Objective: To study the effects of deguelin on proliferation and apoptosis of human breast cancer cell line MCF-7 and on phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Methods: After treatment with 0, 1, 5, 10, 15 and 20 μmol/L of deguelin for 6, 24, 48 and 72 hours, the proliferation inhibition rate of MCF-7 cells was measured by cell counting kit-8 assay. Apoptosis rate of MCF-7 cells was detected with Annexin V-fluorescein isothiocyanate/propidium iodide double staining by flow cytometry and the apoptotic morphology was observed under a transmission electron microscope. After treatment with 0, 1 and 5 μmol/L of deguelin for 6 hours, 5 proteins involved in the PI3K/Akt signaling pathway were examined by Western blot analysis. Results: Deguelin at doses of 5, 10, 15 and 20 μmol/L inhibited the proliferation of MCF-7 cells at 6, 24, 48 and 72 hours. There was a significant difference in each group compared with the control group (P<0.01). The inhibitory effect was more marked with increasing concentration and duration of treatment. There were statistical differences (P<0.05) among 5, 10, 15 and 20 μmol/L groups. However, 1 μmol/L of deguelin had no obvious effects on the proliferation of MCF-7 cells at 6, 24, 48 and 72 hours, showing no significant difference compared with control group (P>0.05). Deguelin at doses of 5, 10, 15 and 20 μmol/L induced apoptosis of MCF-7 cells at 6 hours. There were significant differences (P<0.01) in the early and late apoptosis rate between the treated groups and the control group. The typical apoptotic MCF-7 cells were observed under the transmission electron microscopy. However, 1 μmol/L of deguelin had no apparent effect in inducing apoptosis of MCF-7 cells at 6 hours. After treatment with 5 μmol/L of deguelin for 6 hours the expression of phosphorylated phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (Ser380), phosphorylated 3-phosphoinositide-dependent protein kinase 1 (PDK1) (Ser241), phosphorylated Akt (Thr308) and phosphorylated glycogen synthase kinase-3β (GSK-3β) (Ser9) proteins were significantly reduced in MCF-7 cells, while there was no significant change in the expression of total Akt protein. However, after treatment with 1 μmol/L of deguelin for 6 hours, there was no apparent change in the expression of these 5 proteins. Conclusion: Deguelin can inhibit the phosphorylation of GSK-3β (Ser9) via inhibition of the phosphorylation of PTEN (Ser380) and PDK1 (Ser241) pathway, thus inducing apoptosis and inhibiting proliferation of MCF-7 cells.

Objective:To explore the expression of B7-1,B7-2 and B7-H1 on tumor-infiltrating dendritic cells(TIDC) and on splenic dendritic cells(SDC),and to investigate TIDC-mediated and SDC-mediated T-cell function after blocking B7-H1 expression in these dendritic cells.Methods: The TIDCs and SDCs were isolated from tumor-bearing mice using anti-mouse CD11c magnetic beads.The expression of B7-1,B7-2 and B7-H1 on TIDC and SDC was analyzed using flow cytometer.T cells were co-cultured with TIDCs or SDCs for the mixed lymphocyte reaction(MLR),and monoclonal antibodies to B7-H1 or the isotype control antibodies were added to the MLR cultures.T-cell proliferation was assessed using XTT method and the secretion of IL-10 was detected using ELISA.Results: B7-1 and B7-2 positive TIDCs were significantly less than SDCs(P0.05).T-cell proliferation stimulated by TIDCs was weaker than that stimulated by SDCs;T cells produced more IL-10 after TIDCs stimulation than after SDCs stimulation(P

Objective:To evaluate the effect of Nutlin-3,a MDM2 antagonist,on the pyroptosis in SMMC-7721 cells.Methods: The expression of actived caspase-1(p20) and IL-1β was detected using Western blot analysis.Pyroptosis was investigated by a standard lactate dehydrogenase release assay(LDH).IL-1β content in the cell culture supernatant was quantified by ELISA.Results: Nutlin-3 up-regulated the expression level of actived caspase-1 and IL-1β in SMMC-7721 cells.Meanwhile,Nutlin-3 increased significantly the content of LDH and IL-1β in the cell culture supernatant(P<0.05).Conclusion: Nutlin-3 activated pro-caspase-1,promoted pyroptosis and IL-1β release in SMMC-7721 cells.

