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.

Assimiloside A, a Glycolipid with Immunomodulatory Activity from the Northwestern Pacific Marine Sponge Hymeniacidon assimilis.

Assimiloside A (1), an unprecedented marine glycolipid containing a γ-lactone of 4R,16,26R-trihydroxy C28 fatty acid as an aglycon and a trisaccharide carbohydrate moiety, was isolated from the marine sponge Hymeniacidon assimilis. Its structure was elucidated by NMR spectroscopy, mass spectrometry, chemical transformations, and ECD spectroscopy combined with time-dependent density functional theory calculations. Assimiloside A at nontoxic concentrations of 0.01-0.1 µM was shown to present lysosomal activity stimulation and intracellular reactive oxygen species level elevation in RAW 264.7 murine macrophages.

New Guanidine Alkaloids Batzelladines O and P from the Marine Sponge Monanchora pulchra Induce Apoptosis and Autophagy in Prostate Cancer Cells.

Two new guanidine alkaloids, batzelladines O (1) and P (2), were isolated from the deep-water marine sponge Monanchora pulchra. The structures of these metabolites were determined by NMR spectroscopy, mass spectrometry, and ECD. The isolated compounds exhibited cytotoxic activity in human prostate cancer cells PC3, PC3-DR, and 22Rv1 at low micromolar concentrations and inhibited colony formation and survival of the cancer cells. Batzelladines O (1) and P (2) induced apoptosis, which was detected by Western blotting as caspase-3 and PARP cleavage. Additionally, induction of pro-survival autophagy indicated as upregulation of LC3B-II and suppression of mTOR was observed in the treated cells. In line with this, the combination with autophagy inhibitor 3-methyladenine synergistically increased the cytotoxic activity of batzelladines O (1) and P (2). Both compounds were equally active in docetaxel-sensitive and docetaxel-resistant prostate cancer cells, despite exhibiting a slight p-glycoprotein substrate-like activity. In combination with docetaxel, an additive effect was observed. In conclusion, the isolated new guanidine alkaloids are promising drug candidates for the treatment of taxane-resistant prostate cancer.

New diterpenes from the marine sponge Spongionella sp. overcome drug resistance in prostate cancer by inhibition of P-glycoprotein.

Spongian diterpenes are a group of marine natural compounds possessing various biological activities. However, their anticancer activity is still poorly studied and understood. We isolated six spongian diterpenes from the marine sponge Spongionella sp., including one new spongionellol A and five previously known molecules. The structures were elucidated using a detailed analysis MS and NMR spectra as well as by comparison with previously reported data. Two of them, namely, spongionellol A and 15,16-dideoxy-15α,17ß-dihydroxy-15,17-oxidospongian-16-carboxylate-15,17-diacetate exhibited high activity and selectivity in human prostate cancer cells, including cells resistant to hormonal therapy and docetaxel. The mechanism of action has been identified as caspase-dependent apoptosis. Remarkably, both compounds were able to suppress expression of androgen receptor (AR) and AR-splice variant 7, as well as AR-dependent signaling. The isolated diterpenes effectively inhibited drug efflux mediated by multidrug-resistance protein 1 (MDR1; p-glycoprotein). Of note, a synergistic effect of the compounds with docetaxel, a substrate of p-glycoprotein, suggests resensitization of p-glycoprotein overexpressing cells to standard chemotherapy. In conclusion, the isolated spongian diterpenes possess high activity and selectivity towards prostate cancer cells combined with the ability to inhibit one of the main drug-resistance mechanism. This makes them promising candidates for combinational anticancer therapy.

1-O-Alkylglycerol Ethers from the Marine Sponge Guitarra abbotti and Their Cytotoxic Activity.

