Rho GDP Dissociation Inhibitor Alpha Research Articles

P18: Novel Anticancer Peptide from Induced Tumor-Suppressing Cells Targeting Breast Cancer and Bone Metastasis.

The skeletal system is a common site for metastasis from breast cancer. In our prior work, we developed induced tumor-suppressing cells (iTSCs) capable of secreting a set of tumor-suppressing proteins. In this study, we examined the possibility of identifying anticancer peptides (ACPs) from trypsin-digested protein fragments derived from iTSC proteomes. The efficacy of ACPs was examined using an MTT-based cell viability assay, a Scratch-based motility assay, an EdU-based proliferation assay, and a transwell invasion assay. To evaluate the mechanism of inhibitory action, a fluorescence resonance energy transfer (FRET)-based GTPase activity assay and a molecular docking analysis were conducted. The efficacy of ACPs was also tested using an ex vivo cancer tissue assay and a bone microenvironment assay. Among the 12 ACP candidates, P18 (TDYMVGSYGPR) demonstrated the most effective anticancer activity. P18 was derived from Arhgdia, a Rho GDP dissociation inhibitor alpha, and exhibited inhibitory effects on the viability, migration, and invasion of breast cancer cells. It also hindered the GTPase activity of RhoA and Cdc42 and downregulated the expression of oncoproteins such as Snail and Src. The inhibitory impact of P18 was additive when it was combined with chemotherapeutic drugs such as Cisplatin and Taxol in both breast cancer cells and patient-derived tissues. P18 had no inhibitory effect on mesenchymal stem cells but suppressed the maturation of RANKL-stimulated osteoclasts and mitigated the bone loss associated with breast cancer. Furthermore, the P18 analog modified by N-terminal acetylation and C-terminal amidation (Ac-P18-NH2) exhibited stronger tumor-suppressor effects. This study introduced a unique methodology for selecting an effective ACP from the iTSC secretome. P18 holds promise for the treatment of breast cancer and the prevention of bone destruction by regulating GTPase signaling.

Calreticulin as a special marker to distinguish dental pulp stem cells from gingival mesenchymal stem cells

The construction of protein abundance profiles helps to interpret the clinical applications of stem cells. Dental pulp stem cells (DPSCs) and gingival mesenchymal stem cells (GMSCs) can be isolated from teeth and used as a highly convenient clinical potential material. Here, we aimed to explore commonalities and differences of DPSCs and GMSCs at the protein level. TMT-based quantitative proteomics and two-dimensional gel electrophoresis technology were used in combination to describe the protein profile of DPSCs and GMSCs extracted from the same donor. A total of 2821 proteins were identified by LC-MS/MS, of which 248 differentially abundant proteins (DAPs) were highly expressed in GMSCs while 782 proteins were highly expressed in DPSCs. The biological functions and molecular pathways of DAPs were annotated with GO enrichment and KEGG analysis. The relationship between molecular abundance and cell characteristics including source, proliferation, angiogenesis and inflammation were connected by WGCNA. Special markers, including Calreticulin (CALR), Annexin A5 (ANXA5) and Rho GDP dissociation inhibitor alpha (GDIR1), were proposed to distinguish DPSCs from GMSCs. Our results provide a molecular basis for in-depth understanding of the protein composition and special functions of dental stem cells, and promote the potential clinical application.

A novel preventive mechanism of gentamicin-induced nephrotoxicity by atorvastatin.

