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• Genetic mutation of Arrb2 results in decreased autophagy level in hippocampus. • Genetic mutation of Arrb2 leads to hyperactive Akt-mTOR signaling in hippocampus. • Genetic mutation of Arrb2 remarkably destroys the mitochondrial function of mouse hippocampal neurons. The cytoplasmic multifunctional adaptor protein β-arrestin 2 (Arrb2) is involved in the occurrence of various nervous system diseases, such as Alzheimer's disease and Parkinson's disease. Previous laboratory studies have shown that the expression and function of the Arrb2 gene was increased in valproic acid-induced autistic mice models. However, few reports have examined the possible role of Arrb2 in the pathogenesis of autism spectrum disorder. Therefore, Arrb2 -deficient (Arrb2−/−) mice were further studied to uncover the physiological function of Arrb2 in the nervous system. In this study, we found that Arrb2−/− mice had normal behavioral characteristics compared with wild-type mice. The autophagy marker protein LC3B was decreased in the hippocampus of Arrb2−/− mice compared to wild-type mice. Western blot analysis revealed that deletion of Arrb2 caused hyperactivation of Akt-mTOR signaling in the hippocampus. In addition, abnormal mitochondrial dysfunction was observed in Arrb2−/− hippocampal neurons, which was characterized by a reduction in mitochondrial membrane potential and adenosine triphosphate production and an increase in reactive oxygen species levels. Therefore, this study elucidates the interaction between Arrb2 and the Akt-mTOR signaling pathway and provides insights into the role of Arrb2 in hippocampal neuron autophagy. [ABSTRACT FROM AUTHOR]

Exposure to benzene causes immunosuppression, but the mechanism has not been clarified. In this study, mice were subcutaneously injected with different concentrations (0, 6, 30 and 150 mg/kg) of benzene for four weeks. The lymphocytes of bone marrow (BM), spleen and peripheral blood (PB) and the level of short-chain fatty acids (SCFAs) in mouse intestine were measured. The results showed that benzene exposure led to a reduction in CD3+ and CD8+ lymphocytes in mouse BM, spleen and PB, and CD4+ lymphocytes were increased in mouse spleen but decreased in mouse BM and PB after 150 mg/kg benzene exposure. In addition, Pro-B lymphocytes were reduced in mouse BM in the 6 mg/kg group. Besides, the levels of IgA, IgG, IgM, IL-2, IL-4, IL-6, IL-17a, TNF-α and IFN-γ in mouse serum were reduced after benzene exposure. Furthermore, the levels of acetic, propionic, butyric and hexanoic acid were reduced in mouse intestine, and the AKT-mTOR signaling pathway was activated in mouse BM cells after benzene exposure. Our results demonstrate that benzene induced immunosuppression in mice, and the B lymphocytes in BM are more sensible to benzene-induced toxicity. The reduction in mouse intestinal SCFAs as well as the activation of AKT-mTOR signaling may be related to the occurrence of benzene immunosuppression. Our study provides new insight for further mechanistic research on benzene-induced immunotoxicity. [ABSTRACT FROM AUTHOR]

Oxaliplatin is a platinum analog that can interfere with DNA replication and transcription. Continuous exposure to oxaliplatin results in chemoresistance; however, this mechanism is not well known. In this study, oxaliplatin-resistant (OR) colorectal cancer (CRC) cells of HCT116, HT29, SW480 and SW620 were established by gradually increasing the drug concentration to 2.5 μM. The inhibitory concentrations of cell growth by 50% (IC50) of oxaliplatin were 4.40–12.7-fold significantly higher in OR CRC cells as compared to their respective parental (PT) CRC cells. Phospho-Akt and phospho-mammalian target of rapamycin (mTOR) decreased in PT CRC cells but was overexpressed in OR CRC cells in response to oxaliplatin. In addition, an oxaliplatin-mediated decrease in phospho-AMP-activated protein kinase (AMPK) in PT CRC cells induced autophagy. Contrastingly, an increased phospho-AMPK in OR CRC cells was accompanied by a decrease in LC3B, further inducing the activity of glycolytic enzymes, such as glucose transporter 1 (GLUT1), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), to mediate cell survival. Inhibition of AMPK in OR CRC cells induced autophagy through inactivation of Akt/mTOR pathway and a decrease in GLUT1, PFKFB3, and PFK1. Collectively, targeting AMPK may provide solutions to overcome chemoresistance in OR CRC cells and restore chemosensitivity to anticancer drugs. [ABSTRACT FROM AUTHOR]

Consumers are more inclined to choose beef with a high intramuscular fat content (IMF), which regulated by lots of factors. It is very significant to find a miRNA that plays a key role in the accumulation of IMF. In our study, we found that bta-miR-330 was highly expressed in Japanese black cattle and differentially expressed at intramuscular pre-adipocytes differentiation processes. Furthermore, we transfected the bta-miR-330 mimic & inhibitor in intramuscular pre-adipocytes. The results showed that bta-miR-330 inhibits the proliferation but promotes the adipogenesis of intramuscular pre-adipocytes. Subsequently, our study showed that bta-miR-330 binds to SESN3 , which inhibits the adipogenesis of intramuscular pre-adipocytes. Moreover, we established the mechanism that bta-miR-330 promotes the adipogenesis of intramuscular pre-adipocytes by targeting SESN3 to activate the Akt-mTOR signaling pathway. Overall, our results revealed that bta-miR-330-SESN3 -Akt-mTOR axis plays an important role in adipogenesis of intramuscular pre-adipocytes, which provides a molecular basis for increasing IMF content in beef cattle. [ABSTRACT FROM AUTHOR]

Autophagy impacts the replication cycle of many viruses. Grass Carp Reovirus (GCRV) is an agent that seriously affects the development of the grass carp aquaculture industry. The role of autophagy in GCRV infection is not clearly understood. In this study, we identified that GCRV infection triggered autophagy in CIK cells, which was demonstrated by transmission electron microscopy, the conversion of LC3B I to LC3B II and the level of autophagy substrate p62. Furthermore, we found that GCRV infection activated Akt-mTOR signaling pathway, and the conversion of LC3B I to LC3B II was increased by inhibiting mTOR with rapamycin (Rap) but decreased by activating Akt with insulin. We then assessed the effects of autophagy on GCRV replication. We found that inducing autophagy with Rap promoted GCRV proliferation but inhibiting autophagy with 3 MA or CQ inhibited GCRV replication in CIK cells. Moreover, it was found that enhancing Akt-mTOR activity by insulin, GCRV VP7 protein and viral titers of GCRV were decreased. Collectively, these results indicated that GCRV infection induced autophagy involved in GCRV replication via the Akt-mTOR signal pathway. Thus, new insights into GCRV pathogenesis and antiviral treatment strategies are provided. • GCRV infection induces autophagy in CIK cells. • GCRV infection dysregulates the Akt/mTOR pathway to induce autophagy. • Autophagy promotes GCRV replication through Akt/mTOR pathway. [ABSTRACT FROM AUTHOR]

