70-kDa Ribosomal Protein S6 Kinase Research Articles

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

Glucose Regulates Glucose Transport and Metabolism via mTOR Signaling Pathway in Bovine Placental Trophoblast Cells.

It has been confirmed that improving the energy level of the diet contributed to the greater reproductive performance and birth weight of calves in periparturient dairy cows. To investigate the effect of glucose on nutrient transport during fetal development, the bovine placental trophoblast cells (BPTCs) were cultured in media with different glucose concentrations (1, 2, 4, 8, or 16 mg/mL). Subsequently, the BPTCs were cultured in media with 1, 8 mg/mL glucose and 8 mg/mL glucose plus 100 nmol/L rapamycin (the inhibitor of mTOR pathway). Compared with the 1 mg/mL glucose, the addition of 8 mg/mL glucose stimulated cell proliferation, upregulated the mRNA abundance of the glucose transporter GLUT1 and GLUT4, and increased the activity of glucose metabolism-related enzyme glucose-6-phosphate dehydrogenease (G6PD), lactate dehydrogenase (LDHA) and phosphoglycerate kinase 1 (PGK1), as well as adenosine-triphosphate (ATP) content (p < 0.05).Furthermore, compared with the treatment of 1 mg/mL glucose, adding 8 mg/mL of glucose-upregulated gene expression in the mTOR signaling pathway, including phosphatidylinositol3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase 2 (P70S6K) (p < 0.05).The supplementation of rapamycin downregulated the gene and protein expression of the mTOR signaling pathway, including mTOR, P70S6K, EIF4E-binding protein 1 (4EBP1), hypoxia-inducible factor 1-alpha (HIF-1α) and gene expression of glucose transporter upregulated by 8 mg/mL glucose (p < 0.05). Thus, these results indicated that the addition of 8 mg/mL glucose regulated the glucose transport and metabolism in BPTCs through the mTOR signaling pathway, thereby promoting the supply of nutrients to fetus.

Insulin, dibutyryl-cAMP, and glucose modulate expression of patatin-like domain containing protein 7 in cultured human myotubes.

Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces Pnpla7 expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed PNPLA7 mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of α1-subunit of Na+,K+-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed PNPLA7 mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed PNPLA7 mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.

Ethoxysanguinarine directly targets CIP2A to inhibit proliferation and induce autophagy of SGC7901/DDP cells

This study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stability(DARTS) assay. The pharmacokinetic properties and drug-like activity of Eth were predicted by SwissADME. The results indicated that SGC7901/DDP cells were more sensitive to Eth than SGC7901 cells. Eth significantly inhibited proliferation and colony formation and changed the morphology, roundness, and area of SGC7901/DDP cells. Eth treatment caused the nucleus shrinking and significantly increased the apoptosis rate of the cells. Furthermore, Eth down-regulated the expression of caspase-9 and caspase-3 precursors and promoted the cleavage of PARP, which suggested that Eth induced the apoptosis of SGC7901/DDP cells. The GFP-LC3 in Eth-treated cells showed speckled aggregation. The up-regulated expression of LC3-Ⅱ by Eth indicated that Eth activated the autophagy of SGC7901/DDP cells. Eth down-regulated the expression of P-gp, the phosphorylation of mTOR, P70 S6K, and 4E-BP1, the expression of CIP2A, and the phosphorylation of Akt. Additionally, it increased the activity of PP2A, and had no significant effect on the expression of CIP2A in SGC7901/DDP cells. CIP2A overexpression antagonized the inhibition of cell proliferation and the activation of autophagy by Eth. Molecular docking suggested that Eth bound to CIP2A. The results of DARTS assay further proved the above binding effect. Eth has potential drug-like activity. The above results demonstrated that Eth inhibited the proliferation, induced the apoptosis, and activated the autophagy of SGC7901/DDP cells by targeting CIP2A and then down-regulating PP2A/mTORC1 signaling pathway. This study provided a new target for the treatment of cisplatin-resistant gastric cancer.

Medicinal Chemistry of Pyrazolopyrimidine Scaffolds Substituted with Different Heterocyclic Nuclei.

