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Due to the unsatisfactory therapeutic efficacy and inexorable side effects of small molecule antineoplastic agents, extensive efforts have been devoted to the development of more potent macromolecular agents with high specificity. Gelonin is a plant-derived protein toxin that exhibits robust antitumor effect via inactivating ribosomes and inhibiting protein synthesis. Nonetheless, its poor internalization ability to tumor cells has compromised the therapeutic promise of gelonin. In this study, a tumor acidity-responsive intracellular protein delivery system ─ functional gelonin (Trx-pHLIP-Gelonin, TpG) composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP) and gelonin, was designed and obtained by genetic recombination technique for the first time. TpG could effectively enter into tumor cells under weakly acidic conditions and markedly suppress tumor cell proliferation via triggering cell apoptosis and inhibiting protein synthesis. Most importantly, treatment by intravenous injection into subcutaneous SKOV3 solid tumors in a mouse model showed that TpG was much more effective than gelonin in curtailing tumor growth rates with negligible toxicity. Collectively, our present work suggests that the tumor acidity-targeted delivery manner endowed by pHLIP offers a new avenue for efficient delivery of other bioactive substances to acidic diseased tissues.

Cancer is one of the most serious human diseases, causing millions of deaths worldwide annually, and, therefore, it is one of the most investigated research disciplines. Developing efficient anticancer tools includes studying the effects of different natural enzymes of plant and microbial origin on tumor cells. The development of various smart delivery systems based on enzyme drugs has been conducted for more than two decades. Some of these delivery systems have been developed to the point that they have reached clinical stages, and a few have even found application in selected cancer treatments. Various biological, chemical, and physical approaches have been utilized to enhance their efficiencies by improving their delivery and targeting. In this paper, we review advanced delivery systems for enzyme drugs for use in cancer therapy. Their structure-based functions, mechanisms of action, fused forms with other peptides in terms of targeting and penetration, and other main results from in vivo and clinical studies of these advanced delivery systems are highlighted. [ABSTRACT FROM AUTHOR]

This work describes use of anti-carcinoembryonic antigen antibodies (10H6, T84.66) for targeted delivery of an endosomal escape peptide (H6CM18) and gelonin, a type I ribosome inactivating protein. The viability of colorectal cancer cells (LS174T, LoVo) was assessed following treatment with gelonin or gelonin immunotoxins, with or without co-treatment with T84.66-H6CM18. Fluorescent microscopy was used to visualize the escape of immunoconjugates from endosomes of treated cells, and efficacy and toxicity were assessed invivo in xenograft tumor-bearing mice following single- and multiple-dose regimens. Application of 25 pM T84.66-H6CM18 combined with T84.66-gelonin increased gelonin potency by ~ 1,000-fold and by ~ 6,000-fold in LS174T and LoVo cells. Intravenous 10H6-gelonin at 1.0 mg/kg was well tolerated by LS174T tumor-bearing mice, while 10 and 25 mg/kg doses led to signs of toxicity. Single-dose administration of PBS, gelonin conjugated to T84.66 or 10H6, T84.66-H6CM18, or gelonin immunotoxins co-administered with T84.66-H6CM18 were evaluated. The combinations of T84.66-gelonin + 1.0 mg/kg T84.66-H6CM18 and 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 led to significant delays in LS174T growth. Use of a multiple-dose regimen allowed further anti-tumor effects, significantly extending median survival time by 33% and by 69%, for mice receiving 1 mg/kg 10H6-gelonin + 0.1 mg/kg T84.66-H6CM18 (p = 0.0072) and 1 mg/kg 10H6-gelonin + 1 mg/kg T84.66-H6CM18 (p = 0.0017). Combined administration of gelonin immunoconjugates with antibody-targeted endosomal escape peptides increased the delivery of gelonin to the cytoplasm of targeted cells, increased gelonin cell killing invitro by 1,000–6,000 fold, and significantly increased invivo efficacy. [ABSTRACT FROM AUTHOR]

Cancer is one of the most serious human diseases, causing millions of deaths worldwide annually, and, therefore, it is one of the most investigated research disciplines. Developing efficient anticancer tools includes studying the effects of different natural enzymes of plant and microbial origin on tumor cells. The development of various smart delivery systems based on enzyme drugs has been conducted for more than two decades. Some of these delivery systems have been developed to the point that they have reached clinical stages, and a few have even found application in selected cancer treatments. Various biological, chemical, and physical approaches have been utilized to enhance their efficiencies by improving their delivery and targeting. In this paper, we review advanced delivery systems for enzyme drugs for use in cancer therapy. Their structure-based functions, mechanisms of action, fused forms with other peptides in terms of targeting and penetration, and other main results from in vivo and clinical studies of these advanced delivery systems are highlighted.

