in different kidney disease.

     

Roflumilast enhances cisplatin‐sensitivity and reverses cisplatin‐resistance of ovarian cancer cells via cAMP/PKA/CREB‐FtMt signalling axis

Objective

We previously demonstrated the roflumilast inhibited cell proliferation and increased cell apoptosis in ovarian cancer. In this study, we aimed to investigate the roles of roflumilast in development of cisplatin (DDP)‐sensitive and ‐resistant ovarian cancer.

Methods

OVCAR3 and SKOV3 were selected and the corresponding DDP‐resistant cells were constructed. Cell viability, proliferation, apoptosis, cycle were performed. Expression cAMP, PKA, CREB, phosphorylation of CREB and FtMt were detected. The roles of roflumilast in development of DDP‐sensitive and ‐resistant ovarian cancer were confirmed by xenograft model.

Results

Roflumilast + DDP inhibited cell proliferation, and induced cell apoptosis and G0/G1 arrest in OVCAR3 and SKOV3 cells, roflumilast induced expression of FtMt, the activity of cAMP and PKA and phosphorylation of CREB in ovarian cancer cells and the above‐effect were inhibited by H89. Downregulation of CREB inhibited the roflumilast‐increased DDP sensitivity of ovarian cancer cells, and the roflumilast‐induced FtMt expression and phosphorylation of CREB. Also, roflumilast reversed cisplatin‐resistance, and induced expression of FtMt and activation of cAMP/PKA/CREB in DDP‐resistant ovarian cancer cells. Similarly, treated with H89 or downregulation of CREB inhibited the changes induced by roflumilast. In vivo, roflumilast inhibited the development of SKOV3 or SKOV3‐DDP‐R xenograft models.

Conclusions

Roflumilast enhanced DDP sensitivity and reversed the DDP resistance of ovarian cancer cells via activation of cAMP/PKA/CREB pathway and upregulation of the downstream FtMt expression, which has great promise in clinical treatment.

     

Curcumin enhances the anti‐cancer effects of Paris Saponin II in lung cancer cells

Objectives

To investigate the synergistic mechanisms of Paris Saponin II (PSII) and Curcumin (CUR) in lung cancer.

Materials and Methods

The combination changed the cellular uptake of CUR and PSII, apoptosis, cell cycle arrest and cytokine levels were analysed on different lung cancer cells.

Results

The combination displayed a synergistic anti‐cancer effect through promoting the cellular uptake of CUR on different lung cancer cells. Hoechst H33258 staining and FACS assay indicated that the combination of PSII and CUR induced cell cycle arrest and apoptosis. Western blot and cytokine antibody microarray suggested that the combination activated death receptors such as DR6, CD40/CD40L, FasL and TNF‐α to induce cancer cells apoptosis, and up‐regulated IGFBP‐1 leading to inhibition of PI3K/Akt pathway and increase of p21 and p27, which therefore induced a G2 phase arrest in NCI‐H446 cells. Meanwhile, the combination suppressed PCNA and NF‐κB pathway in 4 kinds of lung cancer cells. They activated the phosphorylation of p38 and JNK, and inhibited PI3K in NCI‐H460 and NCI‐H446 cells, enhanced the phosphorylation of JNK in NCI‐H1299 cells, and increased the phosphorylation of p38 and ERK, and suppressed PI3K in NCI‐H520 cells.

Conclusions

PSII combined with CUR had a synergistic anti‐cancer effect on lung cancer cells. These findings provided a rationale for using the combination of curcumin and PSII in the treatment of lung cancer in future.

     

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.

     

PMA withdrawal in PMA‐treated monocytic THP‐1 cells and subsequent retinoic acid stimulation,modulate induction of apoptosis and appearance of dendritic cells

Objectives

To analyse proliferation, differentiation and apoptosis in THP‐1 cells after stimulation with phorbol 12‐myristate 13‐acetate (PMA) and retinoic acid (RA).

Materials and methods

PMA and RA were used in a three‐step‐procedure: (i) treatment with 6, 30, 60 nm PMA, that induced initial, intermediate and advanced levels of monocyte‐macrophage transition, respectively; (ii) recovery in PMA‐free medium; (iii) incubation with 4 μm RA. Cultures were characterized cytokinetically (flow cytometry/bromodeoxyuridine uptake) and immunocytochemically (static cytometry) for expression of CD14, CD11b (monocyte‐macrophage) and DC‐SIGN (dendritic cell: DCs) markers.

