Rho GDP Dissociation Inhibitor Research Articles

A rho GDP dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.

In this study, we found that a subpopulation of CD4+CD25+ (85% Foxp3+) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4+CD25+ (85% Foxp3+) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4+CD25+Foxp3+D4GDI+ cells do not produce IL-10, TGF-β and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1β, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4+CD25+ (85% Foxp3+) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4+CD25+ (85% Foxp3+) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.

Manganese superoxide dismutase is required to maintain osteoclast differentiation and function under static force.

Bone homeostasis is maintained by the balance of osteoblasts (OBs) and osteoclasts (OCs). Increased activity of OCs not only contributes to pathological bone resorption, such as osteoporosis and periodontitis, but also is responsible for physiological conditions like orthodontic tooth movement (OTM). However, the detailed mechanism by which orthodontic force promotes the formation of OCs is still poorly understood. In this study, we confirmed that static force promoted the differentiation of human cord monocytes (HMNCs) into OCs depending on loading time and magnitude. Protein expression profiles among HMNCs, HMNCs subjected to static force and mature OCs were established via 2-DE and MALDI-TOF-MS analyses. Total respective protein spot numbers of 549 ± 13, 612 ± 19 and 634 ± 16 were detected in each of the gels by image analysis. The five proteins identified were plasminogen activator inhibitor 2 (PAI-2, Spot 1), peroxiredoxin-6 (PRD-6, Spot 3), manganese superoxide dismutase (SOD2, Spot 6), Rho GDP-dissociation inhibitor 2 (Rho-GDI2, Spot 11) and L-lactate dehydrogenase B chain (L-LDH, Spot 15). More importantly, we revealed that SOD2 was required to maintain monocyte differentiation into functional OCs and may become a potential target for regulating the efficiency of OTM in the future.

Overexpression of RhoGDI2 correlates with the progression and prognosis of pancreatic carcinoma.

Rho GDP dissociation inhibitor 2 (RhoGDI2) has been found to be a regulator of tumor metastasis. However, the expression of RhoGDI2 and its clinicopathological significance as well as the pathway of RhoGDI2 in tumor metastasis have yet to be investigated. To investigate the role of RhoGDI2 in the progression and prognosis of pancreatic carcinoma (PC), the expression of RhoGDI2 in human PC tissues was examined and compared with the clinicopathological characteristics and prognosis. Moreover, the relationship between RhoGDI2 and E-cadherin was examined. The results indicated that RhoGDI2 was overexpressed in PC tissues and associated with clinicopathological characteristics, including clinical stage and lymph node metastasis. Patients with a RhoGDI2‑negative expression had a significantly longer survival time than those with a RhoGDI2‑positive expression. Additionally, the expression of RhoGDI2 was negatively correlated with the expression of E-cadherin in PC tissues. Taken together, the findings suggest that RhoGDI2 is important in the progression and prognosis of PC, and may be used as a potential prognostic biomarker and a therapeutic target for PC.

Proteome Profiling of Maize Pollen Coats Reveals Novel Protein Components

The pollen coat, which covers the exine wall of pollen, is essential for initial sexual contact and thus successful fertilization in flowering plants. Pollen coat proteins (PCPs) not only mediate species specificity but are also needed for pollen–stigma recognition and pollen germination on the stigma. Maize (Zea mays L.) is one of the most common cereal crops in the world. To date, only a few PCPs from maize have been identified and characterized. In the present study, we extracted the pollen coat fraction from maize inbred line B73 with chloroform and purified the PCPs via a phenol-based protocol prior to two-dimensional gel electrophoresis (2-DE). By proteomic analysis, 26 protein spots were successfully identified and classified into 12 unique proteins. The protein composition of the maize pollen coat is distinctly different from those of the dicot plants Arabidopsis and rapeseed. Of the proteins identified in this study, eight (including profilins, caleosin, Zea m 2, β-expansin-10, exopolygalacturonase, Rho GDP-dissociation inhibitor 1, Ras-related protein Rab-2-A, and putative subtilase) had not previously been observed in the maize pollen coat. Bioinformatic analysis showed that nine of the PCPs were secreted proteins and that most of them were extracellular. These PCPs are potentially involved in pollen germination and tube growth. The current study extracted and identified the pollen coat proteins of maize, one of the most important crops throughout the world. Proteome profiling of the maize pollen coat revealed many novel protein components potentially involved in pollen–stigma interactions and pollen germination. Our results provide basic knowledge and further the functional characterization of PCPs in wind-pollinated species such as maize.

RhoGDI2 expression in astrocytes after an excitotoxic lesion in the mouse hippocampus.