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing are characterized. The studied variants can serve as the basis for the self-removal of polypeptide tags that can carry an affine label and optimize the process of the obtainment of target proteins and peptides in E. coli cells. They make it possible to synthesize target molecules composed of soluble and insoluble hybrid proteins (fusions), to conduct their affine purification and autocatalytic processing, and to obtain mature target products. The presented variants have a number of features as compared to the known prototypes. In particular, the mutant mini-intein Int4bPRO, which contains the L93P mutation, has temperature-dependent properties. It is able to produce target molecules composed of soluble fusions at a cultivation temperature below 30°C; however, it directs most synthesized fusions into insoluble intracellular aggregates after an increase in temperature to 37°C. The transition of Int4bPRO to an insoluble form is accompanied by the complete inactivation of C-terminal processing. Further application of the standard protein denaturation–renaturation procedures enables efficient reactivation of Int4bPRO and the processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, which contain the point mutation T62N or a combination of D144N and L146T mutations, respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows optimization of the biosynthesis of biologically active target proteins and peptides composed of soluble fusions that are suitable for affine purification and subsequent intein-dependent processing without the use of protein denaturation–renaturation procedures. [ABSTRACT FROM AUTHOR]

Despite substantial advances in its treatment, brain cancer remains a life-threatening disease with a poor survival rate. The main challenges for the conventional chemotherapy include an insufficient efficacy of drugs and toxicity caused by their nonselective mode of action. Recently, great attention has been paid to highly potent macromolecules such as gelonin, a type 1 ribosome-inactivating protein that inhibits protein translation. However, gelonin is poorly internalised into tumour cells and cannot distinguish between cancer and normal cells. To overcome these challenges, we engineered in this study a recombinant gelonin fusion protein with chlorotoxin, known as a brain cancer-homing peptide. The gelonin–chlorotoxin (Gel-CLTX) fusion chimera, produced in Escherichia coli, possessed an equipotent N-glycosidase activity with that of unmodified gelonin and, furthermore, could be selectively internalised into U-87 MG glioma cells over noncancerous glial cells. Consequently, Gel-CLTX displayed substantial inhibition of protein translation in U-87 MG cells, which eventually led to significantly augmented tumouricidal effects. When tested against xenograft tumour-bearing mice, Gel-CLTX showed higher tumour accumulation and inhibition of tumour growth than did gelonin, with a low systemic toxicity. Taken together, our results demonstrate the feasibility of using a fusion strategy for enhanced chemotherapy of brain tumours. [ABSTRACT FROM AUTHOR]

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05–0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Frontotemporal dementia includes a large spectrum of neurodegenerative disorders. SQSTM1, coding for p62 protein, plays a vital role in the pathogenesis of FTD. Here, we report a case of a female patient with SQSTM1 mutation S224X, who was 59 years old when she initially exhibited memory decline, mild personality changes and subtle atrophy of frontal/temporal lobes in magnetic resonance imaging (MRI). Genetic testing revealed a nonsense mutation of the SQSTM1 gene (S224X), resulting in premature termination of protein synthesis and a predicted truncated protein 217 amino acids shorter than the normal protein. Moreover, neither intact nor truncated SQSTM1 proteins was detectable in SQSTM1 S224X mutant overexpressing HEK-cells. We assayed for SQSTM1 cDNA in samples from the patient's peripheral leucocytes and did not detect its mutation. The test of quantitative PCR showed significant decreased level of SQSTM1 mRNA from peripheral leucocytes of the patient compared to five dementia controls. Our results identify a novel pathogenic SQSTM1 S224X mutation in an atypical FTD patient accompanied with loss of SQSTM1/p62 protein expression probably due to SQSTM1 gene haploinsufficiency. [ABSTRACT FROM AUTHOR]

