Mogroside V inhibits H202-induced oxidative damage in MIN6 cells by PI3K/Akt pathway / 中国药理学通报

Aim To investigate the protective effect of mogroside V on hydrogen peroxide ( H,02 )-induced oxidative stress response in mouse islet (3 cells MIN6 and the relation of its mechanism to PI3K/Akt signa¬ling pathway.Methods MIN6 cells were treated with 500 (jimol • L_1 H,(), after mogroside V,and cell via¬bility was detected by MTT.The release of reactive ox¬ygen species ( ROS) and apoptotic percentage of MIN6 cells were determined by flow cytometry.The expres¬sions of apoptosis-related factor Bel-2 , proliferation-re¬lated factor PCNA, protein Akt and p-Akt were deter¬mined by Western blot.Results H,02 restrained the proliferation of MIN6 cells obviously, induced ROS pro¬duction and apoptosis, and reduced the expression of Bel-2 and PCN A.The expressions of protein Akt and p-Akt decreased.After treatment of mogroside V , the release of ROS decreased, and the apoptosis of MIN6 cells was inhibited.The expression levels of apoptosis- related protein Bcl-2 and proliferation-related protein PCN A were reversed.The expressions of protein Akt and P-Akt increased.The viability of MIN6 cells in¬duced by H,0, increased.In addition, mogroside V partly reversed the apoptosis induction and ROS pro¬duction of Akt inhibitor MK2206 (5 jjimol • L"1 ) on MIN6 cells.Conclusions Mogroside V has protec¬tive effect on H202-induced oxidative damage in MIN6 cells and its mechanism is related to PI3K/Akt signa¬ling pathway.

Role of Akt during Islet-1 inducing C3H10T1/2 cells to differentiate specifically into cardiomyocytes / 基础医学与临床

Objective To study the effect and mechanism of Akt during Islet -1 inducing the mesenchymal stem cells of C3H10T 1/2 cells to differentiate specifically into cardiomyocytes.Methods Developing a model of Islet-1 over-expression cells, Lentivirus infection efficiency of cells was detected by flow cytometry detection tech-nology.Cell proliferation was tested by CCK-8.The protein expression of Islet-1, cTnT and p-Akt/T-Akt was measured by Western blot.The mRNA expression of GATA4,Nkx2.5 and Mef2c were tested by real-time PCR. Results With the increasing of MK-2206 concentration, the inhibition rate of cell proliferation increased ( P<0.05) ,and the best inhibition concentration of Akt was 8nmol /L; With prolongation of Islet-1 inducing time,the protein expression of p-Akt/TA-kt reduced ( P<0.05 );The gene expression of myocardial specific transcription factor GATA4, Nkx2.5 andMe f2c increased( P<0.05); treated with MK-2206, the geneexpres-sion of GATA4, Nkx2.5 and Mef2cincreased significantly at the first week and then reduced ( P<0.05) . Conclu isons Akt plays different effects in different differentiation stages during the Islet-1 inducing the cells into cardiacs-pecific differentiation process .

Combination of everolimus and MK-2206 for synergistic inhibition of hepatocarcinoma cell proliferation / 中国药理学与毒理学杂志

OBJECTIVE To examine the synergistic inhibiory effect of combination of mammalian target of sirolimus (Rapamycin) (mTOR) inhibitor everolimus and AKT inhibitor MK-2206 on hepatocar-cinoma cell proliferation. METHODS HepG2 and BEL-7402 cells were treated with sirolimus and evero-limus alone for 0, 1, 3, 6, 12 and 24 h or in combination with insulin-like growth factor 1 receptor (IGF-1R) inhibitor NVP-AEW541 or AKT inhibitor MK2206 for 24 h. p70S6K and AKT kinase activityies were detected by Western blotting. Plate clone formation assay and CCK8 assay were used to detect the growth and proliferation of hepatocarcinoma cells treated with everolimus and MK2206 alone or in combi-nation. RESULTS Sirolimus and everolimus inhibited p70S6K activity while causing feedback activa-tion of AKT kinase activity at different time points (P<0.01). NVP-AEW541 and MK-2206 could inhibit AKT kinase feedback activation by everolimus (P<0.05). Colony formation of hepatocarcinoma cells treated with everolimus and MK-2206 in combination was significantly inhibited compared with everolimus or MK-2206 alone (P<0.01). Everolimus and MK-2206 in combination inhibited the proliferation rate of two types of hepatocarcinoma cancer cells by more than 45％ compared with everolimus used alone (P<0.01). CONCLUSION The resistance of sirolimus and its derivatives in hepatocellular carcinoma cells may be achieved throngh the feedback-activated PI3K/AKT pathway, and the combination therapy can synergistically inhibit the growth and proliferation of hepatocarcinoma cells.

