The chemopreventive properties of chlorogenic acid reveal a potential new role for the microsomal glucose-6-phosphate translocase in brain tumor progression

Background  

Chlorogenic acid (CHL), the most potent functional inhibitor of the microsomal glucose-6-phosphate translocase (G6PT), is thought to possess cancer chemopreventive properties. It is not known, however, whether any G6PT functions are involved in tumorigenesis. We investigated the effects of CHL and the potential role of G6PT in regulating the invasive phenotype of brain tumor-derived glioma cells.     

Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP‐ribose) polymerase cleavage and inhibiting nuclear factor kappa‐B activity

Objectives

Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti‐cancer effects. The study described here has evaluated anti‐cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells.

Materials and methods

Anti‐cancer activity of a tocotrienol‐rich fraction (TRF) and a tocotrienol‐enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (α‐tocopherol, α?, δ? and γ?tocotrienols) were studied using highly aggressive triple negative MDA‐MB‐231 cells and oestrogen‐dependent MCF‐7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP‐ribose) polymerase cleavage and levels of NF‐κB were determined using commercial ELISA kits.

Results

Tocotrienols exerted potent anti‐proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC₅₀ concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP‐ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa‐B (NF‐κB), which in turn can increase sensitivity of cancer cells to apoptosis.

Conclusion

Tocotrienols induced anti‐proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP‐ribose) polymerase cleavage and NF‐κB inhibition in the two human breast cancer cell lines.

     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

Cyanide bystander effect of the linamarase/linamarin killer-suicide gene therapy system

Background

The killer‐suicide system linamarase/linamarin (lis/lin) uses the plant gene linamarase (β‐glucosidase) to convert the cyanogenic glucoside substrate, linamarin, into glucose and cyanide. We have studied the bystander effect associated with this new system mediated by the production of the cyanide ion that diffuses freely across membranes.

Methods

Immunofluorescent staining of cells treated with an anti‐linamarase antibody allowed us to localize the enzyme within the cells. Flow cytometry was used to determine the sensitivity of different mixtures of cells, C6lis and C6gfp (green), to linamarin as a percentage of cell survival.

Results

We demonstrate here that rat glioblastoma C6 cells carrying the linamarase gene (lis), mixed with naive C6 cells and exposed to linamarin, induce generalized cell death. Cells expressing lis efficiently export linamarase, whereas linamarin enters cells poorly by endocytosis; as a result most of the cyanide is produced outside the cells. The study was facilitated by the presence of the green fluorescent protein (gfp) gene in the bystander population. As few as 10% C6lis‐positive cells are sufficient to eliminate the entire cell culture in 96 h.

Conclusions

This bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing production of sufficient cyanide to cause tumor regression. In this report we confirm the potential of the lis/lin gene therapy system as a powerful tool to eliminate tumors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Multiple source genes of HAmo SINE actively expanded and ongoing retroposition in cyprinid genomes relying on its partner LINE

Background  

We recently characterized HAmo SINE and its partner LINE in silver carp and bighead carp based on hybridization capture of repetitive elements from digested genomic DNA in solution using a bead-probe [1]. To reveal the distribution and evolutionary history of SINEs and LINEs in cyprinid genomes, we performed a multi-species search for HAmo SINE and its partner LINE using the bead-probe capture and internal-primer-SINE polymerase chain reaction (PCR) techniques.     

Deferoxamine promotes mesenchymal stem cell homing in noise‐induced injured cochlea through PI3K/AKT pathway

Objective

Over 5% of the world's population suffers from disabling hearing loss. Stem cell homing in target tissue is an important aspect of cell‐based therapy, which its augmentation increases cell therapy efficiency. Deferoxamine (DFO) can induce the Akt activation, and phosphorylation status of AKT (p‐AKT) upregulates CXC chemokine receptor‐4 (CXCR4) expression. We examined whether DFO can enhance mesenchymal stem cells (MSCs) homing in noise‐induced damaged cochlea by PI3K/AKT dependent mechanism.

Materials and Methods

Mesenchymal stem cells were treated with DFO. AKT, p‐AKT protein and hypoxia inducible factor 1‐ α (HIF‐1α) and CXCR4 gene and protein expression was evaluated by RT‐ PCR and Western blot analysis. For in vivo assay, rats were assigned to control, sham, noise exposure groups without any treatment or receiving normal, DFO‐treated and DFO +LY294002 (The PI3K inhibitor)‐treated MSCs. Following chronic exposure to 115 dB white noise, MSCs were injected into the rat cochlea through the round window. Number of Hoechst‐ labelled cells was determined in the endolymph after 24 hours.

