p70S6K Signaling Pathway Research Articles

α-Ketoglutarate Modulates Macrophage Polarization Through Regulation of PPARγ Transcription and mTORC1/p70S6K Pathway to Ameliorate ALI/ARDS.

As tissue-resident cells in the lung, alveolar macrophages display remarkable heterogeneity and play a crucial role in the development and control of septic acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Recent evidence suggests that α-ketoglutarate (α-KG) plays an important role in alternative activation of macrophage (M2) through metabolic and epigenetic reprogramming, and thus possesses anti-inflammatory properties. However, the underlying mechanisms of α-KG's effect on alveolar macrophage polarization and the potential effects of α-KG in ALI/ARDS remain unclear. Here, we examined the effects and mechanisms of α-KG on alveolar macrophage polarization, and investigated the possible effects of α-KG on lipopolysaccharide (LPS)-induced ALI/ARDS in a mouse model. We found that α-KG inhibited M1 macrophage polarization and promoted IL-4-induced M2 macrophage polarization in MH-S cells (a murine alveolar macrophage cell line). Further experiments showed that α-KG down-regulated the expression of M1-polarized marker genes and inhibited the activities of mammalian target of rapamycin complex 1 (mTORC1)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway in M1-polarized MH-S cells. Moreover, our results showed that α-KG promoted IL-4-induced M2 polarization of MH-S cells by augmenting nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ) and increasing expression of relevant fatty acid metabolic genes. Finally, using an LPS-induced ALI/ARDS mouse model, we found that α-KG ameliorated the LPS-induced inflammation and lung pathological damage, as well as α-KG pretreated mice had better clinical scores compared with the LPS group. These findings reveal new mechanisms of α-KG in regulating macrophage polarization which may provide novel strategies for the prevention and treatment of inflammatory diseases, including sepsis and septic ALI/ARDS.

HOXC10 promotes proliferation and attenuates lipid accumulation of sheep bone marrow mesenchymal stem cells

Homeodomain-containing gene C10 (HOXC10), known to regulate cell differentiation and proliferation, is a key negative regulator in the browning of white adipose tissue in mice. Sheep is an important farm animal that provides meat for human consumption, with fat content being an important meat quality determinant; however, there is no report about the role of HOXC10 in sheep adipocytes or adipogenesis. In this study, we investigated the effect of HOXC10 on proliferation and adipogenic differentiation in sheep bone marrow mesenchymal stem cells (sBMSCs). In sBMSCs, HOXC10 overexpression promoted cell proliferation and upregulated the expression of p-PI3K, p-AKT, p-p70S6K, p-MEK, and p-ERK, whereas HOXC10 knockdown was associated with the opposite effects. These results suggested that HOXC10 may promote cell proliferation by activating the MEK/ERK and PI3K/AKT/mTOR/p70S6K signaling pathways. In addition, we found that HOXC10 expression was negatively associated with lipid accumulation in adipogenic-differentiated sBMSCs. HOXC10 overexpression in sBMSCs significantly decreased lipid droplet accumulation and suppressed the expression of adipogenic-specific genes, including ACC, LPL, PPARG, and FABP4, while HOXC10 knockdown was associated with the opposite effects. Furthermore, our study suggested a new regulatory mechanism of the effect of HOXC10 on lipid accumulation and metabolism; HOXC10 may negatively regulate lipid accumulation in adipogenic-differentiated sBMSCs, at least in part, by suppressing LPL expression. Overall, our research not only contributes to a better understanding of the mechanism of lipid accumulation and metabolism in sheep, but also shed light on meat quality control in the future.

Effect of resistance training with Spirulina platensis on PI3K/Akt/mTOR/p70S6k signaling pathway in cardiac muscle

