Bioactive peptide inhibits acute myeloid leukemia cell proliferation by downregulating ALKBH5-mediated m6A demethylation of EIF4EBP1 and MLST8 mRNA.

PURPOSE: N6-methyladenosine (m6A), the most prevalent mRNA modification, plays an essential role in tumorigenesis. Notably, increasing interest has been directed to bioactive peptides (BPs) with antitumor activities. Here, we set out to investigate the potential of the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis on prevention and treatment of acute myeloid leukemia (AML). METHODS: The biological effects of BP on AML cells were detected by MTT and ApoLive-Glo™ multiplex assays. The role of BP in tumor growth was determined by a subcutaneous xenograft model. The ALKBH5/MLST8/EIF4EBP1 axis was identified as a potential BP target in AML via methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq). Western blot, RT-qPCR, MeRIP-qPCR, dual-luciferase reporter and RNA stability assays were performed to validate the function and mode of action of the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis. The clinical relevance of the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis in AML was confirmed by TCGA data analysis. RESULTS: We found that BP can inhibit AML cell proliferation and promote apoptosis in vitro, and repress AML tumor growth in vivo. Mechanistically, we found that BP downregulated ALKBH5 expression, which in turn repressed m6A demethylation of MLST8 and EIF4EBP1 mRNAs. Reduction of the m6A levels of MLST8 and EIF4EBP1 facilitated MLST8 and EIF4EBP1 mRNA decay, resulting in inhibition of AML cell proliferation. Furthermore, we found that the BP-regulated ALKBH5/MLST8/EIF4EBP1 axis closely correlates with AML patient prognosis. CONCLUSIONS: Our data indicate that BP can inhibit acute myeloid leukemia cell proliferation by downregulating ALKBH5-mediated m6A demethylation of EIF4EBP1 and MLST8 mRNAs, which may have potential to prevent and treat this disease.

Upregulated YB-1 protein promotes glioblastoma growth through a YB-1/CCT4/mLST8/mTOR pathway.

Y-box-binding protein 1 (YB-1) is a multifunctional RNA binding protein involved in virtually every step of RNA metabolism. However, the functions and mechanisms of YB-1 in one of the most aggressive cancers, glioblastoma, are not well understood. In this study, we found that YB-1 protein was markedly overexpressed in glioblastoma and acted as a critical activator of both mTORC1 and mTORC2 signaling. Mechanistically, YB-1 bound the 5'UTR of CCT4 mRNA to promote the translation of CCT4, a component of the CCT chaperone complex, that in turn activated the mTOR signaling pathway by promoting mLST8 folding. In addition, YB-1 autoregulated its own translation by binding to its 5'UTR, leading to sustained activation of mTOR signaling. In patients with glioblastoma, high protein expression of YB-1 correlated with increased expression of CCT4 and mLST8 and activated mTOR signaling. Importantly, the administration of RNA decoys specifically targeting YB-1 in a mouse xenograft model resulted in slower tumor growth and better survival. Taken together, these findings uncover a disrupted proteostasis pathway involving a YB-1/CCT4/mLST8/mTOR axis in promoting glioblastoma growth, suggesting that YB-1 is a potential therapeutic target for the treatment of glioblastoma.

CDK1/FBXW7 facilitates degradation and ubiquitination of MLST8 to inhibit progression of renal cell carcinoma.

Recent studies have reported that MLST8 is upregulated in many malignant tumors. Nevertheless, the underlying molecular mechanism is still unclear. The aim of this work was to investigate how MLST8 contributes to the development and progression of clear cell renal cell carcinoma (ccRCC). MLST8 is an oncogenic protein in the TCGA database and ccRCC clinical specimens. We also ascertain that MLST8 interacts with FBXW7, which was universally regarded as an E3 ubiquitin ligase. MLST8 can be degraded and ubiquitinated by tumor suppressor FBXW7. FBXW7 recognizes a consensus motif (T/S) PXX (S/T/D/E) of MLST8 and triggers MLST8 degradation via the ubiquitin-proteasome pathway. Strikingly, the activated cyclin dependent kinase 1 (CDK1) kinase engages in the MLST8 phosphorylation required for FBXW7-mediated degradation. In vitro, we further prove that MLST8 is an essential mediator of FBXW7 inactivation-induced tumor growth, migration, and invasion. Furthermore, the MLST8 and FBXW7 proteins are negatively correlated in human renal cancer specimens. Our findings suggest that MLST8 is a putative oncogene that functions via interaction with FBXW7, and inhibition MLST8 could be a potential future target in ccRCC treatment.