Atorvastatin (ATO) inhibits the synthesis of nonsteroidal isoprenoid compounds and possesses a pleiotropic effect. However, the detailed mechanism of ATO in preventing gentamicin (GM)-induced renal injury remains obscure. Although underlying multifaceted mechanisms involving GM-induced nephrotoxicity were well known, further work on elucidating the essential mechanism was needed. Using a fluorogenic derivatization-liquid chromatography tandem mass spectrometry proteomic method (FD-LC-MS/MS method), we investigated the effects and mechanisms of ATO treatment on GM-induced nephrotoxicity in rats. Consequently, 49 differentially expressed proteins were identified. The most significant mechanisms of nephrotoxicity caused by GM were mitochondrial dysfunction, fatty acid metabolism and oxidative stress. Their upstream regulator was found to be PPARα. The proteins involved in GM nephrotoxicity were sodium-hydrogen exchanger regulatory factor (SLC9A3R1), cathepsin V (CTSV), macrophage migration inhibitory factor (MIF) and RhoGDP dissociation inhibitor alpha (ARHGDIA). After ATO intervention, we observed a reversed enrichment pattern of their expression, especially in CTSV and SLC9A3R1 (P-value<0.05). We predicted that ATO may improve abnormal phospholipid metabolism and phospholipidosis caused by GM and also alleviate cell volume homeostasis and reverse the interference of GM with the transporter. Furthermore, proteomic results also provided clues as to GM-induced nephrotoxicity biomarkers such as CTSV and transthyretin.

Expression of Rho GDP dissociation inhibitor alpha in papillary thyroid carcinoma and its clinical significance

Objective To observe the level of Rho GDP dissociation inhibitor (GDI) alpha (RhoGDIa) expression in papillary thyroid carcinoma (PTC) and explore its clinical significance. Methods The levels of RhoGDIa and microRNA (miRNA, miR)-151-5p expression in 200 cases of PTC tissues and paired normal tissues were detected by real-time quantitative polymerase chain reaction (Real-time PCR), respectively. The relationship between RhoGDIa expression and the clinicopathological features in PTC patients, and between RhoGDIa expression and miR-151-5p levels in PTC tissues was investigated. Results As compared with normal tissues adjacent to cancer, the expression of RhoGDIa mRNA was decreased in 76.5% (153/200) PTC tissues and that of miR-151-5p was increased in 72.5% (145/200) PTC tissues, respectively. The correlation analysis showed that there was a significantly negative correlation between the miR-151-5p level and the expression of RhoGDIa mRNA in PTC tissues (r=-0.925, P=0.000). As compared with PTC without lymph node metastasis, the miR-151-5p level was significantly up-regulated and the expression of RhoGDIa protein was significantly down-regulated in those PTC with lymph node metastasis, and the expression abundance of miR-151-5p was negatively correlated with the level of RhoGDIa protein. The low expression of RhoGDIa mRNA was significantly correlated with gender (P=0.000), number of lesions (P=0.004), local invasion (P=0.002), lymph node metastasis (P=0.000) and clinical stage (P=0.000) in PTC patients. Conclusion RhoGDIa was down-regulated in PTC tissues and closely related to tumor progression, and its expression may be negatively regulated by miR-151-5p. Key words: Papillary thyroid carcinoma; Rho GDP dissociation inhibitor alpha; MicroRNA-151-5p; Lymph node metastasis

Effect of carboxyl-terminal truncated HBx mediated down-regulation of RhoGDIα expression on the metastasis and invasion of hepatocellular carcinoma