Summary Background Resistance training and spirulina change the expression of genes involved in the hypertrophy. We investigated the effect of resistance training with Spirulina platensis on PI3K/Akt/mTOR/p70S6k signaling pathway in rat cardiac muscle. Methods For this purpose, 32 male Sprague–Dawley rats were divided into four groups: control (Co; n = 8), spirulina platensis (SP; n = 8), resistance training (RE; n = 8), spirulina platensis + resistance training (SP +RE; n = 8). The resistance training group practiced five sessions each week for eight weeks. Spirulina 200 mg/kg/day was used in the supplement group and exercise + spirulina platensis group. Twenty-four hours after the last training session, the expression of genes was measured by Real-time PCR. Results Results showed the expression of mTOR increased significantly in the RE group (P = 0.001) and the SP +RE (P = 0.02). The expression of Akt in the RE group increased significantly (P = 0.01). The expression of Pi3k in the RE group (P = 0.001) and the SP+ RE group (P = 0.001) increased significantly. The expression of P70-S6K in the RE group increased significantly (P = 0.05). Conclusion Using spirulina platensis alone does not have an effect on the signaling pathway in the heart. However, the combination of resistance training can affect some of the components of the signaling pathway (PI3K/mTOR). And also, the effect of resistance training alone can have a significant effect on all components of the signaling pathway.

Elevated p70S6K phosphorylation in rat soleus muscle during the early stage of unloading: Causes and consequences

Currently, there is a lack of investigation into the initial signaling events underlying the development of disuse muscle atrophy. The study was aimed to (i) identify an assumed relationship between AMPK dephosphorylation and p70S6K hyperphosphorylation in the initial period of hindlimb unloading (HS), and (ii) assess the signaling consequences of p70S6K hyperphosphorylation following 24-h HS. For experiment 1, rats were treated with AMPK activator (AICAR) for 6 d before HS as well as during 24-h HS. For experiment 2, rats were treated with mTORC1 inhibitor rapamycin during 24-h HS. The key signaling markers implicated in protein turnover were assessed using WB and RT-PCR. One-day HS resulted in a significant upregulation of MuRF-1 and MAFbx expression, increase in p70S6K (Thr389) and IRS-1 (Ser639) phosphorylation and a significant decrease in phosphorylated AMPK, AKT, FOXO3, total IRS-1 content, and HDAC5 nuclear content. AMPK and p70S6K phosphorylation did not differ from control in AICAR-treated unloaded rats. Rapamycin treatment during unloading abolished p70S6K and E3 ligases upregulation and increased HDAC5 nuclear accumulation. The results of the study suggest that mTORC-1/p70S6K signaling pathway in rat soleus muscle is activated following 24-h mechanical unloading. This activation is facilitated by a decrease in AMPK phosphorylation. Increased p70S6K activity at the initial stage of hindlimb unloading could lead to the upregulation of E3 ligases MAFbx/atrogin-1 and MuRF-1 via nuclear export of HDAC5.

Helium low temperature plasma induced HepG2 cells autophagy through ROS-mediated PI3K/AKT/mTOR/P70s6k signaling pathway

Low temperature plasma (LTP) has emerged as a new research hotspot of promising therapy to fight against cancer. Autophagy is a multifunctional process that digests and recycles cellular contents within lysosomes to maintain homeostasis, which is the underlying mechanism of cancer cell death and anti-cancer treatment. This study aimed at investigating whether autophagy of HepG2 cells could be induced by LTP and revealing the related molecular mechanism. For this purpose, the atmospheric pressure plasma jet (APPJ) in helium was utilized to generate plasma-actived medium (PAM) to treat HepG2 cells in vitro. Catalase (CAT), superoxide dismutase (SOD) and 3-methyladenine (3-MA) were respectively added into the PAM as the intervention group. The cell viability, formation of autophagosomes, intracellular reactive oxygen species (ROS) level, expression levels of autophagy-associated proteins and key proteins involved in PI3K/AKT/mTOR pathways were detected. The results showed that LTP inhibited cell viability in a dose- and time-dependent manner. Autophagy was induced through the formation of autophagosomes, conversion of LC3-II/LC3-I, increased expression of beclin 1 and degradation of p62. The mechanism was deduced that LTP enhanced the intracellular ROS level and decreased the phosphorylation level of key proteins in PI3K/AKT/mTOR/p70S6K pathway. However, these effects were blocked by the autophagy inhibitor 3-MA and ROS scavengers (CAT and SOD). The therapeutic efficacy of LTP against HepG2 cells may involve autophagy via suppressing PI3K/AKT/mTOR signaling pathway by LTP-mediated ROS.

