OBJECTIVETo investigate the significance of Rac subfamily members in the gastrointestinal carcinogenesis and progression.

METHODSThe mRNA expression of Rac1, Rac2 and Rac3 in 12 kinds of gastrointestinal cancer cell lines was examined by semi-quantitative RT-PCR. The activities of Rac1 protein in 5 kinds of gastric cancer cell lines were tested by pull-down assay.

RESULTSCompared with the normal gastric mucosa and intestinal epithelial cell line, the mRNA expression of Rac1 and Rac3 was up-regulated in most of gastrointestinal cancer cell lines. The activities of Rac1 protein increased markedly in gastric cancer cell lines.

CONCLUSIONThe increased mRNA expression of Rac1 and Rac3 in gastrointestinal cancer cell lines and the abnormal activation of Rac1 protein in gastric cancer cell lines might be correlated with the carcinogenesis of gastrointestinal cancer.

Cell Line, Tumor ; Gastrointestinal Neoplasms ; metabolism ; Humans ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; rac GTP-Binding Proteins ; genetics ; rac1 GTP-Binding Protein ; analysis ; genetics

BACKGROUNDRecent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.

METHODSA series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133 - U87 cells. The migration and invasive ability of CD133+ and CD133 - U87 cells were determined by cell migration and invasion assays in vitro. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.

RESULTSIn the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14 ± 4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378 ± 0.007), compared to CD133- cells (0.195 ± 0.004) (t = 27.81; P < 0.05). CD133+ cells had stronger ability to migrate (74.34 ± 2.40 vs. 38.72 ± 2.60, t = 42.71, P < 0.005) and invade (52.00 ± 2.28 vs. 31.26 ± 1.82, t = 30.76, P < 0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.

CONCLUSIONSThese data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Line, Tumor ; Glioblastoma ; metabolism ; pathology ; Glioma ; metabolism ; pathology ; Glycoproteins ; metabolism ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Peptides ; metabolism ; rac1 GTP-Binding Protein ; metabolism

The aim of this study was to investigate the role of RhoA/mDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with mDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with mDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with mDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with mDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-mDia1 pathway.
Actins ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; immunology ; metabolism ; Blood Platelets ; metabolism ; Cells, Cultured ; Humans ; Phosphatidylinositol 3-Kinases ; pharmacology ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism ; pharmacology

OBJECTIVETo investigate the role and signalling of microRNA(miR)-182 on regulating high glucose-induced cardiomyocyte hypertrophy.

METHODSThe candidates of miR which might potentially be involved on targeting Rac1 were predicted by applying bioinformatics analysis. The expression of all related candidates miRs was verified by real-time reverse transcription-PCR (RT-PCR) in cardiac tissues of db/db mice and db/m mice. Then the relationship between candidates miR and Rac1 was investigated with Pearson relevant analysis. Neonatal mice cardiomyocytes were cultured and divided into 2 groups: normal glucose group and high glucose group. The level of selected miR and Rac1 in two groups was detected by RT-PCR. Neonatal mice cardiomyocytes were then randomly divided into 4 groups: normal glucose group, selected microRNA mimics control group, high glucose group, high glucose plus selected miR mimics control group. The morphology of cardiomyocyte in each group was detected under light microscope. Furthermore, Rac1, β-MHC and α-SMA expressions were detected in cultured cardiomyocyte treated by high glucose for 48 h after transfecting selected miR mimics by RT-PCR and Western blot.

RESULTSA total of 6 miR candidates potentially targeting Rac1 were screened by bioinformatics, which were miR-182, miR-142-3p, miR-140, miR-101a, miR-429 and miR-200b. Among these candidates, miR-182 and miR-142-3p expression was significantly downregulated in cardiac tissues of db/db mice compared with db/m controls (P < 0.05). MiR-182 was negatively correlated with Rac1 by person analysis (r = -0.891 02). Downregulation of miR-182 and upregulation of Rac1, β-MHC, α-SMA were found in high glucose-induced cardiomyocyte. After transfection of miR-182 mimics, hypertrophic changes were significantly reduced and Rac1 as well β-MHC expression was significantly downregulated in cardiomyocyte incubated with high glucose.

CONCLUSIONMiR-182 might be involved in the regulation of high glucose-induced myocardial hypertrophy process via targeting Rac1.

