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Objective To determine the effects of deoxyelephantopin on mTOR and its related target molecules (Akt/mTOR/P70S6K) in the ER-positive breast cancer cell line. Materials and Methods Primary in silico simulations were determined, and the effects of deoxyelephantopin on the phosphorylation of the Akt/mTOR/P70S6K molecules were evaluated using AlphaScreen-based assays and western blot analysis, respectively. Results Based on the estimated FEB and Ki values, deoxyelephantopin appeared to have a stronger affinity toward P70S6K as compared with Akt and mTOR. Both deoxyelephantopin and control inhibitors were observed to form hydrogen bonds with the same key residue, Leu175 of the P70S6K molecule. Deoxyelephantopin downregulated the p P70S6K protein expression significantly from 18 µM (p < 0.05) and onward. Based on the AlphaScreen assay, deoxyelephantopin produced a concentration-dependent inhibition on the phosphorylation of P70S6K with an IC50 value of 7.13 µM. Conclusion Deoxyelephantopin induced cell death in MCF-7 cells possibly via DNA fragmentation, inhibition of the phosphorylation of P70SK6, and downregulation of the relative p-p70S6K protein expression levels.
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OBJECTIVE：To study the mechanism of improvement effects of Fupi rougan granule （FRG）on hepatic fibrosis model rats. METHODS ：The rats were randomly divided into blank group ，model group ，Colchicine tablet group （chemical positive control ，0.2 mg/kg），Fuzheng huayu capsule group （TCM positive control ，0.415 g/kg），FRG low-dose ，medium-dose and high-dose groups （20，40，80 g/kg），with 10 rats in each group ，except for 11 rats in blank group and model group （one rat was used to judge whether the modeling was successful ）. Except for blank group ，other groups were given intraperitoneal injection of 50％ CCl4 olive oil solution and intragastric administration of 30％ ethanol to induce hepatic fibrosis model. After modeling ， administration groups were given relevant medicine intragastrically ；blank group and model group were given constant volume of normal saline intragastrically ，once a day ，for consecutive 4 weeks. After last administration ，morphology changes of liver tissue in rats were observed. The serum levels of HA ，LN，PCⅢ and Col Ⅳ in rats were detected ，and protein expression of Beclin- 1 and LC3-Ⅱin liver tissue were also determined. mRNA and protein expression of Akt ，AMPK，mTOR，p70S6K were detected in liver tissues of rats. RESULTS ：Compared with blank group ，the structure of hepatic lobules in the model group was disordered ，the proliferation of fibrous tissue was obvious ，and some pseudolobules were formed ；the serum levels of HA ，LN，PCⅢ and Col Ⅳ， the protein expression of Beclin- 1 and LC 3-Ⅱ in liver tissue as well as mRNA and protein expression of Akt ，AMPK，mTOR and p70S6K were increased significantly （P＜0.01）. Compared with model group ，the liver injury of rats in FRG groups was significantly relieved ，and the levels of the above indexes in serum and liver tissue （except for LN and PC Ⅲ in FRG low-dose group） were significantly reduced （P＜0.05 or P＜0.01）. CONCLUSIONS ：FRG can improve hepatic fibrosis in rats ，the mechanism of which may be associated with down-regulating the expression of autophagy associated protein and Akt/AMPK/mTOR/ p70S6K signaling pathway related protein.