Medicinal chemistry of pyrazolopyrimidine scaffolds substituted with different heterocyclic nuclei has attracted great attention due to their wide range of biological activities that have been reported. Pyrazolopyrimidine scaffold is an important privileged heterocycle nucleus in drug discovery. All pharmacological activities of pyrazolopyrimidine scaffold have been mentioned, such as anticancer, anti-inflammatory, antihypertensive, antitubercular, antiviral, antibacterial, antifungal, antidiabetic, and anti-obesity agents. In addition, it was used in both osteoporosis and neurological disorders. The difference in potency and bioavailability of pyrazolopyrimidine derivatives refers to the substituent groups that can increase the activity against specific targets and enhance their selectivity. This review provides an overview of different synthetic pathways, structure activity relationships, and preclinical studies of pyrazolopyrimidine scaffolds substituted with a variety of heterocyclic nuclei, as well as it provides a discussion on the significant biological findings of these important scaffolds. In addition, it provides some insights on the different macromolecular targets that pyrazolopyrimidine scaffold can effectively work on, such as; cyclin dependent kinases; CDK2, CDK7, and CDK9, checkpoint kinases; CHK1 and CHK2 and their correlation with the anticancer activity, PI3Kα, transient receptor potential canonical 6, B-Raf kinase, Interleukin- 1 receptor-associated kinase 4, B-cell lymphoma 6, TRKA-C kinase, potent kDa ribosomal protein S6 kinase, colon cancer cell line (CaCo-2), domain receptor kinase (KDR), HepG-2 carcinoma cell, FLT3. The antibacterial activity against B. subtilis and E. coli and antifungal activity against C. albicans, C. tropicalis, A. niger, and A. clavatus are discussed. This review provides an overview of the different pharmacological activities of the pyrazolopyrimidine scaffold and its correlation with chemical structure. Some exciting new developments in pyrazolopyrimidine scaffolds are also presented in this review.

Targeting Glutamine Metabolism Ameliorates Autoimmune Hepatitis via Inhibiting T Cell Activation and Differentiation.

BackgroundAutoimmune hepatitis (AIH) is mediated by a cascade of T cell-mediated events directed at liver cells and persistent inflammation within the liver can eventually result in liver cirrhosis. Targeting glutamine metabolism has an impact on T cell activation and differentiation. However, the effect of glutamine metabolism blocking upon AIH remains unknown. We use glutaminase antagonist 6-diazo-5-oxo-L-norleucine (DON) for in vitro assays and its prodrug 2-(2-amino-4-methylpentanamido)-DON (JHU083) for in vivo assays to investigate the potential therapeutic effect and molecular mechanism of glutamine metabolism blocking in an AIH murine model.MethodsAIH mice were treated with JHU083 or vehicle before concanavalin A (ConA) administration, and disease severity was examined. Then activation and differentiation [including Th1/Th17 cells and cytotoxic T lymphocytes (CTL)] of T cells from Vehicle-WT, JHU083-AIH and Vehicle-AIH mice were tested. Furthermore, in vitro T cell activation and differentiation were measured using separated splenocytes stimulated with ConA with or without DON. The activation and differentiation of T cells were tested using flow cytometry, qRT-PCR and ELISA. Phosphorylation level of mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase (P70S6K) were examined by western blotting.ResultsJHU083 and DON significantly suppressed the activation of T cells and inhibited the differentiation of Th1/Th17 cells and CTL in vivo and in vitro. Besides, we demonstrated that glutamine metabolism blocking inhibited T cells activation and differentiation through decreasing the mRNA expression of amino acid transporter solute carrier family 7 member 5 (SLC7A5) and mitigating the activation of mTOR signaling.ConclusionsWe proved that targeting glutamine metabolism represents a potential new treatment strategy for patients with AIH and other T cell-mediated disease. Mechanistically, we demonstrated that glutamine metabolism blocking inhibits T cells activation and suppresses the differentiation of Th1/Th17 cells and CTL.

Mesenchymal Stem Cell-Derived Neuron-Like Cell Transplantation Combined with Electroacupuncture Improves Synaptic Plasticity in Rats with Intracerebral Hemorrhage via mTOR/p70S6K Signaling.