Objective:Investigate the relation between the phosphorylation of FOXO1 and the apoptosis and the proliferation of lymphoma cells and to clarify its specific mechanism.Methods:The lymphoma cells Namalwa and Jurkat were treated with PI3K inhibitor wort mannin or etoposide or Wortmannin plus etoposide for different times-pan and at different concentration.The inhibition rates for cell growth of lymphoma cells were examined by XTT assay.Apoptosis were detected by flow cytometry.The expressions of p-Akt,p-FOXO1,FOXO1 and Bim were determined by Western blot analysis.Results:Wortmannin induced apoptosis of Jurkat cells and Namalwa cells and inhibited their survival effectively.The growth inhibition rate and the apoptosis rate of lymphoma cells induced by Wortmannin plus etoposide were higher than those induced by etoposide alone.After treated with Wortmannin,phosphorylation of FOXO1 remarkably reduced and bim markedly increased.Conclusion:The dephosphorylation of FOXO1 inhibits proliferation of Jurkat cells and Namalwa cells,promotes their apoptosis and enhanced the sensitivity of Non-Hodgkin lymphoma cells to etoposide.Bim activated by FOXO1 promotes cell apoptosis.

Objective:To establish a method for isolating tumor infiltrating dendritic cells(TIDC)from mice models of Lewis lung cancer by positive selection using anti-CD11c magnetic beads.Methods:A total of 1.0?10~6 Lewis lung cancer cells were subcutaneously injected into a C57BL/6 mice at the lateral abdominal wall to establish the mouse model of lung cancer.TIDC were isolated positively using anti-mouse CD11c magnetic beads;they were labeled by anti-mouse CD11c,and then the purity of the isolated cells was tested by FACScan flow cytometer.The cells were also double labeled by PE-conjugated MHC-ⅡmAb and FITC-conjugated CD83 mAb or FITC-conjugated CD86 mAb to analyze the phenotype of cells by FACScan flow cytometer.Results:The positive selection using anti-CD11 c magnetic beads isolated(1.73?0.31)?10~6 TIDC from each gram of Lewis lung cancer tissue,which accounted for(2.18?0.29)%of the cells in the tumor tissues.The purity of TIDC was 96.49%.Electron microscope showed that the isolated TIDC had the typical character of DC cells.The positive rates of MHC-Ⅱ,CD83 and CD86 molecules in TIDC surface were(51.25?4.21)%, (3.48?0.34)%and(3.07?0.65)%,respectively.Conclusion:The positive selection using anti-CD11c magnetic beads is highly effective,simple,and economic,and is worth popularizing.

[ ABSTRACT] AIM: To explore the relationship between FRAS 1 protein and brain metastases of non-small cell lung cancer (NSCLC).METHODS:The mRNA expression of FRAS1 in the brain metastatic tumor tissues and primary tumor tissues of NSCLC was detected by qPCR .The protein expression of FRAS 1 in the tumor tissues and normal tissues adjacent to tumor tissues of NSCLC was measured by SP method of immunohistochemistry .The protein expression of FRAS 1 in NSCLC primary tumor tissues with or without brain metastases was also determined .RESULTS:The mRNA expression of FRAS1 in the brain metastatic zone was nearly 10 times higher than that in the primary tumor tissues , and there was sig-nificant difference between the 2 groups (P<0.05).FRAS1 protein was expressed in the NSCLC primary tumor tissues , but was not found in the normal tissues adjacent to primary tumor tissues .The protein expression of FRAS 1 in the NSCLC with brain metastases was significantly higher than that without brain metastases ( P<0.01 ) .CONCLUSION: FRAS1 protein may be associated with the occurrence of NSCLC .The over-expression of FRAS1 protein may be related to brain metastases with NSCLC .

BACKGROUND:Skeletal muscle satelite cels are muscle-derived stem cels with proliferation and differentiation potential distributing between the muscle cel membrane and the base film. Studies have shown that skeletal muscle satelite cels are of efficacy and safety, but the survival rate of the transplanted stem cels is very low, which greatly limits the application of skeletal muscle satelite cels. OBJECTIVE: To observe the effects of Fasudil on apoptosis of skeletal muscle satelite cels induced by H2O2. METHODS: Skeletal muscle satelite cels cultured in vitro were randomly divided into three groups including H2O2group, H2O2+Fasudil group (Fasudil group) and control group. Apoptosis rates were observed by flow cytometry. The concentrations of interleukin-4 and tumor necrosis factor-a in each group were detected by ELISA. Western blot was employed to measure the protein level of Bax in each group. RESULTS AND CONCLUSION: Compared with the H2O2group, a significant decrease was found in the apoptosis rate of cels, protein level of Bax, and concentrations of interleukin-4 and tumor necrosis factor-a in the Fasudil group (alP < 0.05). These findings indicate that Fasudil can play anti-apoptosis protection by inhibiting Rho-kinase signaling pathway, which may be related to the reduced expression of Bax.

