Subject(s)
Dialysis Solutions/adverse effects , Intestinal Mucosa/drug effects , Ischemia/etiology , Peritoneal Dialysis/methods , Pyruvic Acid/analysis , Reperfusion Injury/etiology , Animals , Claudin-1/genetics , Claudin-1/metabolism , Dialysis Solutions/chemistry , Hemodynamics , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Intestinal Absorption , Intestinal Mucosa/blood supply , Intestinal Mucosa/metabolism , Male , Peritoneal Dialysis/adverse effects , Rats , Rats, Sprague-Dawley , Receptors, Purinergic/genetics , Receptors, Purinergic/metabolism , Shock, Hemorrhagic/therapy , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolismABSTRACT
BACKGROUND: This study aimed to investigate the role of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway in protection by peritoneal resuscitation (PR) using pyruvate-peritoneal dialysis solution (PY-PDS) against intestinal injury from hemorrhagic shock (HS) in rats. MATERIALS AND METHODS: Sixty-four rats were assigned to eight groups: group SHAM; group intravenous resuscitation (VR); groups NS, LA, and PY in which the rats were subjected to HS and PR with normal saline (NS), lactate-peritoneal dialysis solution (LA-PDS), and PY-PDS, respectively, combined with VR; and groups DMSO, RPM, and AG490 in which the rats were subjected to HS and VR with pretreatment of dimethyl sulfoxide (DMSO), rapamycin (RPM), and tyrphostin B42 (AG490). RESULTS: At 2 h after HS and resuscitation, the levels of diamine oxidase, 15-F2t-isoprostane, thromboxane B2, and endothelin-1, in the blood and the intestinal mucosal apoptotic index and caspase-3 were lower in groups PY, RPM, and AG490 than in groups VR, NS, LA, and DMSO. Group PY showed lower levels of malondialdehyde and myeloperoxidase and a higher level of superoxide dismutase than groups VR, NS, and LA. Phosphorylated JAK2 and phosphorylated STAT3 levels were lower in groups PY, RPM, AG490, and LA than in groups VR, NS, and DMSO. CONCLUSIONS: The protection mechanism of PR with PY-PDS combined with VR was related to the inhibition of the JAK/STAT signaling pathway during HS and resuscitation. The process might include suppression of oxidative stress, reduction of neutrophil infiltration, regulation of microcirculation, and inhibition of apoptosis.
Subject(s)
Intestinal Diseases/prevention & control , Pyruvic Acid/therapeutic use , Resuscitation/methods , Shock, Hemorrhagic/therapy , Animals , Dialysis Solutions , Drug Evaluation, Preclinical , Intestinal Diseases/etiology , Janus Kinases/antagonists & inhibitors , Janus Kinases/metabolism , Male , Pyruvic Acid/pharmacology , Rats , Rats, Sprague-Dawley , STAT Transcription Factors/antagonists & inhibitors , STAT Transcription Factors/metabolism , Shock, Hemorrhagic/complications , Signal Transduction/drug effectsABSTRACT
OBJECTIVE: To explore the functions of tumor susceptibility gene 101 (TSG101) in the invasion and metastasis of gastric cancer cells by cell culture. METHODS: The TSG101 eukaryotic expression and empty plasmids were transfected into gastric cancer cell line SGC7901. After screening with G418, single cell clone was selected and cultured. The expression of TSG101 was detected by reverse transcription (RT)-PCR and Western blotting. Cells were divided into TSG101 eukaryotic expression plasmid and blank control groups. Then the relationship was examined between TSG101 expression and tumor invasion and metastasis through the invasion, mobile, adhesion and damage scar experiment. RESULTS: The expression levels of TSG101 in mRNA and protein in the TSG101 eukaryotic expression group were significantly higher than those of the plasmid and blank control groups (0.85 ± 0.09 vs 0.55 ± 0.07, 0.45 ± 0.07 and 29.4 ± 1.2 vs 17.0 ± 0.4, 15.9 ± 0.4, all P < 0.05). The cell number of TSG101 eukaryotic expression group through Matrigel, laminin, type IV collagen protein (84 ± 14, 128 ± 10, 62 ± 7) were significantly higher than those of the plasmid group (55 ± 9, 77 ± 10, 31 ± 6) and blank control group (48 ± 8, 76 ± 9, 24 ± 5, all P < 0.01). The number of cells adherent to Matrigel, laminin, type IV collagen protein of the TSG101 eukaryotic expression group (0.97 ± 0.04, 1.34 ± 0.04, 0.90 ± 0.01) were obviously higher than those of the plasmid group (0.53 ± 0.03, 0.75 ± 0.05, 0.42 ± 0.02) and blank control group (0.60 ± 0.03, 0.72 ± 0.03, 0.40 ± 0.01, all P < 0.01). The number of TSG101 eukaryotic expression group cell migrating to membrane lower surface was obviously higher than that of the plasmid group and blank control group (87 ± 13 vs 54 ± 8, 48 ± 7, all P < 0.01). The fusion speed of the TSG101 eukaryotic expression group was faster than that of plasmid and blank control groups after cultivating for 24 and 48 h. CONCLUSIONS: TSG101 expression increases significantly in SGC-7901 cells after a stable transfection of TSG101 eukaryotic expression plasmids. Also the capacities of invasion and metastasis become markedly enhanced.
