OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid β-peptide_1-42 (Aβ_1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aβ_1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aβ aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.
Alzheimer Disease/therapy* ; Amyloid beta-Peptides/genetics* ; Animals ; Autophagy ; Disease Models, Animal ; Hippocampus/metabolism* ; Mammals/metabolism* ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Moxibustion ; Ribosomal Protein S6 Kinases, 70-kDa/pharmacology* ; Signal Transduction ; Sirolimus/pharmacology* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

OBJECTIVETo investigate the effect of rapamycin on the proliferation of rat valvular interstitial cells in primary culture.

METHODSThe interstitial cells isolated from rat aortic valves were cultured and treated with rapamycin, and the cell growth and cell cycle changes were analyzed using MTT assay and flow cytometry, respectively. RT-PCR was used to detect mRNA expression levels of S6 and P70S6K in cells, and the protein expressions level of S6, P70S6K, P-S6, and P-P70S6K were detected using Western blotting.

RESULTSRat aortic valvular interstitial cells was isolated successfully. The rapamycin-treated cells showed a suppressed proliferative activity (P<0.05), but the cell cycle distribution remained unaffected. Rapamycin treatment resulted in significantly decreased S6 and P70S6K protein phosphorylation level in the cells (P<0.05).

CONCLUSIONThe mechanism by which rapamycin inhibits the proliferation of valvular interstitial cells probably involves suppression of mTOR to lower S6 and P70S6K phosphorylation level but not direct regulation of the cell cycle.

Animals ; Blotting, Western ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Heart Valves ; cytology ; Phosphorylation ; Rats ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacology

OBJECTIVETo investigate the effect of peroxisiome proliferator activated receptor-α (PPAR-α) on the regulation of cardiomyocyte hypertrophy and the relationship between the effect of PPAR-α with PI3K/Akt//mTOR signal pathway.

METHODSCardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (Leica). The expressions of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC) and PPAR-α mRNA were detected by qRT-PCR. The protein expressions of Akt, mTOR and P70S6K were detected by Western blot. The expression of PPAR-α was suppressed by RNAi.

RESULTS(1) The expression of PPAR-α was significantly reduced in cardiomyocyte hypertrophy. PPAR-α activator Fenofibrate (Feno) increased the expression of PPAR-α and suppressed cardiomyocyte hypertrophy. The inhibitory effect of Feno on cardiomyocyte hypertrophy was reversed by PPAR-α RNAi. (2) Feno significantly inhibited the increase of the protein expressions of p-Akt, p-mTOR and p-p70S6K in ISO induced cardiomyocyte hypertrophy, which could be blocked by PPAR-α RNAi. (3) PI3K antagonist LY294002 (LY) or mTOR antagonist rapamycin (RAPA) markedly-inhibited cardiomyocyte hypertrophy. The inhibitory effects of LY or RAPA on cardiomyocyte hypertrophy were reversed by PPAR-α RNAi.

CONCLUSIONPPAR-α can negatively regulate cardiomyocyte hypertrophy. The effect might be associated with PPAR-α inhiting PI3K/ Akt/mTOR signal pathway.

Atrial Natriuretic Factor ; metabolism ; Cardiomegaly ; metabolism ; Cells, Cultured ; Fenofibrate ; pharmacology ; Humans ; Isoproterenol ; adverse effects ; Myocytes, Cardiac ; drug effects ; metabolism ; Myosin Heavy Chains ; metabolism ; PPAR alpha ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism

OBJECTIVETo investigate the effect of everolimus(RAD001)combined with all-trans retinoid acid(ATRA) on drug resistance of ATRA-resistance acute promyelocytic leukemia(APL) cell line NB4-R1 and its molecular mechanism.

METHODSAPL NB4-R1 cells were treated with different concentrations of RAD001(1 nmol/L, 10 nmol/L and 100 nmol/L) with ATRA(1μmol/L) for 24, 48 and 72 h, respectively. The differentiation of NB4-R1 cells was analyzed by flow cytometry with CD11b staining and nitro blue tetrozolium(NBT) reduction test. Cell cycle was detected by cell cycle staining kit and apoptosis was detected by flow cytometry with Annexin V/PI staining. Protein expressions of LC-3II, PML-RARα, P-P70S6K and P-4E-BP1 were determined by Western blotting.

