Improved breast cancer cell-specific intracellular drug delivery and therapeutic efficacy by coupling decoration with cell penetrating peptide and SP90 peptide.

Lack of satisfactory specificity towards tumor cells and poor intracellular delivery efficacy are the major drawbacks with conventional cancer chemotherapy. Conjugated anticancer drugs to targeting moieties e.g. to peptides with the ability to recognize cancer cells and to cell penetrating peptide can improve these characteristics, respectively. Combining a tumor homing peptide with an appropriate cell-penetrating peptide can enhance the tumor-selective internalization efficacy of the carrying cargo molecules. In the present study, the breast cancer homing ability of SP90 peptide and the synergistic effect of SP90 with a cell-penetrating peptide(C peptide) were evaluated. SP90 and chimeric peptide SP90-C specifically targeted cargo molecule into breast cancer cells, especially triple negative MDA-MB-231 cell, in a dose- and time-dependent manner, but not normal breast cells and other cancer cells, while C peptide alone had no cell-selectivity. SP90-C increased the intracellular delivery efficiency by 12-fold or 10-fold compared to SP90 or C peptide alone, respectively. SP90 and SP90-C conjugation increased the anti-proliferative and apoptosis-inducing activity of HIV-1 Vpr, a potential novel anticancer protein drug, to breast cancer cell but not normal breast cell by arresting cells in G2/M phase. With excellent breast cancer cell-selective penetrating efficacy, SP90-C appears as a promising candidate vector for targeted anti-cancer drug delivery. SP90-VPR-C is a potential novel breast cancer-targeted anticancer agent for its high anti-tumor activity and low toxicity.

The HIV-derived protein Vpr52-96 has anti-glioma activity in vitro and in vivo.

Patients with actively replicating human immunodeficiency virus (HIV) exhibit adverse reactions even to low irradiation doses. High levels of the virus-encoded viral protein R (Vpr) are believed to be one of the major underlying causes for increased radiosensitivity. As Vpr efficiently crosses the blood-brain barrier and accumulates in astrocytes, we examined its efficacy as a drug for treatment of glioblastoma multiforme (GBM).In vitro, four glioblastoma-derived cell lines with and without methylguanine-DNA methyltransferase (MGMT) overexpression (U251, U87, U251-MGMT, U87-MGMT) were exposed to Vpr, temozolomide (TMZ), conventional photon irradiation (2 to 6 Gy) or to combinations thereof. Vpr showed high rates of acute toxicities with median effective doses of 4.0±1.1 µM and 15.7±7.5 µM for U251 and U87 cells, respectively. Caspase assays revealed Vpr-induced apoptosis in U251, but not in U87 cells. Vpr also efficiently inhibited clonogenic survival in both U251 and U87 cells and acted additively with irradiation. In contrast to TMZ, Vpr acted independently of MGMT expression.Dose escalation in mice (n=12) was feasible and resulted in no evident renal or liver toxicity. Both, irradiation with 3x5 Gy (n=8) and treatment with Vpr (n=5) delayed intracerebral tumor growth and prolonged overall survival compared to untreated animals (n=5; p3x5 Gy<0.001 and pVpr=0.04; log-rank test).Our data show that the HIV-encoded peptide Vpr exhibits all properties of an effective chemotherapeutic drug and may be a useful agent in the treatment of GBM.

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway.

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.

Extracellular HIV-1 viral protein R affects astrocytic glyceraldehyde 3-phosphate dehydrogenase activity and neuronal survival.

Extracellular human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a pleiotropic protein accomplishing several functions within the viral life cycle. While Vpr has been described extensively as an intracellular protein, very little is known about its role as an extracellular protein. In fact, HIV-1 Vpr has been detected in the blood, serum, and cerebrospinal fluid of HIV-1-infected patients, with concentrations increasingly higher in late-stage disease. To determine the role exogenous Vpr plays in HIV-associated central nervous system dysfunction, primary human fetal astrocytes were exposed to recombinant Vpr and a time- and dose-dependent decrease was demonstrated in two fundamental intracellular metabolites (adenosine-5'-triphosphate (ATP) and glutathione (GSH)). Additionally, exposure to exogenous Vpr led to increased caspase activity and secretion of proinflammatory cytokines IL-6 and IL-8 and chemoattractants, monocyte chemotactic protein-1, and migration inhibition factor. Extracellular Vpr also dampened the glycolytic pathway through impairment of glyceraldehyde 3-phosphate dehydrogenase activity, causing a decline in the levels of ATP. The reduction in intracellular ATP increased reactive oxygen species buildup, decreasing GSH concentrations, which affected several genes in the oxidative stress pathway. In addition, exposure of the SK-N-SH neuroblastoma cell line to conditioned medium from exogenous Vpr-treated astrocytes decreased synthesis of GSH, leading to their apoptosis. These observations point to a role that Vpr plays in altering astrocytic metabolism and indirectly affecting neuronal survival. We propose a model that may explain some of the neurological damage and therefore neurocognitive impairment observed during the course of HIV-1 disease.

Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2-arrest.

The HIV-1 accessory protein Vpr induces G2 cell cycle arrest and apoptosis. Previous studies indicate that the induction of G2-arrest requires the localization of Vpr to the nuclear envelope. Here we show that treatment of Vpr-expressing HeLa cells with the caspase 3 inhibitor Z-DEVD-fmk induced accumulation of Vpr at the nuclear lamina, while other proteins or structures of the nuclear envelope were not influenced. Furthermore, Z-DEVD-fmk enhances the Vpr-mediated G2-arrest that even occurred in HIV-1(NL4-3)-infected T-cells. Mutation of Pro-35, which is important for the integrity of helix-α1 in Vpr, completely abrogated the Z-DEVD-fmk-mediated accumulation of Vpr at the nuclear lamina and the enhancement of G2-arrest. As expected, inhibition of caspase 3 reduced the induction of apoptosis by Vpr. Taken together, we could show that besides its role in Vpr-mediated apoptosis induction caspase 3 influences the localization of Vpr at the nuclear envelope and thereby augments the Vpr-induced G2-arrest.

The role of HIV-1 Vpr in promoting the infection of nondividing cells and in cell cycle arrest.

PURPOSE OF REVIEW: The search for the role(s) that HIV-1 Vpr and its HIV2/SIV paralogs Vpr and Vpx play in viral infection and pathogenesis showed that all three engage CRL4 ubiquitin ligase complexes. This association triggers ubiquitination and degradation of cellular substrates. The identity of the ubiquitin ligase substrates is only now beginning to be revealed. This review focuses on recent work that has identified one such substrate and exposed new cellular restrictions to infection. RECENT FINDINGS: Three groups have now described cellular factors that restrict HIV-1 infection in cells of the myeloid lineage. One of these factors, sterile alpha motif- and metal-dependent phosphohydrolase domain-containing protein 1 (SAMHD1), was shown to be depleted through the CRL4 ubiquitin ligase complex in the presence of HIV-2/SIV Vpx. The other restriction can be defeated by Vpx in the absence of at least one part of the ubiquitin ligase complex that triggers SAMHD1 depletion.Another group has shown that the previously described upregulation of natural killer-cell ligands on the surface of HIV-1-infected cells requires the actions of both the cytidine deaminase APOBEC3G and uracil-N-glycosylase 2 in association with HIV-1 Vpr. SUMMARY: As more cellular interaction partners are identified for HIV-1 Vpr and its paralogs from other viruses, details are emerging about Vpr function. The recent findings have highlighted the existence of two new human proteins that can act to combat HIV infection and have revealed how HIV-1 proteins act in concert to modulate the interaction between natural killer cells and HIV-1 infected cells.

Exogenous HIV-1 Vpr disrupts IFN-alpha response by plasmacytoid dendritic cells (pDCs) and subsequent pDC/NK interplay.

HIV Vpr is known for its immunomodulatory capacities including its impairment of NK cell functions. However, the role of pDCs in this context remains elusive. We show that synthetic Vpr substantially inhibits type I IFN production by pDCs without inducing apoptosis in pDCs. Furthermore, we found that exogenous Vpr compromises subsequent pDC/NK interplay as shown by diminished IFN-gamma production by NK cells. Thus, Vpr-mediated dysregulation of IFN-alpha and IFN-gamma production affects key components of the innate immune response supporting an essential role of Vpr in HIV pathogenesis.

Analysis of the potential for HIV-1 Vpr as an anti-cancer agent.

Viral protein R (Vpr) is a 14kD, 96 amino acid accessory protein of the HIV virion that has been demonstrated to have important functions in the viral replication cycle including, among others, the induction of cell cycle arrest and apoptosis in rapidly proliferating cells, which results in immune dysfunction in infected individuals. Several investigators have studied the potential use of the apoptosis inducing and cell cycle arrest effect of Vpr as an anti-tumor therapeutic. In vitro studies have indicated that Vpr is cytotoxic against a large number of different tumor cell types including a number that are p53 independent. Likewise, some in vivo tumor studies using different delivery platforms/methods have indicated an anti-cancer effect mediated by Vpr. Our group has used the aggressive and poorly immunogenic murine melanoma tumor line B16.F10 as a model to deliver, through in vivo electroporation, Vpr expressing plasmids to established tumors and have demonstrated that this treatment regimen can induce growth attenuation and tumor regression in a proportion of the treated mice and appears to be associated with the induction of intratumoral apoptosis. Overall, to date, the data from a number of research groups, including our own, have indicated that Vpr has biological activity against a number of tumors in both in vivo and in vitro models and, as such, may be a potential candidate for testing in human clinical trials. In this report, we summarize the evidence supporting this hypothesis.

