Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

Global Protein Profiling in Processed Immunohistochemistry Tissue Sections.

Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.

Anticancer Activity of the Marine Triterpene Glycoside Cucumarioside A2-2 in Human Prostate Cancer Cells.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), treatment is inevitably hampered by the development of drug resistance. Thus, new drugs are urgently needed. We investigated the efficacy, toxicity, and mechanism of action of the marine triterpene glycoside cucumarioside A2-2 (CA2-2) using an in vitro CRPC model. CA2-2 induced a G2/M-phase cell cycle arrest in human prostate cancer PC-3 cells and caspase-dependent apoptosis executed via an intrinsic pathway. Additionally, the drug inhibited the formation and growth of CRPC cell colonies at low micromolar concentrations. A global proteome analysis performed using the 2D-PAGE technique, followed by MALDI-MS and bioinformatical evaluation, revealed alterations in the proteins involved in cellular processes such as metastatic potential, invasion, and apoptosis. Among others, the regulation of keratin 81, CrkII, IL-1ß, and cathepsin B could be identified by our proteomics approach. The effects were validated on the protein level by a 2D Western blotting analysis. Our results demonstrate the promising anticancer activity of CA2-2 in a prostate cancer model and provide insights on the underlying mode of action.

Immunoproteasomes control activation of innate immune signaling and microglial function.

Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.

Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach.

MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.

Deficiency in FTSJ1 Affects Neuronal Plasticity in the Hippocampal Formation of Mice.

The role of the tRNA methyltransferase FTSJ1 in the brain is largely unknown. We analyzed whether FTSJ1-deficient mice (KO) displayed altered neuronal plasticity. We explored open field behavior (10 KO mice (aged 22-25 weeks)) and 11 age-matched control littermates (WT) and examined mean layer thickness (7 KO; 6 WT) and dendritic spines (5 KO; 5 WT) in the hippocampal area CA1 and the dentate gyrus. Furthermore, long-term potentiation (LTP) within area CA1 was investigated (5 KO; 5 WT), and mass spectrometry (MS) using CA1 tissue (2 each) was performed. Compared to controls, KO mice showed a significant reduction in the mean thickness of apical CA1 layers. Dendritic spine densities were also altered in KO mice. Stable LTP could be induced in the CA1 area of KO mice and remained stable at for at least 1 h, although at a lower level as compared to WTs, while MS data indicated differential abundance of several proteins, which play a role in neuronal plasticity. FTSJ1 has an impact on neuronal plasticity in the murine hippocampal area CA1 at the morphological and physiological levels, which, in conjunction with comparable changes in other cortical areas, might accumulate in disturbed learning and memory functions.

Macrophages: shapes and functions.

Macrophages are cells of the innate immune system and represent an important component of the first-line defense against pathogens and tumor cells. Here, their diverse functions in inflammation and tumor defense are described, and the mechanisms, tools, and activation pathways and states applied are presented. The main focus is on the role and origin of reactive oxygen species (ROS), the important signal pathways TLR/NF-κB, and the M1/​​M2 polarization of macrophages.

Gelsolin Governs the Neuroendocrine Transdifferentiation of Prostate Cancer Cells and Suppresses the Apoptotic Machinery.

BACKGROUND/AIM: Interleukin 6 (IL6) is increased in patients with progressive prostate cancer and induces its transdifferentiation to neuroendocrine prostate cancer. Neuroendocrine prostate cancer has become one of the greatest challenges in treating castration-resistant disease and is linked to poor prognosis. It is necessary to understand better the cellular events associated with IL6-mediated neuroendocrine differentiation to prevent it and identify potential new therapeutic targets. MATERIALS AND METHODS: In the present study, an IL6-inducible neuroendocrine differentiation model established specifically for this purpose was applied using LNCaP cells. Proteomics and western blot analyses were used to identify proteins involved in neuroendocrine differentiation. Subsequently, the role of gelsolin (GSN) in the neuroendocrine differentiation model was characterized (knock-down analyses, microscopic co-localization analyses, apoptosis assay) and GSN expression levels in patient material were investigated. RESULTS: This study revealed that GSN is a crucial factor in the neuroendocrine differentiation process. CONCLUSION: It was shown that siRNA-mediated knock-down of GSN can inhibit neuroendocrine differentiation, making it a valid target for preventing IL6-mediated neuroendocrine differentiation.

Inspired by Sea Urchins: Warburg Effect Mediated Selectivity of Novel Synthetic Non-Glycoside 1,4-Naphthoquinone-6S-Glucose Conjugates in Prostate Cancer.

