Translation of paired box 6 (PAX6) mRNA is IRES-mediated and inhibited by cymarin in breast cancer cells.

Paired box 6 (PAX6) is a member of the PAX family and plays an essential role in cancer cell cycle progression, colony formation, proliferation and invasion. Its expression is upregulated in many cancers including breast cancer, but the process of PAX6 mRNA translation has rarely been studied. We found that PAX6 translation level increased in MCF-7 breast cancer cells treated with the chemotherapeutic drug adriamycin (ADM), which might be attributable to internal ribosome entry site (IRES)-mediated translation. By modifying a bicistronic luciferase plasmid that is widely used to examine IRES activity, we found that the 469-base 5'-UTR of PAX6 mRNA contains an IRES element and that core IRES activity is located between nucleotides 159 and 333. Moreover, PAX6 IRES activity was induced during ADM treatment, which may be the main reason for the elevated level of PAX6 protein. We also found that cymarin, a cardiac glycoside, acts as an inhibitor of PAX6 protein expression by impairing its IRES-mediated translation. Furthermore, MCF-7 cell proliferation was suppressed during treatment with cymarin. These results provide novel insights into the translation mechanism of PAX6 in breast cancer cells and suggest that cymarin is a promising candidate for the treatment of breast cancer via targeting the expression of PAX6.

Simulation of a new respiratory phase sorting method for 4D-imaging using optical surface information towards precision radiotherapy.

BACKGROUND: Respiratory signal detection is critical for 4-dimensional (4D) imaging. This study proposes and evaluates a novel phase sorting method using optical surface imaging (OSI), aiming to improve the precision of radiotherapy. METHOD: Based on 4D Extended Cardiac-Torso (XCAT) digital phantom, OSI in point cloud format was generated from the body segmentation, and image projections were simulated using the geometries of Varian 4D kV cone-beam-CT (CBCT). Respiratory signals were extracted respectively from the segmented diaphragm image (reference method) and OSI respectively, where Gaussian Mixture Model and Principal Component Analysis (PCA) were used for image registration and dimension reduction respectively. Breathing frequencies were compared using Fast-Fourier-Transform. Consistency of 4DCBCT images reconstructed using Maximum Likelihood Expectation Maximization algorithm was also evaluated quantitatively, where high consistency can be suggested by lower Root-Mean-Square-Error (RMSE), Structural-Similarity-Index (SSIM) value closer to 1, and larger Peak-Signal-To-Noise-Ratio (PSNR) respectively. RESULTS: High consistency of breathing frequencies was observed between the diaphragm-based (0.232 Hz) and OSI-based (0.251 Hz) signals, with a slight discrepancy of 0.019Hz. Using end of expiration (EOE) and end of inspiration (EOI) phases as examples, the mean±1SD values of the 80 transverse, 100 coronal and 120 sagittal planes were 0.967, 0,972, 0.974 (SSIM); 1.657 ± 0.368, 1.464 ± 0.104, 1.479 ± 0.297 (RMSE); and 40.501 ± 1.737, 41.532 ± 1.464, 41.553 ± 1.910 (PSNR) for the EOE; and 0.969, 0.973, 0.973 (SSIM); 1.686 ± 0.278, 1.422 ± 0.089, 1.489 ± 0.238 (RMSE); and 40.535 ± 1.539, 41.605 ± 0.534, 41.401 ± 1.496 (PSNR) for EOI respectively. CONCLUSIONS: This work proposed and evaluated a novel respiratory phase sorting approach for 4D imaging using optical surface signals, which can potentially be applied to precision radiotherapy. Its potential advantages were non-ionizing, non-invasive, non-contact, and more compatible with various anatomic regions and treatment/imaging systems.

miR-205-5p contributes to paclitaxel resistance and progression of endometrial cancer by downregulating FOXO1.