TRP14 promotes resistance to cisplatin by inducing autophagy in ovarian cancer

Cisplatin is a common chemotherapeutic agent against ovarian cancer; however, drug resistance is a major limiting factor for its use in clinical treatment. The underlying mechanisms of cisplatin resistance in ovarian cancer have not yet been fully elucidated. Thus, this study aimed to elucidate some of the mechanisms responsible for resistance to cisplatin in ovarian cancer. The results demonstrated that the cisplatin-resistant human ovarian cancer cell lines, SKOV3/DDP and A2780/DDP, exhibited higher autophagy levels than the control ovarian cancer cell lines, SKOV3 and A2780. Moreover, autophagy inhibition by 3-methyladenine or shRNA against autophagy-related gene (ATG)5 potentiated the cytotoxicity induced by cisplatin, whereas autophagy induction by rapamycin (Rapa) increased cell survival. Exposure to cisplatin induced an upregulation in the expression of thioredoxin-related protein of 14 kDa (TRP14). Furthermore, TRP14 knockdown or overexpression decreased or increased the autophagy response and cisplatin resistance, and this effect was reversed by treatment with Rapa or ATG5 knockdown. The findings of this study also suggested that TRP14 induced autophagy and chemoresistance via the 5′AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/p70S6K signaling pathway. Importantly, the data from a tissue array revealed a positive association between TRP14 and Beclin1 in human ovarian cancer and marginal tissues. These findings have identified, for the first time, to the best of our knowledge, that TRP14 induces autophagy and consequently cisplatin resistance in ovarian cancer cells via the AMPK/mTOR/p70S6K signaling pathway. This in turn renders TRP14 as a potential predictor or target in ovarian cancer therapy.

Angiotensin II Receptor Type 1 Antagonists Modulate Vascular Smooth Muscle Cell Proliferation and Migration via AMPK/mTOR

The aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) in the vascular wall are crucial pathological events involved in cardiovascular impairments including hypertension, heart failure, and atherosclerosis. At the molecular level, the mammalian target of rapamycin (mTOR)-ribosomal protein S6 kinase beta-1 (p70S6K) signaling pathway is essential to potentiate VSMC proliferation and migration. Although angiotensin II receptor type 1 ­(AT1-R) antagonists such as valsartan and telmisartan have a significant cardiovascular protective effect, the molecular basis of this class of drugs in VSMC proliferation and migration remains elusive. By using cultured VSMCs, adenosine monophosphate-activated protein kinase (AMPK) α2 knockout mice, and hypertensive rat models, this study investigated whether AT1-R antagonists can inhibit the mTOR-p70S6K signaling pathway in VSMCs and the vascular wall. Valsartan activated AMPK, which in turn suppressed reactive oxygen species production and consequently attenuated VSMC proliferation and migration. In vivo, a clinical dose of telmisartan significantly inhibited the mTOR-p70S6K signaling pathway in the vascular wall of wild-type but not AMPKα2^–/– mice. Furthermore, spontaneously hypertensive rats had significantly elevated phosphorylation of mTOR and p70S6K in the aorta compared to Wistar-Kyoto rats, which were reduced by telmisartan administration. These data suggest that AT1-R antagonists inhibit VSMC proliferation and migration via their regulation of AMPK, mTOR, and p70S6K, which contribute to the cardioprotective effects of these drugs.

Inositol Deficiency Induces Autophagy Impairing via PI3K/Akt/mTOR/p70S6K Signaling (P11-032-19)