Results

Some treatments determined appearance of monocyte/macrophage, dendritic and apoptotic phenotypes, percentages of which were related to PMA dose used in step 1, and dependent on presence/absence of PMA and RA. PMA withdrawal induced dedifferentiation and partial restoration of proliferative activity, specially in 6 and 30 nm PMA‐derived cells. Recovery in the presence of serum (fundamental to DC appearance) indicated that depending on differentiation level, cell proliferation and apoptosis were inversely correlated. Treatment with 30 nm PMA induced intermediate levels of monocytic‐macrophagic differentiation, with expression of alternative means of differentiation and acquisition of DCs without using cytokines, after PMA withdrawal and RA stimulation.

Conclusions

Our experimental conditions favoured differentiation, dedifferentiation and transdifferentiational pathways, in monocytic THP‐1 cells, the balance of which could be related to both cell proliferation and cell death.

     

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.

     

Wnt/β‐catenin signalling pathway mediates high glucose induced cell injury through activation of TRPC6 in podocytes

Objectives

Diabetic nephropathy is a major complication of diabetes and a frequent cause of end‐stage renal disease and recent studies suggest that podocyte damage may play a role in the pathogenesis of this. At early onset of diabetic nephropathy there is podocyte drop‐out, which is thought to provoke glomerular albuminuria and subsequent glomerular injury; however, the underlying molecular mechanisms of this remain poorly understood. Here we report that we tested the hypothesis that early diabetic podocyte injury is caused, at least in part, by up‐regulation of transient receptor potential cation channel 6 (TRPC6), which is regulated by the canonical Wnt signalling pathway, in mouse podocytes.

Materials and methods

Mechanism of injury initiation in mouse podocytes, by high concentration of D‐glucose (HG, 30 mM), was investigated by MTT, flow cytometry, real‐time quantitative PCR, and western blot analysis.

Results

HG induced apoptosis and reduced viability of differentiated podocytes. It caused time‐dependent up‐regulation of TRPC6 and activation of the canonical Wnt signalling pathway, in mouse podocytes. In these cells, blockade of the Wnt signalling pathway by dickkopf related protein 1 (Dkk1) resulted in effective reduction of TRPC6 up‐regulation and amelioration of podocyte apoptosis. Furthermore, reduction of cell viability induced by HG was attenuated by treatment with Dkk1.

Conclusion

These findings indicate that the Wnt/β‐catenin signalling pathway may potentially be active in pathogenesis of TRPC6‐mediated diabetic podocyte injury.

     

SATB2 targeted by methylated miR‐34c‐5p suppresses proliferation and metastasis attenuating the epithelial‐mesenchymal transition in colorectal cancer

Objectives

SATB2 has been shown to be markedly reduced in colorectal cancer (CRC) tissues relative to paired normal controls; however, the mechanism behind remains not well understood. To investigate why SATB2 was down‐regulated in CRC, we attempted to analyse it from the angle of miRNA‐mRNA modulation.

Materials and methods

SATB2 expression was detected in CRC tissues using immunohistochemistry and verified using real‐time PCR on mRNA level, followed by analysis of clinicopathological significance of its expression. Metastatic variation of CRC cells was evaluated both in vivo and in vitro. To find out the potential miRNA that directly regulate the SATB2, luciferase reporter assay was performed following the bioinformatic prediction.

Results

SATB2 was confirmed to be closely linked with the metastasis and shorter overall survival of CRC in our own cases. Silencing of SATB2 was shown to be able to promote the metastatic ability of CRC cells in vivo, enhancing the epithelial‐mesenchymal transition (EMT). Mechanistically, miR‐34c‐5p was identified to be a novel miRNA that can directly modulate the SATB2. It turned out that the promoter of miR‐34c‐5p was methylated, which leads to the repression of miR‐34c‐5p in CRC. Treatment with 5‐Aza‐dC can reasonably and significantly restore the level of miR‐34c‐5p in CRC cells relative to control, thereby down‐regulating the SATB2.

Conclusions

Together, our study revealed that SATB2 targeted by methylated miR‐34c‐5p can suppress the metastasis, weakening the EMT in CRC.

     

In vitro cytotoxicity of Gymnema montanum in human leukaemia HL‐60 cells; induction of apoptosis by mitochondrial membrane potential collapse

Objectives

Gymnema montanum Hook, an Indian Ayurvedic medicinal plant, is used traditionally to treat a variety of ailments. Here, we report anti‐cancer effects and molecular mechanisms of ethanolic extract of G. montanum (GLEt) on human leukaemia HL‐60 cells, compared to peripheral blood mononuclear cells.

