Efficiency of Immunotoxin Cytotoxicity Is Modulated by the Intracellular Itinerary

Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F_v)-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity – presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.     

Combination Gene Delivery of the Cell Cycle Inhibitor p27 with Thymidine Kinase Enhances Prodrug Cytotoxicity

Cytoxicity induced by the herpesvirus thymidine kinase (TK) gene in combination with prodrugs is dependent on cell growth and leads to the elimination of genetically modified cells, thus limiting the duration of expression and efficacy of this treatment in vivo. Here, an effort was made to enhance TK/prodrug efficacy by coexpression of a cyclin-dependent kinase inhibitor (CKI), p27, to render cells resistant to TK/prodrug by inhibiting DNA synthesis. Expression of p27 by transfection substantially reduced cell cycle progression, and its activity was enhanced by mutations designed to stabilize the protein. Coexpression of p27 and TK or a p27/TK fusion protein led to greater prodrug cytotoxicity than that produced by TK alone in the Renca cell line, which is sensitive to bystander killing. Combination gene transfer of this CKI with TK therefore sustained the synthesis of TK by genetically modified cells to enhance the susceptibility of bystander cells to prodrug cytotoxicity and increased the efficacy of this gene transfer approach.     

Evaluation of the Antitumor Effects of BPR1J-340, a Potent and Selective FLT3 Inhibitor,Alone or in Combination with an HDAC Inhibitor,Vorinostat, in AML Cancer

Overexpression or/and activating mutation of FLT3 kinase play a major driving role in the pathogenesis of acute myeloid leukemia (AML). Hence, pharmacologic inhibitors of FLT3 are of therapeutic potential for AML treatment. In this study, BPR1J-340 was identified as a novel potent FLT3 inhibitor by biochemical kinase activity (IC₅₀ approximately 25 nM) and cellular proliferation (GC₅₀ approximately 5 nM) assays. BPR1J-340 inhibited the phosphorylation of FLT3 and STAT5 and triggered apoptosis in FLT3-ITD⁺ AML cells. The pharmacokinetic parameters of BPR1J-340 in rats were determined. BPR1J-340 also demonstrated pronounced tumor growth inhibition and regression in FLT3-ITD⁺ AML murine xenograft models. The combination treatment of the HDAC inhibitor vorinostat (SAHA) with BPR1J-340 synergistically induced apoptosis via Mcl-1 down-regulation in MOLM-13 AML cells, indicating that the combination of selective FLT3 kinase inhibitors and HDAC inhibitors could exhibit clinical benefit in AML therapy. Our results suggest that BPR1J-340 may be further developed in the preclinical and clinical studies as therapeutics in AML treatments.     

Effects of Calphostin C,Specific PKC Inhibitor on TPA-Induced Normal Human Melanocyte Growth,Morphology and Adhesion

Normal human melanocytes, which rarely undergo mitosis in vivo, require many growth factors and growth-stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophosphate-stimulating agents or 12-0-tetrade-canoylphorbol 13-acetate (TPA), to proliferate. TPA, known as a protein kinase C (PKC)-activator, supports normal human melanocyte growth and influences on melanocyte dendrite formation. We have further confirmed the role of the PKC-mediated pathway in the TPA-dependent melanocyte functions—i.e., proliferation, morphology, and adhesion—using Calphostin C (CPC), a highly specific PKC inhibitor. Melanocytes require the continual presence of TPA for growth in culture. Addition of 8 nM TPA to the medium increased melanocyte growth by 198.4 ± 2.3% of that without TPA. The growth induction by TPA was suppressed by the addition of 10 nM CPC at the level comparable to that without TPA without any morphological alterations. Significant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long-term exposure to TPA (more than 5 days) resulted in marked reduction of melanocyte adhesion to plastic cell culture dishes, both uncoated and coated with type IV collagen. By the addition of 10 nM CPC in the adhesion assay, the melanocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA-dependent melanocyte functions. Continuous activation of PKC by TPA is implicated in melanocyte growth stimulation. TPA also has effects on melanocyte morphology, causing the formation of long extended dendrites with little cytoplasm. However, inhibition of PKC activation by CPC does not affect the melanocyte morphology, and CPC reduces melanocyte adhesion to uncoated or type IV collagen coated plastic cell culture dishes.     