ObjectivesOur previous case-control study found that maternal MI deficiency was associated with an increased risk of NTDs. Bioinformatics analysis showed that PI3K/Akt/mTOR/p70S6K signaling pathway might be one of the important regulatory mechanisms of inositol deficiency-induced NTDs. So, we intended to explore the possible mechanisms of PI3K/Akt/mTOR/p70S6K signaling in inositol deficiency-induced neural tube defects (NTDs) in this study. MethodsThe activity of the PI3-kinase in MI deficiency NE-4C cells was detected by the PI3-kinase ELISA kit. Using the method of western blot, we analyzed the activity of the PI3K/Akt/mTore/p70S6K signaling pathway and the level of LC3B. And the LC3B levels were also detected by electron microscopy. ResultsThe result showed that PI3K activity was significantly higher in the Li2CO3-treated group than in the control cells (P < 0.05), and the increased kinase activity was abolished by additional treatment with MI (P < 0.05). We found that PI3K activity decreased dramatically with the increasing MI concentration in a dose-dependent manner when the inositol concentration was below 5 mg/L in vitro enzymology experiment. The result showed that the PI3K/Akt/mTOR/p70S6K signaling pathway significant activated in MI deficient NE-4C cells compare with the controls. We found that the autophagy was significantly impaired by Li2CO3 treatment in NE-4C cells, and the effects were abrogated by combining Li2CO3 with MI or signaling inhibitor (Ly294002 and Rapamycin). Which suggested that MI deficiency induced autophagy impairment through the Akt/mTOR/p70S6K signaling. We next validate the above result in our previous established MI deficiency mouse model and found PI3K activity was significantly elevated in heart, neural and placenta tissues 8 hours after Li2CO3 injection. The maternal serum MI levels correlated with the PI3K activity in the mouse embryonic neural tissue (R = –0.817, P < 0.05). And the activity of Akt/mTOR/p70S6K signaling was over activated accompanied by the reduced level of LC3B. ConclusionsThese results showed that inositol deficiency activated PI3K/Akt/mTOR/p70S6K signaling, thus causing impaired autophagy. Funding SourcesThis study was supported by The National Key Basic Research Program(2018YFC1002500), National Nature Science Foundation of China (81571443 to JW, 81801451 to JG).

Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF-α-Smurf1-Akt-mTOR-P70S6K pathway.

Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial‐mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK‐2) treated with tumour necrosis factor‐alpha (TNF‐α) in various doses or at various time points by Western Blotting or qRT‐PCR. We knockdown or overexpressed Smurf1 in HK‐2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF‐α significantly promoted the development of EMT in a time‐dependent and dose‐dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF‐α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF‐α. Bortezomib can attenuate the Sumrf1‐mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.

428 Itraconazole inhibits the PDGF-D/PI3K/Akt/mTOR pathway in vitro and down-regulates serum PDGF-A of infantile hemangioma via oral administration

The incidence of infantile hemangiomas (IHs) is 8-10% in children. We found that itraconazole, a common anti-fungal agent, can improve or cure IH for the first time. However, the underlying molecular mechanisms are still unclear. Our research showed that itraconazole treatment significantly inhibits the proliferation and promotes apoptosis of the endothelial cells of mouse hemangioma (EOMA) cell line and infantile primary hemangioma endothelial cell (HemEC). Itraconazole also remarkably reduce angiogenesis of HemEC in vitro. We performed transcriptome profiling via mRNA microarrays in HemEC cells upon itraconazole treatment, and identified cytokine-cytokine receptor interaction as the top significantly enriched pathway. Importantly, itraconazole significantly reduced platelet-derived growth factor D (PDGF-D) level, resulting in suppression of PDGFR-β activation and inhibition of its downstream effectors, such as PI3K, Akt, 4E-BP1, p70S6K and mTOR signaling pathway, which are important for cellular growth and survival of IH. Oral itraconazole solution with 5mg/kg/d was administrated in 17 children, aging from 1 to 12 months (average 5.4 months), weighing from 5 to 10.5 kg. The blood samples collected were used to detect the concentration of serum vascular endothelial growth factor (VEGF) and PDGF-A by ELISA kits. The treatment period varied from 13 to 63 days, with total dose of itraconazole 390-2250 mg. Serum PDGF-A concentration was 52879.92 ± 31435.65 pg/mL before treatment and 26289.34 ± 26095.77 pg/mL after treatment (p<0.05), whereas VEGF showed no statistical difference (p>0.05). Both in vitro and in vivo data suggest that PDGF (at least A and D) is a novel target of itraconazole in IH, which need further investigations.

Modafinil protects hippocampal neurons by suppressing excessive autophagy and apoptosis in mice with sleep deprivation.

