Down-regulation Of Akt Phosphorylation Research Articles

Activation of metabotropic glutamate receptor 4 regulates proliferation and neural differentiation in neural stem/progenitor cells of the rat subventricular zone and increases phosphatase and tensin homolog protein expression.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III β-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.

Anti-HER2 antibody therapy using gene-transduced adipocytes for HER2-positive breast cancer.

Although recent advances in molecular target therapy have improved the survival of breast cancer patients, high cost and frequent hospital visits result in both societal and individual burden. To reduce these problems, it has been proposed to produce antibodies in vivo. Here, we constructed gene-transduced human ceiling culture-derived proliferative adipocytes secreting anti-HER2 antibody (HER2-ccdPAs) and evaluated their ability to secrete antibody and mediate an anti-tumor effect. Plasmid lentivirus was used as a recipient for anti-HER2 antibody cDNA and transduced into human proliferative adipocyte. Secretory antibody expression was evaluated by ELISA and western blot. Specific binding of secretory antibody to HER2 was examined by immunofluorescence analysis. Direct and indirect anti-tumor effects of supernatants from HER2-ccdPAs were evaluated using BT474 (HER2+) and MDA-MB-231 (HER2-) breast cancer cell lines. Additionally, whether adipocyte differentiation affects antibody secretion was investigated using supernatant collected from different cell maturation states. Anti-HER2 antibody was identified in the supernatant from HER2-ccdPAs and its production increased with the differentiation into mature adipocyte. Antibodies in supernatants from HER2-ccdPAs bound to HER2-positive breast cancer cells similar to trastuzumab. Supernatant from HER2-ccdPAs inhibited the proliferation of BT474 but not MDA-MB-231 cells, and downregulated AKT phosphorylation in BT474 cells compared with controls. Supernatants from HER2-ccdPAs also had an indirect anti-tumor effect on BT474 cells through ADCC. Additionally, Single inoculation of HER2-ccdPAs showed an anti-tumor effect in BT474 xenograft model. HER2-ccdPAs might be useful for cell-based gene therapy. This system could be a platform for various antibody therapies.

Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages.

Cytokines and chemokines are transcriptionally regulated by inflammatory transcription factors such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor (IRF)-3. A daidzein derivative compound, 8-hydroxydaidzein (8-HD), isolated from soy products, has recently gained attention due to various pharmacological benefits, including anti-inflammatory activities. However, regulation of the inflammatory signaling mechanism for 8-HD is still poorly understood, particularly with respect to the IRF-3 signaling pathway. In this study, we explored the molecular mechanism of 8-HD in regulating inflammatory processes, with a focus on the IRF-3 signaling pathway using a lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [Poly (I:C)] stimulated murine macrophage cell line (RAW264.7). The 8-HD downregulated the mRNA expression level of IRF-3-dependent genes by inhibiting phosphorylation of the IRF-3 transcription factor. The inhibitory mechanism of 8-HD in the IRF-3 signaling pathway was shown to inhibit the kinase activity of IKKε to phosphorylate IRF-3. This compound can also interfere with the TRIF-mediated complex formation composed of TRAF3, TANK, and IKKε leading to downregulation of AKT phosphorylation and reduction of IRF-3 activation, resulted in inhibition of IRF-3-dependent expression of genes including IFN-β, C-X-C motif chemokine 10 (CXCL10), and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Therefore, these results strongly suggest that 8-HD can act as a promising compound with the regulatory function of IRF-3-mediated inflammatory responses.

Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain

A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 μg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

MicroRNA-143 Increases Oxidative Stress and Myocardial Cell Apoptosis in a Mouse Model of Doxorubicin-Induced Cardiac Toxicity.