BACKGROUND: Inflammation plays an important role in vessel proliferation after balloon injury. Reducing inflammatory reaction may lighten the ocurrence and development of the restenosis after angioplasty. Studies have demonstrated that PPAR_Y excitomotor has inhibitory effects on inflammation development. OBJECTIVE: To observe the changes in inflammatory factors after carotid artery balloon injury in rats and the intervention of PPARy excitomotor rosiglitazone. DESIGN, TIME AND SETTING: The randomized, controlled animal experiment was performed at the Central Laboratory of Shenzhen People's Hospital from January to June 2009. MATERIALS: Male SPF SD rats weighing about 350 g were selected to generate models of carotid balloon injury. METHODS: SD rats were equally and randomly divided into 3 groups: the control group, the balloon injury group and the rosiglitazone group. The left common carotid arteries were injured by balloon in the balloon injury group and the rosiglitazone group. The control group received sham operation. The rosiglitazone group was administered rosiglitazone daily by gavage,which began 4 days before operation and continued until harvesting.Accordingly,the control group and the balloon injury group were administered normal saline daily by gavage. MAIN OUTCOME MEASURES: All rats were executed under anesthesia at 14 days after operation, respectively to harvest left common carotid artery samples. The vessels were stained by hematoxylin-eosin, and Neointimal area (NIA) and media area (MA) as well as NIA/MA were calculated. Real time RT-PCR and Western Blot method were used to assay the expression of interleukin (IL)-6, IL-10, IL-17A mRNA and the distribution of nuclear factor (NF)-kB protein. expression levels of IL-6 and IL-17A mRNA in the rosiglitazone group were significantly lower than the balloon injury group, but higher than the Control group( P < 0.05), The expression levels of IL-10 mRNA in the rosiglitazone group were higher than the the rosiglitazone group was down-regulated, and lower than the balloon injury group, but higher than control group (P < 0.05). CONCLUSION: Rosiglitazone can regulate the expression of II-6 IL-10 IL-17A mRNA and the balance of inflammatory factors via NF-kB,inhibit the inflammatory reaction of injured vessels and may contribute to lighten the restenosis of injured vessels.

To explore the mechanism of the absorption enhancement of Angelica dahurica extract (Ade), the absorption mechanism of baicalin in the Scutcllaria water extraction as well as the effect of Angelica dahurica extract on absorption of baicalin were investigated. In order to determine the main absorption site, everted intestinal sac model was used to study the effect of Angelica dahurica extract on the absorption of baicalin at duodenum, jejunum, ileum and colon. In situ single pass intestinal perfusion model was performed to study the absorption of various concentrations of baicalin and the effect of Angelica dahurica extract on the absorption of baicalin at the main absorption site. To authenticate the consequence of perfusion by getting the blood from the hepatic portal vein and determine the concentration of the baicalin in the blood. The result showed that baicalin could be absorbed at all of the four intestinal segments with increasing absorption amount per unit as follows: ileum > colon > jejunum > duodenum. The absorption ofbaicalin in the duodenum significantly increased with Angelica dahurica extract, thus, duodenum was chosen to be the studying site. Apparent permeability values (Papp) and absorption rate constant (Ka) of baicalin in the duodenum increased gradually with higher concentrations. When the concentration of baicalin rises to a certain degree, the absorption increase had a saturable process, the absorption of baicalin may be an active transportation. Baicalin may be not a substrate of P-gp as verapamil which had not significantly affected the Papp and Ka of baicalin. The absorption of baicalin in the duodenum significantly increased (P < 0.01) in the two models with Angelica dahurica extract and the concentration of baicalin in the blood from the hepatic portal vein showed that the Angelica dahurica extract can increase the absorption of baicalin.

Objective Tostudyrecentclinicalefficacyandadversereactionsofpemetrexedincombi-nationwithcisplatininthetreatmentofadvancedbreastcancer.Methods Usingenvelopesandrandomlythe patients were dividing into treatment group and control group.The treatment group with 1 8 cases of advanced breast cancer were treated with pemetrexed and cisplatin while in the control group ,23 cases advanced breast cancer patients were treated with gemcitabine and cisplatin with 21 days as a cycle.The efficacy and adverse re-actions were determined according to the WHO on Response Evaluation Criteria in Solid Tumors therapy and chemotherapytoxicityevaluationcriteria.Results Tumorcontrolrateinthetreatmentgroupwas83.3%(1 5/1 8),while in the control group was 78.3%(1 8/23 ),and the difference was not statistically significant (χ2 =0.000 94,P>0.05);while the adverse reactions differenced between the treatment group and the con-trol group were statistically significant(bone marrow suppression χ2 =9.23;fatigue χ2 =4.96;nausea and vomi-tingχ2=4.98;diarrheaχ2=4.45;skinrashχ2=5.03,P<0.05).Conclusion Pemetrexedcombinedwith cisplatin in patients with advanced breast cancer has a good effect,and the adverse reaction is low.