Protein S6 Kinase Research Articles

RSK4: a new prognostic factor in glioma

Glioma is the most common and fatal brain tumour and has a poor prognosis. Ribosomal S6 protein kinase 4 (RSK4) has been found to be involved in multiple tumour types; however, the role of RSK4 in gliomas and its clinical relevance remain unclear. In the present study, RSK4 expression was found to be significantly increased in glioma tissues compared with matched adjunct non-noncancerous tissues. Moreover, the expression of RSK4 was significantly higher in high-grade (III and IV) glioma tissues than in low-grade (I and II) glioma tissues. The data showed that the expression of RSK4 was significantly correlated with WHO grade, three-year survival rate and five-year survival rate. Kaplan-Meier analyses showed that patients with high RSK4 expression had poor overall survival. In addition, multivariate Cox regression analysis showed that RSK4 might be an independent prognostic factor in glioma patients. Collectively, these results suggest that RSK4 may be a new prognostic factor in glioma patients, and RSK4 is expected to be a potential biomarker and a potential target for glioma therapy.

Deep Brain Stimulation Improves Motor Function in Rats with Spinal Cord Injury by Increasing Synaptic Plasticity

To investigate the effect of deep brain stimulation (DBS) on rats with spinal cord injury (SCI) and its possible molecular mechanism. A rat SCI model was prepared using a modified Allen method. The animals were randomly divided into 3 groups (n= 12 per group): the sham group, the SCI group, and the SCI+ DBS group. Then, DBS was applied to the rats in the SCI+ DBS group for half an hour per day for 4 weeks. Basso, Beattie, and Bresnahan scores were used to assess spinal function. DBS significantly improved hindlimb motor function in SCI rats, and the protein expression levels of brain-derived neurotrophic factor, the mammalian target of rapamycin, tropomyosin-related kinase B, protein kinase B, p70 ribosomal S6 protein kinase, postsynaptic density protein 95, and synaptophysin increased correspondingly. DBS improves motor function in rats with SCI by increasing synaptic plasticity via tropomyosin-related kinase B-protein kinase B-mammalian target of rapamycin pathway.

AMPK decreases ERK1/2 activity and cancer cell sensitivity to nutrition deprivation by mediating a positive feedback loop involving eEF2K.

Nutrition deprivation (ND) is a common feature of the tumor microenvironment. Tumor cells, therefore, frequently develop resistance mechanisms against ND. One of these mechanisms is the activation of the AMP-activated protein kinase (AMPK), which promotes cell survival under ND. AMPK activation promotes the activity of eukaryotic elongation factor 2 kinase (eEF2K), thereby blocking protein synthesis. The results of the present study indicated the inhibiting effect of AMPK activation on mitogen-activated protein kinase (ERK1/2) activity, which in turn downregulates G1/S transition and promotes cell survival by mediating eEF2K under ND. The knockdown of ERK1/2 enhances cancer cell survival under ND. In the presence of nutrients, eEF2k interacts with dual-specificity mitogen-activated protein kinase kinase (MEK)1/2, conferring a positive feedback loop via MEK1/2-ERK1/2-ribosomal protein S6 kinase α-1-eEF2K signaling, leading to the constitutive activation of ERK1/2. By contrast, under acute ND, AMPK activation blocked the interaction between eEF2K and MEK1/2, contributing to the increased resistance of cancer cells to ND. The present findings reveal a previously undiscovered mechanism that uses AMPK activation to mediate ERK1/2-regulated protein synthesis and cell survival by inhibiting eEF2K-MEK1/2 interaction under ND conditions.