Sleep deprivation compromises learning and memory in both humans and animals, and can be reversed by administration of modafinil, a drug promoting wakefulness. Dysfunctional autophagy increases activation of apoptotic cascades, ultimately leading to increased neuronal death, which can be alleviated by autophagy inhibitors. This study aimed to investigate the alleviative effect and mechanism of modafinil on the excessive autophagy occurring in the hippocampus of mice with deficiency of learning and memory induced by sleep deprivation. The Morris water maze was used to assess the effects of modafinil on male C57BL/6Slac mice after 48-hr sleep deprivation. The HT-22 hippocampal neuronal cell line was also used. Nissl staining, transmission electron microscope, immunofluorescence, Western blot, transient transfection, and autophagy inducer were used to study the effect and mechanism of modafinil on hippocampal neurons with excessive autophagy and apoptosis. Modafinil improved learning and memory in sleep-deprived mice, associated with the inhibition of excessive autophage and apoptosis and an enhanced activation of the PI3K/Akt/mTOR/P70S6K signalling pathway in hippocampal neurons. These effects of modafinil were abolished by rapamycin. In addition, modafinil suppressed the aberrant autophagy and apoptosis induced by rapamycin and reactivated PI3K/Akt/mTOR/P70S6K signals in HT-22 cells. These results suggested that modafinil alleviated impaired learning and memory of sleep-deprived mice potentially by suppressing excessive autophagy and apoptosis of hippocampal neurons. This novel mechanism may add to our knowledge of modafinil in the clinical treatment of impaired memory caused by sleep loss.

Differential gene expression analysis of sickle cell anemia in steady and crisis state.

Sickle cell anemia is one of the most prevalent genetic diseases worldwide, showing great clinical heterogeneity. This study compared the gene expression patterns between sickle cell anemia pediatric patients in steady state and in crisis state, as compared to age-paired, healthy individuals. RNA sequencing was performed from these groups of patients/controls using Illumina HiSeq 2500 equipment. The resulting differentially expressed genes were loaded into QIAGEN's ingenuity pathway analysis. The results showed that EIF2 pathway and NRF2-mediated oxidative stress-response pathways were more highly activated both in steady state and in crisis patients, as compared to healthy individuals. In addition, we found increased activation of eIF4 and p70S6K signaling pathways in crisis state compared to healthy individuals. The transcription factor GATA-1 was found exclusively in steady state while SPI was found exclusively in crisis state. IL6 and VEGFA were found only in crisis state, while IL-1B was found exclusively in steady state. The regulator effects analysis revealed IgG1 as an upstream regulator in steady state compared to healthy individuals, resulting in invasion of prostate cancer cell lines as the disease/function outcome. For crisis-state patients versus healthy individuals, two networks of regulator effects revealed STAT1, CD40LG, TGM2, IRF7, IRF4, and IRF1 acting as upstream regulators, resulting in disease/function outcomes, including engulfment of cells and aggregation of blood cells and inflammation of joints. Our results indicated genes and pathways that can provide clues on the molecular events involved in the severity of sickle cell disease.

Astragaloside IV ameliorates high glucose‑induced renal tubular epithelial‑mesenchymal transition by blocking mTORC1/p70S6K signaling in HK‑2 cells.

Astragaloside IV (AST) is the major active saponin in Astragalus membranaceus and, reportedly, has a variety of pharmacological activities. However, the potential of AST to ameliorate high glucose-mediated renal tubular epithelial-mesenchymal transition (EMT) remains undetermined. The aim of the present research was to explore the effect and mechanism of AST in EMT of renal tubular epithelial cells, as an underlying mechanism of renal fibrosis and a vital feature involved in diabetic nephropathy. The effect of AST on the EMT of renal tubular epithelial cells (HK-2) stimulated by high glucose was investigated and it was attempted to elucidate the potential underlying mechanism. The expression of E-cadherin and α-smooth muscle actin were determined by western blotting and immunofluorescence assays. The expression of the mammalian target of rapamycin complex 1 (mTORC1)/ ribosomal protein S6 kinase β-1 (p70S6K) signaling pathway and protein levels of four transcriptional factors (snail, slug, twist and zinc finger E-box-binding homeobox 1) were also determined by western blotting. Additionally, extracellular matrix components, including fibronectin (FN) and collagen type IV (Col IV) were detected by ELISA. The results suggested that the EMT of HK-2 cells and the mTORC1/p70S6K pathway were activated by high glucose. The expression of snail and twist in HK-2 cells was elevated by high glucose. Furthermore, extracellular matrix components, FN and Col IV, were increased in HK-2 cells cultured with high glucose. In turn, treatment with AST reduced EMT features in HK-2 cells, inhibited mTORC1/p70S6K pathway activation, downregulated expression of snail and twist, and reduced secretion of FN and Col IV. In summary, the findings suggested that AST ameliorates high glucose-mediated renal tubular EMT by blocking the mTORC1/p70S6K signaling pathway in HK-2 cells.