Previous studies have shown that the combination of mesenchymal stem cell (MSC) transplantation and electroacupuncture (EA) stimulation is a neuroprotective strategy for treating intracerebral hemorrhage (ICH). However, the underlying mechanisms by which the combined treatment promotes neuroprotection remain unclear. This study was designed to investigate the effects of the combined treatment on synaptic plasticity and elucidate their underlying mechanisms. Therefore, rat ICH models were established by injecting collagenase and heparin, and the animals were randomly divided into model control (MC), EA stimulation (EA), MSC-derived neuron-like cell transplantation (MSC-dNLCs), and MSC-dNLC transplantation combined with EA stimulation (MSC-dNLCs+EA) groups. We observed the ultrastructure of the brain and measured the brain water content (BWC) and the levels of the microtubule-associated protein 2 (MAP2), galactocerebrosidase (GALC), and glial fibrillary acidic protein (GFAP) proteins. We also measured the levels of the phosphorylated mammalian target of rapamycin (mTOR) and 70 kDa ribosomal protein S6 kinase (p70S6K) proteins, as well as the expression of synapse-related proteins. The BWC increased in rats after ICH and decreased significantly in ICH rats treated with MSC-dNLC transplantation, EA stimulation, or combined therapy. Meanwhile, after ICH, the number of blood vessels increased more evidently, but only the combined treatment reduced the number of blood vessels among rats receiving the three treatments. Moreover, the levels of MAP2, GALC, postsynaptic density 95 (PSD95), and synaptophysin (SYP) proteins, as well as the levels of the phosphorylated mTOR and p70S6k proteins, increased in the MSC-dNLCs+EA group compared with those in the MSC-dNLCs and EA groups. Compared with the MC group, GFAP expression was significantly reduced in the MSC-dNLCs, EA, and MSC-dNLCs+EA groups, but the differences among the three treatment groups were not significant. In addition, the number of synapses increased only in the MSC-dNLCs+EA group compared to the MC group. Based on these data, the combination of MSC-dNLC transplantation and EA stimulation exerts a synergistic effect on improving the consequences of ICH by relieving cerebral edema and glial scarring, promoting the survival of neurons and oligodendrocytes, and activating mTOR/p70S6K signaling to enhance synaptic plasticity.

Neuroprotection of Triptolide against Amyloid-Beta1-42-induced toxicity via the Akt/mTOR/p70S6K-mediated Autophagy Pathway.

Triptolide is a natural active compound that has significant neuroprotective properties and shows promising effects in the treatment of Alzheimer's disease (AD). Recent studies have shown that autophagy occurs in AD. In this study, we determined whether autophagy regulated by triptolide ameliorates neuronal death caused by amyloid-Beta1-42 (Aβ1-42). We examined the effects of triptolide on cell viability, autophagy, apoptosis, and the protein kinase B/mammalian target of the rapamysin/70 kDa ribosomal protein S6 kinase (Akt/mTOR/p70S6K) signaling pathway in PC12 cells. The results indicated that triptolide treatment exhibited a cytoprotective effect against cell injury induced by Aβ1-42. Triptolide also reduced apoptosis and enhanced cell survival by decreasing autophagosome accumulation and inducing autophagic degradation. Furthermore, our results also showed that activating the Akt/mTOR/p70S6K mechanism was one reason for the protection of triptolide. Triptolide treatment protected against Aβ1-42-induced cytotoxicity by decreasing autophagosome accumulation, and inducing autophagic degradation in PC12 cells. These findings also suggest that the reduction of autophagosome accumulation observed in triptolide-treated cells was Akt/mTOR/p70S6K pathway dependent. Overall, triptolide exhibits a neuron protective effect and this study provides new insight into AD prevention and treatment.

Bisdemethoxycurcumin Promotes Apoptosis and Inhibits the Epithelial-Mesenchymal Transition through the Inhibition of the G-Protein-Coupled Receptor 161/Mammalian Target of Rapamycin Signaling Pathway in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is one of the leading causes of cancer death in the world and lacks an effective targeted therapy. G-protein-coupled receptor 161 (GPR161) has been demonstrated to perform the functional regulations on TNBC progression and might be a potential new target for TNBC therapy. This study showed the effects of bisdemethoxycurcumin (BDMC) on GPR161 regulation, indicating that BDMC effectively inhibited GPR161 expression and downregulated GPR161-driven signaling. BDMC showed the potent inhibitory effects on TNBC proliferation through suppressing GPR161-mediated mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase (p70S6K) activation. Besides, in this study, we discover the mechanism of GPR161-driven TNBC metastasis, linking to GPR161-mediated twist-related protein 1 (Twist1)/matrix metallopeptidase 9 (MMP9) contributing to the epithelial-mesenchymal transition (EMT). BDMC effectively repressed GPR161-mediated TNBC metastasis via inhibiting Twist1/MMP9-induced EMT. The three-dimensional invasion assay also showed that BDMC significantly inhibited TNBC invasion. The combination treatment of BDMC and rapamycin enhanced the inhibition of TNBC proliferation and metastasis through increasing the blockage of mTOR activation. Furthermore, this study also observed that BDMC activated the caspase 3/9 signaling pathway to induce TNBC apoptosis. Therefore, BDMC could be applicable to anticancer therapy, especially targeting on the GPR161-driven cancer type.

