benzofurans and rocaglamide

Covering: 2006 to 2013. Investigations on the chemistry and biology of rocaglamide, silvestrol and structurally related bioactive compounds from Aglaia species during the period 2006-2013 are reviewed. Included are new phytochemical studies of naturally occurring rocaglamide derivatives, an update on synthetic methods for cyclopenta[b]benzofurans, and a description of the recent biological evaluation and mechanism-of-action studies on compounds of this type.

Topics: Aglaia; Animals; Benzofurans; Humans; Mice; Molecular Structure; Structure-Activity Relationship; Triterpenes

Topics: Aglaia; Animals; Benzofurans; Humans; Meliaceae

During the past few years, a group of cyclopenta[b]benzofurans from the plant genus Aglaia has received broad scientific attention as interesting natural product lead compounds with potential anticancer and insecticidal activities. Since the first cyclopenta[b]benzofuran derivative, rocaglamide, from Aglaia elliptifolia, was found to exhibit antileukemic activity in a murine in vivo model, the genus Aglaia has been subjected to further investigation. Over 40 cyclopenta[b]benzofurans have been tested against different human cancer cell lines, and the cumulative results provide some important clues as to how to improve their activity through modification of their chemical structures. The semisynthesis and total synthesis of the cyclopenta[b]benzofurans have been conducted. Although the ultimate molecular target(s) responsible for their antiproliferative activity has not yet been identified, studies on their cellular mechanism of action have demonstrated that some of these compounds inhibit TNF-alpha or PMA-induced NF-kappaB activity in T-lymphocytes and induce apoptosis in different human cancer cell lines. Based on the published data thus far, cyclopenta[b]benzofurans offer excellent potential as therapeutic agent candidates in cancer chemotherapy, even if much work still remains to be done for their further development.

Topics: Aglaia; Animals; Antineoplastic Agents; Benzofurans; Cell Line, Tumor; Drug Screening Assays, Antitumor; Humans; Mice; Xenograft Model Antitumor Assays

Aglaia (Meliaceae) species are used for treating autoimmune disorders and allergic diseases in Asian countries. Rocaglamide, an extract obtained from Aglaia species, exhibits suppressive effect by regulating the T cell subset balance and cytokine network in cancer. However, whether it can be used in organ transplantation is unknown. In this study, we investigated the antirejection effect and mechanism of action of rocaglamide in a mouse cardiac allograft model.. Survival studies were performed by administering mice with phosphate-buffered saline (PBS) (. Rocaglamide administration significantly prolonged the median survival of the grafts from 7 to 25 days compared with PBS treatment (. We identified a new immunoregulatory property of rocaglamide, wherein it was found to regulate oxidative stress response and reduce inflammatory cell infiltration and organ injury, which have been associated with the inhibition of NF-AT activation in T cells.

Topics: Animals; Benzofurans; Cell Differentiation; Graft Survival; Heart Transplantation; Humans; Mice; Th1 Cells; Th17 Cells

Inhibition of adipogenesis is an important strategy for obesity treatment. Rocaglamide-A (Roc-A) is a natural herbal medicine isolated from the genus Aglaia (family Meliaceae), which has a cyclopenta[b]benzofuran core structure. Roc-A exhibits various pharmacological effects against diverse human cancer cells. However, the exact role of Roc-A during adipogenesis in adipocytes has not been studied at all. In this study, we demonstrate that Roc-A is crucial for reducing adipogenesis via downregulating PPARγ transcriptional activity. Consistently, Western-blot and RT-PCR analyses clearly showed that Roc-A inhibits the expression of PPARγ target genes and lipogenic markers in a dose-dependent manner along with suppression of lipid accumulation, in both 3T3-L1 cells and mouse adipose-derived stem cells. Mechanistically, Roc-A significantly decreased STAT3 phosphorylation in a dose-dependent manner in 3T3-L1 adipocytes. In particular, we confirmed that Roc-A effectively suppressed the expression of genes involved in cell-cycle regulation, such as cyclin A, B, D1, and E1, early during mitotic clonal expansion in 3T3-L1 adipocytes, and this effect was abolished by the JAK2/STAT3 activator FGF2. Taken together, our results demonstrated that Roc-A reduces adipogenesis by inhibiting PPARγ transactivation and STAT3 phosphorylation and thus may serve as a therapeutic target in obesity.