Transforming growth factor beta regulates hepatic progenitor cells migration via PI3K/AKT/mTOR/p70S6K pathway

Objective: To investigate the effect and mechanism of transforming growth factor β (TGFβ) on the migration ability of hepatic progenitor cells in vitro. Methods: Primary hepatic progenitor cells of male wild-type C57BL/6J mice were isolated by two-step perfusion method and stimulated with different concentrations of TGFβ .The morphological changes were observed under phase -contrast microscopy. The effects of TGFβ on migration ability of hepatic progenitor cells were evaluated by scratch test and transwell method. Expression profiling and signaling phospho antibody array detected the signaling pathways involved in the regulation of TGFβ on hepatic progenitor cells. Protein level of PI3K/AKT/mTOR/p70S6K signaling pathway and the localization of each signaling molecules in hepatic progenitor cells were detected. Data comparison between the two groups was performed by independent sample t-test. One-way ANOVA was used for data comparison between multiple groups. Results: TGFβ made the liver progenitor cells from oval to long spindle type. Scratch test showed that the scratch healing rates of 24 h control group, and 2 ng/ml and 10 ng/ml TGF-beta groups were 36.48% ± 4.37%, 57.35% ± 4.60%, and 73.14% ± 5.02% (F = 65.87, P < 0.01), respectively. Transwell test showed that the number of migrating cells in 24 h control group, 2 ng/ml and 10 ng/ml TGF-beta groups were 127 ± 16, 230 ± 18, and 385 ±36 (F = 94.99, P < 0.01), respectively. The results of expression profiling showed that TGFβ regulates gene expression in hepatic progenitor cells, and differentially expressed genes participate in the PI3K-AKT signaling pathway. Signaling phospho antibody array and western blot showed that TGFβ regulated PI3K/AKT/mTOR/p70S6K signaling pathway in hepatic progenitor cells. Concurrently, immunofluorescence assay showed phosphorylation (p) 70s6k, p AKT1 and PI3K and F-actin co-localizations. Conclusion: TGFβ can promote hepatic progenitor cell migration through PI3K/AKT/mTOR/p70S6K pathway, and p70S6K, pAKT1 and PI3K signaling molecules are involved in the regulation of morphology and migration of liver progenitor cells.

Downregulation of long noncoding RNA Sox2ot protects PC-12 cells from hydrogen peroxide-induced injury in spinal cord injury via regulating the miR-211-myeloid cell leukemia-1 isoform2 axis.

This study aimed to investigate the effects and possible mechanisms of long noncoding RNA (lncRNA) Sox2 overlapping transcript (Sox2ot) on hydrogen peroxide (H2 O2 )-induced injury in pheochromocytoma (PC-12) cells. PC-12 cells were treated with H2 O2 to cell injury. The cells were transfected with short-hairpin RNA directed against Sox2ot (sh-Sox2ot), small interfering RNA directed against myeloid cell leukemia-1 (MCL-1) isoform2 (si-MCL-1), a miR-211 mimic, a miR-211 inhibitor, and their negative controls. Under different transfected treatments, cell viability, migration, invasion, and apoptosis as well as the expressions of apoptosis- and autophagy-related proteins were investigated. Besides, the regulatory relationships between Sox2ot and miR-211, miR-211 and MCL-1, as well as between MCL-1 and the protein kinase B (Akt)/mammalian target of the rapamycin (mTOR)/p70 ribosomal S6 protein kinase (p70S6K) signaling pathway were explored. Suppression of Sox2ot inhibited H2 O2 -induced PC-12 cell injury by increasing cell viability, migration, invasion, and decreasing apoptosis and autophagy. Moreover, suppression of Sox2ot increased miR-211 expression and alleviated H2 O2 -induced injury in PC-12 cells possibly via upregulation of miR-211. Furthermore, MCL-1 isoform2 was identified as a direct target of miR-211 and could be negatively regulated by miR-211. Suppression of miR-211 aggravated H2 O2 -induced cell injury by regulation of MCL-1 isoform2. Besides, inhibition of miR-211 suppressed the activation of the Akt/mTOR/p70S6K signaling pathway in H2 O2 -treated PC-12 cells, which was reversed after knockdown of MCL-1 isoform2 at the same time. Our findings indicate that downregulation of Sox2ot may protect PC-12 cells from H2 O2 -induced injury in SCI via targeting the miR-211/MCL-1 isoform2 axis. MCL-1 isoform2 may further regulate the activation of the Akt/mTOR/p70S6K pathway to mediate H2 O2 -induced injury. The Sox2ot-miR-211-MCL-1 isoform2 axis may be a promising therapeutic strategy for SCI.