The phenomenon of high sugar consumption by tumor cells is known as Warburg effect. It results from a high glycolysis rate, used by tumors as preferred metabolic pathway even in aerobic conditions. Targeting the Warburg effect to specifically deliver sugar conjugated cytotoxic compounds into tumor cells is a promising approach to create new selective drugs. We designed, synthesized, and analyzed a library of novel 6-S-(1,4-naphthoquinone-2-yl)-d-glucose chimera molecules (SABs)-novel sugar conjugates of 1,4-naphthoquinone analogs of the sea urchin pigments spinochromes, which have previously shown anticancer properties. A sulfur linker (thioether bond) was used to prevent potential hydrolysis by human glycoside-unspecific enzymes. The synthesized compounds exhibited a Warburg effect mediated selectivity to human prostate cancer cells (including highly drug-resistant cell lines). Mitochondria were identified as a primary cellular target of SABs. The mechanism of action included mitochondria membrane permeabilization, followed by ROS upregulation and release of cytotoxic mitochondrial proteins (AIF and cytochrome C) to the cytoplasm, which led to the consequent caspase-9 and -3 activation, PARP cleavage, and apoptosis-like cell death. These results enable us to further clinically develop these compounds for effective Warburg effect targeting.

Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones.

Treatment of castration-resistant prostate cancer (CRPC) remains challenging due to the development of drug resistance. The Warburg effect describes the ability of cancer cells to consume larger amounts of glucose compared to normal tissues. We identified derivatives of natural 1,4-naphthoquinones to be active in CRPC and further synthetically modified them via glucose conjugation to increase selectivity by Warburg effect targeting. Mechanisms of action were examined by quantitative proteomics followed by bioinformatical analysis and target validation. Four synthesized molecules revealed the highest selectivity towards human CRPC cells, which correlated with higher GLUT-1 activity and expression. The compounds were able to induce pro-apoptotic signs and to inhibit the pro-survival processes and mechanisms of drug resistance (i.e., AR-signaling and autophagy). Proteome analysis suggested a disruption of the mitochondria/oxidative phosphorylation, which was validated by further functional analysis: thus, mitochondria depolarization, elevated levels of cytotoxic ROS, an increase of Bax/Bcl-2 ratio as well as release of mitochondrial AIF and cytochrome C to cytoplasm were observed. In conclusion, glucose-conjugated 1,4-naphthoquinones show potent activity and selectivity in human CRPC exerted via mitochondrial targeting. The compounds can overcome drug resistance against current standard therapies and suppress pro-survival mechanisms. This unique combination of properties makes them new promising candidates for the treatment of CRPC.

Nicotinamide N-Methyltransferase and Its Precursor Substrate Methionine Directly and Indirectly Control Malignant Metabolism During Progression of Renal Cell Carcinoma.

BACKGROUND/AIM: Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults, and new specific biomarkers are urgently needed to define diagnosis and prognosis of patients with RCC as well as monitor the outcome of therapeutic interventions. The enzyme nicotinamide N-methyltransferase (NNMT) is believed to represent such a marker molecule in RCC therapy. MATERIALS AND METHODS: NNMT expression was examined by western blotting in samples from patients with RCC and in RCC cell lines. Effects of NNMT on cell growth and metabolism were assessed using the Hoechst 33342 reagent assay and Vita-Orange cell viability assay. Incubation experiments were performed to study the influence of methionine and interleukin-6 (IL6) on expression of NNMT. RESULTS: In patient samples, NNMT was up-regulated depending on the stage of progression. Investigations in an RCC cell culture model showed that after modulation of NNMT expression, cellular metabolism, but not cell growth was affected. This regulatory function was also dependent on the presence of the NNMT precursor substrate methionine and IL6. CONCLUSION: The metabolism-regulatory activity of NNMT depends on the precursor substrate methionine and the presence of IL6. The function of methionine appears to be dependent on the stage of progression, since in individual RCC cell lines, opposing effects on metabolism were demonstrated. This, in turn, reflects the thoroughly complex situation in the clinic.

Inhibition of calpain-1 stabilizes TCF11/Nrf1 but does not affect its activation in response to proteasome inhibition.

