Role of cerebrospinal fluid-contacting nucleus in sodium sensing and sodium appetite.

The brainstem plays an important role in controlling sodium and water homeostasis. It is a major regulatory site for autonomic and motor functions. Moreover, it integrates cerebrospinal fluid (CSF) signals with neuronal and hormonal signals. Evidence suggests that the CSF-contacting nucleus (CSF-CN) transmits and integrates CSF signals, but, the definitive role of CSF-CN in sodium homeostasis is poorly understood. In this study, we used c-Fos as a marker of neuronal activity and causing colocalization of Nax channel and 5-HT. This proved that CSF-CN played a role in sensing the increase of CSF sodium level. Then, we determined the role of the CSF-contacting nucleus in increasing the sodium appetite of rats. So, we performed targeted lesion of the CSF-contacting nucleus in the brainstem using the cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. The lesion of the CSF-CN showed decreased and degenerative neurons, while sodium appetite have increased and Fos immunocytochemistry detected neuronal activity in the lateral parabrachial nucleus (LPBN), but not in the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT). These results indicate that the CSF-CN plays an important role in sensing CSF sodium level and satiating sodium appetite by influencing the LPBN but not SFO and OVLT. The Nax channel and 5-HT might be the molecular mechanisms through which contribute to sodium homeostasis.

Structurally distinct hybrid polymer/lipid nanoconstructs harboring a type-I ribotoxin as cellular imaging and glioblastoma-directed therapeutic vectors.

A nanoformulation composed of a ribosome inactivating protein-curcin and a hybrid solid lipid nanovector has been devised against glioblastoma. The structurally distinct nanoparticles were highly compatible to human endothelial and neuronal cells. A sturdy drug release from the particles, recorded upto 72 h, was reflected in the time-dependent toxicity. Folate-targeted nanoparticles were specifically internalized by glioma, imparting superior toxicity and curbed an aggressively proliferating in vitro 3D cancer mass in addition to suppressing the anti-apoptotic survivin and cell matrix protein vinculin. Combined with the imaging potential of the encapsulated dye, the nanovector emanates as a multifunctional anti-cancer system.

Intrathecal substance P-saporin in the dog: distribution, safety, and spinal neurokinin-1 receptor ablation.

BACKGROUND: Neurokinin-1 receptors (NK1-rs) located on superficial dorsal horn neurons are essential for integration of nociceptive input. Intrathecal injection of substance P-saporin (SP-SAP) leads to local loss of spinal NK1-r (+) neurons suggesting its potential as a therapeutic agent for chronic pain. The authors determined, in a canine model, effects of lumbar intrathecal SP-SAP. METHODS: Distribution of SP-SAP and Saporin was determined in plasma, lumbar cerebrospinal fluid, and tissue. Safety of intrathecal SP-SAP was determined in four groups (six dogs each) administered 0 (0.9% saline), 1.5, 15, or 150 µg SP-SAP through lumbar intrathecal catheters. Behavioral, physiologic, and biochemical variables were assessed. Spinal tissues were collected at 7 and approximately 90 days, or earlier if significant morbidity developed, and analyzed for NK1-r (+) neuron loss and histopathology. RESULTS: SP-SAP and Saporin were detectable in lumbar cerebrospinal fluid for up to 4 and 24 h, respectively. Animals receiving intrathecal saline, 1.5, or 15 µg of SP-SAP showed no persistent neurologic deficits. Three animals receiving 150 µg of SP-SAP developed pelvic limb paraparesis and were euthanized prematurely. Immunohistochemistry and in situ hybridization cell counts confirmed a significant reduction in NK1-r (+) in superficial dorsal horn neurons from lumbar spinal cord after intrathecal administration of 15 and 150 µg of SP-SAP. A significant loss of NK1-r neurons in the lumbar ventral horn occurred only with 150-µg SP-SAP. CONCLUSION: Intrathecal 15-µg SP-SAP reduced dorsal, but not ventral, NK1-r (+) neurons at the spinal level of delivery with minimal side effects, whereas 150-µg SP-SAP resulted in motor neuron toxicity.

Antitumor activity of a humanized, bivalent immunotoxin targeting fn14-positive solid tumors.

The TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) receptor Fn14 (TNFRSF12A) is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, melanoma, and breast, and therefore it is a potentially unique therapeutic target for these diverse tumor types. We have generated a recombinant protein containing a humanized, dimeric single-chain anti-fibroblast growth factor-inducible 14-kDa protein (Fn14) antibody fused to recombinant gelonin toxin as a potential therapeutic agent (designated hSGZ). The hSGZ immunotoxin is a highly potent and selective agent that kills Fn14-positive (Fn14(+)) tumor cells in vitro. Treatment of cells expressing the MDR protein MDR1 (ABCB1B) showed no cross-resistance to hSGZ. Induced overexpression of Fn14 levels in MCF7 cells through HER2 (ERBB2) signaling translated to an improved therapeutic index of hSGZ treatment. In combination with trastuzumab, hSGZ showed an additive or synergistic cytotoxic effect on HER2(+)/Fn14(+) breast cancer cell lines. Also, hSGZ treatment inhibited Erb3/Akt signaling in HER2-overexpressing breast cancer cells. Pharmacokinetic studies in mice revealed that hSGZ exhibited a biexponential clearance from plasma with a rapid initial clearance (t1/2α = 1.26 hours) followed by a seven-fold longer plasma half-life (t1/2ß = 7.29 hours). At 24, 48, and 72 hours after injection, uptake of the hSGZ into tumors was 5.1, 4.8, and 4.7%ID/g, with a tumor-to-muscle ratio of 5.6, 6.2, and 9.0, respectively. Therapeutic efficacy studies showed significant tumor inhibition effects using an MDA-MB-231/Luc breast cancer xenograft model. Our findings show that hSGZ is an effective anticancer agent and a potential candidate for clinical studies.

Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.

Targeted delivery of immunotoxin by antibody to ganglioside GD3: a novel drug delivery route for tumor cells.

Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.

Saponins modulate the intracellular trafficking of protein toxins.

Type I ribosome inactivating proteins such as saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process. In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of saporin. Binding analysis demonstrated an association of the saponins with saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a saporin-based targeted anti-tumor toxin in combination with characterized saponins.

Ribosome-inactivating proteins: current status and biomedical applications.

Ribosome-inactivating proteins (RIPs) are mainly present in plants and function to inhibit protein synthesis through the removal of adenine residues from eukaryotic ribosomal RNA (rRNA). They are broadly classified into two groups: type I and type II. Type I RIPs are a diverse family of proteins comprising a single polypeptide chain, whereas type II RIPs are heterodimeric glycoproteins comprising an A-chain (functionally equivalent to a type I RIP) linked via a disulphide bond to a B chain, mediating cell entry. In this review, we describe common type I and type II RIPs, their diverse biological functions, mechanism of cell entry, stability in plasma and antigenicity. We end with a discussion of promising applications for RIPs in biomedicine.

Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6.

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from abasic sites in HeLa nuclei after intoxication with saporin-S6.

Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein.

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency.

BACKGROUND: Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008. RESULTS: CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9) through a G4S linker to R9-rGel (E9-G4S-R9-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel. CONCLUSION: We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R9-rGel from CPE-E9-G4S-R9-rGel.

Biodistribution, pharmacokinetics, and nuclear imaging studies of 111In-labeled rGel/BLyS fusion toxin in SCID mice bearing B cell lymphoma.

PURPOSE: We examined the biodistribution and pharmacokinetics of (111)In-labeled rGel/BLyS, a gelonin toxin (rGel)-B lymphocyte stimulator (BLyS) fusion protein. MATERIALS AND METHODS: rGel/BLyS was labeled with In-111 through DTPA with a labeling efficiency >95%. Biodistribution/imaging studies were obtained in severe-combined immunodeficiency mice bearing diffuse large B cell lymphoma OCI-Ly10. Pharmacokinetic studies were performed in BALB/c mice. RESULTS: In vitro, DTPA-conjugated rGel/BLyS displayed selective cytotoxicity against OCI-Ly10 cells and mantle cell lymphoma JeKo cells. In vivo, rGel/BLyS exhibited a tri-exponential disposition with a rapid initial mean distribution followed by an extensive mean distribution and a long terminal elimination phase. At 48 h after injection, uptake of the radiotracer in tumors was 1.25 %ID/g, with a tumor-to-blood ratio of 13. Tumors were clearly visualized at 24-72 h post-injection. Micro-SPECT-CT images and ex vivo analyses confirmed the accumulation of rGel/BLyS in OCI-Ly10 tumors. CONCLUSIONS: (111)In-DTPA-rGel/BLyS are distributed to B cell tumors and induce apoptosis in tumors. Preclinical antitumor studies using rGel/BLyS should use a twice-per-week treatment schedule.