Topics: Adipogenesis; Animals; Benzofurans; Mice; Obesity; PPAR gamma

Hepatitis E Virus (HEV) follows waterborne or zoonotic/foodborne transmission. Genotype 3 HEV infections are worldwide spread, especially in swine populations, representing an emerging threat for human health, both for farm workers and pork meat consumers. Unfortunately, HEV in vitro culture and analysis are still difficult, resulting in a poor understanding of its biology and hampering the implementation of counteracting strategies. Indeed, HEV encodes for only one non-structural multifunctional and multidomain protein (ORF1), which might be a good candidate for anti-HEV drugging strategies. In this context, an in silico molecular modelling approach that consisted in homology modelling to derive the 3D model target, docking study to simulate the binding event, and molecular dynamics to check complex stability over time was used. This workflow succeeded to describe ORF1 RNA Helicase domain from a molecular standpoint allowing the identification of potential inhibitory compounds among natural plant-based flavagline-related molecules such as silvestrol, rocaglamide and derivatives thereof. In the context of scouting potential anti-viral compounds and relying on the outcomes presented, further dedicated investigations on silvestrol, rocaglamide and a promising oxidized derivative have been suggested. For the sake of data reproducibility, the 3D model of HEV RNA Helicase has been made publicly available.

Topics: Animals; Benzofurans; DNA Helicases; Hepatitis E virus; Humans; Reproducibility of Results; RNA Helicases; Swine

Tactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple "translational inhibition" as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical "translation inhibitors" is mediated by comprehensive translational landscape remodeling.

Topics: Animals; Benzofurans; Cell Line, Tumor; Eukaryotic Initiation Factor-4A; Humans; Male; Mice; Mice, Inbred NOD; Primary Cell Culture; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteomics; Ribosomes; Transcriptome; Triterpenes

Covering: up to the beginning of 2020Many natural substances have been transformed again and again with regard to their pharmaceutical-medical potential, including new members of a growing class of natural products, the flavaglines. Important representatives are rocaglamide and silvestrol, isolated from the Aglaia species, which are highlighted here. These products started as potential anti-tumor agents five decades ago and have recently proved to be very promising antiviral agents, especially against RNA viruses. Today they are discussed as potential starting compounds for developing drug candidates and therapeutics.

Topics: Antineoplastic Agents; Antiviral Agents; Benzofurans; Biological Products; Coronavirus; Humans; Molecular Structure; Triterpenes

The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity.

Topics: Benzofurans; Cells, Cultured; DEAD-box RNA Helicases; Enzyme Inhibitors; Eukaryotic Initiation Factor-4A; Female; Humans; Male; Models, Molecular; Molecular Conformation

In this issue of Cell Chemical Biology, Chen et al. (2020) expand the target repertoire of rocaglamide A (RocA) to now include eIF4A2 and DDX3X, converting DEAD-box helicases into dominant-negative translation repressors. These results also highlight how cancer cell sensitivity to RocA is dependent on eIF4A and DDX3X levels.

Topics: Benzofurans; DEAD-box RNA Helicases; DNA Helicases

Flavaglines such as silvestrol (1) and rocaglamide (2) constitute an interesting class of natural products with promising anticancer activities. Their mode of action is based on inhibition of eukaryotic initiation factor 4A (eIF4A) dependent translation through formation of a stable ternary complex with eIF4A and mRNA, thus blocking ribosome scanning. Herein we describe initial SAR studies in a novel series of 1-aminomethyl substituted flavagline-inspired eIF4A inhibitors. We discovered that a variety of N-substitutions at the 1-aminomethyl group are tolerated, making this position pertinent for property and ADME profile tuning. The findings presented herein are relevant to future drug design efforts towards novel eIF4A inhibitors with drug-like properties.