Protein degradation is essential to compensate for the damaging effects of proteotoxic stress. To ensure protein and redox homeostasis in response to proteasome inhibition, the cleavage and nuclear translocation of the endoplasmic reticulum (ER)-bound transcription factor TCF11/Nrf1 (NFE2L1) is crucial for the activation of rescue factors including the synthesis of new proteasomal subunits. Even though TCF11/Nrf1 is an essential transcription factor, the exact mechanisms by which it is activated and stabilized are not fully understood. It was previously shown that the calcium-dependent protease calpain-1 interacts with TCF11/Nrf1 and the TCF11/Nrf1 cleavage site is a potential calpain target. Here, we tested the hypothesis that calpain-1 or -2 cleave TCF11/Nrf1. However, we did not find a role for calpain-1 or -2 in the activation of TCF11/Nrf1 after proteasome inhibition neither by using chemical inhibitors nor siRNA-mediated knockdown or overexpression of calpain subunits. Instead, we found that TCF11/Nrf1 is digested by calpain-1 in vitro and that calpain-1 inhibition slows down the degradation of membrane-bound TCF11/Nrf1 by the proteasome in cultured cells. Thus, we provide evidence that calpain-1 is involved in the degradation of TCF11/Nrf1. Furthermore, we confirmed DDI2 as an essential factor for TCF11/Nrf1 activation and demonstrate an undefined role of DDI2 and calpain-1 in TCF11/Nrf1 stability.

Proteomic-based investigations on the mode of action of the marine anticancer compound rhizochalinin.

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid-like compound, which shows promising in vitro and in vivo activity in human castration-resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration-resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer-associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL-1ß by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC-3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz-treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non-ATP competitive MEK1 inhibitor) and FR180204 (ATP-competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.

The Angiotensin-(1-7)/Mas Axis Improves Pancreatic ß-Cell Function in Vitro and in Vivo.

The angiotensin-converting enzyme 2/angiotensin (Ang)-(1-7)/Mas axis of the renin-angiotensin system often opposes the detrimental effects of the angiotensin-converting enzyme/Ang II/Ang II type 1 receptor axis and has been associated with beneficial effects on glucose homeostasis, whereas underlying mechanisms are mostly unknown. Here we investigate the effects of Ang-(1-7) and its receptor Mas on ß-cell function. Isolated islets from Mas-deficient and wild-type mice were stimulated with Ang-(1-7) or its antagonists and effects on insulin secretion determined. Islets' cytoplasmic calcium and cAMP concentrations, mRNA amounts of Ins1, Ins2, Pdx1, and Mafa and effects of inhibitors of cAMP downstream signaling were determined. Ang-(1-7) was also applied to mice by osmotic pumps for 14 days and effects on glucose tolerance and insulin secretion were assessed. Ang-(1-7) increased insulin secretion from wild-type islets, whereas antagonists and genetic Mas deficiency led to reduced insulin secretion. The Mas-dependent effects of Ang-(1-7) on insulin secretion did not result from changes in insulin gene expression or changes in the excitation-secretion coupling but from increased intracellular cAMP involving exchange protein activated directly by cAMP. Administration of Ang-(1-7) in vivo had only marginal effects on glucose tolerance in wild-type mice but still resulted in improved insulin secretion from islets isolated of these mice. Interestingly, although less pronounced than in wild types, Ang-(1-7) still affected insulin secretion in Mas-deficient islets. The data indicate a significant function of Ang-(1-7) in the regulation of insulin secretion from mouse islets in vitro and in vivo, mainly, but not exclusively, by Mas-dependent signaling, modulating the accessory pathway of insulin secretion via increase in cAMP.

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer.

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) - a novel sphingolipid-like marine compound - was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

Cabazitaxel overcomes cisplatin resistance in germ cell tumour cells.

BACKGROUND: Cisplatin-based chemotherapy is highly effective in metastasized germ cell tumours (GCT). However, 10-30 % of patients develop resistance to cisplatin, requiring salvage therapy. We investigated the in vitro activity of paclitaxel and the novel taxane cabazitaxel in cisplatin-sensitive and -resistant GCT cell lines. METHODS: In vitro activity of paclitaxel and cabazitaxel was determined by proliferation assays, and mode of action of cabazitaxel was assessed by western blotting and two screening approaches, i.e. whole proteome analysis and a human apoptosis array. RESULTS: Activity of paclitaxel and cabazitaxel was not affected by cisplatin resistance, suggesting that there is no cross-resistance between these agents in vitro. Cabazitaxel treatment showed a strong inhibitory effect on colony formation capacity. Cabazitaxel induced classical apoptosis in all cell lines, reflected by cleavage of PARP and caspase 3, without inducing specific changes in the cell cycle distribution. Using the proteomic and human apoptosis array screening approaches, differential regulation of several proteins, including members of the bcl-2 family, was found, giving first insights into the mode of action of cabazitaxel in GCT. CONCLUSION: Cabazitaxel shows promising in vitro activity in GCT cells, independent of levels of cisplatin resistance.