Effect of Artesunate on Akt/GSK-3β/β-catenin Signal Pathway in Human Hepatic Stellate Cells / 中国药师

Objective: To explore the effect of artesunate (Art) on Akt/GSK-3β/β-catenin signal pathway.Methods: Art at different concentrations (0, 12.5, 25, 50 μg·ml-1) was used to treat human hepatic stellate cells (LX-2), and CCK-8 assay was used to detect the cell proliferation to determine the optimal concentration.Art inhibitor (MK-2206) at different concentrations (0~8 μmol·L-1) was given to LX-2 cells, and a Western Blot method was applied to determine the optimal inhibition concentration.Art, MK-2206 and MK-2206+Art were respectively given to LX-2 cells, and a Western Blot method was used to detect the levels of Akt, p-Akt, GSK-3β, p-GSK-3β and β-catenin proteins.Results: CCK-8 assay was used to detect the cell survival rate, and the survival rate was 80% after the 24-hour treatment with 25 μg·ml-1 Art.The results of Western Blot showed that MK-2206 at 6 μmol·L-1 could effectively inhibit the expression of p-Akt.Compared with those of the control group, the levels of Akt, p-Akt, p-GSK-3β and β-catenin protein were significantly different (P＜0.05) in Art (25 μg·ml-1) group, MK-2206 (6 μmol·L-1) group and MK-2206 (6 μmol·L-1) + Art (25 μg·ml-1) group.The expression of GSK-3β and Akt in MK-2206+Art group had no significant difference when compared with that in Art group and MK-2206 group (P＞0.05), while the levels of p-Akt, p-GSK-3β and β-catenin were significantly reduced (P＜0.01).Conclusion: Art exhibits the influence on the relative factors in Wnt/β-catenin signal pathway by Akt/β-catenin, subsequently inhibits the cell proliferation and alleviates the liver fibrosis process.

Effects of Akt Inhibitor MK-2206 on Proliferation and Apoptosis of Lung Adenocarcinoma A549 Cells / 中国药房

OBJECTIVE:To study the effects of Akt inhibitor MK-2206 on the proliferation and apoptosis of lung adenocarcino-ma A549 cells. METHODS:The optical density of A549 cells was detected by MTT assay after treated with 0(blank control),0.5, 1,2.5,5,10,20 and 30 μmol/L MK-2206 for 24 h;after pretreatment with 0(blank control),5,10 and 20 μmol/L MK-2206 for 24 h,morphological changes of A549 cells were observed with inverted microscope. Cell cycle and apoptosis rate were analyzed by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins Cyclin D1,p21 and p27 and apopto-sis-related protein PARP(cf-PARP),cf-caspase-3,Bcl-2 and Bax. RESULTS:Compared with blank control,the optical density of A549 cells decreased,cells shrank and presented vesicular state after treatment of MK-2206;A549 cells arrested in G0/G1 stage, the protein expression of p21 and p27 strengthened while that of Cyclin D1 decreased;the apoptotic rate of cells increased,the ex-pression of cf-PARP,cf-caspase-3 and Bax in cells increased while that of Bcl-2 decreased. All reponse were in concentration-de-pendant manner (P<0.05 or P<0.01). CONCLUSIONS:MK-2206 can inhibit the proliferation of A549 cells,and induce the apoptosis of A549 cells by adjusting the expression of activating caspase-3,down-regulating Bcl-2 and up-regulating Bax.