Estradiol (E2) deficiency arising from menopause is closely related to changes in body composition and declines of muscle mass and strength in elderly women. Whole-body vibration training (WBV) is an emerging approach expected to improve muscle mass and strength of older person, but the underlying mechanisms remain unclear. The balance between protein synthesis and degradation is a determining factor for muscle mass and strength, which is regulated by Akt-mTOR and FoxO1 signal pathway, respectively. In the present study, we firstly determined whether the effects of WBV on muscle mass and strength in ovariectomized female mice was affected by estrogen level, then investigated whether this was associated with Akt-mTOR and FoxO1 signal pathways. We found that (1) WBV, E2 supplementation (E) and WBV combined with E2 supplementation (WBV+E) significantly increased serum estradiol content, quadriceps muscle mass and grip strength in ovariectomized mice, accompanied with alterations of body composition (reducing fat content, increasing lean body mass and lean percent), furthermore, the altered degrees of these indicators by WBV+E were greater than WBV alone; (2) WBV, E and WBV+E remarkably increased the activities of Akt and mTOR and decreased FoxO1 activity, and the changed degrees by WBV+E were greater than WBV alone; (3) Pearson correlation coefficient revealed that serum estradiol content was positively correlated with Akt and mTOR activities, while inversely associated with FoxO1 activity. We concluded that WBV could significantly increase muscle mass and strength in ovariectomized mice, which might achieve through activating Akt-mTOR and suppressing FoxO1 signal pathways, and the improving effect of WBV on muscle mass and strength was better when in the presence of estrogen., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Hematopoietic stem cells (HSCs) are primarily dormant in a cell-cycle quiescence state to preserve their self-renewal capacity and long-term maintenance, which is essential for the homeostasis of hematopoietic system. Dysregulation of quiescence causes HSC dysfunction and may result in aberrant hematopoiesis (e.g., myelodysplastic syndrome and bone marrow failure syndromes) and leukemia transformation. Accumulating evidence indicates that both intrinsic molecular networks and extrinsic signals regulate HSC quiescence, including cell-cycle regulators, transcription factors, epigenetic factors, and niche factors. Further, the transition between quiescence and activation of HSCs is a continuous developmental path driven by cell metabolism (e.g., protein synthesis, glycolysis, oxidative phosphorylation, and autophagy). Elucidating the complex regulatory networks of HSC quiescence will expand the knowledge of HSC hemostasis and benefit for clinical HSC use. Here, we review the current understanding and progression on the molecular and metabolic regulation of HSC quiescence, providing a more complete picture regarding the mechanisms of HSC quiescence maintenance. [ABSTRACT FROM AUTHOR]

Oxaliplatin is a platinum analog that can interfere with DNA replication and transcription. Continuous exposure to oxaliplatin results in chemoresistance; however, this mechanism is not well known. In this study, oxaliplatin-resistant (OR) colorectal cancer (CRC) cells of HCT116, HT29, SW480 and SW620 were established by gradually increasing the drug concentration to 2.5 μM. The inhibitory concentrations of cell growth by 50% (IC50) of oxaliplatin were 4.40–12.7-fold significantly higher in OR CRC cells as compared to their respective parental (PT) CRC cells. Phospho-Akt and phospho-mammalian target of rapamycin (mTOR) decreased in PT CRC cells but was overexpressed in OR CRC cells in response to oxaliplatin. In addition, an oxaliplatin-mediated decrease in phospho-AMP-activated protein kinase (AMPK) in PT CRC cells induced autophagy. Contrastingly, an increased phospho-AMPK in OR CRC cells was accompanied by a decrease in LC3B, further inducing the activity of glycolytic enzymes, such as glucose transporter 1 (GLUT1), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), to mediate cell survival. Inhibition of AMPK in OR CRC cells induced autophagy through inactivation of Akt/mTOR pathway and a decrease in GLUT1, PFKFB3, and PFK1. Collectively, targeting AMPK may provide solutions to overcome chemoresistance in OR CRC cells and restore chemosensitivity to anticancer drugs.

Background/Aims: Hexavalent chromium [Cr(VI)] pollution has become a global concern for both ecosystems and human health. Our previous study revealed Cr(VI) could induce both apoptosis and autophagy in L-02 hepatocytes. Here, we sought to explore the underlying mechanism of Cr(VI)-induced autophagy and its exact role in cell death. Methods: Autophagy ultrastructure was observed under transmission electron microscope (TEM), autophagy flux was measured with double-tagged mCherry-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) assay, long-lived protein degradation assay, and LC3II expression assay in the presence of lysosomal inhibitor, bafilomycin A1 (BafA1). Reactive oxygen species (ROS) level was determined using fluorescent probe dichloro-dihydrofluorescein diacetate (DCFH-DA). The expression levels of Beclin-1, LC3, p62/ SQSTM1, and AKT-mammalian target of rapamycin (mTOR) pathway-related molecules including phosphorylation (p)-AKT, AKT, p-mTOR, and mTOR were examined using real-time polymerase chain reaction (RT-PCR) and western blotting. Apoptosis was determined using Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Results: Our results demonstrated Cr(VI) exposure activated autophagy in L-02 hepatocytes, as evidenced by the accumulation of autophagosomes, the increase of LC3-II and degradation of p62/ SQSTM1, and the enhanced overall degradation of proteins. We also confirmed Cr(VI)-induced LC3-II elevation mainly came from autophagy induction rather than lysosomal degradation impairment. ROS-AKT-mTOR pathway was associated with Cr(VI)-induced autophagy, and ROS scavenger N-acetylcysteine (NAC) pretreatment inhibited Cr(VI)-induced autophagy by alleviating the inhibition of the AKT-mTOR pathway. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine diphosphate (CDP) promoted Cr(VI)-induced apoptotic death. Conclusion: These findings indicated Cr(VI)-induced autophagy protected L-02 hepatocytes from apoptosis through the ROS-AKT-mTOR pathway.

Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication. ATG14/Barkor: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; ER: endoplasmic reticulum; hpi: hours post-infection; IBDV: infectious bursal disease virus; IP: co-immunoprecipitation; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PDPK1: 3-phosphoinositid-dependent protein kinase-1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; vBCL2: viral BCL2 apoptosis regulator. [ABSTRACT FROM AUTHOR]

Abstract Background The non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus, a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells. Methods The active fraction HS7 was prepared by n-hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot. Results The active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 μg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed. Conclusions The active fraction HS7 from n-hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.