BackgroundOxidative stress and myocardial apoptosis are features of doxorubicin-induced cardiac toxicity that can result in cardiac dysfunction. Previous studies showed that microRNA-143 (miR-143) was expressed in the myocardium and had a role in cardiac function. This study aimed to investigate the effects and possible molecular mechanisms of miR-143 on oxidative stress and myocardial cell apoptosis in a mouse model of doxorubicin-induced cardiac toxicity.Material/MethodsMice underwent intraperitoneal injection of doxorubicin (15 mg/kg) daily for eight days to develop the mouse model of doxorubicin-induced cardiac toxicity. Four days before doxorubicin administration, a group of mice was pretreated daily with a miR-143 antagonist (25 mg/kg/day) for four consecutive days by tail vein injection. The study included the use of a miR-143 antagomir, or anti-microRNA, an oligonucleotide that silenced endogenous microRNA (miR), and an agomir to miR-143, and also the AKT inhibitor, MK2206. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblot analysis were used to measure mRNA and protein expression, respectively.ResultsDoxorubicin treatment increased the expression of miR-143, which was reduced by the miR-143 antagomir. Overexpression of miR-143 increased doxorubicin-induced myocardial apoptosis and oxidative stress. The use of the miR-143 antagomir significantly activated protein kinase B (PKB) and AKT, which were reduced in the presence of the AKT inhibitor, MK2206. However, the use of the miR-143 antagomir further down-regulated AKT phosphorylation following doxorubicin treatment and increased AKT activation.ConclusionsIn a mouse model of doxorubicin-induced cardiac toxicity, miR-143 increased oxidative stress and myocardial cell apoptosis following doxorubicin treatment by inhibiting AKT.

Aspirin enhances trophoblast invasion and represses soluble fms-like tyrosine kinase 1 production: a putative mechanism for preventing preeclampsia.

Recent studies suggested that prophylactic aspirin prior to 16 weeks of gestation in high-risk patients may reduce the risk of developing preeclampsia; however, the exact mechanism of aspirin's effect on the pathophysiology of preeclampsia is not clear. This study was designed to investigate the effect of aspirin on trophoblast cell function and its effect on soluble fms-like tyrosine kinase 1 (sFlt-1) production to elucidate the preventive mechanisms for preeclampsia. We used two human trophoblastic cell lines (HTR-8/SVneo and JAR) and freshly isolated cytotrophoblasts from normal and preeclamptic placenta at term to determine the effect of aspirin on trophoblast cell function. Trophoblasts were pretreated with aspirin, and then cell functions and sFlt-1 expression were assessed. Our results showed that aspirin promoted trophoblast invasion not only in HTR-8/SVneo and JAR cells, but also in isolated cytotrophoblasts. sFlt-1 production was repressed by aspirin in a dose-dependent manner. By adding Flt-1 recombinant protein, the trophoblast invasion ability was inhibited in HTR-8/SVneo cells, which was reversed by Flt-1 small interfering ribonucleic acid knockdown. In addition, metalloproteinase 2/9 expression and activity were activated by aspirin but inhibited by sFlt-1. Aspirin also downregulated Akt phosphorylation, and trophoblast invasiveness was facilitated under Akt inhibitor treatment. Aspirin enhances cell invasiveness and inhibits sFlt-1 production in trophoblasts. Moreover, sFlt-1 itself also inhibits trophoblast invasion. Our novel findings suggest that the preeclampsia prevention effect of aspirin may be exerted through these two mechanisms.

MiR-21-3p inhibits autophagy of bovine granulosa cells by targeting VEGFA via PI3K/AKT signaling.