Genome-Wide Identification and Expression Analysis of the BvSnRK2 Genes Family in Sugar Beet (Beta vulgaris L.) Under Salt Conditions

The sucrose non-fermenting-1-related protein kinase 2s (SnRK2s) have been shown to play critical roles in the response to environmental stresses in higher plants. Although the SnRK2 genes family has been identified in various plants, little is reported regarding SnRK2s in sugar beet (Beta vulgaris L.), which is one of the most important crops for both food and sugar production. In the current study, the SnRK2s genes are identified in the sugar beet genome by bioinformatics, and their expression patterns under salinity conditions are tested by the qRT-PCR method. Results showed that a total of six BvSnRK2 genes are identified and characterized from the genome of sugar beet and are further classified into three distinct groups (Group 1, 2, and 3). All BvSnRK2s contained a highly conserved N-terminal kinase region and a greatly divergent C-terminal region. Except for BvSnRK2.4, most of the BvSnRK2 genes were disrupted by eight introns with size ranging from 82 to 2164 bp. Moreover, the expression levels of the BvSnRK2s genes were strongly enhanced by salt treatments, which may be an indicator of potential roles in the response to salinity. The present work is the first systematic analysis of the SnRK2 family genes in sugar beet. The results from this study provide a novel insight for the functional exploration and application of the SnRK2s genes for crop improvement, especially in sugar crops.

Effects of partial fish meal replacement with two fermented soybean meals on the growth of and protein metabolism in the Chinese mitten crab (Eriocheir sinensis)

Fermented soybean meal (FSM) is used as a potential substitute for fishmeal in various aquatic species due to its enriched nutrient profile. This study is aimed to investigate the effect of two different kinds of FSM on growth and protein metabolism in the Chinese mitten crabs (Eriocheir sinensis). One hundred and sixty-eight crabs were randomly assigned to 21 cement pools, each with eight crabs. The crabs in the control group were fed a basic diet, without FSM. Six isonitrogenous and isolipidic diets were prepared by replacing fish meal with two kinds (A and B) of FSM at concentrations of 5 %, 10 %, and 15 %, respectively. The results showed that partial replacement of fish meal with FSM could improve the growth performance of and crude protein content in the Chinese mitten crabs. In addition, increased activities of trypsin, pepsin, Na+/K+-ATPase, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase in the Chinese mitten crabs were observed. Furthermore, the hepatopancreas mRNA levels of protein metabolism-related genes, including protein kinase B (Akt), protein S6 kinase 1 (S6k1), 4E-binding protein 1 (4E-BP1), and oligopeptide transporter 1 (Pept1), were higher in the crabs fed FSM-supplemented diets than in the control crabs. The present study concludes that about 10 and 15 % of fish meal could be substituted by FSM A and B, respectively, without having any deleterious effects on growth performance, protein metabolism-related enzyme activities, and the expression ofand protein synthesis- and metabolism-related genes expressions.

PP1C and PP2A are p70S6K Phosphatases Whose Inhibition Ameliorates HLD12-Associated Inhibition of Oligodendroglial Cell Morphological Differentiation.

Myelin sheaths created by oligodendroglial cells encase neuronal axons to achieve saltatory conduction and protect axons. Pelizaeus-Merzbacher disease (PMD) is a prototypic, hereditary demyelinating oligodendroglial disease of the central nervous system (CNS), and is currently known as hypomyelinating leukodystrophy 1 (HLD1). HLD12 is an autosomal recessive disorder responsible for the gene that encodes vacuolar protein sorting-associated protein 11 homolog (VPS11). VPS11 is a member of the molecular group controlling the early endosome antigen 1 (EEA1)- and Rab7-positive vesicle-mediated protein trafficking to the lysosomal compartments. Herein, we show that the HLD12-associated Cys846-to-Gly (C846G) mutation of VPS11 leads to its aggregate formation with downregulated signaling through 70 kDa S6 protein kinase (p70S6K) in the oligodendroglial cell line FBD-102b as the model. In contrast, wild-type proteins are localized in both EEA1- and Rab7-positive vesicles. Cells harboring the C846G mutant constructs decrease differentiated phenotypes with web-like structures following differentiation, whereas parental cells exhibit them suitably. It is of note that we identify PP1C and PP2A as the protein phosphatases for phosphorylated Thr-389 of p70S6K essential for kinase activation in cells. The respective knockdown experiments or inhibitor treatment stimulates phosphorylation of p70S6K and ameliorates the inhibition of morphological differentiation, as well as the formation of protein aggregates. These results indicate that inhibition of p70S6K phosphatases PP1C and PP2A improves the defective morphological differentiation associated with HLD12 mutation, thereby hinting at amelioration based on a possible molecular and cellular pathological mechanism underlying HLD12.

