Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) downregulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.
Amines/chemistry* ; Antineoplastic Agents/adverse effects* ; Breast Neoplasms/pathology* ; Chitosan/chemistry* ; Doxorubicin/adverse effects* ; Drug Delivery Systems ; Epithelial-Mesenchymal Transition/drug effects* ; Female ; Humans ; MCF-7 Cells ; Neoplasm Metastasis/prevention & control* ; Oxidation-Reduction ; RNA, Small Interfering/administration & dosage* ; Reactive Oxygen Species/metabolism* ; rac1 GTP-Binding Protein/physiology*

The aims of the present study were to evaluate the effects of puerarin on angiotensin II-induced cardiac fibroblast proliferation and to explore the molecular mechanisms of action. Considering the role of HO in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, we hypothesized that modulating catalase activity would be a potential target in regulating the redox-sensitive pathways. Our results showed that the activation of Rac1 was dependent on the levels of intracellular HO. Puerarin blocked the phosphorylation of extracellular regulated protein kinases (ERK)1/2, abolished activator protein (AP)-1 binding activity, and eventually attenuated cardiac fibroblast proliferation through the inhibition of HO-dependent Rac1 activation. Further studies revealed that angiotensin II treatment resulted in decreased catalase protein expression and enzyme activity, which was disrupted by puerarin via the upregulation of catalase protein expression at the transcriptional level and the prolonged protein degradation. These findings indicated that the anti-proliferation mechanism of puerarin was mainly through blocking angiontensin II-triggered downregulation of catalase expression and HO-dependent Rac1 activation.
Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Animals, Newborn ; Catalase ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Fibroblasts ; Gene Expression Regulation ; drug effects ; Heart ; drug effects ; Hydrogen Peroxide ; metabolism ; pharmacology ; Isoflavones ; pharmacology ; Mice ; Myocardium ; cytology ; enzymology ; metabolism ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Neuropeptides ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor AP-1 ; antagonists & inhibitors ; metabolism ; Transcriptional Activation ; drug effects ; rac1 GTP-Binding Protein ; metabolism

OBJECTIVETo investigate the effects of silencing ADP-ribosylation factor 6 (Arf6) on the proliferation, migration, and invasion of prostate cancer cell line PC-3 and the possible molecular mechanisms.

METHODSThree Arf6-specific small interfering RNA (siRNA) were transfected into cultured prostate cancer cell line PC-3. Arf6 expression was examined by real-time PCR and Western blotting. MTT assay, wound healing assay, and Transwell migration and invasion assay were used to observe the effect of Arf6 silencing on the proliferation, migration, and invasion ability of PC-3 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, p-AKT, AKT and Rac1 were detected by Western blotting.

RESULTSTransfection of siRNA-3 resulted in significantly decreased Arf6 mRNA and protein expression with inhibition rates of (91.88±3.13)% and (86.37±0.57)%, respectively. Arf6 silencing by siRNA-3 markedly suppressed the proliferation, migration and invasion of PC-3 cells and reduced the expression levels of p-ERK1/2 and Rac1.

CONCLUSIONSilencing of Arf6 efficiently inhibits the proliferation, migration, and invasion of PC-3 cells in vitro, and the underlying mechanisms may involve the down-regulation of p-ERK1/2 and Rac1.

ADP-Ribosylation Factors ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Humans ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neoplasm Invasiveness ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; Wound Healing ; rac1 GTP-Binding Protein ; metabolism

Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.
Animals ; Arthritis ; Arthritis, Rheumatoid ; metabolism ; prevention & control ; Benzylisoquinolines ; pharmacology ; therapeutic use ; Cell Movement ; drug effects ; Cell Proliferation ; Cells, Cultured ; Disease Models, Animal ; Down-Regulation ; Fibroblasts ; drug effects ; metabolism ; Humans ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Plant Roots ; Protein-Serine-Threonine Kinases ; metabolism ; Signal Transduction ; Stephania ; chemistry ; Synovial Membrane ; cytology ; drug effects ; metabolism ; rac1 GTP-Binding Protein ; metabolism ; rhoA 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