It is well documented that granulosa cell apoptosis is the main reason for follicular atresia and death; however, increasing evidence suggests that autophagy plays an important role in the fate of granulosa cells. miR-21-3p regulates many fundamental biological processes and is pivotal in the autophagy of tumor cells; nevertheless, the autophagy in cattle ovary and how miR-21-3p regulates the follicular cells is unknown. In this study, we aimed to elucidate the autophagy and the role of miR-21-3p in cattle ovary using bovine primary ovarian granulosa cells (BGCs). The results showed the autophagy for the first time in BGCs in large follicle according to autophagic gene transcript of LC3, BECN-1, ATG3, protein expression of LC3, P62 and LC3 puncta, a standard marker for autophagosomes. miR-21-3p was identified as a novel miRNA that repressed BGCs autophagy according to the results from plasmids transfection of miR-21-3p mimics and inhibitor. Meanwhile, VEGFA was confirmed to be a validated target of miR-21-3p in BGCs using luciferase reporter assays and the results of VEGFA expression decreased with transfection of miR-21-3p mimics, while it increased with transfection of miR-21-3p inhibitor. In addition, small interference-mediated knockdown of VEGFA significantly inhibits BGCs autophagy signaling; however, overexpression of VEGFA in BGCs promoted autophagy in the presence of miR-21-3p. Finally, the results of AKT and its phosphorylation suggested that miR-21-3p suppressed VEGFA expression through downregulating AKT phosphorylation signaling. In summary, this study demonstrates that miR-21-3p inhibits BGCs autophagy by targeting VEGFA and attenuating PI3K/AKT signaling.

Prazosin inhibits the growth and mobility of osteosarcoma cells

Background: Osteosarcoma is a primary malignant bone tumor that frequently occurs in adolescents and children, its high aggressiveness and rapid metastasis often resulting in poor prognoses. In previous studies, Prazosin has been shown to possess anti-proliferative properties against prostate cancer and glioblastoma cells. In our study, we investigated Prazosin’s underlying mechanisms and its effects on the biological behaviors of osteosarcoma cells. Methods: Osteosarcoma cell lines MG63 and 143B were treated with different concentrations of Prazosin, and a CCK8 assay assessed its effect on cell viability. Colony formation, Transwell and flow cytometry assays were used to examine its effects on cell proliferation, cell migration, and cell invasion and apoptosis, respectively. The expression of relevant proteins was then examined using western blotting. Results: Our data showed that Prazosin dose-dependently reduced the viability of MG63 and 143B cells and significantly inhibited their clonogenic ability. Moreover, Prazosin attenuated the cell migration and invasion abilities of MG63 and 143B cells when compared with the NC group. It also accelerated cell apoptosis in mitochondrial pathways by regulating Bcl-2/Bax axis and caspase 3. Furthermore, Prazosin treatment inactivated the Akt/mTOR pathway by down-regulating Akt and mTOR phosphorylation (p-Akt, p-mTOR) and the expression of P70 and cyclin D1. Conclusions: Our data highlights the fact that Prazosin inhibits cell growth, inhibits the motility of osteosarcoma cells, and promotes apoptosis, suggesting that Prazosin is a potential anti-cancer agent in osteosarcoma therapy.

Sec3 knockdown inhibits TGF-β induced epithelial-mesenchymal transition through the down-regulation of Akt phosphorylation in A549 cells

The exocyst, an evolutionarily conserved octomeric protein complex, has been demonstrated as an essential component for vesicle tethering during cell exocytosis, and participates in various physiological processes in the cell. Although subunits of the exocyst complex have been reported to be involved in the regulation of TGF-β induced cancer cell migration and epithelial-mesenchymal transition (EMT), the potential function of Sec3 in these regulated processes remains unclear. Here, we show that Sec3 knockdown abolishes TGF-β stimulated A549 lung cancer cell migration in vitro and causes defects in the regulated EMT process. In addition, we find that depletion of Sec3 significantly inhibits TGF-β stimulated Akt phosphorylation in A549 cells, whereas the increase of Smad2 phosphorylation is unaffected. Furthermore, replenishment of an RNAi-resistant form of Sec3 is shown to restore the defects of TGF-β induced cell migration, EMT and Akt signaling activation. In summary, our study provides evidence that Sec3 is involved in TGF-β induced cell migration and EMT processes, presumably through the regulation of PI3K/Akt signaling activation in A549 cancer cells.

Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = −0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.