Phosphoproteomic Analysis of Neonatal Regenerative Myocardium Revealed Important Roles of Checkpoint Kinase 1 via Activating Mammalian Target of Rapamycin C1/Ribosomal Protein S6 Kinase b-1 Pathway

In mammals, regenerative therapy after myocardial infarction is hampered by the limited regenerative capacity of adult heart, whereas a transient regenerative capacity is maintained in the neonatal heart. Systemic phosphorylation signaling analysis on ischemic neonatal myocardium might be helpful to identify key pathways involved in heart regeneration. Our aim was to define the kinase-substrate network in ischemic neonatal myocardium and to identify key pathways involved in heart regeneration after ischemic insult. Quantitative phosphoproteomics profiling was performed on infarct border zone of neonatal myocardium, and kinase-substrate network analysis revealed 11 kinases with enriched substrates and upregulated phosphorylation levels, including checkpoint kinase 1 (CHK1) kinase. The effect of CHK1 on cardiac regeneration was tested on Institute of Cancer Research CD1 neonatal and adult mice that underwent apical resection or myocardial infarction. In vitro, CHK1 overexpression promoted whereas CHK1 knockdown blunted cardiomyocyte proliferation. In vivo, inhibition of CHK1 hindered myocardial regeneration on resection border zone in neonatal mice. In adult myocardial infarction mice, CHK1 overexpression on infarct border zone upregulated mammalian target of rapamycin C1/ribosomal protein S6 kinase b-1 pathway, promoted cardiomyocyte proliferation, and improved cardiac function. Inhibiting mammalian target of rapamycin activity by rapamycin blunted the neonatal cardiomyocyte proliferation induced by CHK1 overexpression in vitro. Our study indicates that phosphoproteome of neonatal regenerative myocardium could help identify important signaling pathways involved in myocardial regeneration. CHK1 is found to be a key signaling responsible for neonatal regeneration. Myocardial overexpression of CHK1 could improve cardiac regeneration in adult hearts by activating the mammalian target of rapamycin C1/ribosomal protein S6 kinase b-1 pathway. Thus, CHK1 might serve as a potential novel target in myocardial repair after myocardial infarction.

Overexpression of CsSnRK2.5 increases tolerance to drought stress in transgenic Arabidopsis

Drought is a major factor limiting crop productivity and quality. Sucrose non-fermenting-1 (SNF1)-related protein kinase 2s (SnRK2s) play critical roles in plant abiotic stress responses, especially in drought stress. However, knowledge regarding the functional roles of SnRK2s in drought stress and their underlying mechanisms is relatively limited in tea plant. In this study, CsSnRK2.5, a PEG 6000- and ABA-induced SnRK2 gene from tea plant, was overexpressed in Arabidopsis to investigate its potential function in drought stress response. The results showed that overexpression of CsSnRK2.5 resulted in enhanced drought tolerance, as indicated by an amelioration of the changes in various physiological indexes, including a decreased rate of water loss and decreased accumulation of ROS and MDA. In addition, CsSnRK2.5 overexpression conferred hypersensitivity to exogenous ABA, and transgenic plants exhibited improved ABA-mediated stomatal closure compared to WT plants. Moreover, the expression of some stress response genes, including AtRAB18 and AtRD29b, was more strongly induced in transgenic plants than in the WT when subjected to ABA and drought treatments. Taken together, our results indicate that CsSnRK2.5 is a positive regulator of ABA-regulated drought stress responses.

A RAF-SnRK2 kinase cascade mediates early osmotic stress signaling in higher plants

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

Overexpression of RSK4 reverses doxorubicin resistance in human breast cancer cells via PI3K/AKT signalling pathway.