Endometrial cancer (EC) is one of the most frequent malignancies occurring in female genital system. miR-205-5p has been reported to involve in the progression of multiple malignancies, including EC. However, the detail function and mechanism of miR-205-5p in chemoresistance of EC have not been defined. qRT-PCR assay was performed to detect miR-205-5p abundance in EC tissues and cell lines. The sensitivity of HEC-1-A and RL95-2 cells to PTX was assessed based on the results of IC50. MTT and flow cytometry (FCM) analyses were carried out to determine cell proliferation and apoptosis. Bioinformatics, luciferase, RNA immunoprecipitation (RIP) and western blot analyses were employed to confirm the true interaction between miR-205-5p and FOXO1. Functional restoration experiments were carried out to explore the regulatory mechanism of miR-205-5p in PTX sensitivity and cell growth. miR-205-5p was upregulated in EC tissues and cell lines compared with respective control. Knockdown of miR-205-5p enhanced PTX-sensitivity of EC cells and induced cell growth, which was reflected by the decreased cell proliferation and increased apoptosis. FOXO1 was identified to be a target of miR-205-5p. Elevated miR-205-5p expression reversed FOXO1-enhanced chemosensitivity and cell growth. miR-205-5p enhanced PTX-resistance and contributed to tumorigenesis of EC cells through directly targeting FOXO1. These data shed light on a novel regulation of miR-205-5p in EC, providing a potential therapeutic target for EC patients with PTX resistance.

Activation of Transient Receptor Potential Vanilloid 4 is Involved in Neuronal Injury in Middle Cerebral Artery Occlusion in Mice.

Transient receptor potential vanilloid 4 (TRPV4) is widely expressed in the central nervous system and can be activated by multiple stimuli during cerebral ischemia. Recently, we reported that intracerebroventricular (icv.) injection of HC-067047, a specific TRPV4 antagonist, reduced brain infarction following 60-min of middle cerebral artery occlusion (MCAO). This study was undertaken to investigate the molecular mechanisms underlying TRPV4-mediated neuronal injury in cerebral ischemia. We demonstrated that TRPV4 expression was upregulated in the ipsilateral hippocampus at 4 to 48 h post-MCAO, peaking at 18 h post-MCAO. Treatment with TRPV4 antagonists (HC-067047 and ruthenium red) dose-dependently reduced brain infarction at 24 h post-MCAO. Phosphorylation of protein kinase B (p-Akt) was downregulated and that of extracellular signal-related kinase (p-ERK) was upregulated at 8 to 24 h post-MCAO, which was markedly blocked by treatment with HC-067047. Icv. injection of GSK1016790A (a TRPV4 agonist), dose-dependently induced hippocampal neuronal death, accompanied by an increase in phosphorylation of the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR). In addition, the level of p-Akt was decreased and that of p-ERK was increased by GSK1016790A-injection, which was sensitive to an NR2B antagonist. The neuronal toxicity of GSK1016790A was blocked by treatment with an NR2B antagonist and a phosphatidylinositol-3-kinase (PI3K) agonist but not by administration of a MAPK/ERK kinase antagonist. We conclude that the activation of TRPV4 is upregulated and involved in neuronal injury during cerebral ischemia and that the neurotoxicity associated with TRPV4-activation is mediated through NR2B-NMDAR and the related downregulation of the Akt signaling pathway.

Lack of JWA Enhances Neurogenesis and Long-Term Potentiation in Hippocampal Dentate Gyrus Leading to Spatial Cognitive Potentiation.