Results

Deferoxamine increased P‐AKT, HIF‐1α and CXCR4 expression in MSCs compared to non‐treated cells. DFO pre‐conditioning significantly increased the homing ability of MSCs into injured ear compared to normal MSCs. These effects of DFO were blocked by LY294002.

Conclusions

Pre‐conditioning of MSCs by DFO before transplantation can improve stem cell homing in the damaged cochlea through PI3K/AKT pathway activation.

     

Molecular mechanisms of Lycoris aurea agglutinin‐induced apoptosis and G2/M cell cycle arrest in human lung adenocarcinoma A549 cells,both in vitro and in vivo

Objectives

Lycoris is aurea agglutinin (LAA) has attracted rising attention due to its remarkable bioactivities. Here, we aimed at investigating its anti‐tumor activities.

Material and Methods

In vitro methods including MTT, cellular morphology observation, FCM and immunoblotting were performed. In vivo methods like detection of tumor volume, body weight and survival ratio, as well as TUNEL staining were performed.

Results and Conclusion

LAA triggers G₂/M phase cell cycle arrest via up‐regulating p21expression as well as down‐regulating cdk‐1cyclinA singling pathway, and induces apoptotic cell death through inhibiting PI3K‐Akt survival pathway in human lung adenocarcinoma A549 cells. While LAA has no significant cytotoxic effect toward normal human embryonic lung fibroblast HELF cells, and moreover, LAA could amplify the antineoplastic effects of cisplatin toward A549 cells. Lastly LAA also bears anti‐cancer and apoptosis‐inducing effects in vivo, and it could decrease the volume and weight of subcutaneous tumor mass obviously as well as expand lifespan of mice. These findings may provide a new perspective for elucidating the complicated molecular mechanisms of LAA‐induced cancer cell growth‐inhibition and death, providing a new opportunity of LAA as a potential candidate anti‐neoplastic drug for future cancer therapeutics.

     

Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy

Background

Thymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear.

Methodology/Principal Findings

In this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis.

Conclusions

These findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies.     

MRI Findings for Frozen Shoulder Evaluation: Is the Thickness of the Coracohumeral Ligament a Valuable Diagnostic Tool?

Background

Recent studies have demonstrated that the coracohumeral ligament (CHL) is shortened and thickened in a frozen shoulder. We analyzed the rate in CHL visualization between patients with frozen shoulder and normal volunteers using Magnetic Resonance Imaging (MRI) to determine the CHL thickness in the patients with a frozen shoulder.

Methods and Findings

There were 72 shoulder joints in 72 patients (50 femles and 22 males with a mean age of 53.5 years) with clinical evidence and MR imaging evidence of frozen shoulder. These were prospectively analyzed to identify and measure the maximum thickness of the CHL. The control group, which included 120 shoulder joints in 60 normal volunteer individuals (30 females and 30 males with a mean age of 50.5 years) was also referred for MR imaging. A chi-square test was used to analyze the data of the rate of CHL visualization between the patients with frozen shoulder and the control group. A two-way ANOVA was used to analyze the mean maximal thickness of CHL. The CHL was visualized in 110 out of 120 shoulders in the control group (91.7%), and in 57 out of 72 shoulders for the frozen shoulder group (79.2%), there was significant difference, using a chi-square test (P<0.05). The CHL was not visualized in 10 out of 120 shoulders in the control group (8.3%), and 15 out of 72 shoulders in the frozen shoulder group (20.8%), there was a significant difference (P<0.05). The CHL thickness (3.99±1.68 mm) in the patients with frozen shoulder was significantly greater than that thickness (3.08±1.32 mm) in the control group, using a two-way ANOVA (P<0.001). The CHL thickness (3.52±1.52 mm, n = 97) in the female shoulders was no significantly greater than that thickness (3.22±1.49 mm, n = 70) in the male shoulders, using a two-way ANOVA (P>0.05).

Conclusions

MR Imaging is a satisfactory method for CHL depiction, and a thickened CHL is highly suggestive of frozen shoulder.     

LincRNA‐p21 suppresses development of human prostate cancer through inhibition of PKM2

Objectives

Previously, we found that long intergenic non‐coding RNA‐p21 (lincRNA‐p21) inhibited the development of human prostate cancer. However, the underlying molecular mechanisms are poorly understood. Here, we attempted to investigate the downstream targets of lincRNA‐p21 in prostate cancer.

Materials and methods

Expression of lincRNA‐p21 and PKM2 was determined by qRT‐PCR and Western blot. Lentivirus expressing shPKM2 or shCtrl was used to explore the role of PKM2 on the enhanced cell proliferation and glycolysis of lincRNA‐p21‐silenced prostate cancer cells. A xenograft mouse model was performed to investigate the effect of PKM2 suppression, glycolytic or mammalian target of rapamycin (mTOR) inhibitor on the tumorigenic capacity of lincRNA‐p21‐silenced prostate cancer cells.