The Rho GDP-dissociation inhibitor (RhoGDI) originally downregulates Rho family GTPases by preventing nucleotide exchange and membrane association. Although RhoGDI2 functions as a metastasis regulator, little is known in glial cells under neuropathological conditions. We monitored RhoGDI2 expression in the mouse brain after administering a kainic acid(KA)-induced excitotoxic lesion. In control, RhoGDI2 immunoreactivity (IR) was evident in the neuronal layer of the hippocampus. However, RhoGDI2 IR was increased in astrocytes markedly throughout the hippocampus at day 3 post-treatment with KA. To further investigate the molecular mechanism of RhoGDI2-induced cellular migration, primary astrocytes were transfected with the flag-tagged RhoGDI2 cDNA. Cell migration assay revealed that RhoGDI2 cDNA transfection inhibits astrocyte migration. Overexpression of RhoGDI2 leads to inhibit protein kinase B (PKB) activation and cdc42 and cAMP-responsive element-binding protein (CREB) phosphorylation. In conclusion, our results suggested for the first time that RhoGDI2 is required for PKB and CREB activation and cdc42 expression in astrocyte migration after KA-mediated excitotoxic lesion in mouse brain.

Role of Rho GDP Dissociation Inhibitor α in Control of Epithelial Sodium Channel (ENaC)-mediated Sodium Reabsorption

The epithelial sodium channel (ENaC) is expressed in the aldosterone-sensitive distal nephron where it performs sodium reabsorption from the lumen. We have recently shown that ENaC activity contributes to the development of salt-induced hypertension as a result of deficiency of EGF level. Previous studies revealed that Rho GDP-dissociation inhibitor α (RhoGDIα) is involved in the control of salt-sensitive hypertension and renal injury via Rac1, which is one of the small GTPases activating ENaC. Here we investigated the intracellular mechanism mediating the involvement of the RhoGDIα/Rac1 axis in the control of ENaC and the effect of EGF on ENaC in this pathway. We demonstrated that RhoGDIα is highly expressed in the cortical collecting ducts of mice and rats, and its expression is down-regulated in Dahl salt-sensitive rats fed a high salt diet. Knockdown of RhoGDIα in cultured cortical collecting duct principal cells increased ENaC subunits expression and ENaC-mediated sodium reabsorption. Furthermore, RhoGDIα deficiency causes enhanced response to EGF treatment. Patch clamp analysis reveals that RhoGDIα significantly decreases ENaC current density and prevents its up-regulation by RhoA and Rac1. Inhibition of Rho kinase with Y27632 had no effects on ENaC response to EGF either in control or RhoGDIα knocked down cells. However, EGF treatment increased levels of active Rac1, which was further enhanced in RhoGDIα-deficient cells. We conclude that changes in the RhoGDIα-dependent pathway have a permissive role in the Rac1-mediated enhancement of ENaC activity observed in salt-induced hypertension.

RNA interference-mediated knockdown of RhoGDI2 induces the migration and invasion of human lung cancer A549 cells via activating the PI3K/Akt pathway.

Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a tumor suppressor gene for cellular migration and invasion. However, the underlying mechanism and effector targets of RhoGDI2 in lung cancer are still not fully understood. In this study, a vector-expressed small hairpin RNA (shRNA) of RhoGDI2 was transfected into the human lung cancer cell line A549. After the successful transfection, the down-regulation of RhoGDI2 promoted the proliferation, migration, and invasion of lung cancer cells in vitro through the increasing expression and activities of the matrix metallopeptidase 9 (MMP-9) and PI3K/Akt pathways. Transiently transfecting the small interfering RNA (siRNA) of MMP-9 into the RhoGDI2 shRNA cells reduced the MMP-9 expression. Both transfecting the siRNA and adding the MMP-9 antibody into the RhoGDI2 shRNA cells led to a decrease in the invasion and migration of the lung cancer cells. The blockade of the PI3K/Akt pathway by LY294002 resulted in abolishment of the effects of RhoGDI2 shRNA in Akt phosphorylation and MMP-9 expression. This result suggests that the down-regulated RhoGDI2 contributed to the migration and invasion of the lung cancer cell line via activating the PI3K/Akt pathway and the ensuing increase in the expression and activity of MMP-9. In conclusion, we report that the shRNA-mediated knockdown of RhoGDI2 induces the invasion and migration of lung cancer due to cross-talk with the PI3K/Akt pathway and MMP-9. Verifying the role and molecular mechanism of the participation of RhoGDI2 in the migration and invasion of lung cancer may provide a target for better treatment.