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content.

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R2 = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.

Sodium-glucose co-transporter-2 (SGLT-2) inhibition reduces glucose uptake to induce breast cancer cell growth arrest through AMPK/mTOR pathway

ObjectiveThe sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate the potential ability of SGLT2 inhibitors to attenuate cancer growth of SGLT2-expressing cancer cells, but there is little known about the effects of SGLT2 inhibitors on breast cancer. The goal in this research was to assess the anticancer activity of SGLT2 inhibitors in breast cancerin vitro and in vivo. MethodsWe test the SGLT2 expression in breast cancer using immunohistochemistry and immunoblot assay. MTT cytotoxicity assay, colony formation assay and human breast cancer cells nude mice xenograft model were performed to detect the effects of SGLT2 inhibitors on cancer cell proliferation and growth. Flow Cytometry assay was performed to determine if the SGLT2 inhibitors induced cell cycle arrest and apoptosis. ResultsWe proved that SGLT2 expresses in breast cancer cell lines and human breast tumor tissue samples. SGLT2 inhibitors Dapagliflozin and Canagliflozin exhibited a potent anti-proliferative effect in breast cancer cells as demonstrated by MTT, clonogenic survival assay in vitro and xenograft growth model in vivo. Furthermore, we found that SGLT2 inhibitors arrested cell cycle in G1/G0 phase and induced cell apoptosis. Western blot analysis demonstrated that treatment with SGLT2 inhibitors increased the phosphorylation of Amp-activated protein kinase (AMPK) and decreased the phosphorylation of 70 kDa ribosomal protein S6 kinase 1 (p70S6K1) in breast cancer cells. ConclusionsThese findings indicate that SGLT2 inhibitor-therapy induced AMPK-mediated cell cycle arrest and apoptosis, which is a potential novel strategy for the treatment of breast cancer.

IGF-1R inhibition induces MEK phosphorylation to promote survival in colon carcinomas

The insulin-like growth factor 1 receptor (IGF-1R) governs several signaling pathways for cell proliferation, survival, and anti-apoptosis. Thus, targeting IGF-1R appears as a reasonable rationale for tumor treatment. However, clinical studies showed that inhibition of IGF-1R has very limited efficacy due to the development of resistance to IGF-1R blockade in tumor cells. Here, we discovered that prolonged treatment of colon cancer cells with IGF-1R inhibitors (BMS-754807 and GSK1838705A) stimulates p70 KDa ribosomal protein S6 kinase 1 (p70S6K1) activation, a well-known kinase signaling for cell survival. We also found that p70S6K1 activation by IGF-1R inhibition is independent of K-Ras and PIK3CA mutations that frequently occur in colon cancer. Besides the increased p70S6K1 phosphorylation, the phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) was elevated in the cells treated with BMS-754807. Interestingly, the increases in MEK1/2 and p70S6K1 phosphorylation were also observed when cells were subjected to the treatment of AKT inhibitor or genetic knockdown of AKT2 but not AKT1, suggesting that AKT2 inhibition stimulates MEK1/2 phosphorylation to activate p70S6K1. Conversely, inhibition of MEK1/2 by MEK1/2 inhibitor (U0126) or knockdown of MEK1 and MEK2 by corresponding mek1 and mek2 siRNA enhanced AKT phosphorylation, indicating mutual inhibition between AKT and MEK. Furthermore, the combination of BMS-754807 and U0126 efficiently decreased the cell viability and increased cleaved caspase 3 and apoptosis in vitro and in vivo. Our data suggest that the treatment of colon tumor cells with IGF-1R inhibitors stimulates p70S6K1 activity via MEK1/2 to promote survival, providing a new strategy for colorectal cancer therapeutics.