Objective To investigate the mechanism of natural carboxyl-terminal truncated HBx (with 31 amino acids deleted at the C-terminal end) (HBxΔ31)-dependent down-regulation of Rho GDP dissociation inhibitor alpha (RhoGDIα) expression and its role in enhancing hepatocellular carcinoma (HCC) metastasis. Methods HepG2 cells with stable expression of wild type HBx and its deletion mutant HBxΔ31 protein were selected as the study subjects. The effects of HBx and HBxΔ31 on RhoGDIα expression in HepG2 cells was detected using real-time quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. A series of deleted and mutated variants of the RhoGDIα promoter were made and individually co-transfected with HBx-and HBxΔ31-expressing vectors or control empty vector into HepG2 cells. The luciferase reporter assay was used to identify the cis-regulatory elements of the RhoGDIα promoter in response to HBxΔ31 regulation. The interaction between Myc-associated zinc finger protein (MAZ) and HBxΔ31 was examined by co-immunoprecipitation experiment. Eelectrophoretic mobility shift assay (EMSA) was performed to determine interaction of MAZ with the RhoGDIα promoter in HBxΔ31-expressing HepG2 cells. The impact of reduced RhoGDIα expression by HBxΔ31 on cell-invasive activity was analyzed by the Matrigel cell invasion assay. In addition, the effects of silencing of shRNA-mediated RhoGDIα in HepG2 or introduction of RhoGDIα by transfection in HBxΔ31-expressing HepG2 cells on cell-invasion were investigated. Results HBxΔ31, but not HBx, suppressed RhoGDIα expression at transcriptional levels. Analysis of the deletion and mutation of RhoGDIα promoter showed that the HBxΔ31 repressive element localized between nt-460 and-242 bp of RhoGDIα promoter and that the transcription factor MAZ binding sites was required for RhoGDIα promoter inactivation regulated by HBxΔ31. In addition, HBxΔ31 represses RhoGDIα expression by enhancing MAZ binding to its promoter through directly associating with MAZ. The cell-migratory and cell-invasive activity were significantly increased in sh-RhoGDIα-expressing HepG2 cells, as compared to control cells (migrated cells number: 58 ± 5 vs. 98 ± 7, invaded cells number: 55 ± 6 vs. 113 ± 6, t= 18.91 and t= 20.12, both P < 0.01). However, ectopic expression of RhoGDIα in HBxΔ31-expressing HepG2 cells significantly decreased cell migration and invasion (migrated cells number: 40 ± 4 vs. 115 ± 5, invaded cells number: 42 ± 4 vs. 102 ± 4, t= 18.14 and t= 16.31, both P < 0.001). Conclusion Carboxyl-terminal truncated HBx deregulates RhoGDIα expression through MAZ, in turn which promotes the invasion and metastasis of HCC. Key words: Carcinoma, hepatocellular; Sequence deletion; Hepatitis B virus; Gnanine nucleotide dissociation inhibitors; Neoplasm metastasis

RGC-32 is expressed in the human atherosclerotic arterial wall: Role in C5b-9-induced cell proliferation and migration

The complement system is an important player in the development of atherosclerosis. Previously reported as a cell cycle regulator, RGC-32 is an essential effector of the terminal complement complex, C5b-9. In this study, our aims were to determine the expression of RGC-32 in the human atherosclerotic arterial wall and to delineate the mechanisms through which RGC-32 affects C5b-9-induced endothelial cell proliferation and migration. We now demonstrate that RGC-32 is expressed in human aortic atherosclerotic wall and that RGC-32 expression increases with the progression of atherosclerosis. Furthermore, silencing of RGC-32 expression abolished C5b-9-induced human aortic endothelial cell (HAEC) proliferation and migration. Of the 279 genes differentially expressed in HAECs after RGC-32 silencing, the genes involved in cell adhesion and cell cycle activation were significantly regulated by RGC-32. RGC-32 silencing caused a significant reduction in the expression of cyclin D1, cyclin D3, Akt, ROCK1, Rho GDP dissociation inhibitor alpha and profilin. These data suggest that RGC-32 mediates HAEC migration through the regulation of RhoA and ROCK1 expression and is involved in actin cytoskeletal organization. Thus, RGC-32 has promising therapeutic potential with regard to angiogenesis and atherosclerosis.

EIF5A1/RhoGDIα pathway: a novel therapeutic target for treatment of spinal cord injury identified by a proteomics approach.

