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Objective To evaluate the inhibitory effect of geniposide on the oxidative damage to in vitro cultured human melanocytes,and to explore the role of PI3K-Akt signaling pathway in this inhibitory effect.Methods Epidermal melanocytes were isolated from the circumcised foreskin of healthy adolescent males,and then subjected to culture.Melanocytes at passage 2-3 were divided to six groups:control group receiving no treatment,geniposide group treated with 125 μmol/L geniposide alone,LY294002 group treated with 5 μmol/L LY294002 alone,H2O2 group treated with 250 μmol/L H2O2 alone,geniposide + H2O2 group firstly treated with 125 μmol/L geniposide for 24 hours followed by 4-hour treatment with 250 μmol/L H2O2,and geniposide + LY294002 + H2O2 group treated with 125 μmol/L geniposide for 24 hours,then with 5 μmol/L LY294002 for 1 hour,followed by 4-hour treatment with 250 μmol/L H2O2.After the above treatment,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cellular proliferative activity,Western blot analysis to determine the protein expression of Akt,phosphorylated Akt (p-Akt),heme oxygenase1 (HO-1) and glutathione peroxidase (GPx-1),and flow cytometry to detect the level of cellular reactive oxygen species (ROS),and biochemical methods were used to evaluate the activity of superoxide dismutase (SOD) and catalase (CAT).Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparison,and Student-Newman-Keuls-q (SNK-q) test for multiple com-parisons.Results Compared with the control group,the H2O2 group showed significantly decreased cellular proliferative activity (P ＜ 0.01),protein expression of p-Akt (P ＜ 0.01),HO-1 (P ＜ 0.01) and GPx-1 (P ＜ 0.05),SOD activity (P ＜ 0.01) and CAT activity (P ＜ 0.01),but significantly increased ROS level (P ＜ 0.01).Compared with the H2O2 group,the geniposide + H2O2 group showed significantly increased cellular proliferative activity (72.98％ ± 8.92％ vs.50.53％ ± 10.85％,P ＜ 0.05),up-regulated protein expression of p-Akt (P ＜ 0.05),HO-1 (P ＜ 0.01) and GPx-1 (P ＜ 0.01),increased SOD activity (6.82 ±1.03 U/mg vs.1.29 ± 0.43 U/mg,P ＜ 0.05) and CAT activity (46.08 ± 4.16 U/mg vs.18.71 ± 3.09 U/mg,P ＜0.05),but decreased ROS level (1 284.33 ± 110.64 vs.2 158.00 ± 222.75,P ＜ 0.01).The proliferative activity of melanocytes was significantly lower in the geniposide + LY294002 + H2O2 group (44.35％ ±14.85％) than in the geniposide + H2O2 group (P ＜ 0.05).In addition,the geniposide + LY294002 + H2O2 group showed significantly decreased protein expression of p-Akt (P ＜ 0.01),HO-1 (P ＜ 0.05) and GPx-1 (P ＜ 0.01),SOD (1.31 ± 0.65 U/mg,P ＜ 0.05) and CAT activity (23.25 ± 5.56 U/mg,P ＜ 0.05),but significantly increased ROS level (1 668.00 ± 62.03,P ＜ 0.05)compared with the geniposide + H2O2 group.Conclusion Through the PI3K-Akt pathway,geniposide can promote the protein expression of HO-1 and GPx-1 in melanocytes,enhance SOD and CAT activity,and antagonize the oxidative damage of melanocytes.
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Objective: To investigate the effect of chemokine CX3C receptor 1 (CX3CR1) on human hepatocellular carcinoma Huh7 cells and its probable machanism. Methods: Hepatocellular carcinoma Huh7 cells were transfected with the recombinant adenovirus Ad-siCX3CR1 targeting and silencing CX 3CR 1 gene. Then the expression of CX3CR1 was detected by reverse transcription-PCR (RT-PCR) and Western blotting. The proliferation, apoptosis, migration and invasion of Huh7 cells were tested by MTT, flow cytometry, wound-healing and Transwell assay, respectively. The expression and phosphorylation of Akt which was related to phosphoinositide 3-kinase (PI3K)-Akt signal pathway were detected by Western blotting. Meanwhile, Huh7 cells transfected with AdsiCX3CR1 were treated with different concentrations of PI3K inhibitor LY294002, then the expression of Akt and cell invasion ability induced by CX3CR1 were detected by Western blotting and Transwell assay, respectively. Results: After Ad-siCX3CR1 was stably transfected into Huh7 cells, the expression of CX3CR1 was significantly inhibited (P < 0.001), and the cell proliferation and migration and invasion abilities were significantly increased (P < 0.001), but the apoptosis rate was significantly decreased (P < 0.01). When the expression of CX3CR1 was silenced, the level of phosphorylated Akt (p-Akt) in Huh7 cells was increased (P < 0.001), and LY294002 could reverse CX3CR1-induced phosphorylation of Akt and invasion of Huh7 cells effectively (P < 0.001). Conclusion: CX 3CR 1 gene silencing can promote proliferation, migration and invasion of hepatocellular carcinoma Huh7 cells, and also can suppress their apoptosis. The activation of PI3K-Akt signaling pathway may be involved in this process.