Research Progress of 70 kDa Ribosomal Protein S6 Kinase (P70S6K) Inhibitors as Effective Therapeutic Tools for Obesity, Type II Diabetes and Cancer.

At present, diseases such as obesity, type Ⅱ diabetes and cancer have brought serious health problems, which are closely related to mTOR pathway. 70 kDa ribosomal protein S6 kinase (p70S6K), as a significant downstream effector of mTOR, mediates protein synthesis, RNA processing, glucose homeostasis, cell growth and apoptosis. Inhibiting the function of p70S6K can reduce the risk of obesity which helps to treat dyslipidemia, enhance insulin sensitivity, and extend the life span of mammals. Therefore, p70S6K has become a potential target for the treatment of these diseases. So far, except for the first p70S6K specific inhibitor PF-4708671 developed by Pfizer and LY2584702 developed by Lilai, all of them are in preclinical research. This paper briefly introduces the general situation of p70S6K and reviews their inhibitors in recent years, which are mainly classified into two categories: natural compounds and synthetic compounds. In particular, their inhibitory activities, structure-activity relationships (SARs) and mechanisms are highlighted.

Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas.

Chronic subdural hematoma (CSDH) is an angiogenic disease that is involved with many inflammatory mediators. Tie2 is predominantly expressed in the embryonic endothelium and plays an important role in the maturation and stabilization of the vasculature. Angiopoietin (Ang)1 and Ang2 are well-known ligands of the Tie2 receptor. We examined the expression of Ang1 and Ang2 in CSDH fluid and the expression of Tie-2 receptor and components of the angiogenic signaling pathways in the outer membrane of CSDH. Twenty-five samples of CSDH fluid and eight samples of outer membrane of CSDH were included. The concentrations of Ang1 and Ang2 in the CSDH fluid were measured using enzyme-linked immunosorbent assay (ELISA) kits. The expression of Tie2, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) mechanistic target of rapamycin (mTOR), GβL, 70 kDa ribosomal protein S6 kinase (p70S6K), eukaryotic initiation factor 4E (eIF-4E), and β-actin was examined by a Western blot analysis. The expression of Tie2, Akt, and mTOR was also examined by immunohistochemistry. The concentration of Ang2 in CSDH fluid was significantly higher than that in the serum or cerebrospinal fluid (CSF), and also higher than that of Ang1 in CSDH fluid. Tie2, PI3K, Akt, mTOR, GβL, p70S6K, and eIF-4E were detected in all cases. In addition, Tie2, Akt, and mTOR were localized in the endothelial cells of vessels in the CSDH outer membrane. Our data suggest that Ang2, although not Ang1, in CSDH fluid promotes angiogenesis in endothelial cells through the Tie2 receptor. The Ang2/Tie2 signaling pathway might therefore be a useful therapeutic target for treating the growth of intractable CSDH.

Deficiency of the oxidative stress–responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy

Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For invitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes invivo and high glucose invitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

Differentiation-inducing factor-1 suppresses cyclin D1-induced cell proliferation of MCF-7 breast cancer cells by inhibiting S6K-mediated signal transducer and activator of transcription 3 synthesis.

Differentiation‐inducing factor‐1 (DIF‐1) has been reported to inhibit the proliferation of various mammalian cells by unknown means, although some possible mechanisms of its action have been proposed, including the activation of glycogen synthase kinase‐3 (GSK‐3). Here, we report an alternative mechanism underlying the action of DIF‐1 in human breast cancer cell line MCF‐7, on which the effects of DIF‐1 have not been examined previously. Intragastric administration of DIF‐1 reduced the tumor growth from MCF‐7 cells injected into a mammary fat pad of nude mice, without causing adverse effects. In cultured MCF‐7, DIF‐1 arrested the cell cycle in G0/G1 phase and suppressed cyclin D1 expression, consistent with our previous results obtained in other cell species. However, DIF‐1 did not inhibit the phosphorylation of GSK‐3. Investigating an alternative mechanism for the reduction of cyclin D1, we found that DIF‐1 reduced the protein levels of signal transducer and activator of transcription 3 (STAT3). The STAT3 inhibitor S3I‐201 suppressed cyclin D1 expression and cell proliferation and the overexpression of STAT3 enhanced cyclin D1 expression and accelerated proliferation. Differentiation‐inducing factor‐1 did not reduce STAT3 mRNA or reduce STAT3 protein in the presence of cycloheximide, suggesting that DIF‐1 inhibited STAT3 protein synthesis. Seeking its mechanism, we revealed that DIF‐1 inhibited the activation of 70 kDa and/or 85 kDa ribosomal protein S6 kinase (p70S6K/p85S6K). Inhibition of p70S6K/p85S6K by rapamycin also reduced the expressions of STAT3 and cyclin D1. Therefore, DIF‐1 suppresses MCF‐7 proliferation by inhibiting p70S6K/p85S6K activity and STAT3 protein synthesis followed by reduction of cyclin D1 expression.