Purpose: Our aim was to compare the effects of a single exercise training mode (resistance exercise) with a combined exercise training (resistance and plyometric exercise) mode on satellite cell activity and anabolic signaling at the molecular level.Methods: Eighteen male weight lifters (20 ± 4 years, BMI 27 ± 6 kg/m2) were randomly assigned to either a series of resistance exercise or a series of combined exercise group. The intensity of the exercise was set at 60% of their 1 RM weight and subjects completed three sets each of six repetitions. The combined exercise group performed three different types of resistance exercise alternating with three different types of plyometric exercise, whereas the resistance exercise group performed only the three different types of resistance exercise which was repeated twice. Muscle biopsies were obtained the vastus lateralis muscle immediately before and 3 h after one bout of exercise.Results: Exercise induced increases in satellite cell activation and myofibrillar protein synthesis following both exercise modes, but the resistance exercise group was superior compared to the combined exercise group in satellite cell activity expressed by Ki67/CD56 (165 vs 232%) and PI3K/Akt protein expression (121 vs 157%), mTOR protein expression (117 vs 288%), p70S6K protein expression (253 vs 809%), and 4E-BP1 protein expression (70 vs 139%) of anabolic signaling pathway.Conclusions: These results suggest that the previous findings showing a greater effect of combined as opposed to a single exercise mode could be the effect of a greater training volume rather than a true-training effect of a combined exercise program. [ABSTRACT FROM AUTHOR]

Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.

Owing to the extraordinary potency in inhibiting protein translation that could eventually lead to apoptosis of tumor cells, ribosome-inactivating proteins (RIPs) such as gelonin have been considered attractive drug candidates for cancer therapy. However, due to several critical obstacles (e.g., severe toxicity issues caused by a lack of selectivity in their mode of action and the low cytotoxicity via poor cellular uptake, etc.), clinical application of RIPs is yet far from being accomplished. To overcome these challenges, in the present study, we engineered gelonin fusion proteins with anti-insulin-like growth factor-1 receptor (IGF-1R) affibody ('IAFF') via the genetic recombinant method and the SpyCatcher/SpyTag-mediated conjugation method. To this end, recombinant gelonin-anti-IGF-1R affibody (rGel-IAFF), gelonin-SpyCatcher (Gel-SpyCatcher) and SpyTag-IAFF fusion proteins were produced from the E. coli expression system, and gelonin-IAFF conjugate was synthesized by mixing Gel-SpyCatcher and SpyTag-IAFF. After preparation of both rGel-IAFF and Gel-IAFF conjugate, their components' functionality was characterized in vitro. Our assay results confirmed that, while both Gel-IAFF and Gel-SpyCatcher retained equipotent N-glycosidase activity to that of gelonin, IAFF was able to selectively bind to IGF-1R overexpressed U87 MG brain cancer cells over low expression LNCaP cells. The results of cellular analyses showed that rGel-IAFF and Gel-IAFF conjugate both exhibited a greater cell uptake in the U87 MG cells than gelonin, but not in the LNCaP cells, yielding a significantly augmented cytotoxicity only in the U87 MG cells. Remarkably, rGel-IAFF and Gel-IAFF conjugate displayed 22- and 5.6-fold lower IC values (avg. IC: 180 and 720 nM, respectively) than gelonin (avg. IC: 4000 nM) in the U87 MG cells. Overall, the results of the present research demonstrated that fusion of gelonin with IAFF could provide an effective way to enhance the anti-tumor activity, while reducing the associated toxicity of gelonin. [ABSTRACT FROM AUTHOR]

Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs. [ABSTRACT FROM AUTHOR]