Effects of the potassium‐sparing diuretic amiloride on chemotherapy response in canine osteosarcoma cells

BackgroundOsteosarcoma (OSA) is a common bone tumor of mesenchymal origin in dogs. Chemotherapy delays metastasis, yet most dogs die of this disease within 1 year of diagnosis. The high metabolic demand of cancer cells promotes proton pump upregulation, leading to acidification of the tumor microenvironment and chemoresistance. The potassium‐sparing diuretic amiloride is among a class of proton pump inhibitors prescribed for refractory heart failure treatment in dogs.ObjectiveWe hypothesized that amiloride treatment improves chemotherapy response by reducing acidification in canine OSA cells. Our objective was to assess the in vitro effects of amiloride on cell viability, apoptosis, and metabolism.MethodsIn vitro study. Assessments of cell viability and apoptosis were performed after single agent or combination treatment, along with calculations of pharmacological synergism using the combination index. Protein signaling during apoptosis was evaluated by Western blotting. Metabolic profiling was performed using a Seahorse bioanalyzer.ResultsAmiloride strongly synergized with doxorubicin in combination treatment and exhibited additive or antagonistic effects with carboplatin in canine OSA cells. Combination treatment with doxorubicin significantly upregulated p53‐mitochondrial signaling to activate apoptosis and downregulate Akt phosphorylation. Amiloride‐treated cells further exhibited metabolic switching with reductions in glycolytic capacity and maximal respiration.Conclusion and Clinical ImportanceAmiloride synergized with doxorubicin to potentiate apoptosis in canine OSA cells. These results justify further investigation into repurposing of amiloride as an oncology drug for the treatment of OSA in dogs.

Therapeutic efficacy of hydrogen‑rich saline alone and in combination with PI3K inhibitor in non‑small cell lung cancer.

The aim of the present study was to investigate the effects of combination therapy of LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), with hydrogen-rich saline on the proliferation and apoptosis of the non-small cell lung cancer (NSCLC) A549 cell line and the mechanisms underpinning this. Excessive production of reactive oxygen species (ROS) may induce DNA mutations, DNA damage, genomic instability and cell proliferation, and ROS are involved in several types of cancer, particularly lung cancer. In a previous study, hydrogen was recognized as an antioxidant in preventive and therapeutic applications. The PI3K/protein kinase B (Akt) pathway is an important signaling pathway that may activate downstream of a series of extracellular signals and impact on cellular processes including cell proliferation, apoptosis and survival. To date, the PI3K/Akt signaling pathway has been indicated as a feasible target for novel antineoplastic drugs. Different strategies combining the two treatment modalities have been used in cancer therapy in order to achieve an improved therapeutic response and longer control of tumor modalities control. The present study investigated the effect of hydrogen-rich saline alone and in combination with the PI3K inhibitor, LY294002, on the proliferation, oxidative stress and apoptosis of NSCLC A549 cells. This combination therapy may be more effective than separate drug treatment; it decreased the malondialdehyde level and increased the superoxide dismutase activity. The combination therapy also enhanced the efficacy of anti-proliferation and apoptosis. Similarly, the results of the present study demonstrated that administration of the two agents in combination may inhibit phospho-Akt activity, and reduce expression of heme oxygenase-1 and nuclear factor-κB p65. The results further suggested that the combination therapy may reduce cell proliferation and promote cell apoptosis by downregulating Akt phosphorylation and inhibiting the PI3K pathway in NSCLC cell lines. Therefore, the present study provided evidence that combined therapy may be a novel therapeutic option for patients with NSCLC.

Natural Stilbenoids Have Anti-Inflammatory Properties in Vivo and Down-Regulate the Production of Inflammatory Mediators NO, IL6, and MCP1 Possibly in a PI3K/Akt-Dependent Manner.