Doxorubicin (DOX) is one of the most effective chemotherapy drugs for the treatment of metastatic breast cancer (BC), but drug resistance becomes an obstacle to treatment. This study aims to investigate the role of Ribosomal S6 protein kinase 4 (RSK4) in regulating BC resistance to DOX. We first used Kaplan-Meier Plotter to identify the prognostic roles of RSK4 in BC. DOX-resistant BC cells (MCF-7/DOX) were constructed and the expression of RSK4 was determined by reverse transcript polymerase chain reaction and western blot. Subsequently, we overexpressed the RSK4 in MCF-7/DOX cells, and measured drug resistance, colony formation, cell migration, invasion ability and cell apoptosis after transfection. In addition, western blot was used to explore the expression of apoptosis-related proteins and BC-resistance protein. Effects of RSK4 on activation of the PI3K/AKT signalling pathway were also tested. Furthermore, tumour xenograft in nude mice was constructed to observe the effect of RSK4 overexpression on tumour growth in vivo. In conclusion, RSK4 was positively correlated with survival rate in BC patients, which is lowly expressed in MCF-7/DOX. Meanwhile, the overexpression of RSK4 may inhibit drug resistance, cell migration, invasion, apoptosis and tumour growth. RSK4 may effectively attenuate DOX resistance in BC by inhibiting the PI3K/AKT signalling pathway.

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition. We have identified inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without potent cytotoxicity. The structure-activity data from this study will be used as a platform to develop novel RSK2 inhibitors.

MAP3Kinase-dependent SnRK2-kinase activation is required for abscisic acid signal transduction and rapid osmotic stress response

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Genome-wide analysis of PYL-PP2C-SnRK2s family in Camellia sinensis

ABSTRACT Abscisic acid (ABA) signaling regulates plant growth and development and participates in response to abiotic stressors. However, details about the PYL-PP2C-SnRK2 gene family, which is the core component of ABA signaling in Camellia sinensis, are unknown. In this work, we identified 14 pyrabactin resistance-likes (PYLs), 84 type 2C protein phosphatase (PP2Cs), and 8 SNF1-related protein kinase 2s (SnRK2s) from C. sinensis. The transcriptomic analysis indicated that PYL-PP2C-SnRK2s were associated with changes of leaf color and the response of C. sinensis to drought and salt stressors. Changes of the expression of Snrk2s were not significant in the process of leaf color change or drought and salt stress response, suggesting that PYLs and PP2Cs may not interact with SnRK2s in C. sinensis during these processes. Finally, Gene Regulatory Network (GRN) construction and interaction networks analysis demonstrated that PYLs and PP2Cs were associated with multiple metabolic pathways during the changes of leaf color.

High doses of cobalt inhibited hair follicle development in Rex Rabbits

<p>An experiment was conducted to investigate the effect of cobalt supplementation on hair follicle development in rabbits. Rex rabbits (30-d-old, n=180) were divided randomly into five equal treatment groups: rabbits fed a basal diet (control, measured cobalt content of 0.27 mg/kg) or rabbits fed a basal diet with an additional 0.1, 0.4, 1.6 or 6.4 mg/kg cobalt (in the form of cobalt sulfate) supplementation (measured cobalt contents of 0.35, 0.60, 1.83 and 6.62 mg/kg, respectively). Treatment with 6.4 mg/kg cobalt significantly decreased hair follicle density (<em>P</em><0.05), while low levels of cobalt (0.1-1.6 mg/kg) had no effect on hair follicle density (<em>P</em>>0.05). The addition of dietary cobalt at the highest level examined (6.4 mg/kg) significantly increased the gene expression of bone morphogenetic protein (BMP) 2 and BMP4 in skin tissue (<em>P</em><0.05), while the mRNA levels of versican, alkaline phosphatase, hepatocyte growth factor, and noggin remained unchanged (<em>P</em>>0.05). Compared with their levels in the control group, dietary cobalt treatment significantly suppressed the protein levels of p-mechanistic target of rapamycin (mTOR) and p-ribosomal protein S6 protein kinase (<em>P</em><0.05) but did not alter the protein levels of p-AMP-activated protein kinase, Wnt10b or p-β-catenin (<em>P</em>>0.05). In conclusion, cobalt at the highest concentration examined inhibited hair follicle development, which may have involved the mTOR-BMP signalling pathway.</p>

Expression, Subcellular Localization, and Interactions of CPK Family Genes in Maize.