RESULTSRAD001 combined with ATRA significantly induced NB4-R1 cells differentiation, but RAD001 or ATRA alone did not enhance NB4-R1 differentiation. The co-treatment induced accumulation of cells in G1 phase and decreased the proportion of cells in S phase. The combined treatment had no effect on cell apoptosis. The differentiation rate of NB4-R1 cells in 100 nmol/L RAD001, 1μmol/L ATRA, RAD001 combined with ATRA and control groups was(2.29±0.57)%,(17.06±2.65)%,(54.47±4.91)% and(2.54±0.53)%, respectively; the proportion of cells in G1 phase was(35.20±11.97)%,(33.54±6.25)%,(53.70±8.73)% and(27.40±6.01)%, respectively; cells apoptosis rate was(2.30±0.14)%,(2.25±0.21)%,(2.40±0.28)% and(1.95±0.07)%, respectively. The combination of RAD001 with ATRA significantly inhibited mTOR signaling downstream proteins P-P70S6K, P-4E-BP1 and enhanced autophagy-related protein LC3-II and Beclin 1. The co-treatment also induced degradation of fusion protein PML-RARα.

CONCLUSIONRAD001 combined with ATRA can induce cell differentiation, inhibit cell cycle, resulting the reverse of drug resistance in NB4-R1 cells, which is associated with increase of autophagy level and degradation of PML-RARα.

Adaptor Proteins, Signal Transducing ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle ; Cell Differentiation ; Cell Line, Tumor ; drug effects ; Drug Resistance, Neoplasm ; Everolimus ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Oncogene Proteins, Fusion ; metabolism ; Phosphoproteins ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Tretinoin ; metabolism

OBJECTIVETo explore the differences in sensitivity to rapamycin of five esophageal squamous cell carcinoma cell lines with different differentiation and the changes of sensitivity of the cells after siRNA-interfered expression of p70S6K.

METHODSEffects of rapamycin on proliferation of ESCC cell lines with different differentiation, EC9706, TE-1, Eca109, KYSE790 and KYSE450 cells, were investigated using cell counting kit 8 (CCK-8) assay, and according to the above results, the EC9706 cells non-sensitive to rapamycin were chosen to be transfected with p70S6K-siRNA. The changes in sensitivity of cells to rapamycin were measured in vitro and in vivo using CCK-8 kit, flow cytometry and tumor formation in nude mice.

RESULTSCCK-8 results showed that all the five cell line cells were sensitive to low concentration of rapamycin (<100 nmol/L), but TE-1 and EC9706 cells, which were with poor differentiation, showed resistance to high concentration of rapamycin. After EC9706 cells were treated with 50, 100, 200, 500 and 1 000 nmol/L rapamycin and p70S6K-siRNA, the proliferation rates of EC9706 cells were (48.67 ± 1.68)%, (15.45 ± 1.54)%, (14.00 ± 0.91)%, (10.97 ± 0.72)% and (2.70 ± 0.32)%, respectively, and were significantly lower than that of cells treated with 50, 100, 200, 500 and 1 000 nmol/L rapamycin and control siRNA [(74.53 ± 1.71)%, (68.27 ± 1.35)%, (71.74 ± 2.44)%, (76.23 ± 1.02)% and (80.21 ± 2.77)%] (P<0.05 for all). The results of flow cytometry showed that the ratios of cells in G1 phase of the p70S6K-siRNA, rapamycin and p70S6K-siRNA+ rapamycin groups were (53.82 ± 1.78)%, (57.87 ± 4.01)% and (73.73 ± 3.68)%, respectively, significantly higher than that in the control group (46.09 ± 2.31)% (P<0.05 for all). The results of tumor formation test in vivo showed that the inhibitory effect of rapamycin on tumor growth was stronger after the cells were transfected with p70S6K-siRNA, and the inhibition rate was 96.5%.

CONCLUSIONESCC cells with different differentiation have different sensitivity to rapamycin, and p70S6K-siRNA can improve the sensitivity of cells to rapamycin in vitro and in vivo.