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AIM: To analyze the effects of salvianolic acid on autophagy and apoptosis of skeletal muscle ischemia-reperfusion injury mice through AKT/mTOR/p70S6K signaling pathway. METHODS: A total of 45 SPF male rats were randomly divided into sham operation group, model group, salvianolic acid group. Model group and salvianolic acid group were prepared for skeletal muscle ischemia-reperfusion injury model. At 72 h, 48 h, 24 h and 15 min before reperfusion, 1 mL of salvianolic acid solution was administered intraperitoneally at a dose of 30 mg/kg. Rats in the sham operation group and the model group were intraperitoneally injected with 1 mL of normal saline. The pathological morphology of the gastrocnemius muscle, the contents of serum CK and LDH, the expression of Akt, p-Akt, p70S6K, p-p70S6K, mTOR and p-mTOR in the gastrocnemius muscle and the expression of Bcl-2 and Bax in the gastrocnemius muscle were observed. RESULTS: At the 0 h, 4 h and 24 h within reperfusion, the W/D ratio of gastrocnemius muscle in the model group were higher than those in the sham operation group. The W/D ratio of the gastrocnemius muscle in the salvianolic acid group was lower than that in the model group (P<0.05). The levels of serum LDH and CK in the model group were higher than those in the sham operation group at 0 h, 4 h and 24 h after reperfusion. The serum levels of LDH and CK in the salvianolic acid group were lower than those in the model group (P<0.05). The expression of Akt protein in the gastrocnemius of the model group was lower than that in the sham operation group, and the expression of p-Akt protein was higher than that in the sham operation group. The protein expressions of Akt, p-Akt, p70S6K, p-p70S6K, mTOR and p-mTOR in the salvianolic acid group were significantly higher than those of the model group (P<0.05). The IOD value quantified the expression of Bax and Bcl-2 protein in the gastrocnemius muscle cells. The Bcl-2 IOD value and Bcl-2/Bax ratio in the model group were lower than those in the sham operation, whlie the Bax IOD value in the model group was higher than that in the sham operation group. The Bcl-2 IOD value and Bcl-2/Bax ratio in the salvianolic acid group were higher than those in the model group, and the Bax IOD value was lower than that in the model group, the difference was statistically significant (P<0.05). CONCLUSION: Salvianolic acid can maintain the homeostasis of Bax and Bcl-2 in bone cells of rats with skeletal muscle ischemia-reperfusion injury and inhibit cell apoptosis. The mechanism may be related to the activation of AKT/mTOR/p70S6K signaling pathway.
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To explore the affect and mechanisms of rapamycin on mesangial cell proliferation and cell cycle, rat mesangial cells (HBZY-1) were cultured and divided into the six groups: normal; normal with platelet derived growth factor (PDGF) 20 ng·mL-1; PDGF + rapamycin 1, 10, 100, 1 000 nmol·L-1. The cell proliferation was measured by MTT in 24 and 48 h; flow cytometry was used to detect the cell cycle phase. Western blot was performed to determine cyclin D1,cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), p27, p70S6K/p-p70S6K protein expression. The p27 mRNA was detect by Real-time PCR. The results showed that rapamycin significantly suppressed PDGF induced glomerular mesangial cells (MCs) proliferation in a dose and time-dependent manner, but with the dose increased (1 to 1 000 nmol·L-1), the time dependence gradually weakened. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G0/G1 phase. PDGF at 20 ng·mL-1 significantly increased the expression of cyclin D1, cyclin E and CDK2, CDK4 (P < 0.05), but rapamycin did not affect the expression of cyclin D1, cyclin E and CDK2, CDK4. Rapamycin can significantly inhibited p70S6K phosphorylation, up-regulated the expression of p27 protein and mRNA. Collectively, rapamycin has the effect of inhibiting the glomerular mesangial cells proliferation of mesangial cells by regulating the transcription of p27 mRNA, increasing its protein expression through the mTORC1/p70S6K pathway, resulting in decreased activity of cyclin-CDK, and blocking cell cycle in G0/G1 phase.
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Aim To investigate the protective effect of resveratrol on oxidative stress injury of cardiomyocytes induced by high fat and its correlation with AMPK/mTC)RCl/p70S6K signaling pathway. Methods The model of cardiomyocyte lipotoxicity injury was established by palmitic acid (PA). Resveratrol pretreatment and MTT assay were used to detect cell proliferation. The expression of reactive oxygen species ( ROS) was detected by fluorescence and flow cytometry, and the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were detected by kit. The protein expression of AMPK/mTORCl/P70S6K signaling pathway and apoptosis related proteins were determined by Western blot. Results MIT results showed that the cell viability decreased significantly at 0. 4 mmol L-1 and 24 h-48 h stimulation time, re-spectively. Fluorescence and flow cytometry showed that the levels of ROS and MDA increased significantly and SOD activity decreased significantly when PA (0. 4 mmol • L"1) stimulated cells for 24 h compared with control group. Western blot showed that the expression of p-AMPK and Bcl-2 decreased and the expression of p-mTORCl, p-p70S6K, Bax and cleaved caspase-3 increased when PA (0.4 mmol • L"1) stimulated cells for 24 h compared with control group. Resveratrol pre-treatment could significantly reverse the above chan-ges. Conclusions Resveratrol can significantly inhibit the apoptosis of cardiomyocytes induced by high fat. Its mechanism may be related to the regulation of AMPK/mTORCl/p70S6K signaling pathway.
