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Objective To investigate the effect of Exosomes derived from lung cancer cells on the migration of secretory cells and homologous tumor cells and to explore the role of PI3K/Akt and SRC signaling pathways in this process.Methods Exosomes were isolated from the supematant post density gradient centrifugation of A549,lung cancer cells.Morphology of the Exosomes was studied using transmission electron microscopy.Protein expression was analyzed using Western blotting.Cell migration was analyzed by a transwell assay.Results The double-membrane-bound Exosomes appeared as discal-shaped structures,30-100 nm in diameter.Western blotting showed that CD9 was abundant in the Exosomes.The Exosomes promoted the migration of A549 cells and their homologous tumor cells,HCC827 in a dose-dependent manner,accompanied by the activation of Akt and SRC.Conclusion The Exosomes derived from A549 (lung cancer) cells promote the migration of the secreting cells and the homologous tumor cells.The mechanism may be correlated with the activation of Akt and SRC.
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Objective To explore the effect of Exosomes isolated from the A549 lung cancer cells on the proliferation of these cells and their ho?mologous tumor cells,HCC827,and the role of the PI3K/Akt and SRC signaling pathways in this process. Methods Exosomes were isolated from the supernatant after density gradient centrifugation of A549 cells. The Exosomes morphology was observed by transmission electron microscopy. The expression of the Exosome?specific proteins was analyzed using Western blotting. Cell proliferation was investigated using the MTT assay. Re?sults The A549?derived Exosomes were 30?100 nm in diameter and had a bilayer membrane.Western blotting showed that CD9 was detected in these Exosomes. The isolated Exosomes promoted the proliferation of the A549 and the HCC827 cells in a dose?and time?dependent manner,ac?companied by the activation of Akt and SRC. Conclusion Exosomes isolated from A549 cells promote the proliferation of the secreting cells and the homologous tumor cells in a dose?and time?dependent manner. The mechanism may be related to the activation of Akt and SRC.
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Arsenic trioxide (ATO) is a strong inducer of apoptosis in malignant hematological cells. Inducible phosphatidyl inositol 3 kinase (PI3K)-Akt activation promotes resistance to ATO. In the present study, we evaluated whether E3 ubiquitin ligase Cbl-b, a negative regulator of PI3K activation, is involved in the action of ATO. The effect of ATO on cell viability was measured by the Trypan blue exclusion assay or by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry and protein expression was assayed by Western blotting. ATO decreased the viability of HL60 cells and induced cellular apoptosis, which was accompanied by transient activation of Akt. The PI3K/Akt inhibitor, LY294002, significantly increased ATO-induced apoptosis (P < 0.05). In addition, ATO up-regulated the expression of Cbl-b proteins. Furthermore, ATO inhibited cell viability with an IC50 of 18.54 μM at 24 h in rat basophilic leukemia-2H3 cells. ATO induced cellular apoptosis with transient activation of Akt and Cbl-b was also up-regulated. Rat basophilic leukemia-2H3 cells transfected with a dominant negative (DN) Cbl-b mutation showed overexpression of Cbl-b (DN) and enhanced Akt activation. Compared with cells transfected with vector, ATO-induced apoptosis was decreased and G2/M phase cells were increased at the same concentration (P < 0.05). The PI3K/Akt inhibitor, LY294002, re-sensitized Cbl-b (DN) overexpressing cells to ATO and reversed G2/M arrest (P < 0.05). Taken together, these results suggest that Cbl-b potentiates the apoptotic action of ATO by inhibition of the PI3K/Akt pathway.