Calcium-dependent protein kinase (CPKs) is a key player in the calcium signaling pathway to decode calcium signals into various physiological responses. cDNA sequences of 9 ZmCPK genes were successfully cloned from all four phylogenetic groups in maize. qRT-PCR analysis showed the expression variation of these selected genes under abscisic acid (ABA) and calcium chloride (CaCl2) treatment. Due to the presence of N-myristoylation/palmitoylation sites, the selected ZmCPK members were localized in a plasma membrane. To clarify whether ZmCPK, a key player in calcium signaling, interacts with key players of ABA, protein phosphatase 2Cs (PP2Cs) and the SNF1-related protein kinase 2s (SnRK2s) and mitogen-activated protein kinase (MAPK) signaling pathways in maize, we examined the interaction between 9 CPKs, 8 PP2Cs, 5 SnRKs, and 20 members of the MPK family in maize by using yeast two-hybrid assay. Our results showed that three ZmCPKs interact with three different members of ZmSnRKs while four ZmCPK members had a positive interaction with 13 members of ZmMPKs in different combinations. These four ZmCPK proteins are from three different groups in maize. These findings of physical interactions between ZmCPKs, ZmSnRKs, and ZmMPKs suggested that these signaling pathways do not only have indirect influence but also have direct crosstalk that may involve the defense mechanism in maize. The present study may improve the understanding of signal transduction in plants.

Abstract C093: Substituted pteridinones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors and their evaluation in acute myeloid leukemia

Abstract The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation and the maintenance of cancer stem cells (CSCs). Several pan-RSK inhibitors have been identified with BI-D1870 and the simple LJH685 and LJI308 analogs being the most selective, potent, and frequently used small molecules to identify the physiological substrates and functional roles for RSK in cells. However, these RSK inhibitors have poor PK profiles, display high clearance and a short plasma half-life and this has prevented their transition to clinical evaluation. Therefore, we designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition, with the overall aim of developing a RSK inhibitor with improved drug-like properties. We have identified inhibitors of recombinant RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the molm-13 acute myeloid leukemia (AML) cell line. The results of our initial structure-activity studies indicate that halogen substitutions on the 4-hydroxyanilino ring can greatly influence RSK inhibitory potency, elimination of C7 chirality does not appear to effect RSK inhibitory potency, removal of the N5-methyl maintains RSK inhibitory activity, and the N7-alkyl substitution is required for potent RSK inhibition. We have identified compounds that potently inhibit RSK2 activity in cells using the nanoBRET assay, and coupled with the results from our cellular viability and toxicity studies our data supports that RSK2 inhibition is independent from acute cytotoxicity in molm-13 cells. This finding was surprising as the RSKs phosphorylate a number of substrates involved in many diverse cellular processes critical for cell survival. Our studies have provided important structural information for the further development of RSK inhibitors and potential insights into structural modifications that may limit metabolism and incorporate isoform selectivity. Further studies are required to determine the impact of RSK inhibition in cancer cells as synergistic combination therapies may need to be identified. Alternatively, RSK inhibitors may be more therapeutically effective against specific cancer cell populations, such as in the elimination of CSCs. Citation Format: Kimberly Casalvieri, Christopher Matheson, Philip Reigan, Donald Backos. Substituted pteridinones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors and their evaluation in acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C093. doi:10.1158/1535-7163.TARG-19-C093

Role of BRAF in Hepatocellular Carcinoma: A Rationale for Future Targeted Cancer Therapies.