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Objective To observe the effect of Acanthus ilicifolius alkaloid A (HBOA) on PI3K/AKT/mTOR/p70S6K signaling pathway in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4), and to explore the mechanism of action of HBOA against liver fibrosis. Methods Rats were randomly divided into normal group, model group, high, medium; and low-dose HBOA groups (100, 50, 25 mg/kg), and colchicine group (0.4 mg/kg). Except for the normal group, the rats in other groups were given with a 50% CCl4 olive oil solution twice a week for 12 weeks to induce a rat model of liver fibrosis. From the ninth week of modeling, the drug-administered group was given the corresponding test drug once daily for 4 weeks. After the experiment, the body mass change and liver index were calculated. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the liver homogenate of each group were detected. The protein expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K in liver tissue was detected by Western blotting. Results Compared with the model group, the body weight of mice of each drug-administered group was significantly increased, and the liver index, and ALT and AST levels were decreased in liver tissue. In addition, HBOA high and medium-dose groups significantly inhibited the protein expression of p-PI3K, p-Akt, p-mTOR, and p-p70S6K. Conclusion HBOA has a protective effect on hepatic fibrosis rats, and its mechanism may be related to the inhibition of PI3K/Akt/mTOR/p70S6K signaling pathway.
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Chronic cerebral hypoperfusion (CCH), which is associated with onset of vascular dementia, causes cognitive impairment and neuropathological alterations in the brain. In the present study, we examined the neuroprotective effect of duloxetine (DXT), a potent and balanced serotonin/norepinephrine reuptake inhibitor, on CCH-induced neuronal damage in the hippocampal CA1 region using a rat model of permanent bilateral common carotid arteries occlusion. We found that treatment with 20 mg/kg DXT could attenuate the neuronal damage, the reduction of phosphorylations of mTOR and p70S6K as well as the elevations of TNF-α and IL-1β levels in the hippocampal CA1 region at 28 days following CCH. These results indicate that DXT displays the neuroprotective effect against CCH-induced hippocampal neuronal death, and that neuroprotective effect of DXT may be closely related with the attenuations of CCH-induced decrease of mTOR/p70S6K signaling pathway as well as CCH-induced neuroinflammatory process.
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Brain , CA1 Region, Hippocampal , Carotid Artery, Common , Cognition Disorders , Dementia, Vascular , Duloxetine Hydrochloride , Models, Animal , Neurons , Neuroprotection , Neuroprotective Agents , Phosphorylation , Ribosomal Protein S6 Kinases, 70-kDaABSTRACT
Objective:To study the relationship between alpha seine/threonine-protein kinase (p-Akt)-serine/ threonine-protein kinase (mTOR)-ribosomal protein S6 kinase (p70S6K) signaling pathway and clinicopathological features or chemoresistance of ovarian cancer.Methods:We checked the p-Akt,mTOR and p70S6K protein levels in 18 tissues with chemoresistance or 25 with chemosensitivity of ovarian cancer by immunohistochemistry technique,and analyzed the relationship between those proteins and clinicopathological features or chemoresistance of ovarian cancer.Results:The levels of p-Akt protein in ovarian serous carcinoma,mucinous carcinoma and endometrioid carcinoma were 77.14%,50.00% and 66.67%,respectively,with no significant difference (P>0.05).The levels of these proteins in well-middle differentiated carcinoma and low differentiated carcinoma were 73.33% and 75.00%,respectively,with no significant difference (P>0.05).The levels of these proteins in Ⅰ-Ⅱ stage carcinoma,and Ⅲ-Ⅳ stage carcinoma were 18.18% and 93.75%,respectively,with significant difference (P0.05).The levels of this protein in well-middle differentiated carcinoma and low differentiated carcinoma were 80.00% and 78.57%,respectively,with no significant difference (P>0.05).The levels of this protein