Role of Akt signal pathway in inhibition of neuronal apoptosis by Chinese medicine Zuogui pill in adult rats treated with monosodium glutamate

AIM: To explore the role of Akt signal pathway in apoptosis of neural cells in adult rats treated with Zuogui Pill, a Chinese medicine. METHODS: Flowcytometry and Western blotting methods were used to investigate the changes of cellular apoptosis rate and Akt signal pathway. RESULTS: Monosodium glutamate (MSG) could increase cellular apoptosis rate and significantly restrained the phosphorylation of Akt (Ser473) and Akt (Thr308), and markedly increased the levels of phospho-FKHR (ser256), GSK-3β (Ser9) and PTEN. Zuogui Pill partly inhibited the above effects of MSG. CONCLUSION: Zuogui pill effectively inhibits the neural apoptosis induced by MSG, and Akt pathway is involved in the neuronal protection of Zuogui pill.     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

miRNA-7 inhibits proliferation of human nasopharyngeal 5-8F carcinoma cells via EGFR/PI3K/Akt pathway

AIM:To investigate the relationship of microRNA-7 (miRNA-7) over-expression and epidermal growth factor receptor (EGFR)/phosphatidylinositol kinase-3 (PI3K)/protein kinase B (PKB, also called Akt) pathway in human nasopharyngeal carcinoma 5-8F cells. METHODS:The 5-8F cells were transfected with miRNA-7 mimics (carrying by Lipofectamine 2000). The expression of miRNA-7 was detected by real-time PCR. The cell proliferation was analyzed by CCK-8 assay. The cell colony-forming capability was determined by cell colony formation test. The expression of EGFR/PI3K/Akt at mRNA and protein levels was examined by real-time PCR and Western blotting. RESULTS:The expression level of miRNA-7 was significantly increased in 5-8F cells compared with negative control (NC) group and control group (P<0.01). The proliferation of NPC 5-8F cells was decreased extremely after tansfected with the miRNA-7 mimics (P<0.01), so did the result of the cell colony-formation test. The expression of EGFR/PI3K/Akt at mRNA and protein levels was significantly down-regulated compared with NC group and control group (P<0.01). CONCLUSION: Over-expression of miRNA-7 significantly inhibits the proliferation and colony-forming ability of nasopharyngeal carcinoma 5-8F cells by down-regulation of EGFR/PI3K/Akt pathway.     

Resveratrol inhibits chondrosarcoma via mitochondrial and PI3K/Akt signaling pathways

AIM：To investigate the inhibitory effects of resveratrol on chondrosarcoma and the relation with mitochondrial and PI3K/Akt pathways. METHODS：Chondrosarcoma SW1353 cells were treated with resveratrol at concentrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h. The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK8 assay and Hoechst 33258 staining, respectively. The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting. The cell migration ability was determined by wound scratch assay. RESULTS：Exposure of the cells to resveratrol resulted in a decrease in the cell viability in a dose- and time-dependent manner (P<0.05). visible nuclei with apoptotic characteristics in resveratrol group were observed. The protein levels of activated caspase-3 and Bax were increased, and Bcl-2 and p-Akt were decreased compared with control group. The total Akt were not significantly changed. Resveratrol also significantly reduced the migration of tumor cells. CONCLUSION：Resveratrol induces apoptosis of chondrosarcoma, which plays a role of part through mitochondrial and PI3K/Akt signaling pathways.     

Effect of gonadotropin-releasing hormone analogue on human breast cancer cell line in vitro

