Inhibition of Arp2/3 Complex after ADP-Ribosylation of Arp2 by Binary Clostridioides Toxins.

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.

FPR2 participates in epithelial ovarian cancer (EOC) progression through RhoA-mediated M2 macrophage polarization.

BACKGROUND: In our previous study, we found that formyl peptide receptor 2 (FPR2) promoted the invasion and metastasis of epithelial ovarian cancer (EOC) and could be a prognostic marker for EOC. In this study, we aimed to study the possible mechanism of FPR2 in promoting EOC progression. METHODS: EOC cell lines with ectopic FPR2 expression and knockdown as well as their control cell lines were established, and the expression change of RhoA in each cell line was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot. Wound healing and Transwell assays were performed to detect the migratory ability of EOCs affected by FPR2 and RhoA. The supernatant of each EOC cell line was used to coculture with macrophages, and then we tested M1 and M2 macrophage biomarkers in the supernatants by flow cytometry. The THP-1 cell line was also induced to differentiate into M1 and M2 macrophages, and FPR2 and RhoA expression in each macrophage cell line was detected by RT-qPCR and Western blot. A tumour xenograft model was established with SKOV3 and SKOV3-shFPR2 cell lines, and tumour volumes and weights were recorded. RESULTS: RhoA expression was significantly increased in EOCs along with the overexpression of FPR2, which showed a positive correlation by Pearson correlation analysis. Ectopic FPR2 expression contributes to the migratory ability of EOCs, and a RhoA inhibitor (C3 transferase) impairs EOC migration. Furthermore, FPR2 stimulated the secretion of Th2 cytokines by EOCs, which induced macrophages to differentiate to the M2 phenotype, while a RhoA inhibitor stimulated the secretion of Th1 cytokines and induced macrophages to differentiate to the M1 phenotype. Moreover, compared with M1 macrophages and THP-1 cells, FPR2 and RhoA expression was significantly upregulated in M2 macrophages. CONCLUSION: FPR2 stimulated M2 macrophage polarization and promoted invasion and metastasis of ovarian cancer cells through RhoA.

Cathepsin Release from Lysosomes Promotes Endocytosis of Clostridium perfringens Iota-Toxin.

Iota-toxin from Clostridium perfringens type E is a binary toxin composed of two independent proteins: actin-ADP-ribosylating enzyme component, iota-a (Ia), and binding component, iota-b (Ib). Ib binds to target cell receptors and mediates the internalization of Ia into the cytoplasm. Extracellular lysosomal enzyme acid sphingomyelinase (ASMase) was previously shown to facilitate the internalization of iota-toxin. In this study, we investigated how lysosomal cathepsin promotes the internalization of iota-toxin into target cells. Cysteine protease inhibitor E64 prevented the cytotoxicity caused by iota-toxin, but aspartate protease inhibitor pepstatin-A and serine protease inhibitor AEBSF did not. Knockdown of lysosomal cysteine protease cathepsins B and L decreased the toxin-induced cytotoxicity. E64 suppressed the Ib-induced ASMase activity in extracellular fluid, showing that the proteases play a role in ASMase activation. These results indicate that cathepsin B and L facilitate entry of iota-toxin via activation of ASMase.

Cultured Cell Line Models of Neuronal Differentiation: NT2, PC12, and SK-N-MC.

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.

Pseudomonas Exotoxin A-Based Immunotherapy Targeting CCK2R-Expressing Colorectal Malignancies: An In Vitro and In Vivo Evaluation.