Spinal cord injury (SCI) is frequently accompanied by a degree of spontaneous functional recovery. The underlying mechanisms through which such recovery is generated remain elusive. In this study, we observed a significant spontaneous motor function recovery 14 to 28 days after spinal cord transection (SCT) in rats. Using a comparative proteomics approach, caudal to the injury, we detected difference in 20 proteins. Two of these proteins, are eukaryotic translation initiation factor 5A1 (eIF5A1) that is involved in cell survival and proliferation, and Rho GDP dissociation inhibitor alpha (RhoGDIα), a member of Rho GDI family that is involved in cytoskeletal reorganization. After confirming the changes in expression levels of these two proteins following SCT, we showed that in vivo eIF5A1 up-regulation and down-regulation significantly increased and decreased, respectively, motor function recovery. In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length while eIF5A1 knockdown reversed these results. We found that RhoGDIα up-regulation and down-regulation rescues the effect of eIF5A1 down-regulation and up-regulation both in vivo and in vitro. Therefore, we have identified eIF5A1/RhoGDIα pathway as a new therapeutic target for treatment of spinal cord injured patients.

MiR-25 promotes hepatocellular carcinoma cell growth, migration and invasion by inhibiting RhoGDI1

MicroRNA (miRNA)-25 is a small non-coding RNA that has been implicated in the tumorigenesis of many cancers, but little is known on the role of miR-25 in HCC metastasis. We hereby found that miR-25 was significantly upregulated in clinical HCC tissues compared with normal liver tissues. We also revealed that miR-25 dramatically stimulates HCC cell growth and activates the epithelial-mesenchymal transition (EMT). MiR-25 is activated by the WNT/β-catenin signaling pathway, and exerts its pro-metastatic function by directly inhibiting the Rho GDP dissociation inhibitor alpha (RhoGDI1). Downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. MiR-25 levels are positively correlated with β-catenin expression, whereas negatively correlated with the level of RhoGDI1 in HCC. Our findings provide new insights into the role of miR-25 in HCC metastasis, and implicate the potential application of miR-25 in HCC therapy.

MiR-483-5p Promotes Invasion and Metastasis of Lung Adenocarcinoma by Targeting RhoGDI1 and ALCAM

The nodal regulatory properties of microRNAs (miRNA) in metastatic cancer may offer new targets for therapeutic control. Here, we report that upregulation of miR-483-5p is correlated with the progression of human lung adenocarcinoma. miR-483-5p promotes the epithelial-mesenchymal transition (EMT) accompanied by invasive and metastatic properties of lung adenocarcinoma. Mechanistically, miR-483-5p is activated by the WNT/β-catenin signaling pathway and exerts its prometastatic function by directly targeting the Rho GDP dissociation inhibitor alpha (RhoGDI1) and activated leukocyte cell adhesion molecule (ALCAM), two putative metastasis suppressors. Furthermore, we found that downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. Importantly, miR-483-5p levels are positively correlated with β-catenin expression, but are negatively correlated with the levels of RhoGDI1 and ALCAM in human lung adenocarcinoma. Our findings reveal that miR-483-5p is a critical β-catenin-activated prometastatic miRNA and a negative regulator of the metastasis suppressors RhoGDI1 and ALCAM.

Influence of ARHGEF3 and RHOA knockdown on ACTA2 and other genes in osteoblasts and osteoclasts.