Rutacecarpine Inhibits Angiogenesis by Targeting the VEGFR2 and VEGFR2-Mediated Akt/mTOR/p70s6k Signaling Pathway.

This study aimed to investigate the effect of Ru (Rut) on angiogenesis, and the underlying regulation mechanism of signal transduction. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, adhesion inhibition experiment, migration inhibition experiment, and chick embryo chorioallantoic membrane (CAM) assays were performed on models of angiogenesis. The potential targets of rutaecarpine (Ru) were reverse screened with Discovery Studio 2017. The interaction between the compound and target were detected by surface plasmon resonance (SPR), enzyme-activity experiment, and Western blot assay. The obtained results confirmed that Ru exhibited modest inhibitory activity against human umbilical vein endothelial cells (HUVECs) (IC50 =16.54 ± 2.4 μM) and remarkable inhibitive effect against the migration and adhesion of HUVECs, as well as significant anti-angiogenesis activities in the CAM assay. The possible targets of vascular endothelial growth factor receptor 2 (VEGFR2) were identified by computer-aided simulation. Results showed a good binding relationship between the ligand and target through molecular docking, and this relationship was confirmed by SPR analysis. Furthermore, enzyme-activity experiment and western blot assay showed that Ru remarkably inhibited the activity of VEGFR2 and blocked the VEGFR2-mediated Akt/ (mTOR)/p70s6k signaling pathway in vitro. Ru can be a potential drug candidate for cancer prevention and cancer therapy.

Down-regulation of Rictor enhances cell sensitivity to PI3K inhibitor LY294002 by blocking mTORC2-medicated phosphorylation of Akt/PRAS40 in esophageal squamous cell carcinoma

PI3K/Akt/mTOR signaling pathway plays a vital role in regulating cell survival, differentiation, metabolism and migration, which is frequently hyperactive in a number of cancers, including esophageal squamous cell carcinoma (ESCC). As the core subunit of mTORC2, Rictor is shown to be amplified in ESCC patients' tissues and plays an important role in regulation of Akt. The objective of this study is to evaluate the effects of Rictor knockdown on cell sensitivity to PI3K inhibitor LY294002 in ESCC cells and ESCC xenografts as well as its mechanisms. We found LY294002 obviously restrained cell proliferation in dose-dependent and time-dependent manners by inhibiting PI3K/Akt/mTOR/p70S6K signaling pathway, whereas triggered mTORC2-medicated phosphorylation of Akt (Ser473)/PRAS40 (Thr246) in ECa109 and EC9706 cells. Stable knockdown of Rictor by shRNA enhanced the inhibitory effects of LY294002 on cell proliferative, migration and colony formation, as well as promoted its effects on cell cycle arrest and cell apoptosis in vitro. Furthermore, stable knockdown of Rictor enhanced the antitumor effects of LY294002 by inhibiting tumor growth and promoting cell apoptosis in vivo. Mechanistic assay revealed that knockdown of Rictor could attenuate LY294002-induced phosphorylation of Akt (Ser473)/PRAS40 (Thr246). Our results provide rationale that combined inhibition of Rictor/mTORC2 and PI3K for the treatment of ESCC.

Asiatic acid inhibits proliferation, migration and induces apoptosis by regulating Pdcd4 via the PI3K/Akt/mTOR/p70S6K signaling pathway in human colon carcinoma cells.