AIM: To investigate the effects of gonadotropin-releasing hormone (GnRH) analogue on the growth of breast cancer cell lines MCF-7 and MDA-MB-231 in vitro and to explore the related mechanisms with PI3K/Akt or ERK/MAPK pathways. METHODS: The proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 treatment with triptorelin was detected by MTT assay and the distribution of the cell cycle was determined by flow cytometry. The phosphorylation of the ERK1/2 and Akt was evaluated by Western blotting. RESULTS: Triptorelin inhibited the proliferation of MCF-7 cells at concentration of 10^-5 mol/L after treated for 192 h or at concentration of 10^-4 mol/L after treated for 168 h and 192 h. Triptorelin inhibited the proliferation of MDA-MB-231 cells at concentration of 10^-4 mol/L after treated for 192 h (P<0.05).Treatment with triptorelin for 192 h at concentration of 10^-4 mol/L had no statistical significance effect on phosphorylation of ERK1/2 and Akt(P>0.05).CONCLUSION: Inhibitory effect of GnRH analogue triptorelin on human breast cancer cells is not just the connection with the down-regulation of pituitary hormone, but also a direct inhibitory effect. The role may not be involved in the activation of ERK/MAPK and PI3K/Akt signaling pathways.     

PI3K/Akt regulates endoplasmic reticulum stress-mediated GRP78 induction

AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODS: PI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR. RESULTS: GRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didnt regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSION: PI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Aerosol delivery of kinase-deficient Akt1 attenuates Clara cell injury induced by naphthalene in the lungs of dual luciferase mice

Conventional lung cancer therapies are associated with poor survival rates; therefore, new approaches such as gene therapy are required for treating cancer. Gene therapies for treating lung cancer patients can involve several approaches. Among these, aerosol gene delivery is a potentially more effective approach. In this study, Akt1 kinase-deficient (KD) and wild-type (WT) Akt1 were delivered to the lungs of CMV-LucR-cMyc-IRES-LucF dual reporter mice through a nose only inhalation system using glucosylated polyethylenimine and naphthalene was administrated to the mice via intraperitoneal injection. Aerosol delivery of Akt1 WT and naphthalene treatment increased protein levels of downstream substrates of Akt signaling pathway while aerosol delivery of Akt1 KD did not. Our results showed that naphthalene affected extracellular signal-regulated kinase (ERK) protein levels, ERK-related signaling, and induced Clara cell injury. However, Clara cell injury induced by naphthalene was considerably attenuated in mice exposed to Akt1 KD. Furthermore, a dual luciferase activity assay showed that aerosol delivery of Akt1 WT and naphthalene treatment enhanced cap-dependent protein translation, while reduced cap-dependent protein translation was observed after delivering Akt1 KD. These studies demonstrated that our aerosol delivery is compatible for in vivo gene delivery.     

Effects of baicalin on CA46 cell xenografts in nude mice

AIM: To study the effects of baicalin on CA46 cell xenografts in nude mice. METHODS: The nude mice with CA46 cell xenografts were treated with drugs via intraperitoneal injection daily, and were divided into 5 groups: negative control group, 15 mg/kg baicalin group, 30 mg/kg baicalin group, 60 mg/kg baicalin group and 4 mg/kg etoposide (VP-16) positive control group. After 12-day treatment, the weight of CA46 cell xenografts stripped from some nude mice in the 5 groups was used to evaluate the effect of baicalin on xenograft growth in the nude mice. The apoptosis, necrosis and pathological changes of the xenograft cells were examined under light microscope and transmission electronic microscope respectively. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway-related proteins extracted from xenografts were determined by Western blotting. The other nude mice with CA46 cell xenografts in the 5 groups continued to be treated with the drugs until death in order to evaluate the effect of balcalin on survival time of the nude mice with CA46 cell xenografts. RESULTS: Baicalin remarkably inhibited the growth of CA46 cell xenografts, induced apoptosis and necrosis of xenograft cells, and reduced the protein expression of phospho-Akt (p-Akt), nuclear factor-kappa B (NF-κB), mammalian target of rapamycin (mTOR) and phospho-mTOR (p-mTOR) in the xenografts after 12-day treatment. Furthermore, baicalin prolonged the survival time of the nude mice with CA46 cell xenografts in a dose-dependent manner. CONCLUSION: Baicalin inhibits the growth and induces apoptosis of CA46 cell xenografts in the nude mice, and prolongs the survival time of the nude mice with CA46 cell xenografts through the mechanism of down-regulating PI3K/Akt/NF-κB and PI3K/Akt/ mTOR signaling pathways.     

Outer membrane protein A of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in RAW264.7 cells

AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.