Osteoporosis is a common bone disease that has a strong genetic component. Genome-wide linkage studies have identified the chromosomal region 3p14-p22 as a quantitative trait locus for bone mineral density (BMD). We have previously identified associations between variation in two related genes located in 3p14-p22, ARHGEF3 and RHOA, and BMD in women. In this study we performed knockdown of these genes using small interfering RNA (siRNA) in human osteoblast-like and osteoclast-like cells in culture, with subsequent microarray analysis to identify genes differentially regulated from a list of 264 candidate genes. Validation of selected findings was then carried out in additional human cell lines/cultures using quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed significant down-regulation of the ACTA2 gene, encoding the cytoskeletal protein alpha 2 actin, in response to RHOA knockdown in both osteoblast-like (P<0.001) and osteoclast-like cells (P = 0.002). RHOA knockdown also caused up-regulation of the PTH1R gene, encoding the parathyroid hormone 1 receptor, in Saos-2 osteoblast-like cells (P<0.001). Other findings included down-regulation of the TNFRSF11B gene, encoding osteoprotegerin, in response to ARHGEF3 knockdown in the Saos-2 and hFOB 1.19 osteoblast-like cells (P = 0.003–0.02), and down-regulation of ARHGDIA, encoding the Rho GDP dissociation inhibitor alpha, in response to RHOA knockdown in osteoclast-like cells (P<0.001). These studies identify ARHGEF3 and RHOA as potential regulators of a number of genes in bone cells, including TNFRSF11B, ARHGDIA, PTH1R and ACTA2, with influences on the latter evident in both osteoblast-like and osteoclast-like cells. This adds further evidence to previous studies suggesting a role for the ARHGEF3 and RHOA genes in bone metabolism.

Differentially expressed proteins in ER+ MCF7 and ER- MDA- MB-231 human breast cancer cells by RhoGDI-α silencing and overexpression.

The consequence of Rho GDP dissociation inhibitor alpha (RhoGDIα) activity on migration and invasion of estrogen receptor positive (ER+) and negative (ER-) breast cancer cells has not been studied using the proteomic approach. Changes in expression of RhoGDIα and other proteins interacting directly or indirectly with RhoGDIα in MCF7 and MDA-MB-231, with different metastatic potentials is of particular interest. ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα. The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells. Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.

Secretomic Analysis of Mouse Choroid Plexus Cell Line ECPC-4 Using Two-Dimensional Gel Electrophoresis Coupled to Mass Spectrometry

Choroid plexus (CP), the main production site of cerebrospinal fluid (CSF), is responsible for the inflammatory mediators involved in meningitis and immune systems. The mouse choroid plexus cell line ECPC-4 is known to be useful for analyses of CP functions. In this study, we performed the secretome analysis of ECPC-4 using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. Twenty-two secreted proteins were identified in the conditioned medium of ECPC-4. They were a secreted protein acidic and rich in cysteine (SPARC), plasminogen activator inhibitor 1 (PAI-1), and others (vinculin, heat shock protein cognate 71 kDa, moesin, tubulin s2A, fascin, calreticulin, gamma actin, alcohol dehydrogenase, malate dehydrogenase, Rho GDP dissociation inhibitor alpha, phosphoglyceratemutase 1, translationally controlled tumor protein, phosphatidylethanolamin binding protein1, peroxiredoxin1 type II, peroxiredoxin1, myosin light polypeptide 6, cofilin-1, nucleoside diphosphate kinase A, peptidyl-prolylcis-trans isomerase A[PPI-A], and galectin1). These proteins could be classified as cytoskeleton/ intermediate filament, protein folding, signal transduction, cell growth, metabolism, and redox regulation groups, suggesting that they could be related to the CP functions. Furthermore, the level of four proteins was changed in the conditioned medium of ECPC-4 treated with lipidA. Proteasome subunit alpha type-1 and nucleoside diphosphate kinase A were significantly increased and gamma-actin and galectin-1 were significantly decreased, as compared withthose in the conditioned medium of non-treated ECPC-4. Also, Western Blotting also validated the changes in proteasome subunit alpha type-1, galectin-1 and nucleoside diphosphate kinase A. Thus, it is suggested that these proteins play an important role in inflammation such as meningitis. ECPC-4, like CP epithelial cells,is useful to analyze the CP functions.