Dynamic modelling of the PI3K/MTOR signalling network uncovers biphasic dependence of mTORC1 activity on the mTORC2 subunit SIN1.

The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GßL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2's activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.

R (-)-methoxetamine exerts rapid and sustained antidepressant effects and fewer behavioral side effects relative to S (+)-methoxetamine.

The newfound antidepressant efficacy of ketamine has provided opportunities for the development of new-generation, rapid-acting, glutamate-based antidepressants. We previously identified that methoxetamine (MXE), a ketamine analog, and an N-Methyl-d-aspartate (NMDA) receptor antagonist, produced rapid and sustained antidepressant effects in mice. MXE (R, S (±)-MXE) is a racemic mixture containing equal parts of S (+)-MXE and R (-)-MXE. However, studies have yet to investigate the antidepressant effects of its enantiomers. Here, we examined the potential antidepressant properties and behavioral side effects of S- and R-MXE in mice. Both S- and R-MXE showed significant NMDA receptor affinity and appreciable inhibitory activity on serotonin transporter. Also, S- and R-MXE (10 mg kg-1) exerted antidepressant effects and increased gamma waves (electroencephalography) but were inhibited by NBQX (an AMPA receptor antagonist). Subsequently, they increased mammalian target of rapamycin phosphorylation and AMPA receptor subunits GluA1 and GluA2 protein levels in the hippocampus or prefrontal cortex. Furthermore, they increased 5HT2a and 5HT2c receptor mRNA levels in the prefrontal cortex, with their antidepressant effects inhibited by ketanserin (a 5HT2a/c receptor antagonist). Taken together, S-MXE and R-MXE elicit antidepressant effects that are probably mediated via glutamatergic and serotonergic mechanisms. Unlike S-MXE, R-MXE did not induce prepulse inhibition deficits, hyperlocomotion, conditioned place preference, and locomotor sensitization, although it acutely altered motor coordination. This suggests that R-MXE induces fewer behavioral side effects and is a safer antidepressant than S-MXE. Overall, this study provides significant implications for future research on the next generation of rapid-acting, glutamate-based antidepressant drugs.

Genomic Database Analysis for Head and Neck Cancer Prevention Targets: MTOR Signal Transduction Pathway.

BACKGROUND: Type II diabetes agents have anticancer effects on head and neck squamous cell carcinoma (HNSCC). The mechanistic target of rapamycin (MTOR) pathway represents a putative target. MATERIALS AND METHODS: We interrogated an Affymetrix HNSCC dataset for MTOR-related gene expression. RESULTS: MTOR expression itself was unchanged, but various related genes demonstrated differential expression. Pathway promoters ras homolog (RHEB), MTOR-associated protein (MLST8), and ribosomal protein S6 kinase B1 (RPS6KB1) were up-regulated. Expression of growth suppressors tuberous sclerosis complex 2 (TSC2), programmed cell death 4 (PDCD4), and BCL2 apoptosis regulator-associated agonist of cell death (BAD) were reduced in HNSCC. Upstream, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), AKT serine/threonine kinase 1 (AKT1), and phosphatase and tensin homolog (PTEN) were up-regulated in cancer. CONCLUSION: Several MTOR pathway promoters and tumor suppressors were found to be differentially expressed, favoring MTOR pathway up-regulation in HNSCC. Genomic databases can be interrogated to identify intervention targets and endpoints in HNSCC trials.

miR-34a/c induce caprine endometrial epithelial cell apoptosis by regulating circ-8073/CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways.

