Rapamycin (Rap) is an immunosuppressant, which is mainly used in the anti-rejection of organ transplantation. Meanwhile, it also shows great potential in the fields of anticancer, neuroprotection and anti-aging. Rap can inhibit the activity of mammalian target of Rap (mTOR). It activates the transcription factor EB (TFEB) to up-regulate lysosomal function and eliminates the inhibitory effect of mTOR on ULK1 (unc-51 like autophagy activating kinase 1) to promote autophagy. Recent research showed that Rap can directly activate the lysosomal cation channel TRPML1 in an mTOR-independent manner. TRPML1 activation releases lysosomal calcium. Calcineurin functions as the sensor of the lysosomal calcium signal and activates TFEB, thus promoting lysosome function and autophagy. This finding has greatly broadened and deepened our understanding of the pharmacological roles of Rap. In this review, we briefly introduce the canonical Rap-mTOR-ULK1/TFEB signaling pathway, and then discuss the discovery of TRPML1 as a new target of Rap and the pharmacological potential of this novel Rap-TRPML1-Calcineurin-TFEB pathway.
Autophagy ; Calcium/metabolism* ; Calcium Channels ; Lysosomes/metabolism* ; Signal Transduction ; Sirolimus

OBJECTIVETo investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells.

METHODSThe in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot.

RESULTSOil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group).

CONCLUSIONSBy downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.

3T3-L1 Cells ; Adipocytes ; drug effects ; metabolism ; Animals ; Cholesterol ; metabolism ; Leptin ; metabolism ; Mice ; PPAR gamma ; genetics ; metabolism ; Sirolimus ; pharmacology

Mammalian target of rapamycin (mTOR) controls cell growth and metabolism in response to nutrients, energy, and growth factors. Recent findings have placed the lysosome at the core of mTOR complex 1 (mTORC1) regulation by amino acids. Two parallel pathways, Rag GTPase-Ragulator and Vps34-phospholipase D1 (PLD1), regulate mTOR activation on the lysosome. This review describes the recent advances in understanding amino acid-induced mTOR signaling with a particular focus on the role of mTOR in insulin resistance.
Amino Acids ; Insulin Resistance* ; Insulin* ; Intercellular Signaling Peptides and Proteins ; Lysosomes ; Metabolism ; Sirolimus*

Fibrosis ; drug therapy ; metabolism ; Humans ; Immunosuppressive Agents ; therapeutic use ; Models, Biological ; Sirolimus ; therapeutic use

OBJECTIVETo investigate the effect of rapamycin on the expression of survivin and caspase-3 at mRNA level in K562 cells and the influence of rapamycin on K562 cell ultrastructure.

METHODSThe effects of rapamycin at various concentration on K562 cell proliferation were analyzed by CCK8; the morphological characteristics of K562 cells was observed by transmission electron microscopy; the expression of survivin and caspase-3 at mRNA level in K562 cells treated with rapamycin was detected by RT-PCR.

RESULTSThe proliferation of K562 cells was significantly inhibited by rapamycin. The apoptosis level of K562 cells increased with increase of rapamycin concentration, the expression of survivin at mRNA level decreased with increase of rapamycin concentration (P < 0.05). The expression of caspase-3 at mRNA level increased with increase of rapamycin concentration.

CONCLUSIONRapamycin can prornote K562 cell apoptosis through up-regulating caspase-3 level and reduceing survivin level.

Apoptosis ; Caspase 3 ; metabolism ; Cell Proliferation ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; K562 Cells ; drug effects ; ultrastructure ; RNA, Messenger ; Sirolimus ; pharmacology

Objective To explore the role of autophagy, apoptosis of neutrophils and neutrophils extracellular traps (NET) formation in systemic lupus erythematosus (SLE). Methods Thirty-six patients with SLE were recruited as research subjects, and 32 healthy controls matched accordingly were enrolled as control subjects. The expression levels of microtubule associated protein 1 light chain 3B (LC3B), autophagy-related gene5(ATG5), P62, B-cell lymphoma 2(Bcl2), Bcl2-related X protein (BAX) in neutrophils were detected by Western blot analysis. Flow cytometry was employed to analyze the expression of LC3B on neutrophils. The expression level of myeloperoxidase(MPO) in plasma was estimated by ELISA. Furthermore, neutrophils were cultured in vitro and stimulated by 100 nmol/L rapamycin and 10 μg/mL lipopolysaccharide (LPS) for 6 hours, respectively. And then, the expression levels of LC3B, ATG5, P62, Bcl2 and BAX in neutrophils were detected by Western blot analysis. The level of MPO in culture supernatant was detected by ELISA. The change of fluorescence intensity of NET in culture supernatant was assayed by Sytox^TM Green staining combined with fluorescence spectrophotometry. Results Compared with healthy controls, the levels of autophagy and apoptosis of neutrophils and NET formation in SLE patients were increased. The level of apoptosis and NET formation was positively associated with neutrophil autophagy. The level of autophagy showed an increase but had no effect on apoptosis and NET formation for neutrophil stimulated by rapamycin. The levels of autophagy and NET formation also increased with no significant effect on apoptosis for neutrophil induced by LPS. Conclusion The autophagy, apoptosis and NET formation of neutrophils increase in SLE patients. The activation of autophagy and NET in neutrophils possibly result from the inflammatory internal environment in SLE patients.
Humans ; Neutrophils ; Extracellular Traps/metabolism* ; Lipopolysaccharides/pharmacology* ; bcl-2-Associated X Protein/metabolism* ; Sirolimus/pharmacology* ; Lupus Erythematosus, Systemic ; Autophagy