in Ⅰ-Ⅱ stage carcinoma,and Ⅲ-Ⅳ stage carcinoma were 27.27% and 96.88%,respectively,with significant difference (P0.05).The levels of this protein in well-middle differentiated carcinoma and low differentiated carcinoma were 93.33% and 78.57%,respectively,with no significant difference (P>0.05).The levels of this protein in Ⅰ-Ⅱ stage carcinoma,and Ⅲ-Ⅳ stage carcinoma were 45.45% and 96.88%,respectively,with significant difference (P<0.05).The levels of p-Akt protein in tissue of chemoresistance and chemosensitivity of ovarian cancer were 88.89% and 64.00%,respectively,with significant difference (P<0.05).The levels of mTOR protein in tissue of chemoresistance and chemosensitivity of ovarian cancer were 94.44% and 68.00%,respectively,with significant difference (P<0.05).The levels of p70S6K protein in tissue of chemoresistance and chemosensitivity of ovarian cancer were 100.00% and 72.00%,respectively,with significant difference (P<0.05).Conclusion:The p-Akt-mTOR-p70S6K signaling pathway may take part in invasion and metastasis of ovarian cancer.The up-regulation of these proteins may be associated with the chemoresistance of ovarian cancer,and these proteins may have potential to be the prognostic markers for the chemoresistance of ovarian cancer.
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OBJECTIVE:To study tanshinone ⅡA inhibiting the proliferation and migration induced by homocysteine(Hcy)of rat aortic vascular smooth muscle cells(VSMCs)and its signal pathway. METHODS:VSMCs were selected for the following ex-periments. In order to validate TanshinoneⅡA inhibiting the proliferation and migration induced by Hcy of VSMCs,VSMCs were di-vided into control group,Hcy group(1 000μmol/L),TanshinoneⅡA low-dose,medium-dose and high-dose groups(5,10,20 μg/L). In mechanism study test,VSMCs were divided into control group,TanshinoneⅡA group(20 μg/L),rapamycin group(20 nmol/L), MHY1485 group(10μmol/L),TanshinoneⅡA+rapamycin group(TanshinoneⅡA,20 μg/L+rapamycin,20 nmol/L),TanshinoneⅡA+MHY1485 group (TanshinoneⅡA,20 μg/L+MHY1485,10 μmol/L). In validation test of P70S6K and p-P70S6K pathway expres-sion,rapamycin and MHY1485 were used for inhibitory test and activation test,respectively. The proliferation of VSMCs was de-termined by ELIASA,and Transwell chambers and wound healing test were employed to test the migratory ability of VSMCs. West-ern blotting were used to investigate the expressions of P21,P27,MMP-2,MMP-9,P70S6K and p-P70S6K in VSMCs. RE-SULTS:In validation test,compared with control group,24,48 h absorbance,migration area,the number of VSMCs penetration and the expression of MMP-2,MMP-9 and p-P70S6K increased significantly,while the expression of P21 and P27 decreased sig-nificantly(P<0.01). Compared with Hcy group,48 h absorbance of TanshinoneⅡA low-dose group,24,48 h absorbance and the expression of MMP-2 of TanshinoneⅡA medium-dose and high-dose groups,migration area,the number of VSMCs penetration, the expression of MMP-9 and p-P70S6K in low-dose,medium-dose and high-dose groups all decreased significantly;the expres-sion of P21 and P27 increased significantly in TanshinoneⅡA medium-dose and high-dose groups(P<0.01);there was no statisti-rapamycin group,while 24,48 h absorbance increased significantly in MHY1485 group(P<0.01). Compared with TanshinoneⅡA group,24,48 h absorbance decreased significantly in TanshinoneⅡA+rapamycin group,while 12,24,48 h absorbance,migration area and the number of VSMCs penetration increased significantly in TanshinoneⅡA+MHY1485 group (P<0.01). In inhibitory test,compared with control group,the expression of p-P70S6K decreased significantly in TanshinoneⅡA group (P<0.01);com-pared with TanshinoneⅡA group,the expression of p-P70S6K decreased significantly in rapamycin group and TanshinoneⅡA+rapa-mycin group(P<0.01);in activation test,the expression of p-P70S6K decreased significantly in TanshinoneⅡA group(P<0.01), compared with TanshinoneⅡA group,the expression of p-P70S6K increased significantly in MHY1485,TanshinoneⅡA+MHY1485 group (P<0.01). CONCLUSIONS:TanshinoneⅡA can inhibit the proliferation and migration of VSMCs by suppressing mTOR/P70S6K signal pathway.