Animals ; Antibiotics, Antineoplastic ; pharmacology ; Carcinoma, Squamous Cell ; drug therapy ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; drug therapy ; metabolism ; pathology ; Humans ; Mice ; Mice, Nude ; RNA, Small Interfering ; Ribosomal Protein S6 Kinases, 70-kDa ; genetics ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; Transfection

We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 microM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.
Animals ; Cell Line ; Cell Proliferation/drug effects/genetics ; Cell Survival/drug effects ; Disease Progression ; Eukaryotic Initiation Factor-4E/*antagonists & inhibitors/genetics ; Female ; Mice ; Mice, Nude ; Mucous Membrane/pathology ; Phosphorylation/drug effects ; RNA Interference ; RNA, Small Interfering ; Ribosomal Protein S6 Kinases, 70-kDa/*antagonists & inhibitors/genetics ; Signal Transduction/drug effects ; Sirolimus/*pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Urinary Bladder Neoplasms/genetics/*pathology ; Urothelium/pathology

We investigated how the dual inhibition of the molecular mechanism of the mammalian target of the rapamycin (mTOR) downstreams, P70S6 kinase (P70S6K) and eukaryotic initiation factor 4E (eIF4E), can lead to a suppression of the proliferation and progression of urothelial carcinoma (UC) in an orthotopic mouse non-muscle invasive bladder tumor (NMIBT) model. A KU-7-luc cell intravesically instilled orthotopic mouse NMIBC model was monitored using bioluminescence imaging (BLI) in vivo by interfering with different molecular components using rapamycin and siRNA technology. We then analyzed the effects on molecular activation status, cell growth, proliferation, and progression. A high concentration of rapamycin (10 microM) blocked both P70S6K and elF4E phosphorylation and inhibited cell proliferation in the KU-7-luc cells. It also reduced cell viability and proliferation more than the transfection of siRNA against p70S6K or elF4E. The groups with dual p70S6K and elF4E siRNA, and rapamycin reduced tumor volume and lamina propria invasion more than the groups with p70S6K or elF4E siRNA instillation, although all groups reduced photon density compared to the control. These findings suggest that both the mTOR pathway downstream of eIF4E and p70S6K can be successfully inhibited by high dose rapamycin only, and p70S6K and Elf4E dual inhibition is essential to control bladder tumor growth and progression.
Animals ; Cell Line ; Cell Proliferation/drug effects/genetics ; Cell Survival/drug effects ; Disease Progression ; Eukaryotic Initiation Factor-4E/*antagonists & inhibitors/genetics ; Female ; Mice ; Mice, Nude ; Mucous Membrane/pathology ; Phosphorylation/drug effects ; RNA Interference ; RNA, Small Interfering ; Ribosomal Protein S6 Kinases, 70-kDa/*antagonists & inhibitors/genetics ; Signal Transduction/drug effects ; Sirolimus/*pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Urinary Bladder Neoplasms/genetics/*pathology ; Urothelium/pathology

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.
Bilirubin/*pharmacology ; Cell Line ; Epithelial Cells/cytology/metabolism ; Humans ; Hydrogen Peroxide/toxicity ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kidney Tubules, Proximal/cytology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Oxygen/*pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/metabolism ; Transcriptional Activation/*drug effects ; Up-Regulation/drug effects

This study is to investigate the antitumor activity of ophiopogonin B (OP-B). MTT assay, flow cytometric analysis, acridine orange staining, Lyso-Tracker Red staining and HeLa-GFP-LC3 transfect cells assay were used to detect the proliferation activity, apoptosis and autophagy of HeLa cells. The results showed that OP-B exerted potent antiproliferative activity on HeLa cells, the cell growth inhibition effect of OP-B was not due to apoptosis and OP-B could induce autophagy of HeLa cells. OP-B also induced the protein expression up-regulation of Beclin-1 and promoted LC3 I transformation LC3 II, which were representative proteins of autophagy. Furthermore, 3-MA, an inhibitor of autophagy, not only inhibited OP-B-mediated autophagy but also almost completely reversed the antiproliferative effect of OP-B, suggesting that the growth inhibition effect of OP-B was autophagy dependent. Western blotting demonstrated that OP-B inhibited the phosphorylation of Akt and its' downstream vital protein, such as mTOR and p70S6K. In addition, OP-B also induced the protein expression up-regulation of PTEN, which is a negative regulation protein for Akt/mTOR signaling pathway. However, OP-B did not affect the protein expression of total Akt. Collectively, the antitumor effects of OP-B were autophagy-dependent via repression Akt/mTOR signaling pathway. Therefore, OP-B is a prospective inhibitor of Akt/mTOR and may be used as an alternative compound to treat cervical carcinoma.
Adenine ; analogs & derivatives ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; Beclin-1 ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; HeLa Cells ; Humans ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Ophiopogon ; chemistry ; PTEN Phosphohydrolase ; metabolism ; Phosphorylation ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-akt ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Spirostans ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; Up-Regulation