Intracellular re-localisation by photochemical internalisation enhances the cytotoxic effect of gelonin--quantitative studies in normal rat liver.

Photochemical internalisation (PCI) is a delivery technology that employs a sub-lethal form of photodynamic therapy (PDT) in which a photosensitiser is activated by light to break down intracellular membranes and release macromolecules into the cytosol where they can be biologically active. Although PCI does enhance the PDT killing of transplanted tumours in mice after local injection of the cytotoxic agent, gelonin, the redistribution of gelonin from intracellular organelles into the cytosol has only previously been demonstrated in vitro. This study is designed to understand the factors controlling the efficacy of PCI in vivo and to document the mechanism of action. Using the photosensitiser AlS(2)Pc in studies on normal rat liver, we have demonstrated in vivo that gelonin is initially taken up into lysosomes, but can be released into the cytosol using PCI. Furthermore, PCI enhances the PDT effect after systemic administration of gelonin (volume of necrosis increased x2.5 when gelonin is given one hour before light), with the remarkably low dose of 5 microg/kg (10,000 times lower than the LD50); in the absence of light, there is no effect with 500 microg/kg. These results suggest that PCI may have a useful role to play in the site specific activation of cytotoxic agents like gelonin, given at a dose level that has no effect in the absence of light.

Environmental enrichment provides a cognitive reserve to be spent in the case of brain lesion.

To experimentally verify the reserve hypothesis, the influence of rearing conditions on the cognitive performances and on dendritic spines following basal forebrain lesions was analyzed. Adult rats reared in enriched or standard conditions were depleted of the cholinergic projection to the neocortex by 192 IgG-saporin injection into Ch4 region of basal forebrain. Their performance in spatial tasks was compared with that of intact animals reared in analogous conditions. Furthermore, number and density of dendritic spines of the layer-III parietal pyramidal neurons were analyzed. Cholinergic depletion of forebrain cortex resulted in impaired performances in most behavioral tasks in animals reared in standard conditions. Conversely, the enriched lesioned animals did not exhibit most deficits evoked by cholinergic lesion, even if some deficits, such as perseverative behaviors, were still present. The pyramidal neurons exhibited an increased spine number and density in the lesioned animals reared in standard conditions. In the enriched lesioned animals, the enhancement of spine number and density elicited by the rearing condition was fully maintained but not further increased in the presence of the lesion. Thus, rearing in an enriched environment results in the development of brain and cognitive reserves that reduce the cognitive impairment following forebrain lesions.

Cytosolic delivery of liposomally targeted proteins induced by photochemical internalization.

PURPOSE: The application of therapeutic proteins is often hampered by limited cell entrance and lysosomal degradation, as intracellular targets are not reached. By encapsulation of proteins into targeted liposomes, cellular uptake via endocytosis can be enhanced. To prevent subsequent lysosomal degradation and promote endosomal escape, photochemical internalization (PCI) was studied here as a tool to enhance endosomal escape. PCI makes use of photosensitising agents which localize in endocytic vesicles, inducing endosomal release upon light exposure. MATERIALS AND METHODS: The cytotoxic protein saporin was encapsulated in different types of targeted liposomes. Human ovarian carcinoma cells were incubated with the photosensitiser TPPS2a and liposomes. To achieve photochemical internalization, the cells were illuminated for various time periods. Cell viability was used as read-out. Illumination time and amount of encapsulated proteins were varied to investigate the influence of these parameters. RESULTS: The cytotoxic effect of liposomally targeted saporin was enhanced by applying PCI, likely due to enhanced endosomal escape. The cytotoxic effect was dependent on the amount of encapsulated saporin and the illumination time. CONCLUSION: PCI is a promising technique for promoting cytosolic delivery of liposomally targeted saporin. PCI may also be applicable to other liposomally targeted therapeutic proteins with intracellular targets.