microRNAs (miRNAs) and circular RNAs (circRNAs) are important for endometrial receptivity establishment and embryo implantation in mammals. miR-34a and miR-34c are highly expressed in caprine receptive endometrium (RE). Herein, the functions and mechanisms of miR-34a/c in caprine endometrial epithelial cell (CEEC) apoptosis and RE establishment were investigated. miR-34a/c downregulated the expression level of centrosomal protein 55 (CEP55) and were sponged by circRNA8073 (circ-8073), thereby exhibiting a negative interaction in CEEC. miR-34a/c induced CEEC apoptosis by targeting circ-8073/CEP55 through the regulation of the RAS/RAF/MEK/ERK and phosphoitide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways. Positive and negative feedback loops and cross-talk were documented between the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. miR-34a/c regulated the levels of RE marker genes, including forkhead box M1, vascular endothelial growth factor, and osteopontin (OPN). These results suggest that miR-34a/c not only induce CEEC apoptosis by binding to circ-8073 and CEP55 via the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways, but may also regulate RE establishment in dairy goats.

STAT3-mediated MLST8 gene expression regulates cap-dependent translation in cancer cells.

Signal transducer and activator of transcription 3 (STAT3) regulates cell growth, cell survival, angiogenesis, metastasis of cancer cells, and cancer immune evasion by regulating gene expression as a transcription factor. However, the effect of STAT3 on translation is almost unknown. We demonstrated that STAT3 acts as a trans-acting factor for MLST8 gene expression and the protein level of mLST8, a core component of mechanistic target of rapamycin complex 1 and 2 (mTORC1/2), positively regulates the mTORC1/2 downstream pathways. Suppression of STAT3 by siRNA attenuated 4E-BP1 phosphorylation, cap-dependent translation, and cell proliferation in a variety of cancer cells. In HCT116 cells, STAT3 knockdown-induced decreases in 4E-BP1 and AKT phosphorylation levels were further attenuated by MLST8 knockdown or recovered by mLST8 overexpression. STAT3 knockdown-induced G2/M phase arrest was partially restored by co-knockdown of 4EBP1, and the attenuation of cell proliferation was enhanced by the expression of an mTORC1-mediated phosphorylation-defective mutant of 4E-BP1. ChIP and promoter mapping using a luciferase reporter assay showed that the -951 to -894 bp of MLST8 promoter seems to include STAT3-binding site. Overall, these results suggest that STAT3-driven MLST8 gene expression regulates cap-dependent translation through 4E-BP1 phosphorylation in cancer cells.

Enhanced mLST8 Expression Correlates with Tumor Progression in Hepatocellular Carcinoma.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway, containing mTOR complex 1 (mTORC1) and mTORC2, is dysregulated in multiple cancers, including hepatocellular carcinoma (HCC). Mammalian lethal with sec-13 protein 8 (mLST8) is a shared constituent of both mTORC1 and mTORC2, yet little is known regarding its role in HCC development. METHODS: mLST8 expression was detected in a total of 186 pairs of HCC and adjacent non-tumor specimens. The correlation between mLST8 level and clinicopathological features or prognostic significance were analyzed. The role of mLST8 on biological functions was also preliminarily studied. RESULTS: The study revealed that the mLST8 level was dramatically higher in HCC specimens than in adjacent non-tumor specimens. mLST8 overexpression positively correlated with tumor size, differentiation, and vessel invasion. Cases with elevated mLST8 level had more unfavorable overall survival (OS) and disease-free survival (DFS) than those with downregulated mLST8 level. Multivariate analysis demonstrated that mLST8 upregulation was an independent predictive marker for OS and DFS. Calibration curves from nomogram models indicated an excellent coherence between nomogram prediction and actual situation. Decision curve analysis proved that mLST8-based nomograms presented much higher predictive accuracy when compared with conventional clinical staging systems. Mechanistically, mLST8 enhanced cell proliferation and invasion through the AKT (protein kinase B) pathway. CONCLUSIONS: Our study demonstrates that mLST8 exerts an oncogenic role in HCC and may become a promising prognostic biomarker and therapeutic target for HCC patients.

MELK promotes Endometrial carcinoma progression via activating mTOR signaling pathway.