Topics: Antineoplastic Agents; Benzofurans; Biological Products; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Eukaryotic Initiation Factor-4A; Humans; Molecular Structure; Structure-Activity Relationship; Triterpenes

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Topics: Antibodies, Anti-Idiotypic; Benzofurans; Cells, Cultured; Eukaryotic Initiation Factor-4A; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Myeloid Cell Leukemia Sequence 1 Protein; Protein Biosynthesis; Proto-Oncogene Proteins c-myc; Receptors, Antigen, B-Cell; RNA Stability; RNA, Messenger; Signal Transduction; Triterpenes

Topics: Aglaia; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Benzofurans; Caspase 3; Cell Cycle; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Mice; Neurofibrosarcoma; Protein Processing, Post-Translational; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

There is still no satisfying method to treat colorectal cancer (CRC) currently. Inspired by cocktail therapy, the combination of 465 nm blue LED irradiation and two multi-target anticancer agents AT406 and Rocaglamide has been investigated as an innovative way to treat colorectal cancer cells in vitro. It showed a strong inhibitory effect on colorectal cancer cells, and its side effects on human normal cells are negligible. When applied to HCT116 cells, it can achieve an apoptotic rate up to 95%. It is also seen to significantly inhibit proliferation of HT29 cells. Furthermore, little to no cell inhibition or damage of normal MRC-5 cells were seen after treatment. The combination of blue LED irradiation and two anti-cancer drugs causes apoptosis of colorectal cancer cells by activating the apoptotic pathway, inhibiting autophagy and proliferation pathways as well as the production of reactive oxygen species (ROS).

Topics: Apoptosis; Azocines; Benzhydryl Compounds; Benzofurans; Cell Proliferation; Combined Modality Therapy; Dose-Response Relationship, Drug; HCT116 Cells; Humans; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

The defining feature of the Nazarov cyclization is a 4π-conrotatory electrocyclization, resulting in the stereospecific formation of functionalized cyclopentanones. The reaction provides access to structural motifs that are found in many natural products and drug targets. Harnessing the full potential of the Nazarov cyclization broadens its utility by enabling the development of new methodologies and synthetic strategies. To achieve these goals through efficient cyclization design, it is helpful to think of the reaction as a two-stage process. The first stage involves a 4π-electrocyclization leading to the formation of an allylic cation, and the second stage corresponds to the fate of this cationic intermediate. With a complete understanding of the discrete events that characterize the overall process, one can optimize reactivity and control the selectivity of the different Stage 2 pathways.In this Account, we describe the development of methods that render the Nazarov cyclization catalytic and chemoselective, focusing specifically on advances made in our lab between 2002 and 2015. The initial discovery made in our lab involved reactions of electronically asymmetric ("polarized") substrates, which cyclize efficiently in the catalytic regime using mild Lewis acidic reagents. These cyclizations also exhibit selective eliminative behavior, increasing their synthetic utility. Research directed toward catalytic asymmetric Nazarov cyclization led to the serendipitous discovery of a 4π-cyclization coupled to a well-behaved Wagner-Meerwein rearrangement, representing an underexplored Stage 2 process. With careful choice of promoter and loading, it is possible to access either the rearrangement or the elimination pathway. Additional experimental and computational studies provided an effective model for anticipating the migratory behavior of substiutents in the rearrangements. Problem-solving efforts prompted investigation of alternative methods for generating pentadienyl cation intermediates, including oxidation of allenol ethers and addition of nucleophiles to dienyl diketones. These Nazarov cyclization variants afford cyclopentenone products with vicinal stereogenic centers and a different arrangement of substituents around the ring. A nucleophilic addition/cyclization/elimination sequence can be executed enantioselectively using catalytic amounts of a nonracemic chiral tertiary amine.In summary, the discovery and development of several new variations on the Naz

Topics: Benzofurans; Biological Products; Catalysis; Copper; Cyclization; Lewis Acids; Quantum Theory; Sesquiterpenes; Stereoisomerism; Thermodynamics

Rocaglates, rocaglamides, and related flavagline natural products exert their remarkable anticancer activity through inhibition of eukaryotic initiation factor 4A (eIF4A) but generally display suboptimal drug-like properties. In our efforts to identify potent drug-like eIF4A inhibitors, we developed synthetic strategies for diastereoselectively functionalizing the C1 position of aza-rocaglamide scaffolds (cf.