This article details the development and validation of an enzyme-linked immunosorbent assay (ELISA) for the quantification of gelonin in mouse plasma. The ELISA was validated for intra- and inter-day variability and for accuracy over a standard curve range of 7.5–100 ng/mL. The assay was then applied to assess gelonin pharmacokinetics in mice. Results from the ELISA were compared to data obtained from a parallel study conducted with125Iodine-labeled gelonin, with quantification via gamma counting. The ELISA demonstrated good precision, as the percent coefficient of variation of quality control samples in intra-day and inter-day validation ranged from 5.4–9.3% and 2.9–7.3%, respectively. Sample recoveries ranged from 98.3–105% of nominal values. The ELISA method yielded lower plasma concentrations of gelonin than found from the less-specific gamma counting method. Consequently, pharmacokinetic analyses yielded significantly higher estimates for volume of distribution (106 ± 31 vs. 55.8 ± 13 mL/kg) and plasma clearance (34.7 ± 6.6 vs. 10.9 ± 2.1 mL/min/kg) for data determined by ELISA vs. by gamma counting. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the applicability of fusion biotoxins combining pore-forming toxins (PFTs) and ribosome-inactivating proteins (RIPs) for the anti-cancer treatment. Methods: Membrane active PFTs tend to destabilize cell membranes of tumor cells, but lack a warhead inducing significant cause of cell death. Cell-impermeable RIPs possess a powerful warhead, yet not able to enter the tumor cells. To address these challenges for anti-tumor effects, we introduced a fusion strategy of conjugating melittin (a PFT) and gelonin (a type 1 RIP) via chemical and recombinant methods, followed by in vitro assays and in vivo animal studies. Results: In vitro characterization results confirmed that the chimeric gelonin-melittin fusion proteins retained equivalent intrinsic activity to that of unmodified gelonin in inhibiting protein translation. However, chemically conjugated gelonin-melittin (cGel-Mel) and recombinant chimeric gelonin-melittin fusion (rGel-Mel) exhibited greater cell uptake, yielding a significantly enhanced cytotoxic activity over treatment of gelonin, melittin or physical mixture of gelonin and melittin. Remarkably, cGel-Mel and rGel-Mel displayed 32- and 10-fold lower IC than gelonin in the cell lines. The superior anti-tumor efficacy of multivalent cGel-Mel to monovalent rGel-Mel suggested that valency could be a crucial factor for the extent of melittin-mediated cell uptake. Tumoricidal effects observed from animal studies were in good accordance with our findings from the cellular assays. Conclusions: This study successfully demonstrated that fusion of biotoxins could provide a simple yet effective way to synergistically augment their anti-tumor activity. [ABSTRACT FROM AUTHOR]

The silk gland in silkworm ( Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval-pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval-pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals. [ABSTRACT FROM AUTHOR]

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5' terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamertoxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC50 of ~60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t1/2 of ~4 h, paving the way for future in vivo experiments. [ABSTRACT FROM AUTHOR]

Toxic gene therapy (or suicidal gene therapy) is gaining enormous interest, specifically for the treatment of cancer. The success of this therapy lies in several crucial factors, including the potency of gene products to kill the transfected tumor cells and the transfection ability of the transfection vehicles. To address the potency problem, in the present study, we engineered two separate mammalian transfection plasmids (pSAP and pGEL) containing genes encoding ribosome inactivating proteins (RIPs), gelonin and saporin. After the successful preparation and amplification of the plasmids, they were tested on various cancer cell lines (HeLa, U87, 9L, and MDA-MB-435) and a noncancerous cell line (293 HEK) using polyethyleneimine (PEI) as the transfection agent. Transfection studies performed under varying gene concentration, incubation time, and gene-to-PEI ratios revealed that, compared to the treatment of pGFP (GFP expression plasmid)/PEI, both pGEL/PEI and pSAP/PEI complexes could induce significantly augmented cytotoxic effects at only 2 μg/mL gene concentration. Importantly, these cytotoxic effects were observed universally in all tested cancer cell lines. Overall, this study demonstrated the potential of pGEL and pSAP as effective gene candidates for the toxic gene-based cancer therapy. [ABSTRACT FROM AUTHOR]

The activation of c-jun N-terminal kinases (JNK) was previously shown to be required for efficient influenza A virus replication, although a detailed mechanism has not been reported. In this study, we found that replication of H5N1 influenza virus was influenced by the JNK inhibitor SP600125. The results of time course experiments suggested that SP600125 inhibited an early post-entry step of viral infection but did not affect nucleocytoplasmic trafficking of the viral ribonucleoprotein complex. The levels of influenza virus genomic RNA (vRNA), but not the corresponding cRNA or mRNA, were specifically reduced by SP600125 in virus-infected cells, indicating that the JNK protein is intimately involved in vRNA synthesis. Additionally, SP600125 affected H5N1 virus protein synthesis, because NS1, PB1, PB2, HA and M1 protein production was impaired. Thus, our data demonstrated a critical role of the JNK protein in the regulation of vRNA and protein synthesis during virus infection. This enhances our understanding of the complicated signal transduction network involved in influenza A virus replication. [ABSTRACT FROM AUTHOR]

Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to evaluate the early response of tumors to a vascular-disrupting agent (VDA) VEGF121/recombinant toxin gelonin (rGel) using dynamic [(18)F]FPPRGD2 positron emission tomography (PET) and kinetic parameter estimation.Procedures: Two tumor xenograft models: U87MG (highly vascularized) and A549 (moderately vascularized), were selected, and both were randomized into treatment and control groups. Sixty-minute dynamic PET scans with [(18)F]FPPRGD2 that targets to integrin αvβ3 were performed at days 0 (baseline), 1, and 3 since VEGF121/rGel treatment started. Dynamic PET-derived binding potential (BPND) and parametric maps were compared with tumor uptake (%ID/g) and the static PET image at 1 h after the tracer administration.Results: The growth of U87MG tumor was obviously delayed upon VEGF121/rGel treatment. A549 tumor was not responsive to the same treatment. BPND of treated U87MG tumors decreased significantly at day 1 (p < 0.05), and the difference was more significant at day 3 (p < 0.01), compared with the control group. However, the tracer uptake (%ID/g) derived from static images at 1-h time point did not show significant difference between the treated and control tumors until day 3. Little difference in tracer uptake (%ID/g) or BPND was found between treated and control A549 tumors. Considering the tracer retention in tumor and the slower clearance due to damaged tumor vasculature after treatment, BPND representing the actual specific binding portion appears to be more sensitive and accurate than the semiquantitative parameters (such as %ID/g) derived from static images to assess the early response of tumor to VDA treatment.Conclusions: Quantitative analysis based on dynamic PET with [(18)F]FPPRGD2 shows advantages in distinguishing effective from ineffective treatment during the course of VEGF121/rGel therapy at early stage and is therefore more sensitive in assessing therapy response than static PET. [ABSTRACT FROM AUTHOR]

The eukaryotic translation elongation factor 1A (eEF1A) is a moonlighting protein that besides to its canonical role in protein synthesis is also involved in many other cellular processes such as cell survival and apoptosis. In a previous work, we identified eEF1A Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and apoptosis of human cancer cells. We proposed that the phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms thus suggesting the formation of a potential eEF1A heterodimer owning regulatory properties. This study aimed at investigating the cellular localization and interaction between two eEF1A isoforms. To this end, we developed chimera proteins by adding at the N-terminal end of both eEF1A1 and eEF1A2 cyan fluorescence protein (mCerulean) and yellow fluorescence protein (mVenus), respectively. The fluorescent eEF1A1 and eEF1A2 chimeras were both addressed to COS-7 cells and found co-localized in the cytoplasm at the level of cellular membranes. We highlighted FRET between the labeled N-termini of eEF1A isoforms. The intra-molecular FRET of this chimera was about 17%. Our results provide novel information on the intracellular distribution and interaction of eEF1A isoforms. [ABSTRACT FROM AUTHOR]

Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products. [ABSTRACT FROM AUTHOR]

VEGF121/rGel, a fusion protein composed of the growth factor VEGF121 and the recombinant toxin gelonin (rGel), targets the tumor neovasculature and exerts impressive cytotoxic effects by inhibiting protein synthesis. We evaluated the effect of VEGF121/rGel on the growth of metastatic MDA-MB-231 tumor cells in SCID mice. VEGF121/rGel treatment reduced surface lung tumor foci by 58% compared to controls (means were 22.4 and 53.3, respectively; P < .05) and the mean area of lung colonies by 50% (210 ± 37 m2vs 415 ± 10 m2 for VEGF121/rGel and control, respectively; P < .01). In addition, the vascularity of metastatic foci was significantly reduced: (198 ± 37 vs 388 ± 21 vessels/mm2 for treated and control, respectively). Approximately 62% of metastatic colonies from the VEGF121/rGel-treated group had fewer than 10 vessels per colony compared to 23% in the control group. The VEGF receptor Flk-1 was intensely detected on the metastatic vessels in the control but not in the VEGF121/rGel-treated group. Metastatic foci present in lungs had a three-fold lower Ki-67 labeling index compared to control tumors. Thus, the antitumor vascular-ablative effect of VEGF121/rGel may be utilized not only for treating primary tumors but also for inhibiting metastatic spread and vascularization of metastases.