The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast–epithelial crosstalk

Healthy kidney structure and environment rely on epithelial integrity and interactions between epithelial cells and other kidney cells. The Ser/Thr kinase 90 kDa ribosomal protein S6 kinase 1 (p90RSK) belongs to a protein family that regulates many cellular processes, including cell motility and survival. p90RSK is predominantly expressed in the kidney, but its possible role in chronic kidney disease (CKD) remains largely unknown. Here, we found that p90RSK expression is dramatically activated in a classic mouse obstructive chronic kidney disease model, largely in the interstitial FSP-1-positive fibroblasts. We generated FSP-1-specific p90RSK transgenic mouse (RSK-Tg) and discovered that these mice, after obstructive injury, display significantly increased fibrosis and enhanced tubular epithelial damage compared with their wt littermates (RSK-wt), indicating a role of p90RSK in fibroblast-epithelial communication. We established an in vitro fibroblast-epithelial coculture system with primary kidney fibroblasts from RSK-Tg and RSK-wt mice and found that RSK-Tg fibroblasts consistently produce excessive H2O2 causing epithelial oxidative stress and inducing nuclear translocation of the signaling protein β-catenin. Epithelial accumulation of β-catenin, in turn, promoted epithelial apoptosis by activating the transcription factor forkhead box class O1 (FOXO1). Of note, blockade of reactive oxygen species (ROS) or β-catenin or FOXO1 activity abolished fibroblast p90RSK-mediated epithelial apoptosis. These results make it clear that p90RSK promotes kidney fibrosis by inducing fibroblast-mediated epithelial apoptosis through ROS-mediated activation of β-catenin/FOXO1 signaling pathway.

Pharmacodynamic Monitoring of mTOR Inhibitors

Pharmacodynamic monitoring may complement routine pharmacokinetic monitoring of mTOR inhibitors (mTORi) in an attempt to better guide individualized sirolimus (SRL) or everolimus (EVR) treatment after organ transplantation. This review focuses on current knowledge about pharmacodynamic biomarkers for personalized mTORi therapies.Different strategies have already been employed in the evaluation of the pharmacodynamics of SRL and EVR as a proxy for their effects on the immune response after transplantation. These include measuring p70S6K (70 kDa ribosomal protein S6 kinase) activity, p70S6K phosphorylation (P-p70S6K), or P-S6 protein expression. Compared to Western blot and ELISA, phosphoflow cytometry can detect phosphorylated proteins and differentiate activation-induced changes of signaling molecules inside the cell from unstimulated populations of identical cells in the same sample. Alternatively, in patients receiving a combined therapy, other pharmacodynamic approach is to consider biomarkers such as NFAT residual expression for calcineurin inhibitors, or to evaluate non-specific effects of the drugs such as lymphocyte proliferation, interleukin synthesis, specific peripheral blood T regulatory subsets or lymphocyte surface antigens, which have the advantage to reflect the overall immunosuppressive status achieved. Although limited, the available data on mTOR pathway biomarkers seem promising. Before clinical implementation, the analytical methodologies must be standardized and cross-validated, and the selected biomarkers will have to demonstrate their clinical utility for SRL or EVR dose individualization in multicenter clinical trials.

Paclitaxel suppresses proliferation and induces apoptosis through regulation of ROS and the AKT/MAPK signaling pathway in canine mammary gland tumor cells.