OBJECTIVE: To explore the protective effect of liraglutide on renal damage in rats with diabetic kidney disease (DKD) through the protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway. MATERIALS AND METHODS: A total of 45 specific pathogen-free male Sprague-Dawley rats were divided into healthy group (no diabetes, n=15), diabetes group (diabetes, n=15), and liraglutide group (diabetes + liraglutide intervention, n=15). The differences in the biochemical indexes, lesion degree, glomerular Nephrin expression level, and mRNA and protein expressions of Akt-mTOR in renal tissues were detected in three groups via hematoxylin-eosin (HE) staining, immunohistochemistry, Western blotting, and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR), respectively. RESULTS: The albumin-to-creatinine ratio (ACR) and levels of serum creatinine (Scr), urine microalbumin (UmAlb), fasting blood glucose (FBG), glycated hemoglobin (HbA1c), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and total cholesterol (TC) in renal tissues were the lowest in healthy group and the highest in diabetes group, while they significantly declined in liraglutide group compared with those in diabetes group. Also, there were statistically significant differences (p<0.05). The level of high-density lipoprotein cholesterol (HDL-C) in renal tissues was the highest in healthy group and the lowest in diabetes group, while it was significantly increased in liraglutide group compared with that in diabetes group. Also, there were statistically significant differences (p<0.05). In healthy group, the mesangial structure and renal tubules were normal, the tubular basement membrane was smooth and intact, and there were no interstitial widening and inflammatory cell infiltration. Compared with diabetes group, the mesangial cell proliferation and vacuolar degeneration were alleviated, while the tubular dilatation or atrophy, fibrous tissues, and inflammatory cells were reduced in liraglutide group. Moreover, the results of immunohistochemical staining revealed that the glomerular Nephrin protein was arranged uniformly and showed the blue-black particles in healthy group. The glomerular Nephrin protein expressed was significantly decreased and arranged disorderly in diabetes group compared with that in healthy group, while it was increased in liraglutide group compared with that in diabetes group (p<0.05). The protein expression of Akt-mTOR in renal tissues was the lowest in healthy group and the highest in diabetes group, while it markedly declined in liraglutide group compared with that in diabetes group, displaying statistically significant differences (p<0.05). Similarly, the mRNA expression of Akt-mTOR in renal tissues was the lowest in healthy group and the highest in diabetes group, while it markedly declined in liraglutide group compared with that in diabetes group, displaying also statistically significant differences (p<0.05). CONCLUSIONS: Liraglutide can significantly reduce the blood glucose and improve the renal function in rats by suppressing the protein expression of AKT-mTOR, thereby exerting a protective effect on renal damage in rats with DKD. [ABSTRACT FROM AUTHOR]

Highlights • Methylphenidate alters Akt-mTOR pathway, including 4E-BP1 and S6K, in rat pheochromocytoma cells. • Methylphenidate changes on the Akt substrates (CREB, FoxO1, and GSK-3β) were also observed. • Methylphenidate alters cell signaling in pheochromocytoma cells and its responses differ according to the time of exposure. Abstract The exponential increase in methylphenidate (MPH) prescriptions in recent years has worried researchers about its misuse among individuals who do not meet the full diagnostic criteria for attention-deficit/hyperactivity disorder (ADHD) such as young children and students in search of cognitive improvement or for recreational reasons. The action of MPH is based mainly on inhibition of dopamine transporter, but the complete cellular effects are still unknown. Based upon prior studies, we attempted to determine whether the treatment with MPH (1μM) influences protein kinase B-mammalian target of rapamycin complex 1 signaling pathways (Akt-mTOR), including translation repressor protein (4E-BP1) and mitogen activated protein kinase (S6K), in rat pheochromocytoma cells (PC12), a well characterized cellular model, in a long or short term. MPH effects on the Akt substrates [cAMP response element-binding protein (CREB), forkhead box protein O1 (FoxO1), and glycogen synthase kinase 3 beta (GSK-3β)] were also evaluated. Whereas short term MPH treatment decreased the pAkt/Akt, pmTOR/mTOR and pS6K/S6K ratios, as well as pFoxO1 immunocontent in PC12 cells, long term treatment increased pAkt/Akt, pmTOR/mTOR and pGSK-3β/GSK-3β ratio. Phosphorylation levels of 4E-BP1 were decreased at 15 and 30 min and increased at 1 and 6 h by MPH. pCREB/CREB ratio was decreased. This study shows that the Akt-mTOR pathway, as well as other important Akt substrates which have been described as important regulators of protein synthesis, as well as being implicated in cellular survival, synaptic plasticity and memory consolidation, was affected by MPH in PC12 cells, representing an important step in exploring the MPH effects. [ABSTRACT FROM AUTHOR]

Background/Aims: Hexavalent chromium [Cr(VI)] pollution has become a global concern for both ecosystems and human health. Our previous study revealed Cr(VI) could induce both apoptosis and autophagy in L-02 hepatocytes. Here, we sought to explore the underlying mechanism of Cr(VI)-induced autophagy and its exact role in cell death. Methods: Autophagy ultrastructure was observed under transmission electron microscope (TEM), autophagy flux was measured with double-tagged mCherry-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) assay, long-lived protein degradation assay, and LC3II expression assay in the presence of lysosomal inhibitor, bafilomycin A1 (BafA1). Reactive oxygen species (ROS) level was determined using fluorescent probe dichloro-dihydrofluorescein diacetate (DCFH-DA). The expression levels of Beclin-1, LC3, p62/ SQSTM1, and AKT-mammalian target of rapamycin (mTOR) pathway-related molecules including phosphorylation (p)-AKT, AKT, p-mTOR, and mTOR were examined using real-time polymerase chain reaction (RT-PCR) and western blotting. Apoptosis was determined using Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Results: Our results demonstrated Cr(VI) exposure activated autophagy in L-02 hepatocytes, as evidenced by the accumulation of autophagosomes, the increase of LC3-II and degradation of p62/ SQSTM1, and the enhanced overall degradation of proteins. We also confirmed Cr(VI)-induced LC3-II elevation mainly came from autophagy induction rather than lysosomal degradation impairment. ROS-AKT-mTOR pathway was associated with Cr(VI)-induced autophagy, and ROS scavenger N-acetylcysteine (NAC) pretreatment inhibited Cr(VI)-induced autophagy by alleviating the inhibition of the AKT-mTOR pathway. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine diphosphate (CDP) promoted Cr(VI)-induced apoptotic death. Conclusion: These findings indicated Cr(VI)-induced autophagy protected L-02 hepatocytes from apoptosis through the ROS-AKT-mTOR pathway. [ABSTRACT FROM AUTHOR]

Oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) exposure to endometrial cells mimics ischemia-reperfusion injury. The present study tests the potential effect of keratinocyte growth factor (KGF) on the process. We show that KGF receptor KGFR is expressed in human endometrial T-HESC cells and primary murine endometrial cells. KGF pre-treatment protected endometrial cells from OGDR, inhibiting cell viability reduction and cell death. KGF attenuated OGDR-induced programmed necrosis in endometrial cells. Significantly, KGF activated Nrf2 signaling, causing Nrf2 Ser-40 phosphorylation, protein stabilization, nuclear translocation to promote anti-oxidant gene ( HO1 , NOQ1 and GCLC ) expression. Nrf2 silencing (by targeted shRNAs) or CRISPR/Cas9 knockout almost abolished KGF-induced endometrial cell protection against OGDR. Furthermore, KGF activated Akt-mTOR signaling in endometrial cells. Whereas Akt-mTOR inhibitors (LY294002, AZD2014 and RAD001) abolished KGF-induced Nrf2 activation and anti-OGDR cytoprotection. Together, KGF protects endometrial cells from OGDR via activating Akt-mTOR-Nrf2 signaling. [ABSTRACT FROM AUTHOR]