JWA (Arl6ip5), a homologous gene of glutamate-transporter-associated protein 3-18 (GTRAP3-18) and addicsin, is highly expressed in hippocampus. We generated systemic and neuronal JWA knockout (JWA-KO and JWA-nKO) mice to investigate the influence of JWA deficiency on spatial cognitive performance, process of neurogenesis, and induction of long-term potentiation (LTP) in hippocampal dentate gyrus (DG). In comparison with wild-type (WT) mice and JWA (loxP/loxP) (control of JWA-nKO) mice, 8-week-old JWA-KO mice and JWA-nKO mice showed spatial cognitive potentiation as assessed by Morris water maze test. In hippocampal DG of JWA-nKO mice, either survival and migration or neurite growth of newborn neurons were significantly enhanced without the changes in proliferation and differentiation of stem cells. In addition, the increase of LTP amplitude and the decline of LTP threshold were observed in DG, but not in CA1 region, of JWA-nKO mice compared to control mice. The levels of hippocampal FAK, Akt, and mTOR phosphorylation in JWA-nKO mice were higher than those in control mice. The PI3K or FAK inhibitor could abolish the enhanced neurogenesis and LTP induction in JWA-nKO mice, which was accompanied by disappearance of the spatial cognitive potentiation. The treatment of JWA-nKO mice with 3'-azido-3'-deoxythymidine (AZT), a telomerase inhibitor, suppressed not only the enhanced neurogenesis but also the enhanced LTP induction in DG, but it did not affect the LTP induction in CA1 region. The results suggest that the JWA deficiency through cascading FAK-PI3K-Akt-mTOR pathway increases the newborn neurons and enhances the LTP induction in hippocampal DG, which leads to the spatial cognitive potentiation.

Sex-related neurogenesis decrease in hippocampal dentate gyrus with depressive-like behaviors in sigma-1 receptor knockout mice.

Male sigma-1 receptor knockout (σ1R(-/-)) mice showed depressive-like phenotype with deficit in the survival of newly generated neuronal cells in the hippocampal dentate gyrus (DG), but female σ1R(-/-) mice did not. The level of serum estradiol (E2) at proestrus or diestrus did not differ between female σ1R(-/-) mice and wild-type (WT) mice. Ovariectomized (OVX) female σ1R(-/-) mice, but not WT mice, presented the same depressive-like behaviors and neurogenesis decrease as male σ1R(-/-) mice. Treatment of male σ1R(-/-) mice with E2 could alleviate the depressive-like behaviors and rescue the neurogenesis decrease. In addition, E2 could correct the decline in the density of NMDA-activated current (INMDA) in granular cells of DG and the phosphorylation of NMDA receptor (NMDAr) subtype 2B (NR2B) in male σ1R(-/-) mice, which was associated with the elevation of Src phosphorylation. The neuroprotection and antidepressant effects of E2 in male σ1R(-/-) mice were blocked by the inhibitor of Src or NR2B. The NMDAr agonist showed also the neuroprotection and antidepressant effects in male σ1R(-/-) mice, which were insensitive to the Src inhibitor. On the other hand, either the deprivation of E2 or the inhibition of Src in female σ1R(-/-) mice rather than WT mice led to a distinct decline in INMDA and NR2B phosphorylation. Similarly, the Src inhibitor could cause neurogenesis decrease and depressive-like behaviors in female σ1R(-/-) mice, but not in WT mice. These results indicate that the σ1R deficiency impairs neurogenesis leading to a depressive-like phenotype, which is alleviated by the neuroprotection of E2.

Pro-neurogenesis and anti-dementia properties of tetradecyl 2,3-dihydroxybenzoate through TrkA receptor-mediated signalling pathways.