The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1 mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI. [ABSTRACT FROM AUTHOR]

The chemical synthesis of a protein from four fragments or more applying native chemical ligation could be achieved stepwise, in one-pot, convergently, or on a solid support. With the increasing demands of applying protein synthesis to highly complex targets, examining these approaches becomes essential to achieve highly efficient synthesis. Different chemical synthetic strategies are compared for the preparation of the H2B protein having different post-translational modifications. The analogues include H2B that is ubiquitinated at Lys34, Lys120, glycosylated at Ser112, and doubly modified with ubiquitin and N-acetylglucosamine. This study demonstrates that the applied convergent strategy for the synthesis of most of these complex targets was better than the one-pot approach in terms of yield and purity. Some guidelines are offered for future synthetic endeavors of similar challenging proteins. [ABSTRACT FROM AUTHOR]

Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids ß-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to +1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3’-UTR. Exploring putative cryptic signals in all 3’-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3’-UTR sequences, but also boost the potential for future genetic adaptations. [ABSTRACT FROM AUTHOR]

The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C-terminal peptidethioesters, key intermediates in NCL. We have developed an N-acylurea (Nbz) approach for the synthesis of thioester peptide precursors that efficiently undergo thiol exchange yielding thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences 0 revealed limitations, such as diacylated products that can not be converted into N-acylurea peptides. Here, we introduce a new N-acylurea linker bearing an o-amino (methyl) aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired N-acyl-N'-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis of all 20 natural amino acids and their NCL reactions. The kinetic analysis of the ligations using model peptides shows the MeNbz peptide rapidly converts to arylthioesters that are efficient at NCL. Finally, we show that the new MeDbz linker can be applied to the synthesis of cysteine-rich proteins such the cyclotides Kalata B1 and MCoTI-II through a one cyclization/folding step in the ligation/folding buffer. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy- trans -2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1–AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1 h) caused HNE–LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR–p70S6K–RPS6 signaling, and stimulated protein synthesis by 27.1±3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1–p70S6K–RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1–p70S6K–RPS6 signaling may be of therapeutic value. [ABSTRACT FROM AUTHOR]

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5’cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3’ UTR motif. Caf20p binds all tested motif-containing 3’ UTRs. Caf20p and the 3’UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3’UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E. [ABSTRACT FROM AUTHOR]

During aging, memory retention and persistence of long-term potentiation (LTP) are impaired, suggesting an aging-related deterioration in mechanisms regulating information storage. Late-phase LTP requires synthesis of proteins at synapses as well as integrated regulation of gene networks. Because aging diminishes the persistence of LTP, primarily by affecting the transition between early and late phases, we assessed whether this was reflected in perturbation of gene networks. Using DNA microarray analysis, we compared LTP-associated gene expression in young (5 months), middle-aged (15 months), and old (22 months) male Sprague–Dawley rats. As expected, we found no significant difference in LTP measured 20 minutes postinduction; however, we found that overall more genes were regulated in the young group. Bioinformatics predicted not only dysregulation of activator protein-1 and nuclear factor k B transcription factor activity and epigenetic modifications but also dysregulation of protein synthesis. Notably, we confirmed an age-related impairment in metabotropic and ionotropic receptor–mediated synaptic protein synthesis. Together, these results demonstrate that LTP-specific gene expression is altered with aging and suggest that dysregulation of synaptic protein synthesis also contributes to the age-dependent reduction in LTP persistence. [ABSTRACT FROM AUTHOR]

Background: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH's modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). Results: Treatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis. Conclusion: Given that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128's complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival. [ABSTRACT FROM AUTHOR]

Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC50 (avg. 54.3 n M) than rGel (avg. IC50: 3640 n M) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 409-419, 2015. [ABSTRACT FROM AUTHOR]