Stilbenoids are a group of polyphenolic compounds found in plants, trees, berries, and nuts. Stilbenoids have been shown to serve an antimicrobial and antifungal function in plants. There is also evidence that as a part of the human diet, stilbenoids play an important role as antioxidants and may have anti-inflammatory effects. The PI3K/Akt pathway is a well-characterized signaling pathway controlling cellular functions involved in growth and cell cycle and in metabolism. There is also increasing evidence to show the involvement of this pathway in the regulation of inflammatory responses. In the present study, an attempt was made to investigate the anti-inflammatory properties of the naturally occurring stilbenoids pinosylvin (1), monomethylpinosylvin (2), resveratrol (3), pterostilbene (4), piceatannol (5), and rhapontigenin (6). Glycosylated derivatives of piceatannol and rhapontigenin, namely, astringin (7) and rhaponticin (8), respectively, were also investigated. In addition to the natural stilbenoids, pinosylvin derivatives (9-13) were synthesized and subjected to the testing of their effects on the PI3K/Akt pathway in inflammatory conditions. The investigated natural stilbenoids (except the glycosylated derivatives) were found to down-regulate Akt phosphorylation, which is a well-acknowledged marker for PI3K activity. It was also found that all of the studied natural stilbenoids had anti-inflammatory effects in vitro. The three most potent stilbenoids, piceatannol, pinosylvin, and pterostilbene, were selected for in vivo testing and were found to suppress inflammatory edema and to down-regulate the production of inflammatory mediators IL6 and MCP1 in carrageenan-induced paw inflammation in mice. When compared to the commercial PI3K inhibitor LY294002, the anti-inflammatory effects appeared to be quite similar. The results reveal hitherto unknown anti-inflammatory effects of natural stilbenoids and suggest that those effects may be mediated via inhibition of the PI3K/Akt pathway.

Synergistic effect of black tea polyphenol, theaflavin-3,3′-digallate with cisplatin against cisplatin resistant human ovarian cancer cells

Theaflavin-3,3′-digallate (TF3) is a phenolic compound extracted from black tea. We previously demonstrated that TF3 selectively inhibited ovarian cancer cells. Ovarian cancer has high death rate because of acquired cisplatin resistance. We aimed to investigate the synergistic effect of TF3 and cisplatin (CDDP) against cisplatin resistant ovarian cancer cells. In the present study, combination treatment with TF3 and CDDP showed a synergistic cytotoxic effect in A2780/CP70 and OVCAR3 cells. Combination treatment showed a synergistic pro-apoptotic effect and synergistically induced G1/S phase cell cycle arrest. Synergistic apoptosis was accompanied by regulating protein expression of cleaved caspase 3/7, cytochrome c, Bax and Bcl-2. Combination treatment induced G1/S phase cell cycle arrest via regulating protein expression of cyclin A2, cyclin D1, cyclin E1 and CDK2/4. Combination treatment could synergistically down-regulate Akt phosphorylation in both cell lines. TF3 may be used as an adjuvant for the treatment of advanced ovarian cancer.

SIRT2 Inhibition Confers Neuroprotection by Downregulation of FOXO3a and MAPK Signaling Pathways in Ischemic Stroke.

Sirtuin 2 (SIRT2) is a family member of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases which appears to have detrimental roles in an array of neurological disorders such as Parkinson's disease (PD) and Huntington's disease (HD). In light of the recently emerging roles of sirtuins in normal physiology and pathological conditions such as ischemic stroke, we investigated the role of SIRT2 in ischemic stroke-induced neuronal cell death. Primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) under in vitro ischemic conditions, and subsequently tested for the efficacy of SIRT2 inhibitors AK1 and AGK2 in attenuating apoptotic cell death caused by OGD. We have also evaluated the effect of SIRT2 inhibition in C57BL/6 mice subjected to 1h middle cerebral artery occlusion (MCAO) followed by 24h reperfusion, which is a model for ischemic reperfusion injury in vivo. Significant reductions in apoptotic cell death were noted in neurons treated with AK1 or AGK2, as evidenced by reduced cleaved caspase-3 and other apoptotic markers such as Bim and Bad. In addition, downregulation of phosphorylated-AKT and FOXO3a proteins of the AKT/FOXO3a pathway, as well as a marked reduction of JNK activity and its downstream target c-Jun, were also observed. When tested in animals subjected to MCAO, the neuroprotective effects of AGK2 in vivo were evidenced by a substantial reduction in ipsilateral infarct area and a significant improvement in neurological outcomes. A similar reduction in the levels of pro-apoptotic proteins in the infarct tissue, as well as downregulation of AKT/FOXO3a and JNK pathway, were also noted. In summary, the current study demonstrated the neuroprotective effects of SIRT2 inhibition in ischemic stroke, and identified the downregulation of AKT/FOXO3a and MAPK pathways as intermediary mechanisms which may contribute to the reduction in apoptotic cell death by SIRT2 inhibition.