Previous studies have demonstrated that asiatic acid (AA), the major component of Centella asiatica, is able to meditate cytotoxic and anticancer effects on various types of carcinoma cells. In order to investigate the molecular mechanism that underlies the antitumor effect of AA, the present study investigated the effects of AA on proliferation, migration and apoptosis of SW480 and HCT116 colon cancer cells. Viability and changes in cell morphology in the cells were assessed by MTT assay and transmission electron microscopy, respectively. Colony formation analysis was used to observe proliferation of the single cell, and migratory ability of the cells was assessed by performing Transwell migration assay. Hoechst 33342 nuclear staining and flow cytometry were used to assess apoptosis in colon carcinoma cells. The expression of proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K signaling pathway and epithelial-mesenchymal transition (EMT) marker were analyzed by western blotting. The present study revealed that proliferation and migration of colon carcinoma cells were inhibited by AA in a dose-dependent and time-dependent manner. Numerous apoptotic bodies were observed, and G2/M and S phase progression were delayed in colon cancer cells treated with AA, but not in the control group. A number of phosphorylated proteins, including PI3K, Akt (Ser473), mTOR, ribosomal protein S6 kinase (p70S6K) downregulated, while the expression of Pdcd4 was upregulated following treatment with AA. Additionally, AA affects expression of EMT markers in a dose-dependent manner. On the basis of these results, it was concluded that AA inhibited proliferation, migration and induced apoptosis of colon cancer cells by regulating Pdcd4 via the PI3K/Akt/mTOR/p70S6K signaling pathway. These observations suggest that AA may be a potential therapeutic agent for the treatment of colon carcinoma.

Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/Akt/mTOR/P70S6K and ERK signaling pathways

Ghrelin is a gastric acyl-peptide that plays an important role in cell proliferation. In the present study, we explored the role of ghrelin in A549 cell proliferation and the possible molecular mechanisms. We found that ghrelin promotes A549 cell proliferation, knockdown of the growth hormone secretagogue receptor (GHSR) attenuated A549 cell proliferation caused by ghrelin. Ghrelin induced the rapid phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, ERK, mammalian target of rapamycin (mTOR) and P70S6K. PI3K inhibitor (LY 294002), ERK inhibitor (PD98059) and mTOR inhibitor (Rapamycin) inhibited ghrelin-induced A549 cell proliferation. Moreover, GHSR siRNA inhibited phosphorylation of PI3K, Akt, ERK, mTOR and P70S6K induced by ghrelin. Akt and mTOR/P70S6K phosphorylation was inhibited by LY 294002 but not by PD98059. These results indicate that ghrelin promotes A549 cell proliferation via GHSR-dependent PI3K/Akt/mTOR/P70S6K and ERK signaling pathways.

ILT3.Fc-CD166 Interaction Induces Inactivation of p70 S6 Kinase and Inhibits Tumor Cell Growth.

The blockade of immune checkpoints by anti-receptor and/or anti-ligand mAb is one of the most promising approaches to cancer immunotherapy. The interaction between Ig-like transcript 3 (ILT3), a marker of tolerogenic dendritic cells, also known as LILRB4/LIR5/CD85k, and its still unidentified ligand on the surface of activated human T cells is potentially important for immune checkpoint blockade. To identify the ILT3 ligand, we generated mAb by immunizing mice with Jurkat acute T cell leukemia, which binds ILT3.Fc to its membrane. Flow cytometry, mass spectrometry, and Biacore studies demonstrated that the ILT3 ligand is a CD166/activated leukocyte cell adhesion molecule. Knockdown of CD166 in primary human T cells by nucleofection abolished the capacity of ILT3.Fc to inhibit CD4+ Th cell proliferation and to induce the generation of CD8+CD28- T suppressor cells. CD166 displays strong heterophilic interaction with CD6 and weaker homophilic CD166-CD166 cell adhesion interaction. ILT3.Fc inhibited the growth of CD166+ tumor cell lines (TCL) derived from lymphoid malignancies in vitro and in vivo. CRISPR-Cas9-based knockout of CD166 from TCL abrogated ILT3.Fc binding and its tumor-inhibitory effect. The mechanism underlying the effect of ILT3.Fc on tumor cell growth involves inhibition of the p70S6K signaling pathway. Blockade of CD166 by ILT3.Fc inhibited progression of human TCL in NOD.Cg-Prkdc Il-2rg/SzJ mice, suggesting its potential immunotherapeutic value.