Silencing of the circ-Arhgap5 RNA protects neuronal PC12 cells against injury and depends on the miR-29a-3p/Rock1 axis.

Circular RNAs (circRNAs) are abundantly expressed in human central nervous system. Here, we explored the role of circ_Arhgap5 in cerebral ischemia/reperfusion (I/R)-induced nerve injury in PC12 cells and its associated mechanism. Cell proliferation ability was assessed by 5-Ethynyl-2'-deoxyuridine (Edu) assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCM) was applied to assess cell apoptotic rate. Cell death was analyzed by lactate dehydrogenase (LDH) assay. Oxygen-glucose deprivation and reoxygenation (OGD/R) up-regulates the expression of circRNA Rho GTPase activating protein 5 (circ_Arhgap5; mmu_circ_0000377) in PC12 cells. OGD/R exposure inhibited the proliferation and induced the apoptosis of PC12 cells, and the silence of circ_Arhgap5 attenuated OGD/R-induced injury in PC12 cells. miR-29a-3p was identified as a target of circ_Arhgap5 in PC12 cells. Circ_Arhgap5 knockdown-mediated protective effects in OGD/R-induced PC12 cells were reversed by the interference of miR-29a-3p. miR-29a-3p interacted with the 3' untranslated region (3'UTR) of Rho-associated coiled-coil-containing protein kinase 1 (Rock1), and circ_Arhgap5 can positively regulate Rock1 expression by sponging miR-29a-3p in PC12 cells. miR-29a-3p overexpression protected PC12 cells against OGD/R-induced damage by down-regulating Rock1. In conclusion, circ_Arhgap5 silencing protected PC12 cells from OGD/R-induced injury through mediating miR-29a-3p/Rock1 axis.

Alpha lipoic acid treatment in late middle age improves cognitive function: Proteomic analysis of the protective mechanisms in the hippocampus.

Alpha lipoic acid (ALA), a powerful antioxidant, has the potential to relieve age-related cognitive impairment and neurodegenerative disease. Clinical randomized controlled studies have demonstrated the cognitive improvement effects of lipoic acid in Alzheimer's disease (AD). In the present study, we examined the effects of ALA on cognitive function in ageing mice and its protective mechanisms. Eighteen-month-old male C57BL6/J mice received ALA or normal saline for 2 months. The Morris water maze test revealed improved cognitive function in animals that received ALA. Furthermore, tandem Mass Tags (TMT) based liquid chromotography with mass spectrometry/mass spectrometry (LC-MS/MS) was established to identify the target proteins. The results showed that 10 proteins were changed significantly. Gene Ontology (GO) analysis indicated that the upregulated proteins were enriched in terminal bouton, synaptic transmission and lipid transporter activity while the down-regulated proteins were involved in nuclear transcription factor-κB binding, apoptosis and mitogen-activated protein kinase binding. Based on the GO results, two upregulated proteins oxysterol-binding protein-related protein 10 (OSBPL10) and oligophrenin 1 (OPHN1), and one downregulated protein, CDK5 regulatory subunit-associated protein 3 (CDK5rap3), were validated through Western blotting. The results were consistent with the proteomic results. Modulation of synaptic transmission, lipid transporter activity and neuroinflammation appears to be the mechanisms of ALA in the aged brain.

ARHGAP9 inhibits colorectal cancer cell proliferation, invasion and EMT via targeting PI3K/AKT/mTOR signaling pathway.