The PPARγ agonist efatutazone delays invasive progression and induces differentiation of ductal carcinoma in situ.

Ductal carcinoma in situ (DCIS) is a pre-invasive lesion of the breast considered a precursor of invasive ductal carcinoma. This study aimed to determine whether activated PPARγ acts as a tumor suppressor in human DCIS progression. We utilized the high-affinity PPARγ agonist, efatutazone, to activate endogenous PPARγ in a well-defined model for the progression of basal (triple negative) DCIS, MCFDCIS cells, cultured under 2D and 3D conditions. We studied the effects of activated PPARγ on DCIS progression in MCFDCIS xenograft and C3(1)/Tag transgenic mice treated with 30mg/kg of efatutazone. In vitro, efatutazone did not alter the MCFDCIS cell proliferation but induced phenotypic and gene expression changes, indicating that activated PPARγ is able to differentiate MCFDCIS cells into more luminal and lactational-like cells. In addition, MCFDCIS tumorsphere formation in 3D was reduced by PPARγ activation. In vivo, efatutazone-treated MCFDCIS tumors exhibited fat deposition along with upregulation of PPARγ responsive genes in both epithelial and stromal compartments, suggesting features of milk-producing mammary epithelial cell differentiation. The efatutazone-treated lesions were less invasive with fewer CD44+/p63+ basal progenitor cells. PPARγ activation downregulated Akt phosphorylation in these tumors, although the ERK pathway remained unchanged. Similar trends in gene expression changes consistent with lactational and luminal cell differentiation were observed in the C3(1)/Tag mouse model after efatutazone treatment. Our data suggest that activation of the PPARγ pathway differentiates DCIS lesions and may be a useful approach to delay DCIS progression.

Expression of the mRNA stability regulator Tristetraprolin is required for lactation maintenance in the mouse mammary gland.

Tristetraprolin (TTP), an mRNA-binding protein that negatively controls levels of inflammatory factors, is highly expressed in the lactating mouse mammary gland. To determine the biological relevance of this expression profile, we developed bi-transgenic mice in which this protein is specifically down-regulated in the secretory mammary epithelium in the secretory mammary epithelium during lactation. Our data show that TTP conditional KO mice produced underweight litters, possibly due to massive mammary cell death induced during lactation without the requirement of additional stimuli. This effect was linked to overexpression of inflammatory cytokines, activation of STAT3 and down-regulation of AKT phosphorylation. Importantly, blocking TNFα activity in the lactating conditional TTP KO mice inhibited cell death and similar effects were observed when this treatment was applied to wild-type animals during 48 h after weaning. Therefore, our results demonstrate that during lactation TTP wards off early involution by preventing the increase of local inflammatory factors. In addition, our data reveal the relevance of locally secreted TNFα for triggering programmed cell death after weaning.

The Flavonoid Jaceosidin from Artemisia princeps Induces Apoptotic Cell Death and Inhibits the Akt Pathway in Oral Cancer Cells.