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

Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.
Albumins/metabolism ; Androstadienes/*administration & dosage/pharmacology ; Animals ; Creatinine/blood ; Desmin/genetics/metabolism ; Diabetes Mellitus, Experimental/*drug therapy/metabolism/pathology ; Diabetic Nephropathies/*drug therapy/metabolism/pathology ; Disease Models, Animal ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Kidney/*pathology ; Membrane Proteins/genetics/metabolism ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Podocytes/*drug effects/metabolism/pathology ; Rats ; Rats, Inbred Strains ; cdc42 GTP-Binding Protein/genetics/metabolism ; rac1 GTP-Binding Protein/genetics/metabolism

OBJECTIVETo observe the subcellular localization of Rac1 and the expression of Tiam1 and Rac1 in gastric carcinoma, in order to reveal the relationship between the distribution of Rac1 and progression of gastric carcinoma.

METHODSBoth carcinoma and adjacent normal tissue of 48 patients with gastric carcinoma were studied in this study. Tissue distribution and expression of Rac1 and Tiam1 were analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR).

RESULTSCompared with that of adjacent non-cancerous gastric mucosa, the expression of Rac1 in cancer tissues was significantly increased. The positive rate of Rac1 expression was 18.8% (9/48 cases) in adjacent non-neoplastic gastric and 79.2% (38/48 cases) in cancer tissues. The positive staining was mainly located in the cell nuclei (31 samples). The real-time PCR results demonstrated that the expression levels of Tiam1 and Rac1 mRNA in cancerous tissues with nuclear localization of Rac1 were evidently increased. Furthermore, nuclear localization of Rac1 was associated with tumor stage and metastasis.

CONCLUSIONSThe majority of gastric cancer tissues show nuclear dislocalization of Rac1 expression, which may be a sign of abnormal activation of Tiam1-Rac1 pathway. It may suggest enhanced invasion ability of the gastric carcinoma.

Adult ; Aged ; Aged, 80 and over ; Cell Nucleus ; metabolism ; Disease Progression ; Female ; Guanine Nucleotide Exchange Factors ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Staging ; Polymerase Chain Reaction ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; T-Lymphoma Invasion and Metastasis-inducing Protein 1 ; rac1 GTP-Binding Protein ; genetics ; metabolism

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

OBJECTIVETo construct lentiviruses carrying dominant negative mutant of Rac1-GTPase (Rac1N17) or the constitutive active mutant of Rac1-GTPase (Rac1L61) and expressing enhanced green fluorescent protein (EGFP) bicistronically.

METHODSThe lentiviral expression plasmid Plenti6/v5-Rac1N17 and Plenti6/v5-Rac1L61 were constructed and identified by restriction enzyme digestion and DNA sequence analysis. The two plasmids were packaged using the ViraPowerTM lentiviral expression system to produce replication-incompetent lentiviruses Rac1L61 and Rac1N17, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different Rac1 mutants were evaluated and the morphology of the transfected PFCs was observed.

RESULTSThe results of DNA sequencing and restriction enzyme analysis demonstrated correct plasmid construction. The packaged lentiviral titer was 1x10(6) TU/ml. Analysis of Rac1 biological activity showed that Rac1N17 lentivirus particles infection significantly inhibited epidermal growth factor-stimulated Rac1 activity in the PFCs, while Rac1L61 lentivirus particles enhanced the Rac1 activity. The transfection efficiency of these Rac1 mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses.

CONCLUSIONThe lentiviruses carrying Rac1 dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs and lend important support for the study of Rac1-GTPase.

3T3 Cells ; Animals ; Animals, Newborn ; Cerebral Cortex ; cytology ; GTP Phosphohydrolases ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; metabolism ; Mice ; Neurons ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction ; Transfection ; rac1 GTP-Binding Protein ; biosynthesis ; genetics