Ricin Trafficking in Cells

The heterodimeric plant toxin ricin binds exposed galactosyls at the cell surface of target mammalian cells, and, following endocytosis, is transported in vesicular carriers to the endoplasmic reticulum (ER). Subsequently, the cell-binding B chain (RTB) and the catalytic A chain (RTA) are separated reductively, RTA embeds in the ER membrane and then retrotranslocates (or dislocates) across this membrane. The protein conducting channels used by RTA are usually regarded as part of the ER-associated protein degradation system (ERAD) that removes misfolded proteins from the ER for destruction by the cytosolic proteasomes. However, unlike ERAD substrates, cytosolic RTA avoids destruction and folds into a catalytic conformation that inactivates its target ribosomes. Protein synthesis ceases, and subsequently the cells die apoptotically. This raises questions about how this protein avoids the pathways that are normally sanctioned for ER-dislocating substrates. In this review we focus on the molecular events that occur with non-tagged ricin and its isolated subunits at the ER–cytosol interface. This focus reveals that intra-membrane interactions of RTA may control its fate, an area that warrants further investigation.     

Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective

Interferon-Stimulated Gene 15 (ISG15), an antagonist of the canonical ubiquitin pathway, is frequently overexpressed in various cancers. In cancer cells, ISG15 is detected as free (intracellular) and conjugated to cellular proteins (ISGylation). Free ISG15 is also secreted into the extracellular milieu. ISGylation has protumor functions and extracellular free ISG15 has immunomodulatory properties in vitro. Therefore, whether ISG15 is a tumor suppressor or tumor promoter in vivo remains controversial. The current study aimed to clarify the role of free ISG15 in tumorigenesis. Breast cancer cells stably expressing control, ISG15, and UbcH8 (ISG15-specific E2 ligase) shRNAs were used to assess the immunoregulatory and antitumor function of free ISG15 in cell culture (in vitro) and in nude mice (in vivo). We show that extracellular free ISG15 suppresses breast tumor growth and increases NK cell infiltration into xenografted breast tumors in nude mice, and intracellular free ISG15 enhances major histocompatibility complex (MHC) class I surface expression in breast cancer cells. We conclude that free ISG15 may have antitumor and immunoregulatory function in vivo. These findings provides the basis for developing strategies to increase systemic levels of free ISG15 to treat cancer patients overexpressing the ISG15 pathway.     

Targeting Notch1 and proteasome as an effective strategy to suppress T-cell lymphoproliferative neoplasms

The T-cell lymphoproliferative neoplasms (T-LPN) are characterized by a poor clinical outcome. Current therapeutics are mostly non-selective and may induce harmful side effects. It has been reported that NOTCH1 activation mutations frequently associate T-LPN. Because anti-Notch1 based therapies such as γ-secretase inhibitors (GSI) are less efficient and induce considerable side effects, we hypothesized that combining low concentrations of GSI and the proteasome inhibitor bortezomib (BTZ) may provide an effective and tolerable approach to treat T-LPN. Hence, we analyzed the in vitro and in vivo effects of GSI-I and BTZ, alone or in combination, against T-LPN. GSI-I and BTZ synergistically decreased cell viability, proliferation, and colony formation, and induced apoptosis in T-LPN cell lines. Furthermore, combining GSI-I and BTZ decreased the viability of primary T-LPN cells from patients. These effects were accompanied by deregulation of Notch1, AKT, ERK, JNK, p38 MAPK, and NF-κB survival pathways. Moreover, combination treatment inhibited T-LPN tumor growth in nude mice. In all experiments, combining low concentrations of GSI-I and BTZ was superior to using a single agent. Our data support that a synergistic antitumor activity exists between GSI-I and BTZ, and provide a rationale for successful utilization of dual Notch1 and proteasome inhibition to treat T-LPN.     