Jaceosidin is a single compound from the Japanese mugwort Artemisia princeps, which is used as a food and a traditional medicinal herb. A. princeps extracts and flavonoid components have been shown to have antihyperglycaemic, antioxidant, and anti-inflammatory properties. Although the anticancer properties of these extracts were recently demonstrated, the related mechanisms have not been characterised. In this study, we investigated the effects of jaceosidin in oral squamous cell carcinoma (OSCC) cells and initially showed selective suppression of proliferation (IC50 = 82.1 μM in HSC-3 cells and 97.5 μM in Ca9.22 cells) and accumulation of cells at the sub-G1 stage of the cell cycle. In addition, jaceosidin increased cleavage of caspase-9 and caspase-3 in OSCC cells, although caspase-8 was not detected. In further experiments, jaceosidin downregulated Akt phosphorylation and ectopic activation of Akt blocked the antiproliferative effects of jaceosidin. Finally, we showed that jaceosidin has no effects on HaCaT normal epithelial cell viability, indicating selective chemotherapeutic potential of jaceosidin and that tumour-specific downregulation of Akt increases apoptosis and inhibits growth in OSCC cells.

Inhibition of CX3CR1 reduces cell motility and viability in pancreatic adenocarcinoma epithelial cells

Increased expression of the chemokine CX3CL1 and its sole receptor, CX3CR1 have been correlated with poor pancreatic cancer patient survival and time to recurrence, as well as with pancreatic perineural invasion. We have previously shown that metastasis of prostate and breast cancer is in part driven by CX3CL1, and have developed small molecule inhibitors against the CX3CR1 receptor that diminish metastatic burden. Here we ask if inhibition of this chemokine receptor affects the phenotype of PDAC tumor cells. Our findings demonstrate that motility, invasion, and contact-independent growth of PDAC cells all increase following CX3CL1 exposure, and that antagonism of CX3CR1 by the inhibitor JMS-17-2 reduces each of these phenotypes and correlates with a downregulation of AKT phosphorylation. These data suggest that PDAC tumor cell migration and growth, elements critical in metastatic progression, may susceptible to pharmacologic intervention.

Long Coding RNA XIST Contributes to Neuronal Apoptosis through the Downregulation of AKT Phosphorylation and Is Negatively Regulated by miR-494 in Rat Spinal Cord Injury.

Recent evidence has suggested that long non-coding RNAs (lncRNAs) may play a significant role in the pathogenesis of several neurological diseases, including spinal cord injury (SCI). However, little is known about the role of lncRNAs in SCI. The aim of the present study was to evaluate the potential functions of lncRNAs in SCI and to identify the underlying mechanisms of action. We firstly analyzed Gene Expression Omnibus (GEO) datasets to investigate aberrantly-expressed lncRNAs which might be involved in the pathogenesis of SCI. The long non-coding RNA X-inactive specific transcript (XIST) was found to be one of the most significantly upregulated lncRNAs in the GEO dataset analysis, and is associated with apoptosis. We, therefore, selected this as a candidate lncRNA and investigated its function. We found that knockdown of lncRNA-XIST by Lv-shRNA had a prominent protective effect on SCI recovery by suppressing apoptosis through reactivation of the PI3K/AKT signaling pathway in rat spinal cord tissue. In particular, our results suggested that lncRNA-XIST may act as a competitive endogenous RNA, effectively becoming a sink for miR-494, leading to derepression of its target gene, phosphatase and tensin homolog deleted on chromosome ten (PTEN). In addition, an inverse relationship between lncRNA-XIST and miR-494 was observed in spinal cord tissues of SCI rats. Further study demonstrated that antagomiR-494 could reverse the protective effects of lncRNA-XIST knockdown on SCI rats through blocking the PTEN/PI3K/AKT signaling pathway. These results suggested that lncRNA-XIST knockdown may play an important role in limiting neuronal apoptosis in rats following SCI, and that the observed protective effects of lncRNA-XIST knockdown might have been mediated by its regulation on the phosphorylation of AKT by competitively binding miR-494. These findings have revealed, for the first time, the importance of the XIST/miR-494/PTEN/AKT signaling axis in the pathogenesis of SCI and suggest that lncRNA-XIST may be a promising molecular target for SCI therapy.