Rutin, a Flavonoid and Principal Component of Saussurea Involucrata, Attenuates Physical Fatigue in a Forced Swimming Mouse Model

This study investigated the antifatigue effects of rutin, a flavonoid extracted from the ethyl acetate extract of S. involucrata. Mice were subjected to a weight-loaded forced swim test (WFST) on alternate days for 3 wk. Rutin was administered orally to the mice for 7 days in dosages of 15, 30, and 60 mg/kg body weight, and several biomarkers of physical fatigue were evaluated: swimming time, change in body weight, lipid peroxidation, lactic acid (LA), glycogen, and the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). On Day 7, the rutin-treated mice had a 3-fold longer exhaustive swimming time than the control mice, as well as significantly reduced blood LA concentrations. The 15, 30, and 60 mg/kg body weight rutin-supplemented groups displayed 11.2%, 22.5%, and 37.7% reduced malondialdehyde (MDA) concentrations, respectively, in brain and muscle tissues compared with the control exercised group. Our results indicated that the administration of rutin protected the mice against the depletion of SOD and GPx activities significantly. Following 7 days of rutin treatment, we sacrificed the mice and analyzed their soleus muscle and brain for peroxisome proliferator-activated receptor-α coactivator (PGC-1α) and sirtuin 1 (SIRT1) mRNA expression. We observed that rutin treatment increased PGC-1α and SIRT1 mRNA and protein expression. The changes in these markers of mitochondrial biogenesis were associated with increased maximal endurance capacity. The application of 2D gel electrophoresis to analyze the rutin-responsive protein profiles in the WFST mouse brain further revealed the upregulation of the CB1 cannabinoid receptor-interacting protein 1, myelin basic protein, Rho GDP dissociation inhibitor (GDI) alpha, and TPI, indicating that rutin might inhibit anxiety through the upregulation of the expression of anxiety-associated proteins. Western blot analysis of MAPK expression further confirmed the antianxiety effects of rutin. Our study results thus indicate that rutin treatment ameliorates the various impairments associated with physical fatigue.

Overexpression of RhoGDI, a novel predictor of distant metastasis, promotes cell proliferation and migration in hepatocellular carcinoma

RhoGDI (Rho GDP-dissociation inhibitor alpha, or RhoGDIα) was identified as a regulator of Rho GTPases, but its role in cancer remains controversial. In this study, increased expression of RhoGDI was detected in hepatocellular carcinoma (HCC) cell lines and tissues with highly metastatic potential. RhoGDI overexpression correlated with postoperative distant metastasis. Enforced expression of RhoGDI in HCC cells significantly enhanced cell proliferation and migration. Conversely, knockdown of RhoGDI caused an inhibition of the aggressive phenotypes of HCC cells. Furthermore, RhoGDI up-regulated Rho, but not Rac, and enhanced PI3K/AKT and MAPK pathway activity. Our findings suggest that RhoGDI overexpression is a predictor of distant metastasis and plays an important role in the progression of HCC.

Pathological Cyclic Strain-Induced Apoptosis in Human Periodontal Ligament Cells through the RhoGDIα/Caspase-3/PARP Pathway

AimHuman periodontal ligament (PDL) cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what mechanism(s) are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis.Materials and MethodsHuman PDL cells were obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expression was evaluated by Western blot analysis.ResultsThe number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIα) decreased. Furthermore, knock-down of RhoGDIα by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibition of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIα protein levels.ConclusionThe overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced apoptosis in human PDL cells through the RhoGDIα/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in human PDL cells.

FERM Domain Containing Protein 7 Interacts with the Rho GDP Dissociation Inhibitor and Specifically Activates Rac1 Signaling