Cholecystokinin-2 receptor (CCK2R) has been proven to be a specific biomarker for colorectal malignancies. Immunotoxins are a valuable class of immunotherapy agents consisting of a targeting element and a bacterial or plant toxin. Previous work demonstrated that targeting CCK2R is a good therapeutic strategy for the treatment of colorectal cancer (CRC). In the present study, we developed a new version of CCK2R-targeting immunotoxin GD9P using a targeted peptide, GD9, as the binding motif and a truncated Pseudomonas exotoxin A (PE38) as the cytokiller. BALB/c nude mice were treated with different doses of GD9P, and pharmacodynamics, pharmacokinetic, and toxicological data were obtained throughout this study. Compared to the parental immunotoxin rCCK8PE38, GD9P exhibited about 1.5-fold yield, higher fluorescence intensity, and increased antitumor activity against human CRC in vitro and in vivo. The IC50 values of GD9P in vitro ranged from 1.61 to 4.55 nM. Pharmacokinetic studies were conducted in mice with a T1/2 of 69.315 min. When tumor-bearing nude mice were treated with GD9P at doses ≥2 mg/kg for five doses, a rapid shrinkage in tumor volume and, in some cases, complete remission was observed. A preliminary safety evaluation demonstrated a good safety profile of GD9P as a Pseudomonas exotoxin A-based immunotherapy. The therapy in combination with oxaliplatin can increase the antitumor efficacy and reduce the toxic side effects caused by chemotherapy. In conclusion, the data support the use of GD9P as a promising immunotherapy targeting CCK2R-expressing colorectal malignancies.

The cytotoxic effect of Clostridioides difficile pore-forming toxin CDTb.

Clostridioides (C.) difficile is clinically highly relevant and produces several AB-type protein toxins, which are the causative agents for C. difficile-associated diarrhea and pseudomembranous colitis. Treatment with antibiotics can lead to C. difficile overgrowth in the gut of patients due to the disturbed microbiota. C. difficile releases large Rho/Ras-GTPase glucosylating toxins TcdA and TcdB, which are considered as the major virulence factors for C. difficile-associated diseases. In addition to TcdA and TcdB, C. difficile strains isolated from severe cases of colitis produce a third toxin called CDT. CDT is a member of the family of clostridial binary actin ADP-ribosylating toxins and consists of two separate protein components. The B-component, CDTb, binds to the receptor and forms a complex with and facilitates transport and translocation of the enzymatically active A-component, CDTa, into the cytosol of target cells by forming trans-membrane pores through which CDTa translocates. In the cytosol, CDTa ADP-ribosylates G-actin causing depolymerization of the actin cytoskeleton and, eventually, cell death. In the present study, we report that CDTb exhibits a cytotoxic effect in the absence of CDTa. We show that CDTb causes cell rounding and impairs cell viability and the epithelial integrity of CaCo-2 monolayers in the absence of CDTa. CDTb-induced cell rounding depended on the presence of LSR, the specific cellular receptor of CDT. The isolated receptor-binding domain of CDTb was not sufficient to cause cell rounding. CDTb-induced cell rounding was inhibited by enzymatically inactive CDTa or a pore-blocker, implying that CDTb pores in cytoplasmic membranes contribute to cytotoxicity.

Lentiviral vector-mediated expression of C3 transferase attenuates retinal ischemia and reperfusion injury in rats.

AIMS: Our previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury. MAIN METHODS: The LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay. KEY FINDINGS: No obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R. SIGNIFICANCE: This study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies.

Naturally occurring hotspot cancer mutations in Gα13 promote oncogenic signaling.

Heterotrimeric G-proteins are signaling switches broadly divided into four families based on the sequence and functional similarity of their Gα subunits: Gs, Gi/o, Gq/11, and G12/13 Artificial mutations that activate Gα subunits of each of these families have long been known to induce oncogenic transformation in experimental systems. With the advent of next-generation sequencing, activating hotspot mutations in Gs, Gi/o, or Gq/11 proteins have also been identified in patient tumor samples. In contrast, patient tumor-associated G12/13 mutations characterized to date lead to inactivation rather than activation. By using bioinformatic pathway analysis and signaling assays, here we identified cancer-associated hotspot mutations in Arg-200 of Gα13 (encoded by GNA13) as potent activators of oncogenic signaling. First, we found that components of a G12/13-dependent signaling cascade that culminates in activation of the Hippo pathway effectors YAP and TAZ is frequently altered in bladder cancer. Up-regulation of this signaling cascade correlates with increased YAP/TAZ activation transcriptional signatures in this cancer type. Among the G12/13 pathway alterations were mutations in Arg-200 of Gα13, which we validated to promote YAP/TAZ-dependent (TEAD) and MRTF-A/B-dependent (SRE.L) transcriptional activity. We further showed that this mechanism relies on the same RhoGEF-RhoGTPase cascade components that are up-regulated in bladder cancers. Moreover, Gα13 Arg-200 mutants induced oncogenic transformation in vitro as determined by focus formation assays. In summary, our findings on Gα13 mutants establish that naturally occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins are putative cancer drivers.

Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Growth Retardation of Poorly Transfectable Tumor by Multiple Injections of Plasmids Encoding PE40 Based Targeted Toxin Complexed with Polyethylenimine.

BACKGROUND: One of the approaches to cancer gene therapy relies on tumor transfection with DNA encoding toxins under the control of tumor-specific promoters. METHODS: Here, we used DNA plasmids encoding very potent anti-ERBB2 targeted toxin, driven by the human telomerase promoter or by the ubiquitous CAG promoter (pTERT-ETA and pCAG-ETA) and linear polyethylenimine to target cancer cells. RESULTS: We showed that the selectivity of cancer cell killing by the pTERT-ETA plasmid is highly dependent upon the method of preparation of DNA-polyethylenimine complexes. After adjustment of complex preparation protocol, cell lines with high activity of telomerase promoter can be selectively killed by transfection with the pTERT-ETA plasmid. We also showed that cells transfected with pTERT-ETA and pCAG-ETA plasmids do not exert any detectable bystander effect in vitro. CONCLUSION: Despite this, three intratumoral injections of a plasmid-polyethylenimine complex resulted in substantial growth retardation of a poorly transfectable D2F2/E2 tumor in mice. There were no significant differences in anti-tumor properties between DNA constructs with telomerase or CAG promoters in vivo.

Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy.

Immunotoxins are a class of targeted cancer therapeutics in which a toxin such as Pseudomonas exotoxin A (PE) is linked to an antibody or cytokine to direct the toxin to a target on cancer cells. While a variety of PE-based immunotoxins have been developed and a few have demonstrated promising clinical and preclinical results, cancer cells frequently have or develop resistance to these immunotoxins. This review presents our current understanding of the mechanism of action of PE-based immunotoxins and discusses cellular mechanisms of resistance that interfere with various steps of the pathway. These steps include binding of the immunotoxin to the target antigen, internalization, intracellular processing and trafficking to reach the cytosol, inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (EF2), and induction of apoptosis. Combination therapies that increase immunotoxin action and overcome specific mechanisms of resistance are also reviewed.

Cytotoxic and apoptotic properties of a novel nano-toxin formulation based on biologically synthesized silver nanoparticle loaded with recombinant truncated pseudomonas exotoxin A.

Bacterial toxins have received a great deal of attention in the development of antitumor agents. Currently, these protein toxins were used in the immunotoxins as a cancer therapy strategy. Despite the successful use of immunotoxins, immunotherapy strategies are still expensive and limited to hematologic malignancies. In the current study, for the first time, a nano-toxin comprised of truncated pseudomonas exotoxin (PE38) loaded silver nanoparticles (AgNPs) were prepared and their cytotoxicity effect was investigated on human breast cancer cells. The PE38 protein was cloned into pET28a and expressed in Escherichia coli, BL21 (DE3), and purified using metal affinity chromatography and was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AgNPs were biologically prepared using cell-free supernatant of E. Coli K12 strain. Nanoparticle formation was characterized by energy dispersive spectroscopy, transmission electron microscopy, and dynamic light scattering. The PE38 protein was loaded on AgNPs and prepared the PE38-AgNPs nano-toxin. Additionally, in vitro release indicated a partial slow release of toxin in about 100 hr. The nano-toxin exhibited dose-dependent cytotoxicity on MCF-7 cells. Also, real-time polymerase chain reaction results demonstrated the ability of nano-toxin to upregulate Bax/Bcl-2 ratio and caspase-3, -8, -9, and P53 apoptotic genes in the MCF-7 tumor cells. Apoptosis induction was determined by Annexin-V/propidium flow cytometry and caspases activity assay after treatment of cancer cells with the nano-toxin. In general, in the current study, the nano-toxin exhibit an inhibitory effect on the viability of breast cancer cells through apoptosis, which suggests that AgNPs could be used as a delivery system for targeting of toxins to cancer cells.