Results

We revealed that lincRNA‐p21 silencing in DU145 and LNCaP cells induced up‐regulation of PKM2 and activation of glycolysis, which could be reversed by PKM2 knockdown or rapamycin treatment. We also found that the proliferation and tumorigenesis of lincRNA‐p21‐silenced prostate cancer cells were significantly inhibited after knocking down PKM2. 3‐bromopyruvate (3‐Brpa) or rapamycin treatment largely decreased the tumour burden. Importantly, PKM2 expression was inversely correlated with the lincRNA‐p21 level and the survival of prostate cancer patients.

Conclusions

We demonstrated that lincRNA‐p21 blunted the prostate cancer cell proliferation and tumorigenic capacity through down‐regulation of PKM2. Therefore, targeting PKM2 or glycolysis might be a therapeutic strategy in prostate cancer patients with lowly expressed lincRNA‐p21.

     

Shikonin Kills Glioma Cells through Necroptosis Mediated by RIP-1

Background and Purpose

Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.

Methods

Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.

Results

Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.

Conclusions

We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.     

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.

     

Saccharin enhances neurite extension by regulating organization of the microtubules

Aims

In the present study, we found that saccharin, an artificial calorie-free sweetener, promotes neurite extension in the cultured neuronal cells. The purposes of this study are to characterize the effect of saccharine on neurite extension and to determine how saccharin enhances neurite extension.

Main methods

The analyses were performed using mouse neuroblastoma N1E-115 cells and rat pheochromocytoma PC12 cells. Neurite extension was evaluated by counting the cells bearing neurites and measuring the length of neurites. Formation, severing and transportation of the microtubules were evaluated by immunostaining and western blotting analysis.

Key findings

Deprivation of glucose increased the number of N1E-115 cells bearing long processes. And the effect was inhibited by addition of glucose. Saccharin increased the number of these cells bearing long processes in a dose-dependent manner and total neurite length and longest neurite length in each cell. Saccharin also had a similar effect on NGF-treated PC12 cells. Saccharin increased the amount of the microtubules reconstructed after treatment with nocodazole, a disruptor of microtubules. The effect of saccharin on microtubule reconstruction was not influenced by dihydrocytochalasin B, an inhibitor of actin polymerization, indicating that saccharin enhances microtubule formation without requiring actin dynamics. In the cells treated with vinblastine, an inhibitor of microtubule polymerization, after microtubule reorganization, filamentous microtubules were observed more distantly from the centrosome in saccharin-treated cells, indicating that saccharin enhances microtubule severing and/or transportation.

Significance

These results suggest that saccharin enhances neurite extension by promoting microtubule organization.     

Moderate and severe malnutrition alters proliferation of spleen cells in rats

Objectives

Previous studies have shown alterations in bone marrow cell proliferation in malnourished rats, during lactation. The objective of this study was to determine in vivo effects of moderate and severe malnutrition on spleen cell proliferation in 21‐day‐old rat pups.

Materials and methods

Spleen cell proliferation was determined following administration of bromodeoxyuridine (BrdUrd) over a time course of 2, 4, 6 and 8 h. Incorporation of BrdUrd was detected using FITC‐conjugated anti‐BrdUrd monoclonal antibodies and total DNA content was detected and evaluated using propidium iodide using flow cytometry.

Results

Proportions of cells in S and G₂/M were reduced in the rats with moderate (MN2^nd) and severe (MN3^rd) malnutrition. BrdUrd incorporation was lower in both groups of malnourished rat. In cells of MN2nd individuals, length of G₁ became shorter, while length of S‐phase increased. In contrast, fraction of cells in proliferation was significantly lower in both groups of malnourished rat, with MN3rd group having lowest percentage of cell population growth. In this study, severe malnutrition did not significantly affect duration of phases of the cell cycle, although fractions of proliferating cells were dramatically reduced.

Conclusion

Moderate malnutrition increased time of cells in DNA synthesis and time of total cell cycle and severe malnutrition reduced growth fraction of spleen cells in malnourished rats during lactation.

     

Feasibility Study of the Elaboration of a Biodegradable and Bioactive Ligament Made of Poly(ε-caprolactone)-pNaSS Grafted Fibers for the Reconstruction of Anterior Cruciate Ligament: In Vivo Experiment

Background

The anterior cruciate ligament rupture is a common injury which mainly affects young and active population. Faced to this problem, the development of synthetic structures for ligament reconstruction is increasing. The most recent researches focused on the development of biodegradable structures that could be functionalized to enhance host integration. This work describes the elaboration of different poly(ε-caprolactone) prototypes for the rat anterior cruciate ligament replacement in order to found the best design for further in vivo assays.