Tetradecyl 2,3-dihydroxybenzoate, termed ABG001, has been reported to enhance neurite outgrowth of PC12 cells. Herein, we report that oral administration of ABG001 for five days to adult male mice could dose-dependently enhance survival and neurite growth of newborn cells in hippocampal dentate gyrus (DG) without changes in cell proliferation and differentiation of progenitor cells. The ABG001 administration (0.5 mg/kg) enhanced the phosphorylation of tyrosine kinase A (TrkA) receptor, which induced increases in the levels of ERK, Akt and mTOR phosphorylation in hippocampus. The pro-neurogenesis of ABG001 was blocked by the TrkA receptor inhibitor K252a. By contrast, the ERK inhibitor U0126 attenuated only the ABG001-increased number of newborn cells, while the PI3K inhibitor LY294002 prevented mainly the ABG001-enhanced neurite growth. In comparison with control mice, the mice treated with ABG001 showed a more preferential spatial cognitive function as assessed by Morris water maze and Y maze tests, which was sensitive to the blockade of TrkA receptor. In addition, a single injection (i.c.v.) of 'aggregated' Aß 25-35 in adult male mice (Aß 25-35-mice) impaired spatial memory, survival and neurite growth of newborn cells in the DG with reduced phosphorylation of Akt and mTOR. The treatment of Aß 25-35-mice with ABG001 could protect the survival and neurite growth of newborn cells through increasing TrkA receptor-induced phosphorylation of Akt and mTOR, which was accompanied by the improvement of spatial cognitive performance.

Simvastatin exerts antiamnesic effect in Aß25-35 -injected mice.

AIM AND METHODS: Simvastatin (SV) is reported to improve cognition and slow the progression of Alzheimer's disease (AD). This study explored the mechanisms underlying the antiamnesic effect of SV in AD using behavior tests, histological examination, western blot analysis, and electrophysiological recording technique in AD model mice created by intracerebroventricular injection (i.c.v.) of Aß25-35 . RESULTS: Chronic administration of SV (40 mg/kg/day) for 11 days after Aß25-35 -injection ameliorated the impairment of acquisition performance and probe trail test in Morris water maze task and alternation behavior in Y maze task in Aß25-35 -mice. Aß25-35 -induced apoptosis of hippocampal CA1 pyramidal cells and Aß25-35 -impaired high-frequency stimulation (HFS)-dependent long-term potentiation (LTP) induction in hippocampal Schaffer collaterale-CA1 synapse were rescued by SV-treatment. SV prevented Aß25-35 -inhibited protein kinase B (Akt) and extracellular signal-related kinase-2 (ERK2) phosphorylation, which was sensitive to α7 nicotinic acetylcholine receptor (α7nAChR) antagonist MLA. SV-induced neuroprotection was attenuated by MLA or phosphatidylinositol-3-kinase (PI3K) antagonist LY294002. SV-rescued LTP induction was blocked by α7nAChR, PI3K or MAPK/ERK kinase (MEK) antagonist. Finally, the antiamnesia of SV in Aß25-35 -mice was attenuated by blockage of SV-induced neuroprotection or SV-rescued LTP induction. CONCLUSION: The antiamnesia of SV in Aß25-35 -mice depends on its neuroprotection and synaptic plasticity improvement.

Transient receptor potential vanilloid 4 mediates hypotonicity-induced enhancement of synaptic transmission in hippocampal slices.

AIM AND METHODS: Changes in cerebrospinal fluid osmotic pressure modulate brain excitability. Transient receptor potential vanilloid 4 (TRPV4), which is sensitive to hypotonic stimulation, is expressed in hippocampus. The present study investigated the effect of hypotonic stimulation on hippocampal synaptic transmission and the role of TRPV4 in hypotonicity-action using electrophysiological recording and pharmacological technique. RESULTS: Accompanied with the decrease in paired pulse facilitation, field excitatory postsynaptic potential (fEPSP) was enhanced by hypotonicity and TRPV4 agonist 4α-PDD in hippocampal slices, which was sensitive to TRPV4 antagonist HC-067047. Hypotonicity-induced increase in fEPSP was blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, but not N-methyl-d-aspartate receptor or N- or P/Q-type voltage-gated calcium channel antagonist. High voltage-gated calcium current (ICa ) in hippocampal CA3 pyramidal neurons was not affected by hypotonicity. AMPA-activated current (IAMPA ) in hippocampal CA1 pyramidal neurons was increased by hypotonicity and 4α-PDD, which was attenuated by HC-067047. Inhibition of protein kinase C or protein kinase A markedly attenuated hypotonicity-increased IAMPA , whereas antagonism of calcium/calmodulin-dependent protein kinase II had no such effect. CONCLUSION: TRPV4 is involved in hypotonicity-induced enhancement of hippocampal synaptic transmission, which may be mediated through promoting presynaptic glutamate release and increasing postsynaptic AMPA receptor function.