Ghrelin, a 28-amino acid peptide hormone, has protective effects on neuronal cells. The present study aimed to examine the neuroprotective effects of ghrelin on the rat retinal ganglion cells in the rotenone-induced in vitro model of Parkinson's disease (PD). Cell viability and cell apoptosis were determined by MTT assay and flow cytometry, respectively. Mitochondrial functions were detected by mitochondrial complex I activity assay and mitochondrial membrane potential (MMP) assay. The mRNA and protein expression levels were determined by qRT-PCR and western blot, respectively. Rotenone significantly suppressed cell viability and increased cell apoptosis, also decreased the mitochondrial complex I activity as well as MMP in rat retinal ganglion cell line (RGC-5). Growth hormone secretagogue receptor ( Ghsr ) siRNA transfection significantly suppressed the expression of Ghsr in RGC-5 cells. Ghrelin treatment attenuated the effects of rotenone-induced changes in cell viability, cell apoptosis and mitochondrial functions in RGC-5 cells. Post-transcriptional suppression by Ghsr siRNA transfection and treatment with GHS-R antagonist, YIL781, both significantly attenuated the effects of ghrelin in RGC-5 cells. Rotenone decreased the protein levels of Bcl-2 and increased the protein levels of Bax, cleaved caspase-3 and cleaved caspase-9, and this effect was reversed by ghrelin treatment. Ghrelin also prevented the inhibitory effects of rotenone on the AKT-mTOR signaling. The effects of ghrelin on the rotenone-induced changes in apoptosis-related protein levels and AKT-mTOR signaling were attenuated by Ghsr siRNA transfection and treatment with YIL781 in the RGC-5 cells. In addition, both rapamycin and AKT inhibitor IV pre-treatment significantly attenuated the effects of ghrelin on rotenone-induced changes in cell viability and cell apoptosis. In conclusion, ghrelin by acting on the GSH-R to protect rat retinal ganglion cells against rotenone via inhibiting apoptosis and restore mitochondrial functions in RGC-5 cells, and this effect was partially associated with the AKT-mTOR signaling pathway in RGC-5 cells. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) still constitutes a public health concern, however, no vaccines or therapies are currently approved for treatment. A fundamental process involved in ZIKV infection is autophagy, a cellular catabolic pathway delivering cytoplasmic cargo to the lysosome for degradation—considered as a primordial form of innate immunity against invading microorganisms. ZIKV is thought to inhibit the Akt-mTOR signaling pathway, which causes aberrant activation of autophagy promoting viral replication and propagation. It is therefore appealing to study the role of autophagic molecular effectors during viral infection to identify potential targets for anti-ZIKV therapeutic intervention.

It is well known that high salt intake is associated with cardiovascular diseases including hypertension. However, the research on the mechanism of obesity due to high salt intake is rare. To evaluate the roles of salt on obesity prevalence, the gene expression of adipogenesis/lipogenesis and adipocytokines secretion according to adipocyte dysfunction were investigated in salt-loading adipocytes. High salt dose-dependently increased the expression of adipogenic/lipogenic genes, such as PPAR-γ, C/EBPα, SREBP1c, ACC, FAS, and aP2, but decreased the gene of lipolysis like AMPK, ultimately resulting in fat accumulation. With SIK-2 and Na+/K+-ATPase activation, salt increased the metabolites involved in the renin-angiotensin-aldosterone system (RAAS) such as ADD1, CYP11β2, and MCR. Increasing insulin dependent insulin receptor substrate (IRS)-signaling, resulting in the insulin resistance, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Akt-mTOR were activated but AMPK(Thr172) was depressed in salt-loading adipocytes. The expression of pro-inflammatory adipocytokines, TNFα, MCP-1, COX-2, IL-17A, IL-6, leptin, and leptin to adiponectin ratio (LAR) were dose-dependently increased by salt treatment. Using the inhibitors of MAPK/ERK, U0126, we found that the crosstalk among the signaling pathways of MAPK/ERK, Akt-mTOR, and the inflammatory adipogenesis can be the possible mechanism of salt-linked obesity. The possibilities of whether the defense mechanisms against high dose of intracellular salts provoke signaling for adipocytes differentiation or interact with surrounding tissues through other pathways will be explored in future research.

Thyroid cancer has long been considered to arise in middle age and progress to more aggressive and lethal cancers after its repeated proliferation. In this research, we aimed at investigating the biological function and the underlying molecular mechanism of Melanoregulin (MREG) in thyroid cancer. It was found that the expression of MREG was significantly downregulated in thyroid cancer tissues. The downregulation of MREG expression was caused by epigenetic methylation. MREG overexpression could suppress the invasion and proliferation of thyroid cancer cells. While MREG knockdown promoted the invasion and proliferation of thyroid cancer cells. Furthermore, the phosphorylation of Akt or mTOR was decreased by MREG overexpression and increased by MREG knockdown. Moreover, Dactolisib (the inhibitor of mTOR) could abrogate silenced MREG induced thyroid cancer cell invasion and proliferation. Taken together, MREG regulates thyroid cancer cell invasion and proliferation through PI3K/Akt-mTOR signaling pathway. MREG may serve as a promising therapeutic strategy for thyroid cancer. [ABSTRACT FROM AUTHOR]

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase, which promotes the flux of carbohydrates into mitochondria and enhances the aerobic oxidation of glucose. DCA has previously been demonstrated to exhibit antitumor properties. The present study revealed that treatment with DCA induced increased levels of autophagy-associated proteins in esophageal squamous carcinoma cells while minimally affecting apoptosis. The present study examined the localization of light chain (LC)-3 by adenovirus infection with a green fluorescent protein (FP)-red FP-LC3 reporter construction and confirmed that DCA treatment induced significant autophagy. Furthermore, the inhibition of DCA-induced autophagy facilitated cell apoptosis and improved the drug sensitivity of esophageal squamous carcinoma cells to DCA and 5-FU (5-fluorouracil). The proliferation of TE-1 cells was markedly inhibited at low concentrations of DCA and 5-FU treatment when subjected to Atg5 mRNA interference, indicating that autophagy performed a protective role in cell survival upon DCA treatment. To determine the underlying mechanism of DCA-induced autophagy, the present study measured alterations in autophagy-associated signaling pathways. Notably, the protein kinase B (Akt)-mechanistic target of rapamycin (mTOR) signaling pathway, an important negative regulator of autophagy, was demonstrated to be suppressed by DCA treatment. These results may direct the development of novel strategies for the treatment of esophageal squamous carcinoma based on the combined use of DCA and autophagy inhibitors. [ABSTRACT FROM AUTHOR]