The Proteasome Inhibitor Bortezomib Inhibits Inflammatory Response of Periodontal Ligament Cells and Ameliorates Experimental Periodontitis in Rats

Background: Periodontitis is a chronic inflammatory disease initiated by bacteria and their virulence factors. Bortezomib (BTZ) is the first proteasome inhibitor for clinical treatment of malignancies. Its anticancer activity is accompanied by an anti‐inflammatory effect. However, there are few reports about its anti‐inflammatory effect and underlying mechanism in periodontal disease, especially on human periodontal ligament cells (hPDLCs), which are considered a promising cell‐based therapy for treating periodontitis. Methods: hPDLCs were treated with lipopolysaccharide (LPS) and pretreated with BTZ. mRNA and protein levels of tumor necrosis factor (TNF)‐alpha, interleukin (IL)‐1β, IL‐6, and IL‐8 were determined. The anti‐inflammatory mechanism of BTZ was studied. Further, experimental rat periodontitis was induced with ligature and LPS injection, and simultaneously and locally treated with BTZ (three injections/week). Four weeks after treatment, microcomputed tomography, immunohistochemical, and histopathologic analyses were performed. Results: Bortezomib administration at safe concentrations (≤1 nM) inhibited production of proinflammatory cytokines in LPS‐stimulated hPDLCs via nuclear factor (NF)‐kappa B, p38/extracellular signal‐regulated kinase, and mitogen‐activated protein kinase/activator protein‐1 pathways. Moreover, in the LPS and ligature‐induced periodontitis rat model, BTZ suppressed expression of TNF‐α, IL‐1β, IL‐6, and IL‐8, reduced the ratio of receptor activator of NF‐κB ligand/osteoprotegerin, and prevented alveolar bone absorption. Conclusion: These findings demonstrate the anti‐inflammatory activity of BTZ against periodontal inflammatory response and present BTZ as a promising therapy for periodontal disease.     

FFPE tissue as a feasible source for gene expression analysis – A comparison of three reference genes and one tumor marker

Background

Formalin-fixation, paraffin-embedding is the standard processing technique for tumor tissue in modern pathology. New techniques such as cryo-conservation allow rapid fixation and long-time storage but come along with increased costs and enlarged storage complexity. However, formalin-fixed, paraffin-embedded (FFPE) tissue is available in a large quantity, making it the ideal material for retrospective studies.The following study was designed to investigate the influence of formalin-fixation on the quality of mRNA and applicability of FFPE-derived mRNA for gene expression analysis. Three potential reference genes for pulmonary tumors with neuroendocrine differentiation were included and tested for their robust expression.

Materials and methods

Eighty specimens collected from 2005 to 2012 at the Institute of Pathology and Neuropathology at the University Hospital Essen were analyzed for their gene expression by using TaqMan^® gene expression assays on demand (AoD). Three distinct potential reference genes (ACTB, GAPDH, HPRT1) were evaluated for their expression, and a proteasome subunit (PSMA1) was included in the analysis as tumor marker and functioned as an internal technical control.

Conclusion

For GAPDH and ACTB, a highly significant correlation and consistent expression between the investigated entities was found, making them reliable reference genes for further research. Additionally, the feasibility for a FFPE tissue-based gene expression analysis was verified by showing that the mRNA quality is sufficient. When standardized FFPE preparation is performed carefully, sufficient mRNA can be isolated for reliable and successful gene expression analysis. That provides the basis the door for large, retrospective studies that correlate molecular and clinical follow-up data.     

Quercetin-mediated Mcl-1 and survivin downregulation restores TRAIL-induced apoptosis in non-Hodgkin's lymphoma B cells

Background

Non-Hodgkin''s B-cell lymphomas account for approximately 70% of B-cell lymphomas. While its incidence is dramatically increasing worldwide, the disease is still associated with high morbidity due to ineffectiveness of conventional therapies, creating an urgent need for novel therapeutic approaches. Unconventional compounds, including polyphenols and the cytokine TRAIL, are being extensively studied for their capacity to restore apoptosis in a large number of tumors, including lymphomas.

Design and Methods

Molecular mechanisms of TRAIL-resistance and reactivation of the apoptotic machinery by quercetin in non-Hodgkin’s lymphoma cell lines were determined by Hoescht, flow cytometry, Western blot, qPCR, by use of siRNA or pharmacological inhibitors of the mitochondrial pathway and by immunoprecipitation followed by post-translational modification analysis.