AIM: In digestive system, colorectal cancer (CRC) is a common malignant tumor. The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of the rapamycin (PI3K/AKT/mTOR) signaling pathway plays a central role in CRC, and the aberrant activation of this pathway is associated with tumorigenesis. We aimed to explore the role of Rho GTPase activating protein 9 (ARHGAP9) in the progression of CRC as well as its regulatory effects on the PI3K/AKT/mTOR pathway. METHODS: The expression of ARHGAP9 in CRC tumor tissues and cell lines were detected using reverse transcription-quantitative PCR (qRT-PCR). 5-ethynyl-2'-deoxyuridine (EdU) assay was applied to test the cell proliferation. Cell migration and invasion were both assessed through transwell assay. Xenograft mouse models were constructed to explore the effects of ARHGAP9 on CRC in vivo. The expressions of PI3K/AKT/mTOR-activating factors and epithelial-mesenchymal transition (EMT)-related factors were all determined using western blot. LY294002 was employed to block PI3K/AKT/mTOR pathway in CRC cells. RESULTS: The expression of ARHGAP9 was down-regulated in CRC tumor tissues and cell lines when compared to normal tissues and cells. The over-expression of ARHGAP9 inhibited cell proliferation, invasion, migration and EMT in CRC cell lines while the knockdown of ARHGAP9 promoted them. In addition, ARHGAP9 up-regulation inhibited the activation of PI3K/AKT/mTOR signaling pathway in CRC cell lines while ARHGAP9 down-regulation led to an opposite effect. The over-expression of ARHGAP9 suppressed CRC tumor growth in vivo. When the PI3K/AKT/mTOR pathway was blocked in CRC cells, the effects of ARHGAP9 knockdown on cell proliferation, migration, invasion and EMT were all overturned. CONCLUSION: ARHGAP9 inhibited the malignant phenotypes of CRC cells via interdicting PI3K/AKT/mTOR signaling pathway.

DOCK4 regulates ghrelin production in gastric X/A-like cells.

PURPOSE: Ghrelin, a gastric hormone, provides a hunger signal to the central nervous system to stimulate food intake. Ghrelin also modulates neuroinflammatory and apoptotic processes. Dedicator of cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor (GEF), is involved in the regulation of neuronal polarization and axon regeneration. However, the effect of DOCK4 on ghrelin production has not been explored. METHODS: The expression of DOCK4 in human and mouse stomach was examined by immunohistochemical staining. The synthesis and secretion of ghrelin in Dock4 null mice were evaluated by real-time quantitative PCR, Western blot and ELISA. The effects of DOCK4 on ghrelin production in mHypoE-42 cells were measured by real-time quantitative PCR and Western blot. RESULTS: We showed that DOCK4 was expressed in both human and mouse gastric ghrelin cells. The mRNA and protein levels of gastric ghrelin, as well as ghrelin secretion, were remarkably diminished in Dock4 null mice. Furthermore, we showed that overexpression of Dock4 significantly stimulated ghrelin expression, while siRNA knockdown of endogenous Dock4 resulted in a marked decrease of ghrelin in mHypoE-N42 cells. CONCLUSIONS: Our results identify DOCK4 as a critical regulator for ghrelin production in gastric X/A-like cells.

Exosomes derived from human umbilical cord mesenchymal stem cells reduce tendon injuries via the miR-27b-3p/ARHGAP5/RhoA signaling pathway.

Tendon injuries are common clinical issues resulted from tissue overuse and age-related degeneration. Previous sutdies have suggested that exosomes secreted by mesenchymal stem cells (MSCs) contribute to tissue injury repair. Here, we provide evidence for a critical role of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes in reducing tendon injury by activating the RhoA signaling. Treatment of primary injured tenocytes with hucMSC exosomes increases cell proliferation and invasion, which correlates with increased RhoA activity. RhoA mediates the effects of hucMSC exosomes, as treatment of primary injured tenocytes with the RhoA inhibitor, CCG-1423, abolishes the effects of hucMSC exosomes on cell proliferation and invasion. Mechanistically, we observe that hucMSC exosomes induce the expression of a microRNA, miR-27b-3p, which targets and suppresses ARHGAP5, a negative regulator of RhoA. Consistent with this observation, ARHGAP5 overexpression suppresses the effects of hucMSC exosomes on cell proliferation and invasion, while knockdown of ARHGAP5 rescues these effects. Finally, we demonstrate the functional significance of our findings using an Achilles tendon injury model and show that treatment with exosomes reduces tendon injury in rats, which correlates with increased RhoA activity and reduced ARHGAP5 expression. Taken together, our findings highlight a critical role of hucMSC exosomes in reducing tendon injury via miR-27b-3p-mediated suppression of ARHGAP5, resulting in RhoA activation, and leading to increased cell proliferation and invasion of primary injured tenocytes.