Topics: Benzofurans; Binding Sites; Biological Products; Eukaryotic Initiation Factor-4A; Humans; Molecular Structure

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

Topics: Adenylyl Imidodiphosphate; Aglaia; Amino Acid Substitution; Benzofurans; Binding Sites; Drug Resistance; Eukaryotic Initiation Factor-4A; HEK293 Cells; Humans; Models, Molecular; Molecular Structure; Mutation; Plant Proteins; Protein Binding; Protein Biosynthesis; Protein Interaction Domains and Motifs; Protein Synthesis Inhibitors; Ribosomes; RNA; Structure-Activity Relationship

A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.

Topics: Animals; Antiviral Agents; Benzofurans; Cell Line; Cell Membrane; Enterovirus A, Human; Enterovirus Infections; Host-Pathogen Interactions; Humans; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Mitochondrial Membranes; Nerve Tissue Proteins; Neurons; Prohibitins; Proteomics; Repressor Proteins; RNA Interference; Specific Pathogen-Free Organisms; Survival Analysis; Virus Internalization; Virus Replication

Cancer metastasis causes approximately 90% of all cancer-related death and independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis implies cancer cell migration and invasion throughout the body. Migration requires the formation of pseudopodia in the direction of movement, but a detailed understanding of this process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human cancer pseudopodia to examine this mechanisms essential to metastasis formation, and identify potential candidates for pharmacological interference with the process. We demonstrate that Prohibitins (PHBs) are significantly enriched in the pseudopodia fraction derived from cancer cells, and knockdown of PHBs, as well as their chemical inhibition through Rocaglamide (Roc-A), efficiently reduces cancer cell migration.

Topics: Antineoplastic Agents; Benzofurans; Cell Line, Tumor; Cell Movement; Gene Knockdown Techniques; Humans; Neoplasm Metastasis; Prohibitins; Proteomics; Pseudopodia; Repressor Proteins

Targeting macroautophagy/autophagy is a novel strategy in cancer immunotherapy. In the present study, we showed that the natural product rocaglamide (RocA) enhanced natural killer (NK) cell-mediated lysis of non-small cell lung cancer (NSCLC) cells in vitro and tumor regression in vivo. Moreover, this effect was not related to the NK cell recognition of target cells or expressions of death receptors. Instead, RocA inhibited autophagy and restored the level of NK cell-derived GZMB (granzyme B) in NSCLC cells, therefore increasing their susceptibility to NK cell-mediated killing. In addition, we further identified that the target of RocA was ULK1 (unc-51 like autophagy activating kinase 1) that is required for autophagy initiation. Using firefly luciferase containing the 5´ untranslated region of ULK1, we found that RocA inhibited the protein translation of ULK1 in a sequence-specific manner. Taken together, RocA could block autophagic immune resistance to NK cell-mediated killing, and our data suggested that RocA was a promising therapeutic candidate in NK cell-based cancer immunotherapy.