Results

Results demonstrate that quercetin, a natural flavonoid, restores TRAIL-induced cell death in resistant transformed follicular lymphoma B-cell lines, despite high Bcl-2 expression levels due to the chromosomal translocation t(14;18). Quercetin rescues mitochondrial activation by inducing the proteasomal degradation of Mcl-1 and by inhibiting survivin expression at the mRNA level, irrespective of p53. Restoration of the TRAIL pathway requires Bax and Bak but is independent of enhanced TRAIL DISC formation.

Conclusions

We demonstrate that inactivation of survivin and Mcl-1 expression by quercetin is sufficient to restore TRAIL sensitivity in resistant non–Hodgkin’s lymphoma B cells. Our results suggest, therefore, that combining quercetin with TRAIL treatments may be useful in the treatment of non–Hodgkin’s lymphoma.     

The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo

Background

Multiple myeloma is characterized by the accumulation of tumor plasma cells in the bone marrow. Despite therapeutic improvements brought by proteasome inhibitors such as bortezomib, myeloma remains an incurable disease. In a variety of human cancers, human immunodeficiency virus protease inhibitors (e.g. nelfinavir) effectively inhibit tumor progression, but their impact on myeloma is unknown. We assessed the in vitro and in vivo effects of nelfinavir on multiple myeloma.

Design and Methods

The effects of nelfinavir (1–10 μM) on proteasome activity, proliferation and viability of myeloma cell lines and plasma cells from patients were assessed by measuring PERK, AKT, STAT3 and ERK1/2 phosphorylation and CHOP expression with immunoblotting or flow cytometry. The in vivo effect was assessed in NOD/SCID mice injected with luciferase expressing human myeloma cell lines and treated with nelfinavir at a dose of 75 mg/kg/day. Tumor progression was evaluated using a bioluminescent system.

Results

Nelfinavir inhibited 26S chymotrypsin-like proteasome activity, impaired proliferation and triggered apoptosis of the myeloma cell lines and fresh plasma cells. It activated the pro-apoptotic unfolded protein response pathway by inducing PERK phosphorylation and CHOP expression. Cell death triggered by nelfinavir treatment correlated with decreased phosphorylation of AKT, STAT3 and ERK1/2. Nelfinavir enhanced the anti-proliferative activity of bortezomib, dexamethasone and histone deacetylase inhibitors and delayed tumor growth in a myeloma mouse model.

Conclusions

These results suggest that nelfinavir, used at a pharmacological dosage, alone or in combination, may be useful in the treatment of myeloma. Our data provide a preclinical basis for clinical trials using nelfinavir in patients with myeloma.     

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.     

Hepatitis C core protein impairs insulin downstream signalling and regulatory role of IGFBP-1 expression

Chronic infection with hepatitis C virus (HCV), mainly genotype 1, has been shown to be associated with insulin resistance and type 2 diabetes. The mechanisms underlying this association are partly understood. Increased levels of tumor necrosis factor (TNF)-α occurring in HCV infection have an important role in HCV-mediated insulin resistance; however, other direct effects of HCV core protein on disrupting insulin signalling have been suggested. The insulin receptor substrate (IRS) proteins are key players in insulin signal transduction and are the major substrates of the insulin receptor. To further elucidate the direct effect of HCV core protein on insulin signalling. We studied the direct effects of HCV core protein in two cell lines transfected with HCV core protein. We found several impairments in the insulin signalling cascade which could be attributed to a significant proteasomal degradation of IRS-1 protein, in a dose-dependent way. In addition, our data show that liver cells transfected by HCV core protein show a marked attenuation of the regulatory inhibitory role of insulin on insulin growth factor binding protein-1 (IGFBP-1) expression. Since IGFBP-1 may have a role in glucose regulation and hepatic insulin sensitivity, this effect of HCV core protein can contribute to insulin resistance in chronic HCV infection. Our data suggest that the degradation of IRS-1 by HCV core protein translates to impaired ability of insulin to inhibit the expression of the target gene IGFBP-1 in the liver and may serve as a novel mechanism for insulin resistance and hyperglycaemia.