Tat-SynGAP improves angiogenesis and post-stroke recovery by inhibiting MST1/JNK signaling.

Small G protein Ras induces the activation of apoptosis-related molecule mammalian Ste20-like kinase1 (MST1)/JNK signal pathway, which is involved in the regulation of tissue damage under pathological conditions such as ischemic stroke. Our previous study indicated that GTPase-activating protein for Ras (SynGAP), a negative regulator of Ras, could bind with postsynaptic density protein-93 (PSD-93) and Tat-SynGAP (670-685aa) small peptide to exhibit neuroprotective role. Here, we report that Tat-SynGAP (670-685aa) reduced cerebral edema at acute cerebral ischemia/reperfusion (I/R), improved integrity of blood-brain barrier, and decreased cortical and striatum neuronal injury. Mechanistically, Tat-SynGAP (670-685aa) not only inhibited the phosphorylation of MST1 and JNK and the cleavage of caspase-3, but also facilitated the expression of angiogenesis related molecules VEGF and Ang-1. In conclusion, Tat-SynGAP (670-685aa) reduces neuronal apoptosis and cerebral infarction volume and maintains vascular stability and blood-brain barrier integrity by inhibiting MST1/JNK signaling pathway.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

TBC1D1 interacting proteins, VPS13A and VPS13C, regulate GLUT4 homeostasis in C2C12 myotubes.

Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.

STARD13-correlated ceRNA network-directed inhibition on YAP/TAZ activity suppresses stemness of breast cancer via co-regulating Hippo and Rho-GTPase/F-actin signaling.

BACKGROUND: Targeting cancer stem cells is critical for suppressing cancer progression and recurrence. Finding novel markers or related pathways could help eradicate or diagnose cancer in clinic. METHODS: By constructing STARD13-correlated ceRNA 3'UTR stable overexpression or knockdown breast cancer cells, we aimed to explore the effects of STARD13-correlated ceRNA network on breast cancer stemness in vitro and in vivo. Further RNA-sequencing was used to analyze transcriptome change in combination with functional studies on candidate signaling. Clinical samples obtained from The Cancer Genome Atlas data were used to validate the correlation between STARD13 and related pathways. Finally, in vitro and in vivo experiments were used to examine the effects of STARD13-correlated ceRNA network on chemotherapy sensitivity/resistance. RESULTS: Here, we revealed that this ceRNA network inhibited stemness of breast cancer. Mechanistically, we found that activation of STARD13-correlated ceRNA network was negatively correlated with YAP/TAZ activity in breast cancer. Specifically, this ceRNA network attenuated YAP/TAZ nuclear accumulation and transcriptional activity via collectively modulating Hippo and Rho-GTPase/F-actin signaling. Finally, we demonstrated that YAP/TAZ transcriptional activity regulated by this ceRNA network was involved in chemoresistance. CONCLUSIONS: Our results uncover a novel mechanism of YAP/TAZ activation in breast cancer and propose the possibility to drive STARD13-correlated ceRNA network to inhibit breast cancer stem cell traits.

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner.

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment.

Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38ß and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes). Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin). All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

ARHGAP21 prevents abnormal insulin release through actin rearrangement in pancreatic islets from neonatal mice.