Engineered Anti-GPC3 Immunotoxin, HN3-ABD-T20, Produces Regression in Mouse Liver Cancer Xenografts Through Prolonged Serum Retention.

BACKGROUND AND AIMS: Treatment of hepatocellular carcinomas using our glypican-3 (GPC3)-targeting human nanobody (HN3) immunotoxins causes potent tumor regression by blocking protein synthesis and down-regulating the Wnt signaling pathway. However, immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. APPROACH AND RESULTS: To address these concerns, we engineered HN3-based immunotoxins to contain various deimmunized Pseudomonas exotoxin (PE) domains. This included HN3-T20, which was modified to remove T-cell epitopes and contains a PE domain II truncation. We compared them to our previously reported B-cell deimmunized immunotoxin (HN3-mPE24) and our original HN3-immunotoxin with a wild-type PE domain (HN3-PE38). All of our immunotoxins displayed high affinity to human GPC3, with HN3-T20 having a KD value of 7.4 nM. HN3-T20 retained 73% enzymatic activity when compared with the wild-type immunotoxin in an adenosine diphosphate-ribosylation assay. Interestingly, a real-time cell growth inhibition assay demonstrated that a single dose of HN3-T20 at 62.5 ng/mL (1.6 nM) was capable of inhibiting nearly all cell proliferation during the 10-day experiment. To enhance HN3-T20's serum retention, we tested the effect of adding a streptococcal albumin-binding domain (ABD) and a llama single-domain antibody fragment specific for mouse and human serum albumin. For the detection of immunotoxin in mouse serum, we developed a highly sensitive enzyme-linked immunosorbent assay and found that HN3-ABD-T20 had a 45-fold higher serum half-life than HN3-T20 (326 minutes vs. 7.3 minutes); consequently, addition of an ABD resulted in HN3-ABD-T20-mediated tumor regression at 1 mg/kg. CONCLUSION: These data indicate that ABD-containing deimmunized HN3-T20 immunotoxins are high-potency therapeutics ready to be evaluated in clinical trials for the treatment of liver cancer.

Engineering a Nanostructured Nucleolin-Binding Peptide for Intracellular Drug Delivery in Triple-Negative Breast Cancer Stem Cells.

Five peptide ligands of four different cell surface receptors (nucleolin, CXCR1, CMKLR1, and CD44v6) have been evaluated as targeting moieties for triple-negative human breast cancers. Among them, the peptide F3, derived from phage display, promotes the fast and efficient internalization of a genetically fused green fluorescent protein (GFP) inside MDA-MB-231 cancer stem cells in a specific receptor-dependent fashion. The further engineering of this protein into the modular construct F3-RK-GFP-H6 and the subsequent construct F3-RK-PE24-H6 resulted in self-assembling polypeptides that organize as discrete and regular nanoparticles. These materials, 15-20 nm in size, show enhanced nucleolin-dependent cell penetrability. We show that the F3-RK-PE24-H6, based on the Pseudomonas aeruginosa exotoxin A (PE24) as a core functional domain, is highly cytotoxic over target cells. The combination of F3, the cationic peptide (RK)n, and the toxin domain PE24 in such unusual presentation appears as a promising approach to cell-targeted drug carriers in breast cancers and addresses selective drug delivery in otherwise difficult-to-treat triple-negative breast cancers.

Astragaloside IV attenuates sepsis-induced intestinal barrier dysfunction via suppressing RhoA/NLRP3 inflammasome signaling.