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Objective To explore the improving effect of L-leucine on memory impairment in plateau and the mecha-nism. Methods After successfully trained in the 8-arm radial maze,50 male Kunming mice were selected and randomly divid-ed into normoxic control group (NC group),model group,and L-leucine (low,medium and high dose) groups.Animals in L-leu-cine groups were intragastrically given 0.473 g?kg-1 ,0.945 g?kg-1 and 1.89 g?kg-1 L-leucine for 7 days and those in NC and model control groups were administered the same volume of purified water for the same period of time.At the 4th day of the treat-ment,the mice in the model control group and L-leucine groups were placed in a large low-pressure and low-oxygen chamber to simulate low-pressure hypoxic environment of the plateau (7 500 m,3 d).The 8-arm radial maze was used to measure the spatial learning and memory ability of mice and dry-wet method to measure the water content of brain tissue.HE staining was employed to observe the cell morphological changes in CA1 region of the hippocampus.The expression levels of mTOR,P70S6K and 4E-BP1 mRNA in the hippocampus were detected by SYBR Green real-time PCR. Results The reference memory error ( RME) ,total error ( TE) ,testing time ( TT) ,and water content of brain tissue were significantly increased,the neuron injury was exacerbated in CA1 region of the hippocampus,and the expression levels of mTOR and P70S6K mRNA were markedly decreased in model control group when compared with those in NC group (P<0.05 or P<0.01).These indexes,however,were significantly improved in L-leu-cine groups,especially in high-dose group. Conclusion L-leucine can improve memory impairment in plateau,and the mecha-nism may involve the activation of mTOR and its downstream substrates (4E-BP1 and P70S6K).
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Objective To investigate the effects of 1,25-dihydroxyvitamin D3[1,25 (OH)2D3] on cell proliferation in hu?man glomerular mesangial cells and it′s effects on the regulation of mTOR/p70s6K signaling pathway in this cell line. Meth?ods The cultured human mesangial cells at passage 3-7 were divided into four groups:control group,VD group (addition of 10-8 mol/L of 1,25-dihydroxyvitamin D3 ),R group (addition of 5 mg/L of rapamycin) and R+VD group(addition of 5 mg/L ra?pamycin combined with 10-8 mol/L of 1,25-dihydroxyvitamin D3). Drug incubation last 48 h. The effect of mesangial cell pro?liferation was measured by CCK-8 colorimetric assay. The cell cycles were measured by flow cytometry. The expression of mTOR and p70s6K were detected by immunofluorescence. Results (1) The absorbance of A450 was higher in control group than that in VD group than that in R group than that in R+VD group. But the inhibition rate (IR) was lower in control group than that in VD group than that in R group than that in R+VD group. All comparisons were of statistic significance. ( 2) Cells in G1 phase were higher while cells in G2/M and S phases as well as proliferation rate (PI) were lower in control group than those in VD group than those in R group than those in R+VD group. All comparisons were of statistic significance except in?dexes between group R and group VD. (3) mTOR and p30s6K expressions in mesangial cells were higher in control group than those in VD group than those in R group than those in R+VD group. All comparisons were of statistic significance ex?cept indexes between group R and group VD. Conclusion 1,25-dihydroxyvitamin D3 might inhibit mesangial cell prolifera?tion significantly through mTOR/p70s6K signaling pathways.