Animals ; Cardiomyopathy, Hypertrophic ; metabolism ; Down-Regulation ; Glucose ; physiology ; Mice ; MicroRNAs ; physiology ; Myocytes, Cardiac ; metabolism ; Neuropeptides ; metabolism ; Rats, Sprague-Dawley ; Transfection ; Up-Regulation ; rac1 GTP-Binding Protein ; metabolism

Brain ; Cholesterol ; chemistry ; Cytoskeleton ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Hemolysin Proteins ; chemistry ; pharmacology ; Humans ; Phalloidine ; pharmacology ; Pseudopodia ; drug effects ; Stress Fibers ; drug effects ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism

This study sought to elucidate whether Rac1 mediates the migration of endothelial cell induced by IL-8. The Transwell chamber motility assay was conducted to disclose the effect of different matrigel dilution and different time of IL-8 treatment on the migration of endothelial cells. The mRNA of Rac1 was detected by RT-PCR. The results demonstrated that when the concentration of Matrigel was 1:2, there is significant difference on the amounts of migration cells than that of the concentration of 1:3 or 1:8; When the dilution of Matrigel was 1:4, 1:5 or 1:6, there is no significant difference on the amounts of migration cells than that of other dilution groups. So we choose the Matrigel concentration as 1:4. With the increase of IL-8 stimulation time, the cells which migrated from upper reservoirs to lower reservoirs progressively increased. After six hours stimulation by IL-8, the expression of Rac1 mRNA in migrated cells was increased, compared with that of other groups. The results suggest that Rac1 may mediate the migration of endothelial cells induced by IL-8. It can also be the foundation for further investigation on the role of Rac1 in the migration of endothelial cells induced by IL-8.
Cell Movement ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Humans ; Interleukin-8 ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; metabolism ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

OBJECTIVETo investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing, in order to provide a reference for enriching basic theory of wound healing and guiding clinical application.

METHODSConstitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW, and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First, equal numbers of cells were inoculated into 24-well plates coated with collagen I (20 µg/mL), collagen IV (20 µg/mL) or fibronectin (10 µg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second, 1000 cells adhered to collagen IV, after being stained with tetramethyl rhodamine isothiocyanate-phalloidin, were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third, ESC with density of 2 × 10(5) cells per well were placed in upper compartment of Transwell chamber, DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope, and the result was denoted as migration rate. Lastly, ESC with density of 7.5 × 10(5) cells per well was inoculated into 6-well plates for 12 hours, and treated with 4 µg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test.

RESULTSCompared with that of blank control, the number of Rac1Q61L-transfected cells adhered to collagen I was significantly increased (t = 5.302,P < 0.05), while the number of Rac1T17N-transfected cells adhered to collagen I, IV, and fibronectin were all obviously decreased (with t value respectively 13.741, 15.676, 8.256, P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%, while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching, the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control [(39 ± 9)% vs. (43 ± 5)%, (6 ± 5)% vs. (18 ± 7)%, with t value respectively 1.027, 4.389, with P value respectively above and below 0.05], while that in Rac1T17N-transfected ESC [(81 ± 9)%, (71 ± 11)%, respectively] was obviously higher as compared with that in blank control (with t value respectively 11.386, 11.726, P values all below 0.05).

CONCLUSIONSRac1 protein may control the migration of ESC by regulating its adhesion, spreading, and chemotaxis, and it plays an active role in wound healing accelerated by ESC.

Cell Movement ; Cell Proliferation ; Epidermis ; cytology ; Epithelial Cells ; Humans ; Mutation ; Stem Cells ; cytology ; Transfection ; Wound Healing ; rac1 GTP-Binding Protein ; genetics ; metabolism

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.
Benzamides ; Cell Adhesion ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Fibronectins ; metabolism ; Humans ; Imatinib Mesylate ; Multiple Myeloma ; metabolism ; pathology ; Piperazines ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

OBJECTIVETo study the role of activation of Rac1 in colorectal cancer cell migration and invasion.

METHODSRac1 L61 plasmid and control plasmid were transfected into colorectal cancer cell line SW480 cells. Pull down assay by Western blotting was carried out to measure the amount of activited Rac1, and transwell permeable supports were used to assess the migration and invasion of SW480 cells with different activitivity of Rac1.

RESULTSThe transfection ratio of SW480 cells was more than 80%. Pull down assay showed that the activited Rac1 was significantly higher in the SW480 cells transfected with Rac1 L61 plasmid than that in the control, and the amount of migrating and invasing SW480 cells transfected with Rac1 L61 plasmid in the Transwell permeable supports were significantly more than those in controls (migrating cell numbers: 43 +/- 9 vs. 22 +/- 5, P < 0.01; invasing cell numbers: 73 +/- 13 vs. 38 +/- 1, P < 0.01).

CONCLUSIONThe activation of Rac1 plays an important role in the migration and invasion of colorectal cancer cells.

Cell Line, Tumor ; Cell Movement ; Colonic Neoplasms ; metabolism ; pathology ; Enzyme Activation ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Neoplasm Invasiveness ; Plasmids ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; rac1 GTP-Binding Protein ; genetics ; metabolism

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.
Cell Hypoxia ; Cells, Cultured ; Cellular Senescence ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Plasminogen Activator Inhibitor 1 ; genetics ; metabolism ; Serum Response Factor ; genetics ; metabolism ; beta-Galactosidase ; metabolism ; rac1 GTP-Binding Protein ; metabolism