Postpartum depression (PPD) has adverse effects on offspring and increases their vulnerability to psychiatric disorders such as depression. Akt-mTOR signaling in the hippocampus is implicated in depression but its role in the behavioral deficits in PPD offspring remains unknown. By using a prepregnancy stress model of PPD in which Balb/c females that experience chronic stress before pregnancy show long-lasting PPD-like behaviors, we tested depression-like behaviors in PPD offspring (PPD-F1) at juvenile and adult ages as well as in the second generation (PPD-F2) produced by cross of male PPD-F1 with naïve females. Hippocampal Akt-mTOR signaling was examined in the F1 and F2 generations of PPD, as well as in PPD-F1 mice treated with a single dose of the antidepressant ketamine. PPD-F1 showed depression-like behaviors at juvenile and adult stages, evidenced by reduced sucrose preference (SP), increased immobility time in the forced swim test (FST), and a longer latency to feed and reduced food consumption in the novelty suppressed feeding (NSF) test. PPD-F1 mice showed Akt-mTOR signaling deficiency in the hippocampus, with down-regulated expression of p-Akt, p-mTOR and p-p70S6K. A single dose of ketamine reversed the behavior deficits and the impairment in Akt-mTOR signaling in PPD-F1. Furthermore, the PPD-F2 mice remained deficient in the SP and NSF test and hippocampal Akt-mTOR signaling, although the performance in FST was normal. The present study demonstrated both long-term and transgenerational effects of PPD on the depression–like behaviors of offspring, and suggested impaired Akt-mTOR signaling may play a part. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of expression levels of S100 calcium-binding protein A10 (S100A10) in lung adenocarcinoma (LUAD) on patient prognosis and the regulatory role of S100A10 in lung cancer cell proliferation and metastasis., Methods: Immunohistochemistry was used to detect the expression levels of S100A10 in LUAD and adjacent tissues, and the relationship between S100A10 expression and clinicopathological parameters and prognosis of the patients was statistically analyzed. The lung adenocarcinoma expression dataset in TCGA database was analyzed using gene enrichment analysis (GSEA) to predict the possible regulatory pathways of S100A10 in the development of lung adenocarcinoma. Lactate production and glucose consumption of lung cancer cells with S100A10 knockdown or overexpression were analyzed to assess the level of glycolysis. Western blotting, CCK-8 assay, EdU-594 assay, and Transwell assays were performed to determine the expression level of S100A10 protein, proliferation and invasion ability of lung cancer cells. A549 cells with S100A10 knockdown and H1299 cells with S100A10 overexpression were injected subcutaneously in nude mice, and tumor growth was observed., Results: The expression level of S100A10 was significantly upregulated in LUAD tissues as compared with the adjacent tissues, and an elevated S100A10 expression level was associated with lymph node metastasis, advanced tumor stage and distant organ metastasis ( P < 0.05), but not with tumor differentiation or the patients' age or gender ( P > 0.05). Survival analysis showed that elevated S100A10 expressions in the tumor tissue was associated with a poor outcome of the patients ( P < 0.001). In the lung cancer cells, S100A10 overexpression significantly promoted cell proliferation and invasion in vitro ( P < 0.001). GSEA showed that the gene sets of glucose metabolism, glycolysis and mTOR signaling pathway were significantly enriched in high expressions of S100A10. In the tumor-bearing nude mice, S100A10 overexpression significantly promoted tumor growth, while S100A10 knockdown obviously suppressed tumor cell proliferation ( P < 0.001)., Conclusion: S100A10 overexpression promotes glycolysis by activating the Akt-mTOR signaling pathway to promote proliferation and invasion of lung adenocarcinoma cells.

Background: Survival of random-pattern skin flaps is important for the success of plastic and reconstructive surgeries. This study investigates isoflurane-induced protection against ischemia of skin flap and the underlying molecular mechanism in this process. Methods: Human umbilical vein endothelial cells (HUVECs) and human skin fibroblast cells were exposed to isoflurane for 4 h. Expression of hypoxia inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) were analyzed up to 24 h post isoflurane exposure using qRT-PCR and western blot, or ELISA analyses. PI3K inhibitors - LY 294002 and wortmannin, mTOR inhibitor - rapamycin, and GSK3β inhibitor - SB 216763 were used respectively to assess the effects of isoflurane treatment and HIF-1α expression. Furthermore, 40 rats were randomly divided into 5 groups (control, isoflurane, scrambled siRNA plus isoflurane, HIF-1α siRNA plus isoflurane, and DMOG) and subjected to random-pattern skin flaps operation. Rats were prepared for evaluation of flap survival and full-feld laser perfusion imager (FLPI) (at 7 day) and microvessel density evaluation (at 10 day). Results: Isoflurane exposure induced expression of HIF-1α protein, HO-1 and VEGF mRNA and proteins in a time-dependent manner. Both LY 294002 and wortmannin inhibited phospho-Akt, phospho-mTOR, phospho-GSK 3β and HIF-1α expression after isoflurane exposure. Both wortmannin and rapamycin inhibited isoflurane-induced phospho-4E-BP1 (Ser 65) and phospho-P70s6k (Thr 389) and HIF-1α expression. SB 216763 pre-treatment could further enhance isoflurane-induced expression of phospho-GSK 3β (Ser 9) and HIF-1α protein compared to the isoflurane-alone cells. In animal experiments, isoflurane alone, scrambled siRNA plus isoflurane, or DMOG groups had significantly upregulated vascularity and increased survival of the skin flaps compared to the controls. However, HIF-1α knockdown abrogated the protective effect of isoflurane preconditioning in rats. Conclusions: Isoflurane preconditioning improves survival of skin flaps by up the regulation of HIF-1α expression via Akt-mTOR and Akt-GSK 3β signaling pathways.

In the present study, we investigate the function of ROS-AKT-mTOR axis on the apoptosis, proliferation and autophagy of MC3T3-E1 cells, and the proliferation of MC3T3-E1 cells after autophagy inhibition under high glucose conditions. MC3T3-E1 cells cultured in vitro were divided into the following groups: normal control group, N-acetylcysteine (NAC) group, 11.0 mM high glucose group, 11.0 mM high glucose + NAC group, 22.0 mM high glucose group, 22.0 mM high glucose + NAC group, CQ group, 22.0 mM high glucose + CQ group, 3-MA group and 3-MA + 22.0 mM high glucose group. ROS production was measured by DCFH-DA fluorescent probe. Cell proliferation was measured by MTT assay. Cells in different groups were stained with Annexin V-FITC/PI, and then apoptosis rate was detected by flow cytometry. Nucleus morphology was observed under fluorescence microscope after being incubated with Honchest33258. Protein expression was measured using Western blotting and immunofluorescence. Cell apoptosis and proliferation in high glucose group were increased and decreased, respectively, in a dose-dependent manner. Autophagy was significantly induced in high glucose group, even though different concentration of glucose induced autophagy in different stages of autophagy. ROS production in MC3T3-E1 cells was remarkably increased in high glucose group, but not in a dose-dependent manner. NAC, as an antioxidant, reduced ROS production and ameliorated cell apoptosis, proliferation abnormity and autophagy caused by high glucose. Expression of p-AKT and p-mTOR proteins were dramatically decreased in high glucose group, and NAC reversed their expression. In addition, 3-MA, an inhibitor of autophagy, significantly decreased the proliferation of MC3T3-E1 cells. When cocultured with 22.0 mM glucose that induced autophagy, proliferation of MC3T3-E1 cells was not affected compared to 22.0 mM high glucose group. Our present findings reveal that high glucose affects apoptosis, proliferation and autophagy of MC3T3-E1 cells through ROS-AKT-mTOR axis. In addition, autophagy inhibition does not affect the proliferation of MC3T3-E1 cells under high glucose conditions. [ABSTRACT FROM AUTHOR]