RhoGDI2 is associated with HGF-mediated tumor invasion through VEGF in stomach cancer.

RhoGDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of tumor metastasis; however, its role in cancer remains controversial. The aims of this study were to analyze RhoGDI2 in gastric cancer growth and metastasis, and to determine its possible signaling pathway. The level of expression of RhoGDI2 was further confirmed by real time RT-RCR and Western blot analysis. Transfection of cells with RhoGDI2 shRNA resulted in no effects of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with RhoGDI2 shRNA, compared with control cells, were able to invade across a Matrigel membrane barrier. The role of RhoGDI2 in the level of HGF-induced up-regulation of vascular endothelial growth factor (VEGF) was measured by knockdown of RhoGDI2 with RhoGDI2 shRNA and a chromatic immuno-precipitation assay. The levels of RhoGDI2 and VEGF were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up-regulation of VEGF was repressed by RhoGDI2 knockdown. HGF-induced upregulation of phosphorylated ERK and P38 levels was inhibited in RhoGDI2 knockdown cells. HGF enhanced the binding activity of RhoGDI2 to the VEGF promoter in control cells, but not in RhoGDI2-shRNA cells. Findings of this study also showed a statistically significant difference in the mean RhoGDI2 level before and after surgery (p < 0.01) and the mean level of RhoGDI2 before surgery showed a statistically significant difference depending on lymphatic, neural invasion and stage (p < 0.05). In conclusion, RhoGDI2 might play an important role in up-regulation of VEGF induced by HGF and contributes to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.

RhoGDI facilitates geranylgeranyltransferase-I-mediated RhoA prenylation

Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.

Rho GDP dissociation inhibitor beta promotes cell proliferation and invasion by modulating the AKT pathway in hepatocellular carcinoma.

Rho GDP dissociation inhibitor (GDI) beta, (RhoGDI2), has been identified as a proto-oncogene that is upregulated in human cancers, but the role of RhoGDI2 in hepatocellular carcinoma (HCC) remains unclear. In the present study, we investigated the RhoGDI2 expression level in HCC tissues and the function of RhoGDI2 in HCC cell growth and metastasis. We examined the RhoGDI2 mRNA expression level in 64 sets of HCC tissue and their adjacent nontumor tissue counterparts using quantitative real-time polymerase chain reaction. In vitro proliferation and invasion assays were conducted to determine the effect of RhoGDI2 on the ability of HCC cells to proliferate and invade, respectively. Western blot analysis was conducted to examine expression levels of RhoGDI2p-AKT, MMP-2, and MMP-9 in HCC cells. RhoGDI2 mRNA was significantly overexpressed in the HCC specimens compared with the nonneoplastic liver specimens, and the RhoGDI2 mRNA and protein levels were higher in the HCC cell lines, especially the highly metastatic cell lines 97L and 97H. To further investigate the role that RhoGDI2 plays in HCC, we overexpressed RhoGDI2 using a lentivirus-mediated overexpression technique in two HCC cell lines (Huh7 and 7721) that endogenously express a low level of RhoGDI2. Stable cells overexpressing RhoGDI2 demonstrated a significant increase in cell proliferation and invasion. Furthermore, our additional findings indicated that RhoGDI2-mediated cellular invasion requires the PI3K/Akt signaling-dependent expression of matrix metalloproteinases (MMPs). Our findings suggest that RhoGDI2 plays an important role in HCC growth and invasion and should be considered a novel HCC therapeutic target candidate.

TAT-RhoGDI2, a novel tumor metastasis suppressor fusion protein: expression, purification and functional evaluation.

Rho GDP dissociation inhibitor 2 (RhoGDI2) was identified as a functional metastasis suppressor in human bladder cancer, suggesting that increasing the RhoGDI2 level may represent a promising therapeutic strategy. It has been shown that the transactivator of transcription (TAT) protein from HIV-1 is able to efficiently deliver various biological molecules into several cell types. In this study, TAT peptide was fused with the N-terminus of RhoGDI2, and the resulting TAT-RhoGDI2 fragment was inserted into the pGEX-6p-1 plasmid and expressed as a glutathione S-transferase (GST)/TAT-RhoGDI2 fusion protein in Escherichia coli BL21(DE3) cells. A two-step purification strategy involving glutathione sepharose chromatography and PreScission protease cleavage was developed to purify TAT-RhoGDI2; subsequently, the identification of the involved macromolecules was achieved by Western blot. The final product, TAT-RhoGDI2, was obtained at a concentration of 112 mg/L. This is the first report on the efficient production of bioactive TAT-RhoGDI2 through a gene-engineering approach in E. coli. Using flow cytometry, we found that the TAT-RhoGDI2 fusion proteins could penetrate into bladder cancer cells with an extremely high efficiency. In vitro scratch and transwell assay and the migration/invasion behavior of UMUC3 cells were strongly reduced by the treatment with TAT-RhoGDI2. These studies support the use of the TAT-RhoGDI2 protein in tumor metastasis therapy.