Materials and methods

HL‐60 cells were treated with different concentrations of GLEt (10–50 μg/ml) and cytotoxicity was assessed by MTT assay. Levels of lipid peroxidation, antioxidants, mitochondrial membrane potential and caspase‐3 were measured. Further, apoptosis was studied using annexin‐V staining and the cell cycle was analyzed by flow cytometry.

Results

GLEt had a potent cytotoxic effect on HL‐60 cells (IC₅₀‐20 μg/ml), yet was not toxic to normal peripheral blood mononuclear cells. Exposure of HL‐60 cells to GLEt led to elevated levels of malonaldehyde formation, but to reduced glutathione, superoxide dismutase, catalase and glutathione peroxidase activities (P < 0.05). Induction of apoptosis was confirmed by observing annexin‐V positive cells, associated with loss of mitochondrial membrane potential. Cell cycle arrest at G0/G1 was observed in GLEt‐treated HL‐60 cells, indicating its potential at inducing their apoptosis.

Conclusions

Findings of the present study suggest that G. montanum induced apoptosis in the human leukaemic cancer cells, mediated by collapse of mitochondrial membrane potential, generation of reactive oxygen species and depletion of intracellular antioxidant potential.

     

Ginsenoside Rh2 inhibits prostate cancer cell growth through suppression of microRNA‐4295 that activates CDKN1A

Objectives

Ginsenoside Rh2 (GRh2) has demonstrative therapeutic effects on a variety of diseases, including some tumours. However, the effects of GRh2 on prostate cancer (PC) cell growth remain unknown, and were, thus, addressed in the present study.

Materials and methods

PC3 and DU145 PC cell lines were exposed to GRh2. Cell proliferation was assessed in an MTT assay and by BrdU incorporation. Apoptosis of the cells were assessed by TUNEL staining. Total RNA was assessed by RT‐qPCR. Protein levels were assessed by Western blotting. Bioinformatics and dual luciferase reporter assay were applied to determine the functional binding of miRNA to mRNA of target gene.

Results

GRh2 dose‐dependently decreased PC cell proliferation, but did not alter cell apoptosis. Mechanistically, GRh2 dose‐dependently increased the protein, but not mRNA of a cell‐cycle suppressor CDKN1A in PC cells, suggesting the presence of microRNA (miRNA)‐mediated protein translation control of CDKN1A by GRh2. In all candidate miRNAs that bind to 3′‐UTR of CDKN1A, miR‐4295 was specifically found to be suppressed dose‐dependently by GRh2 in PC cells. Moreover, miR‐4295 bound CDKN1A to suppress its protein translation. Furthermore, cell proliferation in PC cells that overexpressed miR‐4295 did not alter in response to GRh2.

Conclusions

GRh2 may inhibit PC cell growth through suppression of microRNA‐4295 that activates CDKN1A.

     

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.

     

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.

     

4,6,4′‐trimethylangelicin shows high anti‐proliferative activity on DU145 cells under both UVA and blue light

Objectives

Furocoumarins (psoralens and angelicins) have been already used under ultraviolet A light (UVA) for the treatment of skin diseases and cutaneous T‐cell lymphoma. Besides their high anti‐proliferative activity, some severe long‐term side effects have been observed, for example genotoxicity and mutagenicity, likely strictly related to the formation of crosslinks. It has been demonstrated that blue light (BL) activation of 8‐methoxypsoralen, an FDA‐approved drug, leads to less mutagenic monoadducts in the DNA. So far, in this work the less toxic and more penetrating BL is proposed to activate 4,6,4′‐trimethylangelicin (TMA), an already known UVA photoactivatable compound.

Materials and methods

Photocleavage, crosslink formation and oxidative damage were detected in pBR322 plasmid DNA treated with 300.0 μmol/L TMA activated with various exposures of BL. Anti‐proliferative activity, reactive oxygen species (ROS) formation and activation status of some signalling pathways involved in cell growth and apoptosis were verified on DU145 cells treated with 5.0 μmol/L TMA plus 2.0 J/cm² of BL.

Results

Under BL‐TMA, no mutagenic crosslinks, no photocleavage and neither photooxidative lesions were detected on isolated plasmid DNA. TMA showed high anti‐proliferative activity on DU145 cells through induction of apoptosis. Besides ROS generation, the proapoptotic effect seemed to be related to activation of p38 and inhibition of p44/42 phosphorylation. Interestingly, the decrease in nuclear β‐catenin was coupled with a significant dropping of CD44‐positive cells.

Conclusion

Overall, our results indicate that TMA can be activated by BL and may be considered for targeted phototherapy of prostate cancer lesions.