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Purpose To investigate the expression of reactive oxygen species (ROS) and PI3K/AKT signaling pathway associated gene protein p70S6K1, VEGF in prostate cancer and adjacent tissues, and to determine their relationship with micro-vascular density ( MVD) , as well as the relationship between themselves. Methods The expression of ROS was detected by immunofluorescence, and the expression of p70S6K1, VEGF and MVD counting were detected by immunohistochemistry. Results Compared to adjacent tis-sues, the expression of ROS, p70S6K1, VEGF and MVD in PCa were increased considerablely (P<0. 05). In prostate cancer the ex-pression of ROS were positively correlated to the expression of p70S6K1, VEGF protein, and the expression of ROS and p70S6K1, VEGF were positively correlated to the number of MVD. Conclusion (1)The expression level of ROS in prostate cancer tissues was significantly higher than adjacent tissues. (2) The expression of PI3K/AKT pathway associated gene protein p70S6K1, VEGF protein and MVD counting in prostate cancer tissue was higher than the adjacent tissues, suggesting PI3K/AKT signaling pathways may play a role in tumor angiogenesis. (3) The expression of ROS and PI3K/AKT pathway associated gene protein p70S6K1, VEGF and MVD counting in prostate cancer tissues was positively correlated, suggesting that ROS may play a positive regulatory role for the development of prostate cancer by PI3K/AKT signaling pathway.
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Aim To study the mechanism of cucurb-itacin E ( CuE )-induced autophagy in HeLa cells. Methods Improved MTT assay was adopted to meas-ure the effect of CuE on cell proliferation. Western blot was used to determine the phosphorylation levels of downstream signaling proteins of mTORC1 and the ex-pression of autophagy associated proteins. ResultsCuE inhibited the proliferation of HeLa cells in a dose-dependent manner, and the 24-h IC50 of CuE was 4. 01μmol· L-1 . CuE significantly inhibited the phospho-rylation of p70 S6 K in a time-and dose-dependent man-ner as evidenced by decreased phosphorylation levels of the mTORC1 substrate. Meanwhile, the expression of LC3-II, a marker for autophagosome formation, was elevated by CuE treatment, and was further increased in the presence of chloroquine. Furthermore, CuE re-duced the levels of p62/SQSTM1 . These results indi-cated that CuE induced autophagy in HeLa cells. The decreased levels of phosphorylated ULK1 S757 were posi-tively correlated with autophagy induction in HeLa cells. Conclusion CuE is likely to induce autophagy through inhibiting mTORC1 activity.
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Objective To evaluate the expression and function of mammalian target of rapamycin (mTOR) and its substrate kinase P70S6K in mTOR/P70S6K signal pathway in cervical carcinogenesis. Methods The expression of mTOR and P70S6K in normal cervix uteri or cervical cancer was detected by the immunohistochemistry and RT-PCR methods. Results Compared to those in normal cervix uteri, both mRNA and protein of mTOR/P70S6K in cervical carcinomas were significantly increased (P<0.05). There was a positive correlation between mTOR gene and P70S6K gene expression in cervical cancer (r=0.746, P<0.001). Conclusion High expression of mTOR and P7OS6K may play an important role in the pathogenesis and development of cervical cancer.