Subject(s)
DNA-Binding Proteins/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Transcription Factors/genetics , Cell Line, Tumor , Humans , Neoplasm Invasiveness , Neoplasm Metastasis , Plasmids , RNA, Messenger/genetics , TransfectionABSTRACT
OBJECTIVE: To study the expressions and activities of Rho GTPases in hypoxia and its relationship with tumor angiogenesis. METHODS: Three tumor cell lines were used in this study: gastric cancer cell lines AGS, SGC7901 and hepatocellular carcinoma cell line HepG2. Expression level of Rac1 mRNA was detected by semi-quantitative RT-PCR. Activity of Rac1 was determined by pull-down assay and expression of HIF-1alpha, VEGF, p53 and PTEN protein was detected by Westernblot. RESULTS: The expression level of Rac1 mRNA was significantly increased in hypoxia compared to normoxia. Pull-down assay showed that hypoxia-induced activity of Rac1 was elevated in a time-dependent manner and climaxed at 3 hours. The expressions of HIF-1alpha and VEGF protein were up-regulated, while those of PTEN and p53 protein were down-regulated. CONCLUSION: These results indicate that hypoxia enhances Rac1 expression which might be involved in tumor angiogenesis by reacting with hypoxia-responsive genes.
Subject(s)
Hepatoblastoma/metabolism , Liver Neoplasms/metabolism , Neovascularization, Pathologic , Stomach Neoplasms/metabolism , rac1 GTP-Binding Protein/biosynthesis , rho GTP-Binding Proteins/biosynthesis , Cell Hypoxia , Cell Line, Tumor , Hepatoblastoma/blood supply , Hepatoblastoma/pathology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Liver Neoplasms/blood supply , Liver Neoplasms/pathology , PTEN Phosphohydrolase/biosynthesis , PTEN Phosphohydrolase/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Stomach Neoplasms/blood supply , Stomach Neoplasms/pathology , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/genetics , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/genetics , rac1 GTP-Binding Protein/genetics , rho GTP-Binding Proteins/geneticsABSTRACT
OBJECTIVE: To investigate the expression status of Mitogen-activated Protein Kinase Phosphatase-1 in oxygen-deprived gastric cancer cell line SGC7901 and its role in HIF-1 regulation. METHODS: The expression of MKP-1 in gastric cell line SGC7901 was detected with Western blot and semiquantity RT-PCR; Eukaryotic sense expression vector was constructed based on DNA recombination technology. Transfections of SGC7901 were performed using liposome; The luciferase activity was determined using Dual Luciferase Reporter System and the levels of VEGF in SGC7901cells under normoxia and hypoxia were measured by ELISA. RESULTS: (1) Semiquantity RT-PCR and Western blot suggested that the expression of MKP-1 was elevated in oxygen-deprived gastric cancer cell line SGC7901; (2) 48 hours after transfection, the phosphorylated form of HIF-1alpha in cell line transfected with recombinant plasmids was lower compared with that in cell line transfected with empty vectors after 12 hours of exposure to hypoxia; (3) There was very low luciferase activity under nomoxia while under hypoxia luciferase activity increased in a time-dependent manner and at all time points there was significant lower luciferase activity in SGC7901 cells than in cells transfected with empty plasmids. (4) At different points of time course, the expression of VEGF in SGC7901 was significantly higher under hypoxia than that in SGC7901 under nomorxia, while under hypoxia, the expression of VEGF in SGC7901 transfected with recombinant plasmids was significantly lower than that in SGC7901 transfected with empty vectors at all time points as indicated. CONCLUSION: The expression of MKP-1 in SGC7901 was elevated under hypoxia, which could downregulate the HIF-1 trans-activition activity thereby repressing the expression of downstream target gene VEGF.