Subject(s)
Animals , Humans , Rats , Apoptosis/drug effects , Arsenicals/pharmacology , Oxides/pharmacology , /antagonists & inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-cbl/pharmacology , Ubiquitin-Protein Ligases/pharmacology , Blotting, Western , Flow Cytometry , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
Objective To investigate the relationship between the sensitivity of gastric cancer cells to arsenic trioxide（As2O3）and the level of reactive oxygen species（ROS）production.Methods The viability of gastric cancer cell lines MGC803,BGC823 and SGC7901 treated with As2O3 was determined by MTT assay.ROS levels of the gastric cancer cells before and after the treatment of As2O3 were detected by flow cytometry.Results Cell growth was significantly inhibited by As2O3 in time-and dose-dependent manner in three gastric cancer cell lines.The IC50（72 h）of As2O3 for MGC803,BGC823 and SGC7901 was about 2.8,3.1 and 10.2 μmol/L,respectively.IC50（72 h）of MGC803 and BGC823 was lower than that of SGC7901（P 0.01）,gastric cancer cell line SGC7901 was less sensitive than the others.The inherent ROS level of MGC803,BGC823 and SGC7901 was 20.3±2.0,64.2±3.3 and 57.7±2.0.After treatment with As2O3 5 μmol/L for 24 h,the peak level of ROS in MGC803 and BGC823 cells increased to 100.8±3.8 and 103.5±2.3,compared with inherent ROS level,the difference had statistical significance（P 0.001,P 0.01）,but the inherent ROS level in SGC7901 cells was 56.5±2.4（P 0.05）.Conclusion The sensitivity of gastric cancer cells to arsenic trioxide is associated with the increase of reactive oxygen species level.
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Objective To investigate the role of phosphoinositide 3-kinases （PI3K）/Akt signaling pathway in TRAIL-induced cell apoptosis, and the effect of oxaliplatin on TRAIL-induced apoptosis in gastric cancer BGC823 cells. Methods Cell proliferation was roeasured using MTT assay. Cell apoptosis was determined by flow eytoroetry. The expression of Akt and phospbor-Akt were determined by Western blotting. Results 100 ng/mL TRAIL caused little cell apoptosis in BGC823 cells. TRAIL activated P13K/Akt pathway. Pretreated with PI3K in- hibitor LY294002 （25 μmol/L）for 1 h followed by exposure to TRAIL for 16 h,the cell apoptosis was obviously higher （12.7%±3,1% vs 3.5%±1.1% ,P 〈 0.05） than that without the treatment of LY294002. Treatment with 38 μg/mL oxaliplatin blocked the activation of P13K/ Akt signaling, and enhanced the sensitivity of cells to TRAIl,, the rate of cell apoptosis increased to 35.5%±4.5% （P 〈 0.05 ）. Conclusion Oxaliplatin enhanced the sensitivity of gastric cancer BGC823 cells to TRAIL by inhibiting TRAIL-induced the activation of PI3K/Akt pathway.
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Objective To investigate the role of nuclear factor kappa-B（NF-κB）signaling pathway in tumor necrosis factor α（TNF-α）-induced apoptosis in gastric cancer cells.Methods Human gastric cancer cell line SGC7901 was treated with TNF-α for 24 hours.MTT assay,flowcytometry and Western blot was used to detect the cell viability and apoptosis.Transient transfection was performed by using Lipofectamine 2000 reagent.The cells of experimental group and control group were respectively transfected with mutant IκB cDNA and vectors.Results Under the treatment of 100,1 000 or 10 000 U/ml TNF-α,the cell viability of SGC7901 cells was（99.2±0.6）%,（92.0±2.7）% and（97.9±2.2）%,respectively.Further study showed that TNF-α engagement led to rapid activation of NF-κB signaling pathway.Blockage of TNF-α-induced NF-κB activation by transient transfection with a mutant IκB enhanced the sensitivity of cells towards TNF-α-induced apoptosis.Conclusion In human gastric cancer cells,activation of NF-κB signaling pathway by TNF-α might be responsible for the resistance to TNF-α-induced apoptosis.Blockage of NF-κB significantly enhanced the apoptosis induced by TNF-α.
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Background and purpose: FGFR2 is a receptor tyrosine kinase and c-Cbl is a new RING finger type of ubiquitin ligase in the ubiquitin-proteasomes path. The purpose of this study was to evaluate the expression and significance of FGFR2 and c-Cbl in gastric carcinoma. Methods: The expression of FGFR2 and c-Cbl were detected by immunohistochemical method of SP. Results: The positive expression rates of FGFR2 and c-Cbl were 77.4%,71.0% in gastric carcinoma, respectively, both were higher than those normal tissue (P<0.05);The expression of FGFR2 and c-Cbl were positively correlated with depth of invasion and TNM staging, and there was a positive relationship between the expressions of FGFR2 and c-Cbl. Conclusion. The expressions of FGFR2 and c-Cbi were associated with some clinicopathologic features in gastric carcinoma, indicating that their expression may be the prognostic factors for gastric carcinoma.