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Objective To investigate the antiproliferative effect of rosiglitazone, a thiazolidinedione (TZD) on autosomal dominant polycystic kidney disease (ADPKD) cystic lining epithelial cells and to explore the underlying molecular mechanism. Methods ADPKD cysticlining immortalized epithelial (WT9-12) cells were stimulated by rosiglitazone with different concentrations. After treatment, MTT method was performed to detect the level of proliferation; flow cytometry was used to determine the cell cycle distribution and the apoptosis rate. Western blotting was used to detect the protein expressions of mTOR, p70S6K, 4E-Bp1, PPARγ PPARγ siRNA was transfected into WT9-12 cells to knock down the expression of PPARγ Results Treatment of WT9-12 cells with rosiglitazone resulted in a dose-dependent and time-dependent strong inhibition of cell proliferation, an accumulation of cells in the G0/G1 phase (rosiglitazone 50 μmol/L 65.43%,rosiglitazone 100 μmol/L 64.02%, control 49.65% ) and 6% apoptosis at high concentration (rosiglitazone 200 μmol/L). Rosiglitazone reduced the phosphorylation of p70S6K in a dosedependent and time-dependent manner. The levels of phosphorylated mTOR and 4E-Bp1, the latter being a downstream substrate of mTOR related mRNA translation initiation, were not changed by rosiglitazone. Cells were pre-incubated with GW9662, a PPARγ antagonist, before the treatment with rosiglitazone, the inhibition of p70S6 kinase phosphorylation by rosiglitazone was partially prevented by GW9662 (P＜0.01). Then PPARγ siRNA was transfected into WT9-12 cells, in contrast to untransfected control or cells transfected with an irrelevant siRNA, rosiglitazone did not cause an obvious inhibition of p70S6 kinase phosphorylation in PPARγ knock-down.Conclusion Rosiglitazone inhibits the proliferation of ADPKD cystic lining epithelial cells, and down-regulates p70S6 kinase phosphorylation through mTOR-independent and PPARγ-dependent signal pathway.
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AIM To investigate whether Aβ deposit in Alzheimer disease(AD) impairs signal transduction pathway responsible for neuronal survival.METHODSThe rats were randomly divided into six groups:control group and Aβ25-35 group,Aβ25-35+ibuprofen groups (7.5 and 15 mg·kg-1,respectively),Aβ25-35+ibuprofen+LY294002 group,and Aβ25-35+LY294002 group.Rats were given ibuprofen (7.5 and 15 mg·kg-1 daily,ig) for 3 weeks prior to and 1 week after icv single dose of Aβ25-35 (10 μL,1 mmol·L-1).LY294002 was injected icv 1 h before the injection of Aβ25-35.Seven days after Aβ25-35 injection,the hippocampal expressions of P53,Bax,Fas ligand (FasL),Bcl-2 proteins,phospho-Akt/PKB,and phosphorylated 70 ku ribosomal protein S6 kinase (p70S6K) and caspase 3 were determined in the brain tissue preparations from CA1 area with Western blot.The activity of caspase 3 was measured using a caspase 3 colorimetric activity assay kit.RT-PCR was used to show the change of p70s6k mRNA level.RESULTS Aβ25-35 icv injection significantly down-regulated phosphorylated Akt/PKB from 1.32±0.14 to 0.69±0.08 and p70S6K from 0.769±0.028 to 0.479±0.032 in hippocampal CA1 region.These changes were accompanied by increased expressions of the proapoptotic proteins P53,Bax,and FasL and decreased expression of the anti-apoptotic protein Bcl-2 in rat hippocampus.In addition,caspase 3 activity was significantly enhanced in hippocampal CA1 region in Aβ25-35-treated rats compared with control rats.Ibuprofen can reverse these Aβ25-35-induced changes.CONCLUSION Down-regulated anti-apoptotic PI3K/Akt/p70S6K signaling pathway induced by Aβ25-35 in rat hippocampus may contribute to the neuronal damage in AD.Ibuprofen prevents Aβ25-35-induced down-regulation of PI3K/Akt/p70S6K signaling pathway.
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Background and purpose:TGF-?/EGFR autocrine loop is markedly activated in wide malignant tumor. It is closely correlated with the tumorigenesis and development of different cancers. Our study is to investigate the biological behavior and signaling molecule changes in TGF- induced ovarian cancer cell line Caov-3, and to study the effect and molecular mechanism of the activation of TGF- /EGFR autocrine loop in ovarian cancer.Methods:The tetrazolium-based colorimetry assay was used to evaluate the cell growth treated by TGF-?. The vitro invasion assay was used to examine the invasiveness of Caov-3 treated by TGF-?.Expression of EGFR、 ERK1/2、PI3K、AKT、P70S6K were determined by western blotting.Results:Exogenous TGF-? enhanced significantly the proliferation and invasiveness of Caov-3 cells. The proliferation rate of Caov-3 was in a dose-dependent manner to TGF-? within the concentration of 0.5~25ng/ml.Exogenous TGF-? up-regulated the expression of EGFR、PI3K、AKT、P70S6K but not ERK1/2.Conclusions:The activation of TGF-?/EGFR autocrine loop could promote the growth,invasion and metastasis of ovarian cancer through PI3K/AKT/P70S6K signaling survival pathway.