Effects of extracts of Prunella Vulgaris L. on proteome of human lung adenocarcinoma cell line A549

To explore the effect of extracts of Prunella vulgaris L.on proteome of human lung adenocarcinoma cell line A549 by two-dimensional electrophoresis and mass spectrometry and elucidate the mechanism of anti-lung adenocarcinom effect of Prunella vulgaris L.at the level of proteome. The proliferative activity of human lung adenocarcinoma cell line A549 was evaluated by methyl thiazolyl tetrazolium (MTT) colorimetric assay. According to the difference of culture medium, all subjects were divided into the experimental group with culture medium of extracts of Prunella vulgaris L. (300 µg/ml) and the control group with culture medium of DMSO (0.3%). Proteins were isolated by two-dimensional electrophoresis and proteomic maps acquired by silver staining. And proteomic analysis was processed by Image Master 2D Quant Platinum 6.0. The proteins with > 2-fold differences were used to analyze by mass spectrometry and confirmed by Western blot. The expressions of inositol 1, 4, 5-triphosphate receptor-interacting protein-like 2 precursor, heat shock cognate protein 70, serine-threonine kinase receptor-associated protein, tropomyosin 2(β) isoform 1, cyclin B3, MED12L protein and macrophin 1 isoform 2 were higher in experimental group than those in control group (ratio (medicial/normal) 2.051 93, 1 000 001, 2.203 08, 5.042 01, 15.178 00, 1 000 001, 1 000 001) . And the expressions of enolase 1, M2-type pyruvate kinase, heat shock protein 27, Rho GDP-dissociation inhibitor 1, heat shock protein β1, TapasinERP57 heterodimer chain A, inorganic pyrophosphatase and mitochondrial Cysteinyl-tRNA synthetase 2 (putative) were lower in experimental group than those in control group (ratio (medicial/normal) 0.485 18, 0.491 53, 0.465 43, 0.454 71, 0.499 34, 0.450 36, 0.494 62, 0.437 33). The extracts of Prunella vulgaris L.have multi-target and multi-pathway effects on anti-lung adenocarcinoma. And its possible mechanisms may be due to the regulation of steady state of calcium ion, cell cycle and its steady state and the inhibition of tumor cell proliferation and metastasis.

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.

Comparative proteome analysis of peripheral neutrophils from sulfur mustard-exposed and COPD patients

Respiratory disorders in sulfur mustard (SM)-exposed and chronic obstructive pulmonary disease (COPD) patients are mostly associated with neutrophilic inflammation, severe airflow limitation, and oxidative stress. The objective of this study was to establish whether neutrophil (PMN) proteomes in these diseases were similar or differed. Blood neutrophil proteomes from healthy, SM-exposed, and COPD subjects were analyzed using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS). Elastase activity was determined kinetically. The results showed that levels of S100 calcium-binding protein (CBP) A12, S100 CBP A8, glyceraldehyde-3-phosphate dehy-drogenase, superoxide dismutase, and protein disulfide isomerase proteins – as well as elastase activity – were significantly increased in PMN from ‘diseased’ hosts compared to in cells from healthy controls. In contrast, coactosin-like protein, RhoGDP dissociation inhibitor, and actin isoforms were significantly decreased in diseased subjects’ PMN compared to PMN of healthy controls. Moreover, serpin B1 and coronin-1A were expressed only in PMN of the healthy subjects. Lastly, S100 CBP A9, endoplasmic reticulum (ER)-60 protease, and glutathione-S-transferase isoforms were differentially expressed in the cells from the SM-exposed and COPD subjects. These results show that serpin B1, an efficient inhibitor of neutrophil serine proteases, was not detectable, and elastase activity significantly increased in PMN from both SM-exposed and COPD patients. It seems that, apart from inflammation and oxidative stress, a protease:anti-protease imbalance exists within PMN of both COPD and SM-exposed patients.

Association of RhoGDI2 with HGF-mediated tumor invasion through VEGF in stomach cancer.

e22007 Background: Rho GDP dissociation inhibitor 2 (Rho GDI2) has been identified as a regulator of tumor metastasis, but its role in cancer remains controversial. The aims of this study were to a...

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.