The cytotoxicity-bioassay-guided fractionation of the ethanol extract from the marine sponge Guitarra abbotti, whose 1-O-alkyl-sn-glycerol ethers (AGEs) have not been investigated so far, led to the isolation of a complex lipid fraction containing, along with previously known compounds, six new lipids of the AGE type. The composition of the AGE fraction as well as the structures of 6 new and 22 previously known compounds were established using 1H and 13C NMR, GC/MS, and chemical conversion methods. The new AGEs were identified as: 1-O-(Z-docos-15-enyl)-sn-glycerol (1), 1-O-(Z-docos-17-enyl)-sn-glycerol (2), 1-O-(Z-tricos-15-enyl)-sn-glycerol (3), 1-O-(Z-tricos-16-enyl)-sn-glycerol (4), 1-O-(Z-tricos-17-enyl)-sn-glycerol (5), and 1-O-(Z-tetracos-15-enyl)-sn-glycerol (6). The isolated AGEs show weak cytotoxic activity in THP-1, HL-60, HeLa, DLD-1, SNU C4, SK-MEL-28, and MDA-MB-231 human cancer cells. A further cytotoxicity analysis in JB6 P+ Cl41 cells bearing mutated MAP kinase genes revealed that ERK2 and JNK1 play a cytoprotective role in the cellular response to the AGE-induced cytotoxic effects.

Streptocinnamides A and B, Depsipeptides from Streptomyces sp. KMM 9044.

The bacterium Streptomyces sp. KMM 9044 from a sample of marine sediment collected in the northwestern part of the Sea of Japan produces highly chlorinated depsiheptapeptides streptocinnamides A (1) and B (2), representatives of a new structural group of antibiotics. The structures of 1 and 2 were determined using nuclear magnetic resonance and mass spectrometry studies and confirmed by a series of chemical transformations. Streptocinnamide A potently inhibits Micrococcus sp. KMM 1467, Arthrobacter sp. ATCC 21022, and Mycobacterium smegmatis MC2 155.

Novel GSK-3ß Inhibitor Neopetroside A Protects Against Murine Myocardial Ischemia/Reperfusion Injury.

Recent trends suggest novel natural compounds as promising treatments for cardiovascular disease. The authors examined how neopetroside A, a natural pyridine nucleoside containing an α-glycoside bond, regulates mitochondrial metabolism and heart function and investigated its cardioprotective role against ischemia/reperfusion injury. Neopetroside A treatment maintained cardiac hemodynamic status and mitochondrial respiration capacity and significantly prevented cardiac fibrosis in murine models. These effects can be attributed to preserved cellular and mitochondrial function caused by the inhibition of glycogen synthase kinase-3 beta, which regulates the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced, through activation of the nuclear factor erythroid 2-related factor 2/NAD(P)H quinone oxidoreductase 1 axis in a phosphorylation-independent manner.

Gracilosulfates A-G, Monosulfated Polyoxygenated Steroids from the Marine Sponge Haliclona gracilis.

Seven new polyoxygenated steroids belonging to a new structural group of sponge steroids, gracilosulfates A-G (1-7), possessing 3ß-O-sulfonato, 5ß,6ß epoxy (or 5(6)-dehydro), and 4ß,23-dihydroxy substitution patterns as a common structural motif, were isolated from the marine sponge Haliclona gracilis. Their structures were determined by NMR and MS methods. The compounds 1, 2, 4, 6, and 7 inhibited the expression of prostate-specific antigen (PSA) in 22Rv1 tumor cells.

Marine alkaloid monanchoxymycalin C: a new specific activator of JNK1/2 kinase with anticancer properties.

Monanchoxymycalin C (MomC) is a new marine pentacyclic guanidine alkaloid, recently isolated from marine sponge Monanchora pulchra by us. Here, anticancer activity and mechanism of action was investigated for the first time using a human prostate cancer (PCa) model. MomC was active in all PCa cell lines at low micromolar concentrations and induced an unusual caspase-independent, non-apoptotic cell death. Kinase activity screening identified activation of mitogen-activated protein kinase (MAPK) c-Jun N-terminal protein kinase (JNK1/2) to be one of the primary molecular mechanism of MomC anticancer activity. Functional assays demonstrated a specific and selective JNK1/2 activation prior to the induction of other cell death related processes. Inhibition of JNK1/2 by pretreatment with the JNK-inhibitor SP600125 antagonized cytotoxic activity of the marine compound. MomC caused an upregulation of cytotoxic ROS. However, in contrast to other ROS-inducing agents, co-treatment with PARP-inhibitor olaparib revealed antagonistic effects indicating an active PARP to be necessary for MomC activity. Interestingly, although no direct regulation of p38 and ERK1/2 were detected, active p38 kinase was required for MomC efficacy, while the inhibition of ERK1/2 increased its cytotoxicity. In conclusion, MomC shows promising activity against PCa, which is exerted via JNK1/2 activation and non-apoptotic cell death.