An immunotoxin composed of gelonin, a basic ribosome-inactivating protein, type I of 30 kDa, isolated from the seeds of the Indian plant Gelonium multiflorum and methotrexate (MTX) has been studied as a potential tool of gelonin delivery into the cytoplasm of MTX-responsive cells. On the average, about five molecules of methotrexate were chemically coupled to gelonin via an N-hydroxysuccinimide ester of the drug. The MTX-gelonin conjugate was able to reduce the viability of MCF-7 cells in a dose-dependent manner with ID50 of 10 n m, whereas gelonin or MTX alone showed none or very little effects. Besides its ribosome-inactivating activity, which is about ten-fold lower in an in vitro translation assay (IC50 of 50.5 ng/ml as compared to 4.6 ng/ml), the conjugate also significantly induced direct and oxidative DNA damage as shown by the alkaline comet assay. Hence, MTX-gelonin conjugates are promising candidates for the treatment of MTX-responsive cancers. [ABSTRACT FROM AUTHOR]

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC 50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor. [ABSTRACT FROM AUTHOR]

Prediction of microbial CP (MCP) synthesis in the rumen is an integral part of the MP system. For the NRC beef model, MCP is calculated as 0.13 multiplied by TDN intake (TDNI), with adjustment for physically effective NDF (peNDF) concentrations less than 20%. Despite its application for nearly 2 decades, MCP predictions using this approach have not been extensively evaluated. We assembled a database of 285 treatment means from 66 published papers using beef cattle and dairy or dairy x beef crossbred steers, fed diets with a wide range of TDN, CP, and ether extract (EE) concentrations, in which MCP synthesis was measured. Fat-free TDN (FFTDN) concentration was calculated by subtracting 2.25 x percent EE from the TDN concentration. Based on initial model selection procedures indicating that DMI and concentrations of TDN, FFTDN, and CP were significantly (P < 0.04) related to MCP synthesis, linear and quadratic effects of TDNI and FFTDN intake (FFTDNI) and CP intake (CPI) were considered as potential independent variables. Mixed model regression methods were used to fit 1-, 2-, and 3-independent variable models based on either TDNI or FFTDNI (e.g., TDNI only, TDNI and CPI, and TDNI, CPI, and the quadratic effect of TDNI; or FFTDNI only, FFTDNI and CPI, and FFTDNI, CPI, and the quadratic effect of FFTDNI). Tme ruminal OM digested (TROMD; g/d) was highly related (r² = 0.84 using citation-adjusted data) to MCP synthesis. Similarly, both TDNI and FFTDNI were highly related to citation-adjusted TROMD (r² > 0.96) and MCP synthesis (r² > 0.89). Models with FFTDNI were slightly more precise with slightly smaller prediction errors than those with TDNI. Randomly dividing the citations into Development (60%) and Evaluation (40%) data sets indicated that models such as those derived from the overall database accounted for 46 to 56% of the variation in MCP synthesis, with neither mean nor linear bias (P ≥ 0.26). In contrast, calculating MCP as 0.13 x TDNI, with or without adjustment for peNDF concentration, resulted in overprediction of MCP (P < 0.001 for both mean and linear bias). Cross-validation using 5,000 randomly drawn training and testing data sets yielded results similar to the Development/Evaluation approach. Recommended equations are provided, but the errors of prediction associated with these empirical regression equations were on the order of 25 to 30% of the mean MCP. [ABSTRACT FROM AUTHOR]

Gougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities. gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed that gouR plays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of the gouL-to-gouB operon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions of gouL, gouM, and gouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export in Streptomyces graminearus. [ABSTRACT FROM AUTHOR]

The origin of the MHC class I-presented peptides are thought to be primarily from newly synthesized but defective proteins, termed defective ribosomal products. Most of the data supporting this concept come from studies in which inhibitors of protein synthesis were found to rapidly block Ag presentation even when cells contained a pool of mature proteins. However, these data only indirectly address the origin of presented peptides, and in most studies, the contribution of mature functional proteins to the class I peptide pool has not been directly quantified. In this report, we address the efficiency and contribution of mature proteins using a tetracycline-inducible system to express Ags that are conditionally stabilized upon ligand binding. This system circumvents the use of general inhibitors of protein synthesis to control Ag expression. Moreover, by controlling Ag stabilization, we could investigate whether the degradation of mature Ags contributed to Ag presentation at early and/or late time points. We show that mature proteins are the major contributor of peptides presented on class I for two distinct antigenic constructs. Furthermore, our data show that the protein synthesis inhibitors used previously to test the contribution of defective proteins actually block Ag presentation in ways that are independent from blocking Ag synthesis. These data suggest that for the constructs we have analyzed, mature functional proteins, rather than defective ribosomal products, are the predominant source of MHC class I- presented peptides. [ABSTRACT FROM AUTHOR]