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Aim To investigate effects of Sevoflurane on PI_3-K/Akt/P~(70S6K) cell-survival signal transduction pathways after neuron ischemia-reperfusion,and explore neuroprotection mechanisms of sevoflurane.Methods Newborn(24～48 h)Wister rats were decapitated and hippocampus tissue was dissected and cut into 1 mm?1 mm?1 mm pieces.Then digestion with 0.125% trypsin,centrifuged at 800 r?min~(-1) for 5 min at 4℃,and suspended in a medium containing DMEM supplemented to 25 mmol?L~(-1) glucose,10% fetal bovine serum,10% horse serum,and 2 mmol?L~(-1) glutamine.Cells were plated at 1.0?10~5?ml~(-1) on poly-Dlysine-treated 96-well(100 ?l/well)plates as well 6-well(2 ml/well) plates.Cultures were treated with 10 ?mol?L~(-1) cytosine arabinoside on day 4 in culture to minimize glial growth.One-half of the medium was replaced twice a week with medium containing DMEM(4.5 g?L~(-1) glucose)/F12(1 ∶1),5% fetal bovine serum and 5% horse serum.Cells were used after 7 days. For ischemia-reperfusion(oxygen glucose deprivation,OGD)experiments,cultures were washed three times in a glucose-free balanced salt solution(BSS)and placed in deoxygenated glucose-free medium and sealed under 95% N_2-5% CO_2 in an anaerobic chamber equilibrated to 37℃ and 100% humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37℃ in 95% O_2-5% CO_2.Experimental group cells were respectively carried out OGD,OGD+2% Sevoflurane,OGD+2% Sevoflurane +10 ?mol?L~(-1) LY294002,OGD+2% Sevoflurane +10 ?mol?L~(-1) Triciribin,and OGD+2% Sevoflurane +10 nmol?L~(-1) Rapamycin.Control cells were cultured normally.Group Sevo was carried out OGD meanwhile anesthesized with 2% sevoflurane.Group LY,Tri and Rap cells was carried out OGD meanwhile culture medium was added 10 ?mol?L~(-1) LY294002,10 ?mol?L~(-1) Triciribin or 10 nmol?L~(-1) Rapamycin,and anesthesized with 2% sevoflurane.Compound remained present throughout the duration of the experiment until analysis 24 h later.Neuron viability and apoptosis were measured.The protein expression of PI_3-K,Akt and P~(70S6K) were detected.Results Sevoflurane enhanced expression of PI_3-K,Akt and P~(70S6K),meanwhile increased neuron viability and decreased neuron apoptosis(vs group I/R,P
ABSTRACT
Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.
Subject(s)
Humans , Phosphatidylinositol 3-Kinase/metabolism , Drug Synergism , Enzyme Activation , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/enzymology , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Stem Cell Factor/pharmacology , Tacrolimus Binding Protein 1A/metabolismABSTRACT
Objective:To observe expression and diversity of glycogen synthase (GSK)-3 and ribosome S6 protein kinase (P70S6K) in skeletal muscle of insulin resistance (IR) rat induced by high fat food and to approach the effect and significance of GSK-3 and P70S6K in IR occurrence.Methods:Forty Wistar rats aged four-week were randomly divided into normal control, high-glucose, high-fat and high-fat high-glucose groups. 8 weeks after feeding, insulin sensitivity of the rats was evaluated by hyperinsulinism euglycaemic clamps technique (ring clamp experiment), GSK-3 and P70S6K in rat skeletal muscle were measured by Western blot and epididymis fat pad weight, blood glucose, insulin, triglyceride (TG), cholesterin (TC), free fatty acids (FFAs) and hsCRP levels were also determined.Results:IR was induced, body weight (P