Paclitaxel is a diterpenoid compound, derived from the pacific yew (Taxus brevifolia) berry, which exhibits antineoplastic effects against various types of cancer. However, the antitumor effects and the molecular mechanisms of paclitaxel on canine CHMm cells remain to be elucidated. The aim of the present study was to investigate the antitumor effects of paclitaxel on CHMm cells and identify relevant signal transduction pathways modulated by paclitaxel using multiple methods including MTT assay, flow cytometry, acridine orange/ethidium bromide staining, transmission electron microscopy, determination of cellular reactive oxygen species (ROS), superoxide dismutase (SOD) and malondiadehyde (MDA) and western blotting, the data indicated that paclitaxel decreased cell viability, induced G2/M-phase cell cycle arrest, suppressed the expression of cyclin B1 and induced apoptosis in a dose-dependent manner. In addition, paclitaxel upregulated the expression of Bax and cytochrome c, but reduced expression of apoptosis regulator Bcl-2, resulting in activation of caspase-3, chromatin condensation, karyopyknosis, intracellular vacuolization, increased production of ROS and MDA, and decreased activity of SOD. However, these effects were inhibited when CHMm cells were treated with N-acetyl-L-cysteine. Furthermore, treatment with paclitaxel inhibited the level of of phospho (p)-RAC-α serine/threonine-protein kinase (AKT) and p-ribosomal protein S6 kinase proteins, and promoted phosphorylation of P38 mitogen-activated protein kinase (MAPK) and p-90 kDa ribosomal protein S6 kinase 1 proteins in CHMm cells. It was observed that paclitaxel in combination with pharmacological inhibitors of the P38 and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathways (SB203580 and LY294002, respectively) exerted synergistic inhibitory effects on the proliferation of the CHMm cells. The results of the present study demonstrated that paclitaxel inhibited tumor cell proliferation by increasing intrinsic apoptosis through inhibition of the PI3K/AKT signaling pathway and activation of MAPK signaling pathway in CHMm cells.

Investigating the Role of mTORC1 in the Androgen‐Mediated Regulation of Skeletal Muscle Growth

Androgens play an important role in regulating skeletal muscle growth largely through changes in muscle protein balance (synthesis + breakdown). Despite this, the underlying molecular mechanism(s) by which androgens alter protein balance and subsequent muscle growth are poorly defined. Previous research has implicated signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in the androgen‐mediated regulation of protein balance as phosphorylation of mTORC1 substrates that control both protein synthesis and protein breakdown have been shown to be altered by androgen depletion/treatment. However, the role of mTORC1 in the androgen‐mediated regulation of muscle growth in vivo has yet to be defined. To define this role, 10‐week old male mice were randomized into four groups of equal body weight, subjected to a sham or castration surgery, then allowed to recover for 7 weeks to induce muscle atrophy in the castrated groups. Following the recovery, sham and castrated mice were implanted with either a control pellet or a pellet designed to release rapamycin at a rate of ~3 mg/kg of body weight/day over a period of 60 days. One‐week post implantation, a group of castrated mice implanted with either control pellets or rapamycin pellets began receiving weekly, intramuscular injections of nandrolone decanoate (ND) to induce muscle regrowth. All other sham and castrated mice received vehicle only. Mice were treated with vehicle or ND for 6 weeks (42 days). The tibialis anterior (TA) was harvested for analysis. The efficacy of the rapamycin pellets to inhibit mTORC1 was confirmed by the inability of insulin to increase phosphorylation of mTORC1 substrates [i.e. 70 kDa ribosomal protein S6 kinase 1 (Thr389), eIF4E binding protein 1 (Ser65), and uncoordinated like kinase 1(Ser757)] in mice receiving rapamycin even though induction of Akt (Thr308) phosphorylation was unaffected. Castration reduced TA wet weight and muscle fiber cross sectional area (CSA), but these values were restored to sham levels with ND administration, even in the presence of rapamycin. Further, the protein content of the TA was also decreased by castration, and ND administration restored this value to sham levels even in the presence of rapamycin. These changes in protein content were not due to changes in the translational capacity of the muscle as RNA content was similar across treatments. In all, these data indicate that androgens alter protein balance and muscle regrowth in an mTORC1‐independent manner.Support or Funding InformationStart‐up funds from the University of Central FloridaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.