Urupocidin C: a new marine guanidine alkaloid which selectively kills prostate cancer cells via mitochondria targeting.

New bicyclic guanidine alkaloid, urupocidin C (Ur-C) along with the previously known urupocidin A (Ur-A) were isolated from the rare deep-sea marine sponge Monanchora pulchra, harvested in Northwestern Pacific waters. The unique structure of Ur-C was elucidated using 1D and 2D NMR spectroscopy as well as mass spectra. We discovered a promising selectivity of both alkaloids for human prostate cancer (PCa) cells, including highly drug-resistant lines, compared to non-malignant cells. In cancer cells, marine derived compounds were able to induce G1- and S-cell cycle arrest as well as caspase-mediated cell death. For the first time we have identified mitochondrial targeting as a central mechanism of anticancer action for these and similar molecules. Thus, treatment with the isolated alkaloids resulted in mitochondrial membrane permeabilization consequently leading to the release of cytotoxic mitochondrial proteins to cellular cytoplasm, ROS upregulation, consequent activation of caspase-9 and -3, followed by PARP cleavage, DNA fragmentation, and apoptosis. Moreover, synergistic effects were observed when Ur-A and Ur-C were combined with clinically approved PARP inhibitor olaparib. Finally, these alkaloids exhibited additive effects in combination with docetaxel and androgen receptor inhibitor enzalutamide, both applied in PCa therapy. In conclusion, urupocidin-like compounds are promising lead molecules for the development of new drugs for the treatment of advanced PCa.

Synthesis and anticancer activity of the derivatives of marine compound rhizochalin in castration resistant prostate cancer.

Development of resistance to standard therapies complicates treatment of advanced prostate cancer. Alternative splicing variants of the androgen receptor (AR), e.g. AR-V7 can mediate resistance to AR-targeting substances abiraterone and enzalutamide. Semi-synthetic marine natural compound rhizochalinin decreases the expression of AR-V7 in human castration-resistant prostate cancer cells and thus resensitizes cells to enzalutamide. In the current study, we modified the structure of rhizochalin in order to determine structure-activity relationships (SAR) and optimize anticancer properties. Thus, we synthesized new 18-hydroxy- and 18-aminorhizochalins and its aglycones. All compounds exhibited anticancer properties in human castration-resistant prostate cancer cells, induced apoptosis and G2/M cell cycle arrest, and were capable of autophagy inhibition. SAR analysis showed an increase of pro-apoptotic activity in the row 18-amino < 18-hydroxy < 18-keto derivatives. In general, aglycones were more cytotoxic compared to glycosides. The sugar elimination was critical for the ability to suppress AR-signaling. Rhizochalinin (2) and 18-hydroxyrhizochalinin (4) were identified as the most promising derivatives and are promoted for further development.

Absolute Configuration of the Cytotoxic Marine Alkaloid Monanchocidin A.

The absolute configuration of the cytotoxic guanidine alkaloid monanchocidin A with 11 stereogenic centers from the marine sponge Monanchora pulchra was determined as 5 R, 8 S, 10 S, 13 R, 14 S, 15 R, 19 R, 23 R, 37 S, 42 S, 43 R after extensive reductive degradation and conversion of the resulting alcohols to MTPA derivatives.

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.

Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro.