The FERM domain containing protein 7 gene (FRMD7) associated with the X-linked disorder idiopathic congenital nystagmus (ICN) is involved in the regulation of neurite elongation during neuronal development. Members of the Rho family of small G-proteins (Rho GTPases) are key regulators of the actin cytoskeleton and are implicated in the control of neuronal morphology. The Rho GDP dissociation inhibitor alpha, RhoGDIα, the main regulator of Rho GTPases, can form a complex with the GDP-bound form of Rho GTPases and inhibit their activation. Here, we demonstrate that the full length of the mouse FRMD7, rather than the N-terminus or the C-terminus alone, directly interacts with RhoGDIα and specifically initiates Rac1 signaling in mouse neuroblastoma cell line (neuro-2a). Moreover, we show that wild-type human FRMD7 protein is able to activate Rac1 signaling by interacting with RhoGDIα and releasing Rac1 from Rac1-RhoGDIα complex. However, two missense mutations (c.781C>G and c.886G>C) of human FRMD7 proteins weaken the ability to interact with RhoGDIα and release less Rac1, that induce the activation of Rac1 to a lesser degree; while an additional mutant, c.1003C>T, which results in a C-terminal truncated protein, almost fails to interact with RhoGDIα and to activate Rac1 signaling. Collectively, these results suggest that FRMD7 interacts with one of the Rho GTPase regulators, RhoGDIα, and activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth. We predict that human mutant FRMD7 thus influences Rac1 signaling activation, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.

RhoGDIα downregulates androgen receptor signaling in prostate cancer cells

Treatment of primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state due to aberrant activation of androgen receptor (AR). Understanding the mechanisms leading to the aberrant activation of AR is critical to develop effective therapy. We have previously identified Rho GDP Dissociation Inhibitor alpha (GDIα) as a novel suppressor in prostate cancer. In this study, we examine the effect of GDIα on AR signaling in prostate cancer cells. GDIα was transiently or stably transfected into several prostate cancer cell lines including LNCaP, C4-2, CWR22Rv1, and DU145. The regulation of AR expression by GDIα was analyzed by qRT-PCR and Western blot. AR activity was measured by luciferase reporter assays and electrophoretic mobility shift analysis (EMSA). Immunofluorescence assay was performed to study AR nuclear translocation. The interaction between GDIα and AR was examined by co-immunoprecipitation assays. In this study, we have identified GDIα as a negative regulator of AR signaling pathway. Overexpression of GDIα downregulates AR expression at both mRNA and protein levels. Overexpression of GDIα is able to prevent AR nuclear translocation and inhibit transactivation of AR target genes. Co-immunoprecipitation assays showed that GDIα physically interacts with the N-terminal domain of AR. GDIα suppresses AR signaling through inhibition of AR expression, nuclear translocation, and recruitment to androgen-responsive genes. GDIα regulatory pathway may play a critical role in regulating AR signaling and prostate cancer growth and progression.

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2μM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P<0.05) when tested in Wistar rats allotransplanted with C6 glioma cells.

Chemopreventive mechanisms of methionine on inhibition of benzo(a)pyrene–DNA adducts formation in human hepatocellular carcinoma HepG2 cells

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)–DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P–DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [ 3H] B[a]P, but increased the cellular discharge of [ 3H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20–500 μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P–DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.

Overexpression of Rho GDP-dissociation inhibitor alpha predicts poor survival in oral squamous cell carcinoma

Oral cancer has emerged as one of the fastest growing malignancies in Taiwan. However, biomarkers that reliably predict clinical outcomes have yet to be identified. This study was aimed to identify tumor-associated proteins that could be prognostic biomarkers for oral cancer. We compared the protein expression between oral squamous cell carcinoma (OSCC) tissues and adjacent non-cancerous matched tissues (NCMTs) by proteomics. We found that Rho GDP-dissociation inhibitor alpha (RhoGDIα) was differentially expressed in frozen cancerous samples and OSCC cell lines but not in NCMTs. Furthermore, our results indicated that RhoGDIα was selectively upregulated in 78 OSCC tissue sections (p<0.001), and this high expression was significantly correlated with increased tumor size (p<0.05) and poor overall survival (p<0.01). There was a trend that RhoGDIα expression was localized in the cytoplasm of cancer cells but was localized in the plasma membrane of NCMTs. Finally, expression of RhoGDIα was validated to be an independent prognostic indicator for overall survival (p<0.01). These results have identified a novel biomarker that may be useful for prediction of poor prognosis in OSCC patients.