Abstract: The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargos into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2μg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents. [Copyright &y& Elsevier]

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 factorinducible 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 (t½α ¼ 1.26 hours) followed by a seven-fold longer plasma half-life (t½b ¼ 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. [ABSTRACT FROM AUTHOR]

Plant-derived Type I toxins are candidateanticancer therapeuticsrequiring cytosolic delivery into tumor cells. We tested a conceptfor two-stage delivery, whereby tumor cells precoated with an antibody-targetedgelonin toxin were killed by exposure to endosome-disrupting polymernanoparticles. Co-internalization of particles and tumor cell-boundgelonin led to cytosolic delivery and >50-fold enhancement of toxinefficacy. This approach allows the extreme potency of gelonin to befocused on tumors with significantly reduced potential for off-targettoxicity. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in ∼60% of the melanoma cell lines we tested, including both B-Raf WT and B-RafV600E lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested. We generated both a chemical conjugate composed of the recombinant gelonin (rGel) toxin and the anti-Fn14 antibody ITEM-4 (designated ITEM4-rGel) and a humanized, dimeric single-chain antibody of ITEM-4 fused to rGel (designated hSGZ). Both ITEM4-rGel and hSGZ were highly cytotoxic to a panel of different melanoma cell lines. Mechanistic studies showed that both immunotoxins induced melanoma cell necrosis. In addition, these immunotoxins could upregulate the cellular expression of Fn14 and trigger cell-signaling events similar to the Fn14 ligand TWEAK. Finally, treatment of mice bearing human melanoma MDA-MB-435 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared with controls. We conclude that Fn14 is a therapeutic target in melanoma and the hSGZ construct appears to warrant further development as a therapeutic agent against Fn14-positive melanoma. [ABSTRACT FROM AUTHOR]

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. The biological activity of BLyS is mediated by three cell surface receptors: BR3/BAFF-R, TACI and BCMA. The expression of these receptors is highly restricted to B cells, both normal and malignant. A BLyS-gelonin fusion toxin (BLyS-gel) was generated consisting of the recombinant plant-derived toxin gelonin fused to the N-terminus of BLyS and tested against a large and diverse panel of B-NHL cell lines. Interestingly, B-NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursoracute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with low picomolar EC50 values. BLyS receptor expression did not guarantee sensitivity to BLyS-gel, even though the construct was internalized by both sensitive and resistant cells. Resistance to BLyS-gel could be overcome by treatment with the endosomotropic drug chloroquine, suggesting BLyS-gel may become trapped within endosomal/lysosomal compartments in resistant cells. BLySgel induced cell death was caspase-independent and shown to be at least partially mediated by the "ribotoxic stress response." This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment was shown to localize to sites of disease, rapidly reduce tumor burden, and significantly prolong survival in xenograft mouse models of disseminated BCP-ALL, DLBCL, and MCL. Together, these findings suggest BLyS has significant potential as a targeting ligand for the delivery of cytotoxic "payloads" to malignant B cells. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and a major cause of mortality from hematopoietic malignancies in adults. A substantial number of patients become drug resistant during chemotherapy, necessitating the development of alternative modes of treatment. rGel (recombinant Gelonin)/BlyS (B-lymphocyte stimulator) is a toxin-cytokine fusion protein used for selective killing of malignant B-cells expressing receptors for B-cell-activating factor (BAFF/BLyS) by receptor-targeted delivery of the toxin, Gelonin. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection. Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice. Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation. Thus, a combination treatment with the B-cell-specific fusion toxin rGel/BLyS and the mobilizing agent AMD3100 could be an effective alternative approach to chemotherapy for the treatment of primary and relapsed ALL. [ABSTRACT FROM AUTHOR]