Devasis Chatterjee

Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines

While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-κB (NF-κB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-κB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-κB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-κB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-κB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-κB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-κB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.

Raf Kinase Inhibitor Protein Interacts with NF-κB-Inducing Kinase and TAK1 and Inhibits NF-κB Activation

ABSTRACT The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-κB) in response to stimulation with tumor necrosis factor alpha (TNF-α) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-κB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-κB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-κB (IκB). In vitro kinase assays showed that RKIP antagonizes the activation of the IκB kinase (IKK) activity elicited by TNF-α. RKIP physically interacted with four kinases of the NF-κB activation pathway, NF-κB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKα, and IKKβ. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-κB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

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 addressed.

Inhibition of the Raf–MEK1/2–ERK1/2 Signaling Pathway, Bcl-xL Down-Regulation, and Chemosensitization of Non-Hodgkin’s Lymphoma B Cells by Rituximab

Rituximab (Rituxan, IDEC-C2B8) has been shown to sensitize non-Hodgkin’s lymphoma (NHL) cell lines to chemotherapeutic drug-induced apoptosis. Rituximab treatment of Bcl-2–deficient Ramos cells and Bcl-2–expressing Daudi cells selectively decreases Bcl-xL expression and sensitizes the cells to paclitaxel-induced apoptosis. This study delineates the signaling pathway involved in rituximab-mediated Bcl-xL down-regulation in Ramos and Daudi NHL B cells. We hypothesized that rituximab may interfere with the extracellular signal-regulated kinase (ERK) 1/2 pathway, leading to decreased Bcl-xL expression. Rituximab (20 μg/mL) inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK) 1/2 and reduced the phosphorylation of the components of the ERK1/2 pathway (Raf-1, MEK1/2, and ERK1/2) and decreased activator protein-1 DNA binding activity and Bcl-xL gene expression. These events occurred with similar kinetics and were observed 3 to 6 hours after rituximab treatment. Rituximab-mediated effects were corroborated by using specific inhibitors of the ERK1/2 pathway, which also reduced Bcl-xL levels and sensitized the NHL B cells to paclitaxel-induced apoptosis. Previous findings implicated a negative regulatory role of the Raf-1 kinase inhibitor protein (RKIP) on the ERK1/2 pathway. Rituximab treatment of NHL B cells significantly up-regulated RKIP expression, thus interrupting the ERK1/2 signaling pathway through the physical association between Raf-1 and RKIP, which was concomitant with Bcl-xL down-regulation. These novel findings reveal a signaling pathway triggered by rituximab, whereby rituximab-mediated up-regulation of RKIP adversely regulates the activity of the ERK1/2 pathway, Bcl-xL expression, and subsequent chemosensitization of drug-refractory NHL B cells. The significance of these findings is discussed.

Raf-1 Kinase Inhibitor Protein: Structure, Function, Regulation of Cell Signaling, and Pivotal Role in Apoptosis

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains the major obstacle in the successful treatment of cancer patients. Tumor cells acquire resistance to apoptotic stimuli and it has been demonstrated that conventional therapies exert their cytotoxic activities primarily by inducing apoptosis in the cells. Resistance to radiation and chemotherapeutic drugs has led to the development of immunotherapy and gene therapy approaches with the intent of overcoming resistance to drugs and radiation as well as enhancing the specificity to eliminate tumor cells. However, cytotoxic lymphocytes primarily kill by apoptosis and, therefore, drug-resistant tumor cells may also be cross-resistant to immunotherapy. To evade apoptosis, tumor cells have adopted various mechanisms that interfere with the apoptotic signaling pathways and promote constitutive activation of cellular proliferation and survival pathways. Thus, modifications of the antiapoptotic genes in cancer cells are warranted for the effectiveness of conventional therapies as well as novel immunotherapeutic approaches. Such modifications will avert the resistant phenotype of the tumor cells and will render them susceptible to apoptosis. Current studies, both in vitro and preclinically in vivo, have been aimed at the modification and regulation of expression of apoptosis-related gene products and their activities. A novel protein designated Raf-1 kinase inhibitor protein (RKIP) has been partially characterized. RKIP is a member of the phosphatidylethanolamine-binding protein family. RKIP has been shown to disrupt the Raf-1-MEK1/2 [mitogen-activated protein kinase-ERK (extracellular signal-regulated kinase) kinase-1/2]-ERK1/2 and NF-kappaB signaling pathways, via physical interaction with Raf-1-MEK1/2 and NF-kappaB-inducing kinase or transforming growth factor beta-activated kinase-1, respectively, thereby abrogating the survival and antiapoptotic properties of these signaling pathways. In addition, RKIP has been shown to act as a signal modifier that enhances receptor signaling by inhibiting G protein-coupled receptor kinase-2. By regulating cell signaling, growth, and survival through its expression and activity, RKIP is considered to play a pivotal role in cancer, regulating apoptosis induced by drugs or immune-mediated stimuli. Overexpression of RKIP sensitizes tumor cells to chemotherapeutic drug-induced apoptosis. Also, induction of RKIP by drugs or anti-receptor antibodies sensitizes cancer cells to drug-induced apoptosis. In this review, we discuss the discovery, structure, function, and significance of RKIP in cancer.