BACKGROUND: Endometrial carcinoma (EC) is one of the most common gynecological malignancies among women. Maternal embryonic leucine Zipper Kinase (MELK) is upregulated in a variety of human tumors, where it contributes to malignant phenotype and correlates with a poor prognosis. However, the biological function of MELK in EC progression remains largely unknown. METHODS: We explored the MELK expression in EC using TCGA and GEO databases and verified it using clinical samples by IHC methods. CCK-8 assay, colony formation assay, cell cycle assay, wound healing assay and subcutaneous xenograft mouse model were generated to estimate the functions of MELK and its inhibitor OTSSP167. qRT-PCR, western blotting, co-immunoprecipitation, chromatin immunoprecipitation and luciferase reporter assay were performed to uncover the underlying mechanism concerning MELK during the progression of EC. FINDINGS: MELK was significantly elevated in patients with EC, and high expression of MELK was associated with serous EC, high histological grade, advanced clinical stage and reduced overall survival and disease-free survival. MELK knockdown decreased the ability of cell proliferation and migration in vitro and subcutaneous tumorigenesis in vivo. In addition, high expression of MELK could be regulated by transcription factor E2F1. Moreover, we found that MELK had a direct interaction with MLST8 and then activated mTORC1 and mTORC2 signaling pathway for EC progression. Furthermore, OTSSP167, an effective inhibitor, could inhibit cell proliferation driven by MELK in vivo and vitro assays. INTERPRETATION: We have explored the crucial role of the E2F1/MELK/mTORC1/2 axis in the progression of EC, which could be served as potential therapeutic targets for treatment of EC. FUNDING: This research was supported by National Natural Science Foundation of China (No:81672565), the Natural Science Foundation of Shanghai (Grant NO:17ZR1421400 to Dr. Zhihong Ai) and the fundamental research funds for central universities (No: 22120180595).

Cross-talk between ribosome biogenesis, translation, and mTOR in CD133+ 4/CD44+ prostate cancer stem cells.

OBJECTIVE: To investigate the gene expression profile of CSCs and to explore the key pathways and specific molecular signatures involved in the characteristic of CSCs. MATERIALS AND METHODS: CD133+ /CD44+ CSCs and bulk population (non-CSCs) were isolated from DU-145 cells using fluorescence-activated cell sorting (FACS). We used Illumina HumanHT-12 v4 Expression to investigate gene expression profiling of CSCs and non-CSCs. Protein-protein interaction (PPI) network analysis was performed using the STRING database. Biomarkers selected based on gene expression profiling were visually analyzed using immunofluorescence staining method. An image analysis program, ImageJ®, was used for the analysis of fluorescence intensity. RESULTS: In microarray analysis, we found that many ribosomal proteins and translation initiation factors that constitute the mTOR complex were highly expressed. PPI analysis using the 33 genes demonstrated that there was a close interaction between ribosome biogenesis, translation, and mTOR signaling. The fluorescence amount of mTOR and MLST8 were higher in CSCs compared to non-CSCs. CONCLUSIONS: The increase in a number of genes associated with ribosome biogenesis, translation, and mTOR signaling may be important to evaluate prognosis and determine treatment approach for prostate cancer (PCa). A better understanding of the molecular pathways associated with CSCs may be promising to develop targeted therapies to prolong survival in PCa.

Expression and activity of AIM2-inflammasome in rheumatoid arthritis patients.

INTRODUCTION: AIM2 inflammasome activation leads to the release of IL-ß, which plays an important role in rheumatoid arthritis pathogenesis. In this work, we evaluated AIM2 expression and activity in RA patients and healthy controls. METHODS: AIM2 and RANKL expression were evaluated by flow cytometry. Inflammasome activity was determined in monocyte cultures stimulated with synthetic DNA by measuring IL-1ß levels in supernatants using an ELISA assay. The caspase-1 expression in monocytes was measured by western blot, the POP3 expression was analysed by qPCR, and serum levels of IFN-γ were evaluated using ELISA assay. RESULTS: We observed a diminution of CD14+AIM2+ cells in RA patients, associated with disease activity and evolution. Likewise, the levels of IL-1ß were increased in monocyte cultures un-stimulated and stimulated with LPS from RA patients with DAS28 ≥ 4. The Caspase-1 activity and RANKL + monocytes in RA patients were slightly increased. Finally, augmented POP3 expression and diminished IFN-γ serum levels were detected in RA patients. CONCLUSION: Our results showed that the monocytes from RA patients were prone to release IL-1ß in the absence of the AIM2 inflammasome signal. The down-regulation of AIM2 to a systemic level in RA patients might be a consequence of augmented POP3 expression and might imply the survival of pro-inflammatory cells contributing to the inflammation process.

Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly.

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.

Disruption of the Scaffolding Function of mLST8 Selectively Inhibits mTORC2 Assembly and Function and Suppresses mTORC2-Dependent Tumor Growth In Vivo.

mTOR is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. Here we assessed mLST8 loss in a panel of normal and cancer cells and observed little to no impact on assembly or activity of mTORC1. However, mLST8 loss blocked mTOR association with mTORC2 cofactors RICTOR and SIN1, thus abrogating mTORC2 activity. Similarly, a single pair of mutations on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly and activity without affecting mTORC1. We also discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor SIN1. In PTEN-null prostate cancer xenografts, mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation and decreased tumor cell proliferation and tumor growth in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation that could enable therapeutic mTORC2-selective inhibition as a therapeutic strategy. SIGNIFICANCE: These findings show that mLST8 functions as a scaffold to maintain mTORC2 integrity and kinase activity, unveiling a new avenue for development of mTORC2-specific inhibitors.

Magnesium transporter protein solute carrier family 41 member 1 suppresses human pancreatic ductal adenocarcinoma through magnesium-dependent Akt/mTOR inhibition and bax-associated mitochondrial apoptosis.

The aim of this study was to identify the function of the Mg2+ transporter protein solute carrier family 41 member 1 SLC41A1 in pancreatic ductal adenocarcinoma and the underlying mechanisms. A total of 27 solute carrier proteins were differentially expressed in pancreatic ductal adenocarcinoma. Three of these proteins were correlated with clinical outcomes in patients, among which SLC41A1 was downregulated in tumour. Overexpression of SLC41A1 suppressed orthotopic tumour growth in a mouse model and reduced the cell proliferation, colony formation, and invasiveness of KP3 and Panc-1 cells, which may have been associated with the increased population of apoptotic-prone cells. Overexpression of SLC41A1 reduced the mitochondrial membrane potential, induced Bax while suppressed Bcl-2 expression. Suppression of Bax abrogated the tumour-suppressive effects of SLC41A1. Furthermore, overexpression of SLC41A1 promoted Mg2+ efflux and suppressed Akt/mTOR activity, which is the upstream regulator of Bax and Bcl-2. An increase in Akt activity and supplementation with Mg2+ abolished SLC41A1-induced tumour suppression. The results of this study suggest that SLC41A1 may be a potential target for the treatment of pancreatic ductal adenocarcinoma.

Regulation of the expression of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks: Effects of leucine and glycine-leucine peptide.

This study was conducted to analyse the effects of leucine (Leu) and glycine (Gly)-Leu peptide on expressions of key signalling molecules in mTOR pathway of skeletal muscle satellite cells in neonatal chicks. The 4-day-old male AA broilers with similar weight were selected to obtain the broiler skeletal muscle satellite cells with the two-step method of collagenase-I and trypsin digestion. The satellite cells were subjected to primary culture in vitro, and they were cultured in DMEM medium with the Leu concentration of 0.2 mM and 2 mM as well as with the Gly-Leu peptide concentration of 0.2 mM and 2 mM. The experiment lasted for 5 days. The results showed that TOR, S6K1 and 4E-BP1 mRNA expressions in the medium with Leu concentration of 2 mM were significantly higher than that in 0.2 mM group (p < 0.05). There was no difference between the medium with Gly-Leu concentration of 2 mM and 0.2 mM on the TOR, S6K1 and 4E-BP1 mRNA expressions (p > 0.05). In conclusion, Leu significantly increases TOR, S6K1 and 4E-BP1 mRNA expressions of skeletal muscle satellite cells, but Gly-Leu peptide has no effect on them.

Taurine Promotes Milk Synthesis via the GPR87-PI3K-SETD1A Signaling in BMECs.