PKC regulation of the human equilibrative nucleoside transporter,hENT1

Regulation of nucleoside transporters is poorly understood. We show that acute stimulation of protein kinase C (PKC) causes a rapid increase in S-(4-nitrobenzyl)-6-thioinosine-sensitive (human equilibrative nucleoside transporter 1, hENT1) nucleoside uptake, in human cultured cells, which is not due to increased metabolism and which can be blocked by PKC inhibitors. Use of isoform-specific inhibitors indicates that PKC delta and/or epsilon (but not alpha, beta or gamma) are responsible for the acute effects. Down-regulation of PKC decreases hENT1-dependent uridine uptake. These are the first data to show rapid PKC delta/epsilon-dependent stimulation of hENT1 transport by a mechanism that may involve activation of transporters at the membrane possibly by post-translational modification of the protein.     

Kitazin P,Inhibitor of Phosphatidylcholine Biosynthesis in Pyricularia oryzae

The effect of organophosphorus fungicide, Kitazin P (IBP, S-benzyl diisopropyl phosphorothiolate), on lipid biosynthesis of Pyricularia oryzae was investigated. Addition of IBP to the mycelial cells suspension of P. oryzae induced a striking decrease in incorporation of methionine-methyl-¹⁴C, S-adenosylmethionine-methyl-¹⁴C, and glycerol-1-¹⁴C into phosphatidylcholine, which is the most abundant phospholipid in P. oryzae, but incorporation of choline-methyl-¹⁴C into phosphatidylcholine and that of methionine-methyl-¹⁴C into simple lipids were not affected. Incorporation of methionine-methyl-¹⁴C into phosphatidylcholine is found to be directly proportional to mycelial cells growth of P. oryzae. Enzymes responsible for the biosynthesis from glycerol to phosphatidylcholine through Greenberg’s pathway, except phospholipid N-mefhyltransferase, were not inhibited by IBP. IBP concentration required for 50% inhibition of phospholipid N-methyltransferase was 40 ppm. IBP had no effect on activities of glycerokinase, glycerophosphate acyltransferase, phosphatidic acid cytidyltransferase, phosphatidylserine synthetase, and phosphatidylserine decarboxylase, respectively. Therefore, the specific inhibition of conversion from phosphatidylethanolamine to phosphatidylcholine by the transmethylation of S-adenosylmethionine might be regarded as one of the modes of action of IBP.     

Treatments that Enhance Flowering in the Post-inductive Period of a Short-day Plant, Chenopodium rubrum L.

Flowering was promoted in young plants of Chenopodium rubrumL. by application of growth inhibitors such as 5-fluorodeoxyuridine(FUDR) and (2-chloroethyl) trimethylammonium chloride (CCC),growth substances (indol-3yl-acetic acid, IAA), by the removalof roots and by drought. All the treatments were effective onlyduring the post-inductive period and at the threshold levelof photoperiodic induction. The response of plants was strictlytime-dependent. The experimental data indicate that the stimulationof flowering is usually accompanied by inhibition of leaf initiationand growth. The treatments probably produced variation in thequantitative expression of flowering by causing a shift in emphasisin the development of leaf and bud primordia at the shoot apex.The dynamic analysis of differentiation of the shoot apex indicatesa correlation between the morphological stage of the shoot apexand its responsiveness to the treatments.     

Antitumor Activity of the Combination of Topotecan and Tyrosyl-DNA-Phosphodiesterase 1 Inhibitor on Model Krebs-2 Mouse Ascite Carcinoma

Molecular Biology - Topotecan is a cytostatic drug from the camptothecin group, it acts by inhibiting topoisomerase 1 (TOP1). Tyrosyl-DNA phosphodiesterase 1 (TDP1) is capable of interfering with...     

Protective Effects of SKF-96365, a Non-Specific Inhibitor of SOCE,against MPP+-Induced Cytotoxicity in PC12 Cells: Potential Role of Homer1

Parkinson''s disease (PD) is the most common neurodegenerative movement disorder, characterized by loss of dopominergic (DA) neurons in substantia nigra pars compacta (SNpc), and can be experimentally mimicked by the neurotoxin MPP⁺ in vitro models. In this study, we investigated the potential protective effect of SKF-96365, a non-specific inhibitor of SOCE (store-operated calcium entry), on MPP⁺ induced cytotoxicity in PC12 cells. We found that pretreatment with SKF-96365 (10 µM and 50 µM) 30 min before injury significantly increased cell viability, decreased LDH release, prevented nuclear damage, and inhibited apoptotic cell death in MPP⁺ stressed PC12 cells. The results of calcium image using the ratiometric calcium indicator Fura-2-AM also showed that SKF-96365 reduced the intracellular calcium overload induced by MPP⁺ in PC12 cells. In addition, SKF-96365 decreased the expression of Homer1, a more recently discovered postsynaptic scaffolding protein with calcium modulating function, following MPP⁺ administration in PC12 cells, while had no statistically significant effects on endoplasmic reticulum (ER) calcium concentration. Furthermore, overexpression of Homer1 by using recombinant lentivirus partly reversed protective effects of SKF-96365 against MPP⁺ injury. The ER Ca²⁺ release was further amplified and ER calcium recovery was delayed by Homer1 upregulation in PC12 cells following MPP⁺ insult. Taken together, these data suggest that SKF-96365 protects PC12 cells against MPP⁺ induced cytotoxicity, and this protection may be at least in part on the inhibition of intracellular calcium overload and suppression of Homer1-mediated ER Ca²⁺ release.     