Blocking autophagy magnifies MK-2206-induced DNA damage in SGC-7901 cells / 中国病理生理杂志

AIM:To investigate the effect of MK-2206, an inhibitor of protein kinase B (Akt), on the DNA damage of SGC-7901 cells.METHODS: SGC-7901 cells were treated with different concentrations of MK-2206, and phosphorylated histone H2AX (γ-H2AX) foci formation was detected by immunofluorescence staining .Western blot analy-sis was used to exam the levels of DNA damage-related protein.The expression of LC3-Ⅱ was determined to evaluate the change of autophagy .RESULTS:MK-2206 treatment increased the formation of γ-H2AX foci and histone H2AX phospho-rylation in the SGC-7901 cells.The levels of DNA damage response protein were also increased .In addition, MK-2206-treated SGC-7901 cells increased the expression of LC 3-II, a hallmark of autophagy .Inhibition of autophagy significantly enhanced MK-2206-mediated histone H2AX phosphorylation.CONCLUSION:MK-2206 induces DNA damage and auto-phagy in SGC-7901 cells.Blocking autophagy potentiates the response of MK-2206-induced DNA damage .

MK-2206, an inhibitor of Akt, induced cell apoptosis and autophagy in U2 OS cells / 中国病理生理杂志

AIM:To observe the effect of MK-2206, an inhibitor of Akt, on the cell apoptosis and autophagy of U2OS cells.METHODS:The cell viability was detected by MTT assay .The cell apoptosis was analyzed by TdT-media-ted dUTP nick end labeling assay .The expression of LC3-II was examined by Western blotting .RESULTS:MK-2206 in-hibited the cell viability in a dose-dependent manner .MK-2206 induced the cell apoptosis via activation of caspase-3, caspase-9 and PARP.MK-2206 treatment substantially induced the U 2OS cell autophagy by increasing in the levels of LC 3-II.Blockage of autophagy using chloroquine magnified MK-2206-induced cell death in U2OS cells.CONCLUSION:The Akt inhibitor MK-2206 induces cell apoptosis and autophagy .Blocking autophagy magnifies MK-2206-induced the inhibi-tion of the viability in U2OS cells.

Effects of Akt inhibitor MK2206 on proliferation and apoptosis of breast cancer cells / 肿瘤

Objective: To study the effects of Akt inhibitor MK2206 on the proliferation and apoptosis of human breast cancer cell lines T47D and MDA-MB-231, and to elucidate the possible mechanism of such effects. Methods: Human breast cancer T47D and MDA-MB-231 cells were treated with different concentrations of MK2206, respectively. The inhibitory effect of MK2206 on cell proliferation was examined by MTT assay. The apoptosis rates of T47D and MDA-MB-231 cells induced by MK2206 were detected by flow cytometry (FCM). The expression levels of caspase-3, poly (ADP-ribose) polymerase (PARP), bcl-2, bax, phosphorate-AKT (p-AKT) and total Akt (T-Akt) proteins were detected by Western blotting. Results: The proliferation of T47D and MDA-MB-231 cells were inhibited after treatment with MK2206 at 0.1, 1, 10 and 100 nmol/L for 24 h, respectively. The apoptosis rates of T47D and MDAMB-231 cells were increased induced by MK2206. The expression levels of caspase-3, PARP and bax proteins were up-regulated, while the expression levels of bcl-2 and p-Akt proteins were downregulated. All of these effects occurred in a dose-dependent manner. MK2206 had no significant effect on the expression of T-Akt. Conclusion: MK2206 can inhibit the proliferation and induce the apoptosis in human breast cancer cell lines T47D and MDA-MB-231. These effects may be related with the downregulation of p-AKT and the inhibition of PI3K-Akt signaling pathway.