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PURPOSE: To examine the expression of AKT and PTEN in a series of HER2-positive primary invasive breast tumors using immunohistochemistry, and to associate these expression profiles with classic pathologic features such as tumor grade, hormone receptor expression, lymphatic vascular invasion, and proliferation. METHODS: A total of 104 HER2-positive breast carcinoma specimens were prepared in tissue microarrays blocks for immunohistochemical detection of PTEN and phosphorylated AKT (pAKT). Original histologic sections were reviewed to assess pathological features, including HER2 status and Ki-67 index values. The associations between categorical and numeric variables were identified using Pearson's chi-square test and the Mann-Whitney, respectively. RESULTS: Co-expression of pAKT and PTEN was presented in 59 (56.7%) cases. Reduced levels of PTEN expression were detected in 20 (19.2%) cases, and these 20 tumors had a lower Ki-67 index value. In contrast, tumors positive for pAKT expression [71 (68.3%)] were associated with a higher Ki-67 index value. CONCLUSION: A role for AKT in the proliferation of HER2-positive breast cancers was confirmed. However, immunohistochemical detection of PTEN expression did not correlate with an inhibition of cellular proliferation or control of AKT phosphorylation, suggesting other pathways in these mechanisms of control. .
OBJETIVOS: Avaliar a expressão imuno-histoquímica de AKT e PTEN em uma série de carcinomas mamários invasivos HER2-positivos, e associar seus padrões de expressão com variáveis anatomopatológicas clássicas, como grau histológico, expressão de receptores hormonais, embolização vascular linfática e atividade proliferativa. MÉTODOS: Um total de 104 amostras de carcinomas mamários invasivos HER2-positivos foram preparadas em blocos de microarranjos de tecido para detecção imuno-histoquímica de PTEN e AKT fosforilada (pAKT). Cortes histológicos originais foram revistos para avaliação das características anatomopatológicas, incluindo o estado do HER2 e a avaliação da expressão de Ki-67. As associações entre as variáveis categóricas e as numéricas foram feitas com o uso dos testes do chi-quadrado de Pearson e Mann-Whitney, respectivamente. RESULTADOS: Co-expressão de pAKT e PTEN foi identificada em 59 (56,7%) casos. Expressão reduzida de PTEN foi detectada em 20 (19,2%) casos, e esses 20 tumores mostraram menores valores de Ki-67. Por outro lado, tumores positivos para pAKT [71 (68,3%)] apresentaram células positivas para valores mais altos de Ki-67. CONCLUSÕES: O papel de AKT na proliferação de carcinomas mamários HER-2 positiva foi confirmada. Entretanto, a detecção imuno-histoquímica de PTEN não se correlacionou com inibição da proliferação celular ou controle da fosforilação de AKT, sugerindo outras vias nesses mecanismos de controle. .
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , /genetics , Oncogene Protein v-akt/physiology , PTEN Phosphohydrolase/geneticsABSTRACT
Objective To observe the therapeutically effect of kangnao liquid on Pi3k mRNA and Aktm RNA ex-pressions in rats with focal cerebral ischemia-reperfusion (I/R) injury. Methods 180 male SD rats were randomly divided into 6 groups:sham operated group, model group, three kangnao liquid groups (high-dose, medium-dose and low-dose) and nimodipine group. Rats in kangnao liquid groups were administrated with kangnao liquid of 24 g/(kg · d), 12 g/(kg · d) and 6 g/(kg · d), orally once a day. Rats in nimodipine group were given nimodipine 1 mg/(kg · d). Rats in model group and sham group were treated with the same volume of distilled water for 7 days. The animal model of middle cerebral artery occlusion (MCAO) was established by a monofilament method from right internal carotid artery. The neurological evaluation was per-formed 24 h after reperfusion. The in situ hybridization was used to investigate the expression levels of Pi3k mRNA and Akt mRNA in rats on 12 h, 24 h, 48 h, 72 h and 168 h after ischemia for 2 h. Results Compared with model group, neurological functions were improved significantly in kangnao liquid groups. The expression levels of Pi3k mRNA and Akt mRNA were al-so significantly higher in kangnao liquid groups than those of model group. The expression levels of Pi3k mRNA and Akt mRNA were significantly higher in nimodipine group than those of model group, but which were lower compared with those of high-dose and medium-dose kangnao liquid groups. Conclusion Kangnao liquid can protect nerve cells by enhancing the expressions of Pi3k mRNA and Akt mRNA in rats with cerebral ischemia-reprefusion injury.
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Objective: To explore the role of v-akt murine thymoma viral oncogene homolog 2 (AKT2) and p27 in the chemoresistance of multicellular spheroids (MCS) of ovarian cancer to cisplatin. Methods: MCS of A2780 were formed by three-dimensional cell culture. Western blotting was applied to detect the expressions of AKT2 and p27. Then the interfering plasmid of AKT2 was constructed and transfected into the MCS of A2780 cells. The cell apoptosis after treatment with different concentrations of cisplatin was detected by flow cytometry (FCM) and the sensitivity of cells to cisplatin was evaluated by MTT assay. Results: Western blotting indicated that the expressions of AKT2 and p27 were higher in MCS than that in monolayer cells. The expressions of AKT2 and p27 in AKT2 shRNA-transfected MCS were significantly lower than that in MCS without transfection or transfected with empty vector. FACS analysis indicated that after treatment with various concentrations of cisplatin the apoptotic ratio of MCS was significantly lower than that in monolayer cells. AK72 shRNA transfection significantly increased the apoptotic ratio of MCS compared with those without transfection or transfected with empty vector. MTT assay showed that the sensitivity of MCS to cisplatin was increased after transfection with AK'12 shRNA. Conclusion: Interference with AKT2 shRNA down-regulated p27 and consequently reversed the drug resistance of ovarian cancer to cisplatin.