Guanidine alkaloids from sponges Monanchora spp. represent diverse bioactive compounds, however, the mechanisms underlying bioactivity are very poorly understood. Here, we report results of studies on cytotoxic action, the ability to inhibit EGF-induced neoplastic transformation, and the effects on MAPK/AP-1 signaling of eight rare guanidine alkaloids, recently isolated from the marine sponge Monanchora pulchra, namely: monanchocidin A (1), monanchocidin B (2), monanchomycalin C (3), ptilomycalin A (4), monanchomycalin B (5), normonanchocidin D (6), urupocidin A (7), and pulchranin A (8). All of the compounds induced cell cycle arrest (apart from 8) and programmed death of cancer cells. Ptilomycalin A-like compounds 1-6 activated JNK1/2 and ERK1/2, following AP-1 activation and caused p53-independent programmed cell death. Compound 7 induced p53-independent cell death without activation of AP-1 or caspase-3/7, and the observed JNK1/2 activation did not contribute to the cytotoxic effect of the compound. Alkaloid 8 induced JNK1/2 (but not ERK1/2) activation leading to p53-independent cell death and strong suppression of AP-1 activity. Alkaloids 1-4, 7, and 8 were able to inhibit the EGF-induced neoplastic transformation of JB6 P⁺ Cl41 cells. Our results suggest that investigated guanidine marine alkaloids hold potential to eliminate human cancer cells and prevent cancer cell formation and spreading.

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.

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.

Monanchoxymycalins A and B, New Hybrid Pentacyclic Guanidine Alkaloids from the Far-Eastern Marine Sponge Monanchora pulchra.

The new pentacyclic guanidine alkaloids, monanchoxymycalin A (1) and monanchoxymycalin B (2) were isolated from the Far-Eastern marine sponge Monanchora pulchra. Their structures were assigned on the basis of detailed analysis of lD- and 2D-NMR spectroscopic and mass spectrometric data. Compounds 1 and 2 exhibited potent cytotoxic activities against cervical epithelioid carcinoma HeLa cells and breast adenocarcinoma MDA-MB231 cells.

Absolute Configuration and Body Part Distribution of the Alkaloid 6-epi-Monanchorin from the Marine Polychaete Chaetopterus variopedatus.

As a result of the first study on secondary metabolites from the cosmopolitan bioluminescent marine tube polychaete Chaetopterus variopedatus, a new bicyclic guanidine alkaloid, 6-epi-monanchorin (1), along with the previously known monanchorin (2) were isolated. The structure of 1 was elucidated by spectroscopic and chemical methods, including a cleavage of the C1-07 bond to obtain a secondary alcohol (3), which was used to determine the absolute configurations by Mosher's method. It was found that 1 and 2 were mainly accumulated in a secreted mucus special organ of the worm (food net), where green and blue-green microalgae were detected. A biosynthetic pathway to 6-epi-monanchorin and monanchorin from dietary polyeic fatty acid precursors was proposed.

Marine alkaloid Monanchocidin a overcomes drug resistance by induction of autophagy and lysosomal membrane permeabilization.

Monanchocidin A (MonA) is a novel alkaloid recently isolated from the marine sponge Monanchora pulchra. The compound reveals cytotoxic activity in genitourinary cancers including cisplatin-sensitive and -resistant germ cell tumor (GCT) cell lines, hormone-sensitive and castration-resistant prostate carcinoma cell lines and different bladder carcinoma cell lines. In contrast, non-malignant cells were significantly less sensitive. MonA is highly synergistic with cisplatin in GCT cells. Induction of autophagy at lower and lysosomal membrane permeabilization (LMP) at higher concentrations were identified as the dominating modes of action. Cytotoxicity and protein degradation could be inhibited by 3-methyladenine, an inhibitor of autophagy. LMP was confirmed by loss of acridine orange staining of lysosoms and by release of cathepsin B. In conclusion, MonA exerts cytotoxic activity by mechanisms different from "classical" apoptosis, and could be a promising new compound to overcome resistance to standard therapies in genitourinary malignancies.

Pyridine Nucleosides Neopetrosides A and B from a Marine Neopetrosia sp. Sponge. Synthesis of Neopetroside A and Its ß-Riboside Analogue.

Neopetrosides A (1) and B (2), new naturally occurring ribosides of nicotinic acid with extremely rare α-N-glycoside linkages and residues of p-hydroxybenzoic and pyrrole-2-carboxylic acids attached to C-5', were isolated from a marine Neopetrosia sp. sponge. Structures 1 and 2 were determined by NMR and MS methods and confirmed by the synthesis of 1 and its ß-riboside analogue (3). Neopetroside A (1) upregulates mitochondrial functions in cardiomyocytes.