AIMS: ARHGAP21 is a Rho GTPase-activating protein (RhoGAP) that associates with many proteins and modulates several cellular functions, including actin cytoskeleton rearrangement in different tissues. However, it is unknown whether ARHGAP21 is expressed in pancreatic beta cells and its function in these cells. Herein, we assess the participation of ARHGAP21 in insulin secretion. MAIN METHODS: Neonatal mice were treated with anti-sense oligonucleotide against ARHG AP21 (AS) for 2 days, resulting in a reduction of the protein's expression of about 60% in the islets. F-actin depolimerization, insulin secretion,mRNA level of genes involved in insulin secretion, maturation and proliferation were evaluated in islets from both control and AS-treated mice. KEY FINDINGS: ARHGAP21 co-localized with actin inMIN6 beta cells and with insulin in neonatal pancreatic islets. F-actin was reduced in AS-islets, as judged by lower phalloidin intensity. Insulin secretion was increased in islets from AS-treated mice, however no differences were observed in the GSIS (glucose-stimulated insulin secretion). In these islets, the pERK1/2 was increased, as well as the gene expressions of VAMP2 and SNAP25, proteins that are present in the secretory machinery. Maturation and cell proliferation were not affected in islets from AS-treated mice. SIGNIFICANCE: In conclusion, our data show, for the first time, that ARHGAP21 is expressed and participates in the secretory process of pancreatic beta cells. Its effect is probably via pERK1/2, which modulates the rearrangement of the cytoskeleton. ARHGAP21 also controls the expression of genes that encodes proteins of the secretory machinery.

DLC-1 induces mitochondrial apoptosis and epithelial mesenchymal transition arrest in nasopharyngeal carcinoma by targeting EGFR/Akt/NF-κB pathway.

Loss of deleted in liver cancer-1 (DLC-1) can induce apoptosis and inhibit the mobility, migration and metastasis in several cancers. Previously, we revealed that ectopic expression of DLC-1 can suppress proliferation, mobility, migration and tumorigenesis in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms accounting for the roles of DLC-1 in NPC are still obscure. In the present work, we attempted to study and uncover the mechanisms underlying the functions of DLC-1 in NPC. The apoptosis of 5-8F-DLC-1 cells, established previously, was analyzed by mitochondrial membrane potentials assay and flow cytometer analysis. And the antibodies involving pathways such as mitochondrial-associated apoptosis, epithelial mesenchymal transition and metastasis were applied to detect and compare the expression level of targeted proteins. The obvious apoptosis of 5-8F-DLC-1 cells was observed reflected by mitochondrial depolarization and lower ratio in cell viability. Subsequently, the activation of mitochondrial apoptosis was verified by the increased expressions of Bax, Apaf1, cleave-caspases and cleave-PARP, etc, and the decreased expressions of Bcl-2, Bcl-xL, Mcl-1, Survivin, etc, in 5-8F-DLC-1 cells. Then, the inhibited epithelial mesenchymal transition of 5-8F-DLC-1 cells was validated by upregulated expression of E-cadherin and downregulated expression of N-cadherin, Snail, Vimentin. Subsequently, downregulated expressions of proteins such as FAK, RhoA, ROCK1 and cdc25 related to cell adhesion and cytoskeleton organization were also observed. And expressions of MMPs were inhibited in 5-8F-DLC-1 cells. At last, the inhibited activity of EGFR/Akt/NF-κB axis was revealed by the decreased expressions of phosho-EGFR, phosho-Akt, phosho-p38MAPK, phosho-IKKα and phosho-p65. Here, we systematically explored the mechanisms underlying the negative roles of DLC-1 in NPC cells. For the first time, we confirmed that the ectopic expression DLC-1 can induce mitochondrial apoptosis, inhibit EMT and related processes by targeting the EGFR/Akt/NF-κB pathway, which, beyond all doubt, offered beneficial guidelines for other studies and laid a good foundation for its clinical applications ultimately.

Pseudomonas aeruginosa exotoxin T induces potent cytotoxicity against a variety of murine and human cancer cell lines.

In patients with malignancy, the major barrier to achieving complete response is emergence of resistance to current chemotherapeutic agents. One of the major mechanisms by which tumour cells become resistant to therapies is by altering cellular drug targets through mutations and/or deletions. Resistance by this mechanism is achieved more easily if the drug has limited cellular targets and/or processes. We hypothesized that as Pseudomonas aeruginosa exotoxin T (ExoT) targets six proteins that are required for cancer cell survival and proliferation, it is highly unlikely for cancer cells to develop resistance to this toxin. We assessed ExoT's cytotoxicity against multiple invasive and highly resistant tumour cell lines in order to evaluate its potential as a chemotherapeutic agent. Our data demonstrated that ExoT induced potent cytotoxicity in all tumour cell lines that we examined. Collectively, our data highlighted the potential of ExoT as a possible chemotherapeutic candidate for the treatment of cancer.