Taurine, a ß-aminosulfonic acid, exerts many cellular physiological functions. It is still unknown whether taurine can regulate milk synthesis in the mammary gland. Therefore, in this study we investigated the effects and mechanism of taurine on milk synthesis in mammary epithelial cells (MECs). Bovine MECs (BMECs) cultured in FBS-free OPTI-MEMImedium were treated with taurine (0, 0.08, 0.16, 0.24, 0.32, and 0.4 mM). Taurine treatment led to increased milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c protein expression, in a dose-dependent manner, with an apparent maximum at 0.24 mM. Gene function study approaches revealed that the GPR87-PI3K-SETD1A signaling was required for taurine to increase the mTOR and SREBP-1c mRNA levels. Taurine stimulated GPR87 expression and cell membrane localization in a dose dependent manner, suggesting a sensing mechanism of GPR87 to extracellular taurine. Collectively, these data demonstrate that taurine promotes milk synthesis via the GPR87-PI3K-SETD1A signaling.

Sodium fluoride induces splenocyte autophagy via the mammalian targets of rapamycin (mTOR) signaling pathway in growing mice.

Fluoride is known to impair organism's development and function via adverse effects, and autophagy plays a regulation role in human or animal health and disease. At present, there are no reports focused on fluoride-induced autophagy in the animal and human spleen. The objective of this study was to investigate sodium fluoride (NaF)-induced splenocyte autophagy and the potential mechanism via regulation of p-mTOR in growing mice by using the methods of transmission electron microscopy (TEM), immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. A total of 240 ICR mice were equally allocated into four groups with intragastric administration of distilled water in the control group and 12, 24, 48 mg/kg NaF solution in the experimental groups for 42 days. Results revealed that NaF increased autophagosomes or autolysosomes in spleen. Simultaneously, the autophagy marker LC3 brown punctate staining was increased with NaF dosage increase. On the other hand, NaF caused inhibition of mTOR activity, which was characterized by down-regulation of PI3K, Akt and mTOR mRNA and protein expression levels. And the suppression of mTOR activity in turn resulted in the significantly increased of ULK1 and Atg13 expression levels. Concurrently, NaF increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, Atg16L1, Atg12, Atg5 and decreased the mRNA and protein expression levels of p62. The above-mentioned findings verify that NaF induces autophagy via mTOR signaling pathway. The inhibition of mTOR activity and alteration of autophagy-related genes and proteins are the potential molecular mechanism of NaF-induced splenocyte autophagy.

Vanadium quercetin complex attenuates mammary cancer by regulating the P53, Akt/mTOR pathway and downregulates cellular proliferation correlated with increased apoptotic events.

Flavonoid metal ion complexes have been deliberated in recent years and are considered as a new class of medicinal agents with enhanced therapeutic activity and low toxicity. Our study deals with chemotherapeutic effects of vanadium, when coordinated with the flavonoid quercetin on a defined model of chemically induced rat mammary carcinogenesis in vivo and on human breast cancer cell line MCF-7 in vitro. The characterization of the complex was achieved through UV-Visible, IR, and Mass spectra and antioxidant activity was assessed by DPPH, FRAP and ABTS methods. In vitro studies established that the complex upregulated the expressions of p53, Caspase 3 and 9, whereas down regulating Akt, mTOR and VEGF expressions and also induced apoptosis and DNA fragmentation in a dose dependent manner. Acute and Sub-acute toxicity was performed to determine safe doses. 7,12-Dimethylbenz(α)anthracene (0.5 mg/100 g body weight) was used for induction of breast cancer in female Sprague-Dawley rats via single tail vein injection. The histopathological analysis after 24 weeks of carcinogenesis study depicted substantial repair of hyperplastic lesions. TUNEL assay showed an increase in apoptotic index (0.14 ± 0.03; 0.15 ± 0.01) in vanadium-quercetin treated groups as compared to the carcinogen control (0.02 ± 0.01) along with upregulation of Bcl-2 and downregulation of Bax and p53. Immunohistochemical analysis also exhibited decrease in cell proliferation in the vanadium-quercetin treated groups (11.3 ± 0.12; 11.8 ± 0.10). Thus, results from both in vivo and in vitro studies revealed that vanadium-quercetin complex could be a potential candidate for development of approved drug for breast cancer in the near future.