Intestinal barrier dysfunction is a trigger for sepsis progression. NLRP3 inflammasome and RhoA contribute to sepsis and intestinal inflammation. The current study aimed to explore the effects of Astragaloside IV (AS-IV), a bioactive compound from Astragalus membranaceus, on sepsis-caused intestinal barrier dysfunction and whether NLRP3 inflammasome and RhoA are involved. Septic mice modeled by cecal ligation and puncture (CLP) operation were administered with 3 mg/kg AS-IV intravenously. AS-IV decreased mortality, cytokines release, I-FABP secretion, intestinal histological score and barrier permeability, and increased tight junction (TJ) expression in intestine in CLP model. Also, in Caco-2 cells subjected to lipopolysaccharide (LPS), 200 µg/mL AS-IV co-incubation reduced cytokines levels and enhanced in vitro gut barrier function without cytotoxicity. Subsequently, NLRP3 inflammasome and RhoA were highly activated both in intestinal tissue in vivo and in Caco-2 cells in vitro, both of which were significantly suppressed by AS-IV treatment. In addition, the benefits of AS-IV on Caco-2 monolayer barrier were largely counteracted by RhoA agonist CN03 and NLRP3 gene overexpression, respectively. Furthermore, LPS-induced NLRP3 inflammasome activation was abrogated by RhoA inhibitor C3 exoenzyme. However, NLRP3 knockdown by siRNA hardly affected RhoA activation in Caco-2 cells. These data suggest that AS-IV protects intestinal epithelium from sepsis-induced barrier dysfunction via inhibiting RhoA/NLRP3 inflammasome signal pathway.

Preparation of Diphtheria and Pseudomonas Exotoxin A Immunotoxins and Evaluation of Their Cytotoxicity Effect on SK-BR-3, BT-474, and MDA-MB-231 Breast Cancer Cell Lines.

Immunotoxin targeted therapy is a promising way of cancer therapy that is made from a toxin attached to an antibody which target a specific protein presented on cancer cells. In this study, we introduce immunotoxins comprising of truncated pseudomonas exotoxin A (PEA) and diphtheria toxin (DT) conjugated to trastuzumab. The effectiveness of 20 and 30 µg/ml immunotoxins and trastuzumab were studied on SK-BR-3 and BT-474 HER2/neu positive breast cancer cell lines by a cell death assay test. The produced immunotoxins have the potential to reduce the therapeutic dose of the trastuzumab and in the same time achieve higher efficiency.

Recombinant Pierisin-5 Induces Apoptosis and Differential Expression of Bcl-2, Bax, and p53 in Human Cancer Cells.

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.

Evaluating the influence of common antibiotics on the efficacy of a recombinant immunotoxin in tissue culture.

OBJECTIVE: Recombinant immunotoxins (RITs) are antibody-toxin fusion proteins that can selectively eliminate populations of cells expressing specific surface receptors. They are in evaluation as therapeutic agents for cancer. RITs based on Pseudomonas exotoxin A (PE) are in use clinically for the treatment of hairy cell leukemia, and under trial for the treatment of other cancers. In an effort to improve the efficacy of PE-based RITs, we evaluated the potential of combination therapy with several common antibiotics (tetracycline, chloramphenicol, streptomycin, linezolid, fusidic acid, and kanamycin) on human cell lines HEK293, OVCAR8, and CA46. Antibiotics were selected based on their potential to inhibit mitochondrial protein synthesis and disrupt energy metabolism in cancer cells. RESULTS: Tetracycline, chloramphenicol, linezolid, and fusidic acid alone killed cultured human cells at high concentrations. At high but nontoxic concentrations of each antibiotic, only chloramphenicol treatment of the Burkitt's lymphoma cell line CA46 showed enhanced cytotoxicity when paired with an anti-transferrin receptor/PE RIT. This result, however, could not be replicated in additional Burkitt's lymphoma cell lines Ramos and Raji. Although the six antibiotics we tested are not promising candidates for RIT combination therapy, we suggest that fusidic acid could be considered independently as a potential cancer therapeutic.

HER2-Specific Targeted Toxin DARPin-LoPE: Immunogenicity and Antitumor Effect on Intraperitoneal Ovarian Cancer Xenograft Model.

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

Endocytosis of CF in marginal cells of stria vascularis regulated by ROCK and MLCK signaling cascade, but not G-proteins.

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis. Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope. Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade. Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.