The present study was designed to identify the difference between two rapamycin biosynthetic gene clusters from Streptomyces hygroscopicus ATCC29253 and Actinoplanes sp. N902-109 by comparing the sequence and organization of the gene clusters. The biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus ATCC29253 was reported in 1995. The second rapamycin producer, Actinoplanes sp. N902-109, which was isolated in 1995, could produce more rapamycin than Streptomyces hygroscopicus ATCC29253. The genomic map of Actinoplanes sp. N902-109 has been elucidated in our laboratory. Two gene clusters were compared using the online software anti-SMASH, Glimmer 3.02 and Subsystem Technology (RAST). Comparative analysis revealed that the organization of the multifunctional polyketide synthases (PKS) genes: RapA, RapB, RapC, and NRPS-like RapP were identical in the two clusters. The genes responsible for precursor synthesis and macrolactone modification flanked the PKS core region in N902-109, while the homologs of those genes located downstream of the PKS core region in ATCC29253. Besides, no homolog of the gene encoding a putative type II thioesterase that may serve as a PKS "editing" enzyme accounted for over-production of rapamycin in N902-109, was found in ATCC29253. Furthermore, no homologs of genes rapQ (encoding a methyltransferase) and rapG in N902-109 were found in ATCC29253, however, an extra rapM gene encoding methyltransferase was discovered in ATCC29253. Two rapamycin biosynthetic gene clusters displayed overall high homology as well as some differences in gene organization and functions.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Biosynthetic Pathways ; Micromonosporaceae ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Multigene Family ; Sequence Alignment ; Sirolimus ; metabolism ; Streptomyces ; chemistry ; genetics ; metabolism

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.
Humans ; Induced Pluripotent Stem Cells ; Sirolimus/metabolism* ; Caspase 9/metabolism* ; RNA, Guide, CRISPR-Cas Systems ; Pluripotent Stem Cells/metabolism* ; Cell Differentiation ; Puromycin/metabolism*

OBJECTIVE: To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro. METHODS: MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. RESULTS: HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation. CONCLUSION Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.
Emodin/pharmacology* ; AMP-Activated Protein Kinases/metabolism* ; Podocytes ; Caspase 3/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Signal Transduction ; Apoptosis ; Sirolimus/pharmacology* ; Glucose/metabolism* ; Autophagy

The 5'-AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and energy homeostasis in mammalian tissues. Metabolic adaptation is a critical step in ensuring cell survival during metabolic stress. Because of its critical role in the regulation of glucose homeostasis and carbohydrate, lipid, and protein metabolism, AMPK is involved in many human diseases, including cancers. Although AMPK signaling was originally characterized as a tumor-suppressive signaling pathway, several lines of evidence suggest that AMPK plays a much broader role and cannot simply be defined as either an oncogenic regulator or tumor suppressor. Notably, several recent studies demonstrated that the antitumorigenic effects of many indirect AMPK activators, such as metformin, do not depend on AMPK. Conversely, activation of AMPK induces the progression of cancers, emphasizing its oncogenic effect. Bladder cancer can be divided into two groups: non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). The molecular mechanisms underlying these two types of cancer are distinct: NMIBC is associated with activation of the Ras pathway, whereas MIBC is characterized by loss of major tumor suppressors. Importantly, both pathways are connected to the mammalian target of rapamycin (mTOR) pathway. In addition, our recent metabolomic findings suggest that beta-oxidation of fatty acids is an important factor in the development of bladder cancer. Both mTOR and beta-oxidation are tightly associated with the AMPK pathway. Here, I summarize and discuss the recent findings on the two distinct roles of AMPK in cancer, as well as the relationship between bladder cancer and AMPK.
AMP-Activated Protein Kinases* ; Cell Survival ; Fatty Acids ; Glucose ; Homeostasis ; Humans ; Metabolism ; Metabolomics ; Metformin ; Sirolimus ; Stress, Physiological ; Urinary Bladder Neoplasms*