Expression of phosphorylated raf kinase inhibitor protein (pRKIP) is a predictor of lung cancer survival

<p>Abstract</p> <p>Background</p> <p>Raf-1 kinase inhibitor protein (RKIP) has been reported to negatively regulate signal kinases of major survival pathways. RKIP activity is modulated in part by phosphorylation on Serine 153 by protein kinase C, which leads to dissociation of RKIP from Raf-1. RKIP expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. The prognostic power has typically been based on total RKIP expression and has not considered the significance of phospho-RKIP.</p> <p>Methods</p> <p>The present study examined the expression levels of both RKIP and phospho-RKIP in human lung cancer tissue microarray proteomics technology.</p> <p>Results</p> <p>Total RKIP and phospho-RKIP expression levels were similar in normal and cancerous tissues. phospho-RKIP levels slightly decreased in metastatic lesions. However, the expression levels of phospho-RKIP, in contrast to total RKIP, displayed significant predictive power for outcome with normal expression of phospho-RKIP predicting a more favorable survival compared to lower levels (P = 0.0118); this was even more pronounced in more senior individuals and in those with early stage lung cancer.</p> <p>Conclusions</p> <p>This study examines for the first time, the expression profile of RKIP and phospho-RKIP in lung cancer. Significantly, we found that phospho-RKIP was a predictive indicator of survival.</p

Regulation of RKIP Function by Helicobacter pylori in Gastric Cancer

Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium that infects more than half of the world’s population and is a major cause of gastric adenocarcinoma. The mechanisms that link H. pylori infection to gastric carcinogenesis are not well understood. In the present study, we report that the Raf-kinase inhibitor protein (RKIP) has a role in the induction of apoptosis by H. pylori in gastric epithelial cells. Western blot and luciferase transcription reporter assays demonstrate that the pathogenicity island of H. pylori rapidly phosphorylates RKIP, which then localizes to the nucleus where it activates its own transcription and induces apoptosis. Forced overexpression of RKIP enhances apoptosis in H. pylori-infected cells, whereas RKIP RNA inhibition suppresses the induction of apoptosis by H. pylori infection. While inducing the phosphorylation of RKIP, H. pylori simultaneously targets non-phosphorylated RKIP for proteasome-mediated degradation. The increase in RKIP transcription and phosphorylation is abrogated by mutating RKIP serine 153 to valine, demonstrating that regulation of RKIP activity by H. pylori is dependent upon RKIP’s S153 residue. In addition, H. pylori infection increases the expression of Snail, a transcriptional repressor of RKIP. Our results suggest that H. pylori utilizes a tumor suppressor protein, RKIP, to promote apoptosis in gastric cancer cells

Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addresse

Differentiation of Human Malignant Melanoma Cells that Escape Apoptosis After Treatment with 9-Nitrocamptothecin In Vitro

AbstractAfter in-vitro exposure to 0.05 μmol/L 9-nitrocamptothecin (9NC) for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1 B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G1-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells

Induction of Raf-1 Kinase Inhibitory Protein (RKIP) by the Proteasome Inhibitor NPI-0052 and Reversal of B-NHL Resistance to Apoptosis.

Abstract Patients with B-cell malignancies initially respond to conventional cytotoxic therapies. However, many patients experience relapses and recurrences. Novel therapeutics are being explored in the reversal of resistance such as TRAIL. TRAIL has been shown to be selectively cytotoxic to few tumors and both TRAIL and agonist TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies are currently being explored clinically in unresponsive cancer patients. We have recently found that treatment of TRAIL resistant B-NHL cell lines such as Ramos with the novel proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals) sensitizes tumor cells to TRAIL-induced apoptosis. The concentration of NPI-0052 achieving optimal sensitization to TRAIL was in the range of 1–5 nM. In contrast 400 fold more VelcadeTM (bortezomib) was necessary to achieve a similar level of apoptosis. The mechanism of NPI-0052- mediated sensitization to TRAIL was examined. Treatment of Ramos with NPI-0052 inhibited significantly the constitutively activated NF-κB concomitantly with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) resulting in up-regulation of DR5 expression. The role of YY1 in NPI-0052-induced sensitization to TRAIL was corroborated by the use of YY1siRNA as treatment with the YY1siRNA mimicked NPI-0052 induced DR5 up-regulation and sensitization to TRAIL. Studies examining the inhibition of the NF-κB pathway by NPI-0052 revealed a novel finding demonstrating the induced expression of the metastatic suppressor gene, Raf-1 kinase inhibitory protein (RKIP). RKIP has been reported to inhibit NF-κB activity and, thus, its induction by NPI-0052 contributed to the inhibition of NF-κB activity. The role of RKIP in NPI-0052 induced sensitization to TRAIL was corroborated by the use of RKIPsiRNA and transfectants mimicked NPI-0052 mediated inhibition of YY1, DR5 upregulation and sensitization to TRAIL mediated apoptosis. The above findings suggest that NPI-0052-induced inhibition of NF-κB results at least from two distinct mechanisms, namely, inhibition of p-IκBα degradation by the proteasome and by the induction of RKIP expression. The apoptosis induced by combination of NPI-0052 and TRAIL was the result of the activation of the type II mitochondrial pathway. It has been reported that the RKIP levels are reduced in many tumors (e.g. breast, prostate, ovarian) and the expression levels are of prognostic significance. Hence, the present findings demonstrating NPI-0052 induced RKIP expression may be therapeutically relevant in the prevention of metastasis. In addition, NPI-0052 should facilitate the reversal of tumor resistance when used in combination with subtoxic levels of cytotoxics.</jats:p