The few therapeutic strategies for advance hepatocellular carcinoma (HCC) on poor knowledge of its biology. For several years, sorafenib, a tyrosine kinase inhibitors (TKI) inhibitor, has been the approved treatment option, to date, for advanced HCC patients. Its activity is the inhibition of the retrovirus-associated DNA sequences protein (RAS)/Rapidly Accelerated Fibrosarcoma protein (RAF)/mitogen-activated and extracellular-signal regulated kinase (MEK)/extracellular-signal regulated kinases (ERK) signaling pathway. However, the efficacy of sorafenib is limited by the development of drug resistance, and the major neuronal isoform of RAF, BRAF and MEK pathways play a critical and central role in HCC escape from TKIs activity. Advanced HCC patients with a BRAF mutation display a multifocal and/or more aggressive behavior with resistance to TKI. Moreover, also long non-coding RNA (lnc-RNA) have been studied in epigenetic studies for BRAF aggressiveness in HCC. So far, lnc-RNA of BRAF could be another mechanism of cancer proliferation and TKI escape in HCC and the inhibition could become a possible strategy treatment for HCC. Moreover, recent preclinical studies and clinical trials evidence that combined treatments, involving alternative pathways, have an important role of therapy for HCC and they could bypass resistance to the following TKIs: MEK, ERKs/ribosomal protein S6 kinase 2 (RSK2), and phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR). These initial data must be confirmed in clinical studies, which are currently ongoing. Translational research discoveries could create new strategies of targeted therapy combinations, including BRAF pathway, and they could eventually bring light in new treatment of HCC.

Rapamycin enhanced the antitumor effects of doxorubicin in myelogenous leukemia K562 cells by downregulating the mTOR/p70S6K pathway.

Chronic myelogenous leukemia (CML) is a common hematological malignancy. Some patients progressing to the blast phase develop chemotherapeutic drug resistance. In the authors' previous study, it was found that the mammalian target of rapamycin (mTOR) pathway was activated in CML and that rapamycin inhibited the proliferation of K562 cells. Targeting the mTOR pathway may be used in combination with chemotherapeutic drugs to enhance their efficacy and overcome multidrug resistance. The aim of the present study was to investigate the effects of rapamycin and doxorubicin on K562 cell proliferation following the combination treatment, and further focus on confirming whether rapamycin enhanced the antitumor effects of doxorubicin by downregulating the mTOR/ribosomal protein S6 kinase (p70S6K) pathway. It was found that rapamycin and doxorubicin significantly decreased the viability of K562 cells. The apoptotic cells were more frequently detected in rapamycin and doxorubicin treatment groups (25.50±1.25%). Both drugs decreased Bcl-2 and increased Bax expression in K562 cells. Rapamycin and doxorubicin also reduced the phosphorylation levels of mTOR and p70S6K. Meanwhile, p70S6K-targeting small interfering (si)RNA and doxorubicin inhibited cell proliferation and regulated key factors of the cell cycle. In addition, the exposure of cells to p70S6K siRNA and doxorubicin significantly increased cell apoptosis, as compared with single treatment. These results suggested that rapamycin could enhance the antitumor effects of doxorubicin on K562 cells by downregulating mTOR/p70S6K signaling. Targeting the mTOR/p70S6K pathway may be a new therapeutic approach for leukemia.

Role of mammalian target of rapamycin in regulating HIF-1α and vascular endothelial growth factor signals in glaucoma

Hypoxia inducible factor subtype 1α (HIF-1α) in retinal tissues is involved in the development of glaucoma. This study examined the role played by mammalian target of rapamycin (mTOR) in regulating expression of HIF-1α and its downstream pathway, vascular endothelial growth factor (VEGF). Glaucoma was induced by chronic elevation of intraocular pressure using laser burns in rats. ELISA and western blot analysis were employed to determine the levels of HIF-1α, VEGF and mTOR in retinal tissues of eyes with high intraocular pressure. In results, HIF-1α, VEGF and VEGF receptor subtype 2 were increased in laser eyes. The p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4, p70 ribosomal S6 protein kinase 1 were also amplified in retina of laser eyes. Blocking mTOR using rapamycin attenuated HIF-1α-VEGF pathways, accompanied with downregulation of apoptotic Caspase-3. Our data revealed potential signalling pathways engaged in the development of glaucoma, including the activation of mTOR and HIF-1α-VEGF mechanism.