Sigma-1 receptor knockout impairs neurogenesis in dentate gyrus of adult hippocampus via down-regulation of NMDA receptors.

AIMS: This study investigated the influence of sigma-1 receptor (σ1 R) deficiency on adult neurogenesis. METHODS: We employed 8-week-old male σ1 R knockout (σ1 R(-/-) ) mice to examine the proliferation and differentiation of progenitor cells, and the survival and neurite growth of newborn neurons in hippocampal dentate gyrus (DG). RESULTS: In comparison with wild-type (WT) littermates, the numbers of 24-h-old BrdU(+) cells and Ki67(+) cells in σ1 R(-/-) mice increased, while the number of 28-day-old BrdU(+) cells decreased without changes in proportion of BrdU(+) /NeuN(+) cells and BrdU(+) /GFAP(+) cells. The neurite density of newborn neurons was slightly reduced in σ1 R(-/-) mice. In DG granular cells, N-methyl-d-aspartate (NMDA)-activated current (INMDA ) and phosphorylation of NMDA receptor (NMDAr) NR2B were reduced in σ1 R(-/-) mice without the alteration of NR2B expression and membrane properties compared to WT mice. The NR2B antagonist abolished the difference in INMDA between σ1 R(-/-) mice and WT mice. The application of NMDAr agonist in σ1 R(-/-) mice prevented the over-proliferation of cells and reduction in newborn neurons, but it had no effects on the hypoplastic neurite. The administration of NMDAr antagonist in WT mice enhanced the cell proliferation and depressed the survival of newborn neurons. CONCLUSION: The σ1 R deficiency impairs neurogenesis in DG through down-regulation of NMDArs.

Activation of Transient Receptor Potential Vanilloid 4 Increases NMDA-Activated Current in Hippocampal Pyramidal Neurons.

The glutamate excitotoxicity, mediated through N-methyl-d-aspartate receptors (NMDARs), plays an important role in cerebral ischemia injury. Transient receptor potential vanilloid 4 (TRPV4) can be activated by multiple stimuli that may happen during stroke. The present study evaluated the effect of TRPV4 activation on NMDA-activated current (INMDA) and that of blocking TRPV4 on brain injury after focal cerebral ischemia in mice. We herein report that activation of TRPV4 by 4α-PDD and hypotonic stimulation increased INMDA in hippocampal CA1 pyramidal neurons, which was sensitive to TRPV4 antagonist 10 µ M/2 µ 1/mouse [DOSAGE ERROR CORRECTED] and NMDAR antagonist AP-5, indicating that TRPV4 activation potentiates NMDAR response. In addition, the increase in INMDA by hypotonicity was sensitive to the antagonist of NMDAR NR2B subunit, but not of NR2A subunit. Furthermore, antagonists of calcium/calmodulin-dependent protein kinase II (CaMKII) significantly attenuated hypotonicity-induced increase in INMDA, while antagonists of protein kinase C or casein kinase II had no such effect, indicating that phosphorylation of NR2B subunit by CaMKII is responsible for TRPV4-potentiated NMDAR response. Finally, we found that intracerebroventricular injection of 10 µ m/2 µ 1/mouse [DOSAGE ERROR CORRECTED] after 60 min middle cerebral artery occlusion reduced the cerebral infarction with at least a 12 h efficacious time-window. These findings indicate that activation of TRPV4 increases NMDAR function, which may facilitate glutamate excitotoxicity. Closing TRPV4 may exert potent neuroprotection against cerebral ischemia injury through many mechanisms at least including the prevention of NMDAR-mediated glutamate excitotoxicity.