Pharmacological screening and enzymatic assays for apoptosis.

Mitochondria play a central role in the intrinsic pathway of apoptosis. In response to many pro-apoptotic stimuli, mitochondria undergo an irreversible process called mitochondrial membrane permeabilization (MMP). The detection of MMP in isolated mitochondria is most often based on assays that monitor either the loss of the inner transmembrane potential (DYm; classically with Rhodamine 123), permeability transition (PT, cyclosporin A-sensitive matrix swelling), or the release of critical pro-apoptotic intermembrane space effectors. To gain complementary information on MMP mechanisms, we have systematically used three additional assays optimized for the 96-well microplate format: (1) inner membrane permeability, (2) VDAC-associated NADH reductase activity, and (3) ATP/ADP translocase activity. We report that ad hoc combinations of ANT and VDAC ligands, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mastoparan and Vpr52-96 peptide and PT inhibitors, permit to explore relationships between enzymatic functions of sessile mitochondrial proteins (i.e. ANT, VDAC) and MMP. These assays should be useful tools to investigate mitochondrial apoptosis, decipher the implication of inner and outer membrane permeabilization and provide a multi-parametric approach for drug discovery.

Dynamical pathway analysis.

BACKGROUND: Although a great deal is known about one gene or protein and its functions under different environmental conditions, little information is available about the complex behaviour of biological networks subject to different environmental perturbations. Observing differential expressions of one or more genes between normal and abnormal cells has been a mainstream method of discovering pertinent genes in diseases and therefore valuable drug targets. However, to date, no such method exists for elucidating and quantifying the differential dynamical behaviour of genetic regulatory networks, which can have greater impact on phenotypes than individual genes. RESULTS: We propose to redress the deficiency by formulating the functional study of biological networks as a control problem of dynamical systems. We developed mathematical methods to study the stability, the controllability, and the steady-state behaviour, as well as the transient responses of biological networks under different environmental perturbations. We applied our framework to three real-world datasets: the SOS DNA repair network in E. coli under different dosages of radiation, the GSH redox cycle in mice lung exposed to either poisonous air or normal air, and the MAPK pathway in mammalian cell lines exposed to three types of HIV type I Vpr, a wild type and two mutant types; and we found that the three genetic networks exhibited fundamentally different dynamical properties in normal and abnormal cells. CONCLUSION: Difference in stability, relative stability, degrees of controllability, and transient responses between normal and abnormal cells means considerable difference in dynamical behaviours and different functioning of cells. Therefore differential dynamical properties can be a valuable tool in biomedical research.

HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis.

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell death in mammalian and fission yeast cells, suggesting that Vpr may affect a conserved cellular process. It is unclear, however, whether Vpr-induced yeast cell death mimics Vpr-mediated apoptosis in mammalian cells. We have recently identified a number of Vpr suppressors that not only suppress Vpr-induced cell death in fission yeast, but also block Vpr-induced apoptosis in mammalian cells. These findings suggest that Vpr-induced cell death in yeast may resemble some of the apoptotic processes of mammalian cells. The goal of this study was to develop and validate a fission yeast model system for future studies of apoptosis. Similar to Vpr-induced apoptosis in mammalian cells, we show here that Vpr in fission yeast promotes phosphatidylserine externalization and induces hyperpolarization of mitochondria, leading to changes of mitochondrial membrane potential. Moreover, Vpr triggers production of reactive oxygen species (ROS), indicating that the apoptotic-like cell death might be mediated by ROS. Interestingly, Vpr induces unique morphologic changes in mitochondria that may provide a simple marker for measuring the apoptotic-like process in fission yeast. To verify this possibility, we tested two Vpr suppressors (EF2 and Hsp16) that suppress Vpr-induced apoptosis in mammalian cells in addition to a newly identified Vpr suppressor (Skp1). All three proteins abolished cell death mediated by Vpr and restored normal mitochondrial morphology in the yeast cells. In conclusion, Vpr-induced cell death in fission yeast resembles the mammalian apoptotic process. Fission yeast may thus potentially be used as a simple model organism for the future study of the apoptotic-like process induced by Vpr and other proapoptotic agents.