Anti-migratory activity of marine alkaloid monanchocidin A - proteomics-based discovery and confirmation.

Monanchocidin A (MonA) is a novel marine alkaloid with promising anti-cancer properties. We recently demonstrated its high efficacy in human urogenital cancers including germ cell tumors. Here, we applied a global proteome screening approach to investigate molecular targets and biological processes affected by MonA in the human cisplatin-resistant germ cell cancer cell line NCCIT-R. Bioinformatical analysis of the proteomics data predicted an effect of MonA on cancer cell migration. Thus, proteins known to be involved in cancer cell migration and invasion were chosen for further validation. The protein alterations identified by proteomics resulted from both, regulation of the total protein expression and post-transcriptional modifications. Among others, regulation of an isoform of vimentin, up-regulation of multiple apolipoprotein E isoforms, and inhibition of hypusination of eukaryotic translation initiation factor 5A-1 were found upon treatment with MonA. Further functional analyses were performed and revealed decreased cell migration and colony formation of cancer cells treated with MonA at non-cytotoxic and non-antiproliferative concentrations. This work provides further insights into the molecular mechanisms behind MonA bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anti-cancer agents.

Isoform 1 of TPD52 (PC-1) promotes neuroendocrine transdifferentiation in prostate cancer cells.

The tumour protein D52 isoform 1 (PC-1), a member of the tumour protein D52 (TPD52) protein family, is androgen-regulated and prostate-specific expressed. Previous studies confirmed that PC-1 contributes to malignant progression in prostate cancer with an important role in castration-resistant stage. In the present work, we identified its impact in mechanisms leading to neuroendocrine (NE) transdifferentiation. We established for long-term PC-1 overexpression an inducible expression system derived from the prostate carcinoma cell line LNCaP. We observed that PC-1 overexpression itself initiates characteristics of neuroendocrine cells, but the effect was much more pronounced in the presence of the cytokine interleukin-6 (IL-6). Moreover, to our knowledge, this is the first report that treatment with IL-6 leads to a significant upregulation of PC-1 in LNCaP cells. Other TPD52 isoforms were not affected. Proceeding from this result, we conclude that PC-1 overexpression enhances the IL-6-mediated differentiation of LNCaP cells into a NE-like phenotype, noticeable by morphological changes and increased expression of typical NE markers, like chromogranin A, synaptophysin or beta-3 tubulin. Immunofluorescent staining of IL-6-treated PC-1-overexpressing LNCaP cells indicates a considerable PC-1 accumulation at the end of the long-branched neuron-like cell processes, which are typically formed by NE cells. Additionally, the experimentally initiated NE transdifferentiation correlates with the androgen receptor status, which was upregulated additively. In summary, our data provide evidence for an involvement of PC-1 in NE transdifferentiation, frequently associated with castration resistance, which is a major therapeutic challenge in the treatment of advanced prostate cancer.

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

BACKGROUND: Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. METHODS: Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. RESULTS: Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. CONCLUSIONS: The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth.

The marine triterpene glycoside frondoside A exhibits activity in vitro and in vivo in prostate cancer.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), outcome of patients remains poor due to the development of drug resistance. Thus, new drugs are urgently needed. We investigated efficacy, toxicity and mechanism of action of marine triterpene glycoside frondoside A (FrA) using CRPC cell lines in vitro and in vivo. FrA revealed high efficacy in human prostate cancer cells, while non-malignant cells were less sensitive. Remarkably, proliferation and colony formation of cells resistant to enzalutamide and abiraterone (due to the androgen receptor splice variant AR-V7) were also significantly inhibited by FrA. The marine compound caused cell type specific cell cycle arrest and induction of caspase-dependent or -independent apoptosis. Up-regulation or induction of several pro-apoptotic proteins (Bax, Bad, PTEN), cleavage of PARP and caspase-3 and down-regulation of anti-apoptotic proteins (survivin and Bcl-2) were detected in treated cells. Global proteome analysis revealed regulation of proteins involved in formation of metastases, tumor cell invasion, and apoptosis, like keratin 81, CrkII, IL-1ß and cathepsin B. Inhibition of pro-survival autophagy was observed following FrA exposure. In vivo, FrA inhibited tumor growth of PC-3 and DU145 cells with a notable reduction of lung metastasis, as well as circulating tumor cells in the peripheral blood. Increased lymphocyte counts of treated animals might indicate an immune modulating effect of FrA. In conclusion, our results suggest that FrA is a promising new drug for the treatment of mCRPC. Induction of apoptosis, inhibition of pro-survival autophagy, and immune modulatory effects are suspected modes of actions.