Depletion of RhoGDI2 expression inhibits the ability of invasion and migration in pancreatic carcinoma.

Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of tumor metastasis, although its role in tumor progression remains controversial. In this study, we examined the expression of RhoGDI2 in PC tissues and cell lines. To investigate the function of RhoGDI2 in PC cells, RhoGDI2 expression was depleted in PANC-1 and Patu8988 cells by small interfering RNA (siRNA). RhoGDI2 was found to be overexpressed in pancreatic carcinoma (PC) tissues and PC cell lines. Additionally, the results showed that depletion of RhoGDI2 significantly inhibited cell motility and invasion in vitro, but did not affect cell proliferation. The clinical study together with the experimental data confirmed that RhoGDI2 modulated the expression of matrix metalloproteinase 2 (MMP2). Taken together, findings of the present study indicated that RhoGDI2 is involved in pancreatic tumor malignancy and metastasis. Thus, RhoGDI2 is a potential target for the gene therapy of PC.

Comparative proteomic profiling of triple-negative breast cancer reveals that up-regulation of RhoGDI-2 is associated to the inhibition of caspase 3 and caspase 9

There are no targeted therapeutic modalities for triple-negative breast cancer (TNBC), thus it is associated with poor prognosis and worst clinical outcome. Here, our aim was to identify deregulated proteins in TNBC with potential therapeutic applications. Proteomics profiling of TNBC and normal breast tissues through two-dimensional electrophoresis and ESI-MS/MS mass spectrometry revealed the existence of 16 proteins (RhoGDI-2, HSP27, SOD1, DJ1, UBE2N, PSME1, FTL, SH3BGRL, and eIF5A-1) with increased abundance in carcinomas. We also evidenced for the first time the deregulation of COX5, MTPN and DB1 proteins in TNBC that may represent novel tumor markers. Particularly, we confirmed the overexpression of the Rho-GDP dissociation inhibitor 2 (RhoGDI-2) in distinct breast cancer subtypes, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Remarkably, targeted disruption of RhoGDI-2 by RNA interference induced mitochondrial dysfunction, and facilitated caspase-3 and -9 activation in two breast cancer cell lines. Moreover, suppression of RhoGDI-2 resulted in a robust sensitization of breast cancer cells to cisplatin therapy. In conclusion, we identified novel proteins deregulated in TNBC, and confirmed the overexpression of RhoGDI-2. We propose that RhoGDI-2 inhibition may be exploited as a potential therapeutic strategy along cisplatin-based chemotherapy in breast cancer. There are no useful biomarkers neither targeted therapeutic modalities for triple-negative breast cancer, which highly contributes to the poor prognosis of this breast cancer subtype. In this work, we used two-dimensional electrophoresis and ESI-MS/MS spectrometry to identify novel deregulated proteins in breast cancer tissues. Particularly, our results showed that RhoGDI-2, a protein that has been associated to metastasis and poor survival in human cancers, is overexpressed in different subtypes of breast tumors, as well as in metastatic cell lines derived from lung, prostate, and breast cancer. Our data also provided novel insights about the role of RhoGDI-2 in apoptosis through intrinsic pathway inhibition. Importantly, they suggested that targeted modulation of RhoGDI-2 levels might be a useful strategy for breast cancer therapy.

Host differentially expressed genes during association with its defensive endosymbiont.

Mutualism, a beneficial relationship between two species, often requires intimate interaction between the host and symbiont to establish and maintain the partnership. The colonial marine bryozoan Bugula neritina harbors an as yet uncultured endosymbiont, "Candidatus Endobugula sertula," throughout its life stages. The bacterial symbiont is the putative source of bioactive complex polyketide metabolites, the bryostatins, which chemically defend B. neritina larvae from predation. Despite the presence of "Ca. Endobugula sertula" in all life stages of the host, deterrent bryostatins appear to be concentrated in reproductive portions of the host colony, suggesting an interaction between the two partners to coordinate production and distribution of the metabolites within the colony. In this study, we identified host genes that were differentially expressed in control colonies and in colonies cured of the symbiont. Genes that code for products similar to glycosyl hydrolase family 9 and family 20 proteins, actin, and a Rho-GDP dissociation inhibitor were significantly downregulated (more than twice) in antibiotic-cured non-reproductive zooids compared to control symbiotic ones. Differential expression of these genes leads us to hypothesize that the host B. neritina may regulate the distribution of the symbiont within the colony via mechanisms of biofilm degradation and actin rearrangement, and consequently, influences bryostatin localization to bestow symbiont-associated protection to larvae developing in the reproductive zooids.

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.