Characterization of the interaction of P1,P4-diadenosine 5'-tetraphosphate with luciferase

Adenylated dinucleotides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P(2) receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(n)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles.     

Pyrimidine and Purine Analogues,Effects on Cell Cycle Regulation and the Role of Cell Cycle Inhibitors to Enhance Their Cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.     

Investigation of the Cytotoxicity of Antimicrobial Peptide P40 on Eukaryotic Cells

The in vitro cytotoxicity of the antimicrobial peptide P40 was investigated. The food grade bacteriocin nisin was also analyzed for comparison. VERO cells were treated with different concentrations (0.02–2.5 μg ml⁻¹) of nisin and P40, and cell viability and plasma membrane integrity were checked by MTT, neutral red uptake (NRU), and lactate dehydrogenase (LDH) assays. In MTT and NRU assays the EC₅₀ to the purified peptide P40 were 0.30 and 0.51 μg ml⁻¹, while values found to nisin were 0.35 and 0.79 μg ml⁻¹, respectively. In the LDH assay, the EC₅₀ was 0.57 and 0.62 μg ml⁻¹ for P40 and nisin, respectively. The peptide P40 revealed higher hemolytical activity (19%) when compared to nisin (4.9%) at the highest concentration tested (2.5 μg ml⁻¹). Relatively few studies about the cytotoxicity of antimicrobial peptides are available. The determination of the cytotoxicity of antimicrobial peptides is an essential step to warrant their safe use.     

Combination of RNase Binase and AKT1/2 Kinase Inhibitor Blocks Two Alternative Survival Pathways in Kasumi-1 Cells

Molecular Biology - Treatment of malignant neoplasms often requires the use of combinations of chemotherapeutic agents. However, in order to select combinations that are effective against specific...     

Design,Expression, Purification and Characterization of the Recombinant Immunotoxin 4D5 scFv-TRAIL

International Journal of Peptide Research and Therapeutics - Immunotoxins are chimeric proteins that combine antibodies and toxins in targeted cancer therapy. Despite the promising application...     

Cyclic sulfamide HIV-1 protease inhibitors, with sidechains spanning from P2/P2' to P1/P1'

Previous studies of HIV protease inhibitors have shown that it is possible to elongate the P1/P1' sidechains to reach the S3/S3' binding sites. By analogy, we expected that it would be possible to design inhibitors reaching between the S1/S1' and S2/S2' binding sites. Molecular modeling suggested that this could be achieved with appropriate ortho-substitution of the P2/P2' benzyl groups in our cyclic sulfamide inhibitors. Four different spacer groups were investigated. The compounds were smoothly prepared from tartaric acid in five steps and exhibit low to moderate activity, the most potent inhibitor possessing a Ki value of 0.53 microM.     

Immunolesioning of Glutamate Receptor GluR1-Containing Neurons in the Rat Neostriatum Using a Novel Immunotoxin

1. To investigate the potency of a novel immunotoxin that is specific for glutamate receptor GluR1, a subunit of the -amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type receptor channel, immunolesioning was performed.2. A ribosome-inactivating protein, trichosanthin (TCS), was isolated and conjugated to the goat anti-rabbit IgG antibody molecule. The anti-rabbit antibody–TCS complex was preincubated with GluR1-specific rabbit antibody to produce a GluR1-specific immunotoxin. The immunotoxin was unilaterally administered into either the neostriatum or the lateral ventricle of rats.3. Immunoreactivity for GluR1 or GluR4 was revealed in perfuse-fixed sections of the neostriatum obtained from the lesioned and control animals by immunocytochemistry. After ventricular or striatal injections of the immunotoxin, depletions of GluR1-immunoreactive neurons, the presumed GABAergic interneurons in the neostriatum, were found. Depletions of GluR4-immunoreactive perikarya, the presumed same subpopulation of striatal interneurons, were also found. In addition, no change in the pattern of distribution of immunoreactivity for GluR2 or glial fibrillary acidic protein was found in the lesioned neostriatum. These results indicate that the novel GluR1 immunotoxin is potent and specific.4. In addition, striatal application of the immunotoxin caused a greater depletion in the number of GluR1-immunoreactive neurons. The present results also indicate that the route of immunotoxin application may be important in producing specific lesions.