A WXW motif is required for the anticancer activity of the TAT-RasGAP317-326 peptide.

TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.

RLIP76 regulates HIF-1 activity, VEGF expression and secretion in tumor cells, and secretome transactivation of endothelial cells.

This study was undertaken to reveal the mechanisms by which RLIP76 regulates endothelial cell angiogenic responses. RLIP76 is an effector of the angiogenic modulator, R-Ras. RLIP76 is overexpressed in many tumors, required for tumor angiogenesis, and blockade of RLIP76 results in tumor regression in multiple models. We report here that RLIP76 was required for expression and secretion of vascular endothelial growth factor (VEGF) in carcinoma and melanoma cells. Conditioned medium derived from RLIP76-depleted tumor cells, but not control knockdown cells, could not stimulate proliferation, migration, or Matrigel cord formation in endothelial cell cultures, which indicates that RLIP76 regulates angiogenic components of the tumor cell secretome. Recombinant VEGF added to conditioned medium from RLIP76-knockdown tumor cells restored these endothelial cell functions. Transcriptional activity of hypoxia-inducible factor 1 (HIF-1), which drives VEGF expression, was blocked in RLIP76-depleted tumor cells. RLIP76 was required for PI3-kinase activation, known to regulate HIF-1, in these cells. However, HIF-1α expression and nuclear localization were unaffected by RLIP76 knockdown, which suggests that RLIP76 regulates HIF-1 at the functional level. Thus, RLIP76 regulates tumor cell transactivation of endothelial cells via control of VEGF expression and secretion, providing a new important link in the mechanism of tumor cell induction of angiogenesis.

Inhibition of cell migration and invasion mediated by the TAT-RasGAP317-326 peptide requires the DLC1 tumor suppressor.

TAT-RasGAP(317-326), a peptide corresponding to the 317-326 sequence of p120 RasGAP coupled with a cell-permeable TAT-derived peptide, sensitizes the death response of various tumor cells to several anticancer treatments. We now report that this peptide is also able to increase cell adherence, prevent cell migration and inhibit matrix invasion. This is accompanied by a marked modification of the actin cytoskeleton and focal adhesion redistribution. Interestingly, integrins and the small Rho GTP-binding protein, which are well-characterized proteins modulating actin fibers, adhesion and migration, do not appear to be required for the pro-adhesive properties of TAT-RasGAP(317-326). In contrast, deleted in liver cancer-1, a tumor suppressor protein, the expression of which is often deregulated in cancer cells, was found to be required for TAT-RasGAP(317-326) to promote cell adherence and inhibit migration. These results show that TAT-RasGAP(317-326), besides its ability to favor tumor cell death, hampers cell migration and invasion.

TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak.

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway.

Use of a peptide enhancing the ability of radiation therapy to kill cancer cells: a patent evaluation of WO2012016918.

BACKGROUND: Faulty apoptosis is a known mechanism that leads to resistance to radiotherapy. The application (WO2012016918A1) deals with a peptide useful for disrupting this resistance mechanism and enhancing the efficiency of radiotherapy. METHODS: A peptide consisting essentially of the N2 sequence of the RasGAP protein is conjugated to the HIV-TAT(48-57) cell permeation sequence. The DNA sequence encoding the peptide (TAT-RasGAP(317-326)) is synthesized and introduced into the host cells. RESULTS: TAT-RasGAP(317-326) is demonstrated to potentiate the efficacy of γ-irradiation-mediated cell killing both in tumor cell lines and in mouse tumor models, disregarding the status of p53, but not in non-cancer cells. CONCLUSION: TAT-RasGAP(317-326) peptide favors apoptosis of tumor cells, but not normal cells in response to radiotherapy. The invention provides a specific method that is probably to be used in cancers that are radio-resistant.