Background: Accumulating evidence suggests that growth hormone (GH) may play a major role in the regulation of postnatal neurogenesis, thus supporting the possibility that it may be also involved in promoting brain repair after brain injury. In order to gain further insight on this possibility, in this study we have investigated the pathways signaling the effect of GH treatment on the proliferation and survival of hippocampal subgranular zone (SGZ)-derived neurospheres. Results: Our results demonstrate that GH treatment promotes both proliferation and survival of SGZ neurospheres. By using specific chemical inhibitors we have been also able to demonstrate that GH treatment promotes the activation of both Akt-mTOR and JNK signaling pathways, while blockade of these pathways either reduces or abolishes the GH effects. In contrast, no effect of GH on the activation of the Ras-ERK pathway was observed after GH treatment, despite blockade of this signaling path also resulted in a significant reduction of GH effects. Interestingly, SGZ cells were also capable of producing GH, and blockade of endogenous GH also resulted in a decrease in the proliferation and survival of SGZ neurospheres. Conclusions: Altogether, our findings suggest that GH treatment may promote the proliferation and survival of neural progenitors. This effect may be elicited by cooperating with locally-produced GH in order to increase the response of neural progenitors to adequate stimuli. On this view, the possibility of using GH treatment to promote neurogenesis and cell survival in some acquired neural injuries may be envisaged. [ABSTRACT FROM AUTHOR]

Purpose: The pancreatic cancer has extremely low overall 5-year survival, and gemcitabine is the only approved single agent for pancreatic cancer treatment. Methods: In the present study, we investigated the potential effect of perifosine, a novel Akt inhibitor on gemcitabine-induced anti-pancreatic cancer effect both in vivo and in vitro. Results: We showed that sub-cytotoxic low concentration of perifosine dramatically enhanced gemcitabine-induced cytotoxicity in cultured pancreatic cancer cells. Perifosine inhibited Akt-mammalian target of rapamycin and Erk-mitogen-activated protein kinase activation in pancreatic cancer cells. Meanwhile, perifosine suppressed the hedgehog signaling, as it inhibited glioma-associated oncogenes (Gli) 1 activation and decreased its target protein patched 1 (PTCH1) expression. Our data demonstrated that perifosine blocked p70S6K1 (S6K1) activation, thus disrupting S6K1-Gli1 association and subsequent Gli1 activation. The reduction of S6K1 or Gli1 expression by target siRNAs inhibited PTCH1 expression and enhanced gemcitabine-induced cytotoxicity in pancreatic cancer cells. Significantly, perifosine dramatically enhanced gemcitabine-mediated antitumor effect in a PANC-1 xenograft severe combined immunodeficiency mice model. Conclusions: In summary, we conclude that perifosine sensitizes gemcitabine-mediated anti-pancreatic cancer efficiency through regulating multiple signaling pathways. [ABSTRACT FROM AUTHOR]