Regulation of Tumor Cell Sensitivity to TRAIL-Induced Apoptosis by the Metastatic Suppressor Raf Kinase Inhibitor Protein via Yin Yang 1 Inhibition and Death Receptor 5 Up-Regulation

Raf-1 kinase inhibitor protein (RKIP) has been implicated in the regulation of cell survival pathways and metastases, and is poorly expressed in tumors. We have reported that the NF-κB pathway regulates tumor resistance to apoptosis by the TNF-α family via inactivation of the transcription repressor Yin Yang 1 (YY1). We hypothesized that RKIP overexpression may regulate tumor sensitivity to death ligands via inhibition of YY1 and up-regulation of death receptors (DRs). The TRAIL-resistant prostate carcinoma PC-3 and melanoma M202 cell lines were examined. Transfection with CMV-RKIP, but not with control CMV-EV, sensitized the cells to TRAIL-mediated apoptosis. Treatment with RKIP small interfering RNA (siRNA) inhibited TRAIL-induced apoptosis. RKIP overexpression was paralleled with up-regulation of DR5 transcription and expression; no change in DR4, decoy receptor 1, and decoy receptor 2 expression; and inhibition of YY1 transcription and expression. Inhibition of YY1 by YY1 siRNA sensitized the cells to TRAIL apoptosis concomitantly with DR5 up-regulation. RKIP overexpression inhibited several antiapoptotic gene products such as X-linked inhibitor of apoptosis (XIAP), c-FLIP long, and Bcl-xL that were accompanied with mitochondrial membrane depolarization. RKIP overexpression in combination with TRAIL resulted in the potentiation of these above effects and activation of caspases 8, 9, and 3, resulting in apoptosis. These findings demonstrate that RKIP overexpression regulates tumor cell sensitivity to TRAIL via inhibition of YY1, up-regulation of DR5, and modulation of apoptotic pathways. We suggest that RKIP may serve as an immune surveillance cancer gene, and its low expression or absence in tumors allows the tumor to escape host immune cytotoxic effector cells.

Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging

Mesenchymal stem cells (MSCs) contribute to the recovery of tissue injury, providing a paracrine support. Cell-derived extracellular vesicles (EVs), carrying membrane and cytoplasmatic constituents of the cell of origin, have been described as a fundamental mechanism of intercellular communication. We previously demonstrated that EVs derived from human MSCs accelerated recovery following acute kidney injury (AKI) in vivo. The aim of the present study was to investigate the biodistribution and the renal localization of EVs in AKI. For this purpose, two methods for EV labeling suitable for in vivo tracking with optical imaging (OI), were employed using near infrared (NIR) dye (DiD): i) labeled EVs were generated by MSCs pre-incubated with NIR dye and collected from cell supernatants; ii) purified EVs were directly labeled with NIR dye. EVs obtained with these two procedures were injected intravenously (i.v.) into mice with glycerol-induced AKI and into healthy mice to compare the efficacy of the two labeling methods for in vivo detection of EVs at the site of damage. We found that the labeled EVs accumulated specifically in the kidneys of the mice with AKI compared with the healthy controls. After 5 h, the EVs were detectable in whole body images and in dissected kidneys by OI with both types of labeling procedures. The directly labeled EVs showed a higher and brighter fluorescence compared with the labeled EVs produced by cells. The signal generated by the directly labeled EVs was maintained in time, but provided a higher background than that of the labeled EVs produced by cells. The comparison of the two methods indicated that the latter displayed a greater specificity for the injured kidney.

Inverse Association between Raf Kinase Inhibitory Protein and Signal Transducers and Activators of Transcription 3 Expression in Gastric Adenocarcinoma Patients: Implications for Clinical Outcome

Purpose: Raf Kinase Inhibitory Protein (RKIP) plays a pivotal role in cancer by regulating apoptosis induced by chemotherapeutic agents, or immune-mediated stimuli and is a metastasis suppressor protein. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is frequently activated in gastric adenocarcinomas, thereby promoting tumor growth. We examined the expression patterns of RKIP and STAT3 with regard to human gastric cancer, predicting that elevated RKIP status may favor clinical outcome. Experimental Design: Tissue microarrays were created from samples from 143 patients with gastric adenocarcinomas. The microarrays were immunohistochemically stained for RKIP and STAT3, and the intensity and extent of the staining was semiquantitatively scored. Results: In intestinal-type gastric adenocarcinomas, RKIP and STAT3, expression were inversely associated. Cytoplasmic RKIP expression directly correlated with patient survival. Nuclear STAT3 expression inversely correlated with survival. In the diffuse tumor type, no significant correlation was found between RKIP and patient outcome. In the intestinal-type gastric adenocarcinoma, multivariate analysis adjusted for treatment types revealed RKIP and tumor stage to be significant independent predictors of survival. In the diffuse tumor type, stage was the only significant predictor of survival. Conclusion: These results indicate the predictive and protective role of cytoplasmic RKIP expression in gastric adenocarcinoma of the intestinal subtype. In contrast, nuclear STAT3 expression is associated with poor patient prognosis in the intestinal subtype. Significantly, we show an inverse association between RKIP and STAT3 and a positive correlation between RKIP and patient survival.

Microvesicle Induction of Prostate Specific Gene Expression in Normal Human Bone Marrow Cells

PURPOSE Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. MATERIALS AND METHODS We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 μM polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. RESULTS Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. CONCLUSIONS Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles.

Mesenchymal stromal cell-derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells.

Mesenchymal stromal cells (MSCs) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 h to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 h post irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% dimethyl sulfoxide at −80 °C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.