Here we studied expression and potential functions of Gαi3 in cervical cancer. The bioinformatics analysis together with the results from local patients' tissues revealed that Gαi3 expression was remarkably elevated in human cervical cancer tissues and different cervical cancer cells, and was associated with poor overall survival and poor disease-specific survival of patients. Gαi3 depletion resulted in profound anti-cervical cancer activity. In primary or immortalized cervical cancer cells, Gαi3 shRNA or CRISPR/Cas9-caused Gαi3 knockout/KO largely hindered cell proliferation and migration, and provoked apoptosis. On the contrast, ectopic Gαi3 overexpression further enhanced cervical cancer proliferation and migration. Akt-mTOR activation in primary cervical cancer cells was significantly reduced after Gαi3 silencing or KO, but was augmented following Gαi3 overexpression. Further studies revealed that the transcription factor GATA4 binding to Gαi3 promoter region was significantly enhanced in cervical cancer tissues and cells. Gαi3 expression was decreased by GATA4 shRNA, but upregulated following GATA4 overexpression. In vivo , the growth of cervical cancer xenografts was robustly suppressed after Gαi3 silencing or KO. Gαi3 depletion and Akt-mTOR inactivation were detected in Gαi3-silenced/-KO cervical cancer xenograft tissues. Together, upregulated Gαi3 is a valuable oncotarget of cervical cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The Akt-mTOR signaling pathway plays a central role in regulating intracellular protein metabolism, and the NF-κB signaling pathway plays a vital role in developing skeletal muscle atrophy. In this study, we took the three dipeptides Val-Leu (VL), Leu-Leu (LL), and Leu-Gln (LQ) identified from corn peptides as the research objects and proved that peptides could promote C2C12 cell differentiation and protein synthesis by activating the Akt-mTOR signaling pathway. On the other hand, the dipeptides also inhibited the classical pathway of the NF-κB signaling pathway, down-regulated the expression of two E3 ubiquitin ligases MAFbx and MURF-1, attenuated apoptosis induced by TNF-α, and thus inhibited protein degradation in cells. In conclusion, the dipeptides isolated from corn peptides showed the effect of increasing protein accumulation in skeletal muscle, suggesting the application potential of corn peptides in promoting the growth of skeletal muscle mass. In addition, this experiment compared the different characteristics of the effects of the dipeptide VL and its corresponding amino acid mixture on the Akt-mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Survival of random-pattern skin flaps is important for the success of plastic and reconstructive surgeries. This study investigates isoflurane-induced protection against ischemia of skin flap and the underlying molecular mechanism in this process. Methods: Human umbilical vein endothelial cells (HUVECs) and human skin fibroblast cells were exposed to isoflurane for 4 h. Expression of hypoxia inducible factor-1α (HIF-1α), heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) were analyzed up to 24 h post isoflurane exposure using qRT-PCR and western blot, or ELISA analyses. PI3K inhibitors - LY 294002 and wortmannin, mTOR inhibitor - rapamycin, and GSK3β inhibitor - SB 216763 were used respectively to assess the effects of isoflurane treatment and HIF-1α expression. Furthermore, 40 rats were randomly divided into 5 groups (control, isoflurane, scrambled siRNA plus isoflurane, HIF-1α siRNA plus isoflurane, and DMOG) and subjected to random-pattern skin flaps operation. Rats were prepared for evaluation of flap survival and full-feld laser perfusion imager (FLPI) (at 7 day) and microvessel density evaluation (at 10 day). Results: Isoflurane exposure induced expression of HIF-1α protein, HO-1 and VEGF mRNA and proteins in a time-dependent manner. Both LY 294002 and wortmannin inhibited phospho-Akt, phospho-mTOR, phospho-GSK 3β and HIF-1α expression after isoflurane exposure. Both wortmannin and rapamycin inhibited isoflurane-induced phospho-4E-BP1 (Ser 65) and phospho-P70s6k (Thr 389) and HIF-1α expression. SB 216763 pre-treatment could further enhance isoflurane-induced expression of phospho-GSK 3β (Ser 9) and HIF-1α protein compared to the isoflurane-alone cells. In animal experiments, isoflurane alone, scrambled siRNA plus isoflurane, or DMOG groups had significantly upregulated vascularity and increased survival of the skin flaps compared to the controls. However, HIF-1α knockdown abrogated the protective effect of isoflurane preconditioning in rats. Conclusions: Isoflurane preconditioning improves survival of skin flaps by up the regulation of HIF-1α expression via Akt-mTOR and Akt-GSK 3β signaling pathways. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Overexpression and/or overactivation of sphingosine kinase 1/2 (SphK1/2) is important for tumorigenesis and progression of cervical cancer. The current study examined the potential activity and signaling mechanisms of SKI-V, a non-lipid small molecule SphK inhibitor, against cervical cancer cells. In different primary and immortalized cervical cancer cells, SKI-V exerted significant anti-cancer activity by inhibiting cell viability, colony formation, proliferation, cell cycle progression and cell migration. Significant apoptosis activation was detected in SKI-V-treated cervical cancer cells. Significantly, SKI-V also provoked programmed necrosis cascade in cervical cancer cells, as it induced mitochondrial p53-cyclophilin-D-adenine nucleotide translocator-1 (ANT1) complexation, mitochondrial membrane potential collapse, reactive oxygen species production and the release of lactate dehydrogenase into the medium. Further, SKI-V blocked SphK activation and induced ceramide accumulation in primary cervical cancer cells, without affecting SphK1/2 expression. SKI-V-induced cytotoxicity in cervical cancer cells was largely inhibited by sphingosine-1-phosphate or the SphK1 activator K6PC-5, but was sensitized by adding the short-chain ceramide C6. Moreover, SKI-V inhibited Akt-mTOR (mammalian target of rapamycin) activation in primary cervical cancer cells, and its cytotoxicity was mitigated by a constitutively-active Akt. In vivo , daily intraperitoneal injection of SKI-V significantly inhibited subcutaneous primary cervical cancer xenograft growth in nude mice. Together, the SphK inhibitor SKI-V suppresses cervical cancer growth in vitro and in vivo ., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Sustained activation of multiple receptor tyrosine kinases (RTKs) simultaneously is vital for tumorigenesis and progression of osteosarcoma (OS). Gαi proteins recruitment to various RTKs mediates downstream oncogenic signaling activation. The expression, functions and underlying mechanisms of Gαi3 in human OS were examined. Expression of Gαi3 is robustly elevated in human OS tissues and is correlated with a poor overall survival. In patient-derived primary OS cells and immortalized lines (MG63 and U2OS), Gαi3 depletion, by shRNA and CRISPR/Cas9 strategies, robustly suppressed cell viability, proliferation and migration, while provoking G1-S arrest and apoptosis activation. Conversely, Gαi3 overexpressing ectopically can accelerate proliferation and migration of OS cells. In OS cells, Gαi3 immunoprecipitated with VEGFR2, FGFR, PGDFR and EGFR, mediating downstream cascade transduction. Akt-mTOR activation in primary OS cells was potently inhibited by Gαi3 shRNA, knockout or dominant negative mutation, but augmented after Gαi3 overexpression. In vivo studies showed that Gαi3 shRNA AAV (adeno-associated viruses) intratumoral injection largely inhibited the growth of subcutaneous xenografts of primary OS cells. Moreover, the growth of the Gαi3-knockout primary OS xenografts was much slower than that of OS xenografts with empty vector. In Gαi3-depleted OS xenografts tissues, Gαi3 downregulation and Akt-mTOR inactivation were detected. Taken together, overexpressed Gαi3 mediates RTK-Akt signaling to drive OS progression., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Betel-nut, a popular masticatory among Southeast Asian populations is a class I carcinogen, previously associated with dyslipidemia and aberrant lipid metabolism, and is reported to be used more frequently by females, than males. This study investigates the potential of repurposing the anti-diabetic drug, vildagliptin, a dipeptidyl peptidase-4 inhibitor, for alleviating the oncogenic condition in female Swiss Albino mice administered an aqueous extract of betel-nut (AEBN) orally (2 mg ml−1) for 24 weeks. Tissues were investigated by histopathological, immunohistochemical and apoptosis assays. Biochemical analyses of oxidative stress markers and lipid profile were performed using different tissues and sera. The expressions of different proteins involved in lipid metabolism and oncogenic pathways were evaluated by Western blotting. AEBN induced carcinogenesis primarily in the liver by significantly impairing AMPK signaling, inducing oxidative stress, activating Akt/mTOR signaling, increasing Ki-67 immunoreactivity and cyclin D1 expression, and significantly diminishing apoptosis. Co-administration of AEBN with vildagliptin (10 mg kg−1 body weight) for 8 weeks reduced liver dysplasia, and significantly decreased free palmitic acid, increased free oleic acid, normalized lipid profile, decreased oxidative stress, cyclin D1 expression, Ki-67 immunoreactivity, and Bcl2 expression, and increased the ratio of apoptotic/non-apoptotic cells. Mechanistically, vildagliptin elicited these physiological and molecular alterations by restoring normal AMPK signaling and reducing the cellular expressions of FASN and HMGCR, restoring AMPK-dependent phosphorylation of p53 at Ser-15 and reducing Akt/mTOR signaling. These results indicate that vildagliptin may alleviate betel-nut induced carcinogenesis in the liver of female mice. [ABSTRACT FROM AUTHOR]

The early metastasis of cervical cancer is a multistep process requiring the cancer cells to adapt to the signal input from different tissue environments, including hypoxia. Hypoxia-induced epithelial-to-mesenchymal transition (EMT) plays a critical role in the ability to invade surrounding tissues. However, the molecular mechanisms underlying EMT in cervical cancer remain to be elucidated. Herein, we show that hypoxia-inducible factor-1alpha (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) are recruited to the human coilin-interacting nuclear ATPase protein (hCINAP) promoter and initiate hCINAP expression in hypoxia. Ablation of hCINAP decreased the migratory capacity and EMT of cervical cancer cells under hypoxic conditions. Furthermore, hCINAP regulated EMT through the Akt-mTOR signaling pathway, and inhibits hypoxia-induced p53-dependent apoptosis. Our data collectively show that hCINAP may have essential roles in the metastasis of cervical cancer and could be a potential target for curing cervical cancer.

Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS 3 ), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) still constitutes a public health concern, however, no vaccines or therapies are currently approved for treatment. A fundamental process involved in ZIKV infection is autophagy, a cellular catabolic pathway delivering cytoplasmic cargo to the lysosome for degradation—considered as a primordial form of innate immunity against invading microorganisms. ZIKV is thought to inhibit the Akt-mTOR signaling pathway, which causes aberrant activation of autophagy promoting viral replication and propagation. It is therefore appealing to study the role of autophagic molecular effectors during viral infection to identify potential targets for anti-ZIKV therapeutic intervention. [ABSTRACT FROM AUTHOR]

It is well known that high salt intake is associated with cardiovascular diseases including hypertension. However, the research on the mechanism of obesity due to high salt intake is rare. To evaluate the roles of salt on obesity prevalence, the gene expression of adipogenesis/lipogenesis and adipocytokines secretion according to adipocyte dysfunction were investigated in salt-loading adipocytes. High salt dose-dependently increased the expression of adipogenic/lipogenic genes, such as PPAR-γ, C/EBPα, SREBP1c, ACC, FAS, and aP2, but decreased the gene of lipolysis like AMPK, ultimately resulting in fat accumulation. With SIK-2 and Na+/K+-ATPase activation, salt increased the metabolites involved in the renin-angiotensin-aldosterone system (RAAS) such as ADD1, CYP11β2, and MCR. Increasing insulin dependent insulin receptor substrate (IRS)-signaling, resulting in the insulin resistance, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Akt-mTOR were activated but AMPK(Thr172) was depressed in salt-loading adipocytes. The expression of pro-inflammatory adipocytokines, TNFα, MCP-1, COX-2, IL-17A, IL-6, leptin, and leptin to adiponectin ratio (LAR) were dose-dependently increased by salt treatment. Using the inhibitors of MAPK/ERK, U0126, we found that the crosstalk among the signaling pathways of MAPK/ERK, Akt-mTOR, and the inflammatory adipogenesis can be the possible mechanism of salt-linked obesity. The possibilities of whether the defense mechanisms against high dose of intracellular salts provoke signaling for adipocytes differentiation or interact with surrounding tissues through other pathways will be explored in future research. [ABSTRACT FROM AUTHOR]

Background: The non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus , a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells., Methods: The active fraction HS7 was prepared by n -hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot., Results: The active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 μg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed., Conclusions: The active fraction HS7 from n -hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.

Dermatofibrosarcoma protuberans (DFSP) is a cutaneous mesenchymal tumor of intermediate malignancy and fibroblastic/myofibroblastic differentiation. Fibrosarcomatous (FS) component is a high-grade component of DFSP. The detailed oncogenic difference between DFSP and FS components is not clear. We thus investigated the Akt-mTOR pathway in both components. We used 65 tumor samples obtained from 65 patients. The phosphorylation of Akt-mTOR pathway proteins (Akt, mTOR, 4EBP1, and S6RP) and PDGFRα/β was assessed by immunohistochemical staining, the results of which were confirmed by Western blotting. The immunohistochemical results were as follows: in ordinary DFSP components, p-PDGFRα-positive tumors were 41.9% (18/43 cases), p-PDGFRβ 55.8% (24/43 cases), p-Akt 51.2% (22/43 cases), p-mTOR 39.5% (17/43 cases), p-4EBP1 46.5% (20/43 cases), and p-S6RP 41.8% (18/43 cases); in DFSP components of FS-DFSP, 52.6% (10/19 cases), 47.4% (9/19 cases), 52.6% (10/19 cases), 36.8% (7/19 cases), 52.6% (10/19 cases), and 52.6% (10/19 cases); and in FS components, 45.5% (10/22 cases), 36.4% (8/22 cases), 72.7% (16/22 cases), 54.5% (12/22 cases), 72.7% (16/22 cases), and 68.2% (15/22 cases), respectively. There were significant positive correlations of the phosphorylation of most of the Akt-mTOR pathway proteins (p-Akt, p-mTOR, p-4EBP1, and p-S6RP) with each other (P < .05). Phospho-PDGFRβ was well correlated with the phosphorylation of Akt-mTOR pathway proteins in DFSP components of ordinary and FS-DFSPs, but these correlations were weaker in FS components. This study suggested the association of activation of Akt-mTOR pathway proteins and PDGFR with the progression of DFSP to FS. The Akt-mTOR pathway is thus a potential therapeutic target in imatinib-resistant DFSP/FS., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase, which promotes the flux of carbohydrates into mitochondria and enhances the aerobic oxidation of glucose. DCA has previously been demonstrated to exhibit antitumor properties. The present study revealed that treatment with DCA induced increased levels of autophagy-associated proteins in esophageal squamous carcinoma cells while minimally affecting apoptosis. The present study examined the localization of light chain (LC)-3 by adenovirus infection with a green fluorescent protein (FP)-red FP-LC3 reporter construction and confirmed that DCA treatment induced significant autophagy. Furthermore, the inhibition of DCA-induced autophagy facilitated cell apoptosis and improved the drug sensitivity of esophageal squamous carcinoma cells to DCA and 5-FU (5-fluorouracil). The proliferation of TE-1 cells was markedly inhibited at low concentrations of DCA and 5-FU treatment when subjected to Atg5 mRNA interference, indicating that autophagy performed a protective role in cell survival upon DCA treatment. To determine the underlying mechanism of DCA-induced autophagy, the present study measured alterations in autophagy-associated signaling pathways. Notably, the protein kinase B (Akt)-mechanistic target of rapamycin (mTOR) signaling pathway, an important negative regulator of autophagy, was demonstrated to be suppressed by DCA treatment. These results may direct the development of novel strategies for the treatment of esophageal squamous carcinoma based on the combined use of DCA and autophagy inhibitors.

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. In spite of advances in diagnosis and therapy, the prognosis of patients with GBM has remained dismal. The fast recurrence and multi-drug resistance are some of the key challenges in combating brain tumors. Glioma stem cells (GSCs) which are considered the source of relapse and chemoresistance, the need for more effective therapeutic options is overwhelming. In our present work, we found that combined treatment with temozolomide (TMZ) and metformin (MET) synergistically inhibited proliferation and induced apoptosis in both glioma cells and GSCs. Combination of TMZ and MET significantly reduced the secondary gliosphere formation and expansion of GSCs. We first demonstrated that MET effectively inhibited the AKT activation induced by TMZ, and a combination of both drugs led to enhanced reduction of mTOR, 4EBP1 and S6K phosphorylation. In addition, the combination of the two drugs was accompanied with a powerful AMP-activated protein kinase (AMPK) activation, while this pathway is not determinant. Xenografts performed in nude mice demonstrate in vivo demonstrated that combined treatment significantly reduced tumor growth rates and prolonged median survival of tumor-bearing mice. In conclusion, TMZ in combination with MET synergistically inhibits the GSCs proliferation through downregulation of AKT-mTOR signaling pathway. The combined treatment of two drugs inhibits GSCs self-renewal capability and partly eliminates GSCs in vitro and in vivo. This combined treatment could be a promising option for patients with advanced GBM.