Topics: Animals; Autophagy; Autophagy-Related Protein-1 Homolog; Benzofurans; Carcinoma, Non-Small-Cell Lung; Cell Degranulation; Granzymes; Humans; Killer Cells, Natural; Lung Neoplasms; Male; Mice, Inbred C57BL; Mice, SCID; Models, Biological; Protein Biosynthesis; Receptors, Death Domain

Rocaglamide has been reported to sensitize several cell types to TRAIL-induced apoptosis. In recent years, advances in synthetic techniques have led to generation of novel rocaglamide analogs. However, these have not been extensively analyzed as TRAIL sensitizers, particularly in TRAIL-resistant renal cell carcinoma cells. Evaluation of rocaglamide and analogs identified 29 compounds that are able to sensitize TRAIL-resistant ACHN cells to TRAIL-induced, caspase-dependent apoptosis with sub-µM potency which correlated with their potency as protein synthesis inhibitors and with loss of cFLIP protein in the same cells. Rocaglamide alone induced cell cycle arrest, but not apoptosis. Rocaglates averaged 4-5-fold higher potency as TRAIL sensitizers than as protein synthesis inhibitors suggesting a potential window for maximizing TRAIL sensitization while minimizing effects of general protein synthesis inhibition. A wide range of other rocaglate effects (e.g. on JNK or RAF-MEK-ERK signaling, death receptor levels, ROS, ER stress, eIF4E phosphorylation) were assessed, but did not contribute to TRAIL sensitization. Other than a rapid loss of MCL-1, rocaglates had minimal effects on mitochondrial apoptotic pathway proteins. The identification of structurally diverse/mechanistically similar TRAIL sensitizing rocaglates provides insights into both rocaglate structure and function and potential further development for use in RCC-directed combination therapy.

Topics: Apoptosis; Benzofurans; Carcinoma, Renal Cell; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Kidney Neoplasms; RNA, Messenger; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

KRAS is one of the most frequently mutated oncogenes in human non-small cell lung cancers (NSCLCs). RAS proteins trigger multiple effector signalling pathways including the highly conserved RAF-MAPK pathway. CRAF, a direct RAS effector protein, is required for KRAS-mediated tumourigenesis. Thus, the molecular mechanisms driving the activation of CRAF are intensively studied. Prohibitin 1 (PHB1) is an evolutionarily conserved adaptor protein and interaction of CRAF with PHB1 at the plasma membrane is essential for CRAF activation. Here, we demonstrate that PHB1 is highly expressed in NSCLC patients and correlates with poor survival. Targeting of PHB1 with two chemical ligands (rocaglamide and fluorizoline) inhibits epidermal growth factor (EGF)/RAS-induced CRAF activation. Consistently, treatment with rocaglamide inhibited proliferation, migration and anchorage-independent growth of KRAS-mutated lung carcinoma cell lines. Surprisingly, rocaglamide treatment inhibited Ras-GTP loading in KRAS-mutated cells as well as in EGF-stimulated cells. Rocaglamide treatment further prevented the oncogenic growth of KRAS-driven lung cancer allografts and xenografts in mouse models. Our results suggest rocaglamide as a RAS inhibitor and that targeting plasma membrane-associated PHB1 with chemical ligands would be a viable therapeutic strategy to combat KRAS-mediated NSCLCs.

Topics: Animals; Benzofurans; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; EGF Family of Proteins; Gene Expression Regulation, Neoplastic; Humans; Ligands; Lung Neoplasms; Mice; Mice, Knockout; Molecular Targeted Therapy; Prohibitins; Proto-Oncogene Proteins p21(ras); raf Kinases; ras Proteins; Repressor Proteins; Signal Transduction; TNF Receptor-Associated Factor 3; Xenograft Model Antitumor Assays

The natural product Rocaglamide (1), isolated from the tree Aglaia elliptifolia, is a compelling but also challenging lead structure for crop protection. In laboratory assays, the natural product shows highly interesting insecticidal activity against chewing pests and beetles, but also phytotoxicity on some crop plants. Multi-step syntheses with control of stereochemistry were required to probe the structure-activity relationship (SAR), and seek simplified analogues. After a significant research effort, just two areas of the molecule were identified which allow modification whilst maintaining activity, as will be highlighted in this paper.

Topics: Benzofurans; Insecticides; Molecular Structure; Structure-Activity Relationship

Activating BRAF mutations promote constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway and are common in a variety of human malignancies, including melanoma and colon cancer. Several small molecule BRAF inhibitors such as vemurafenib have been developed and demonstrate remarkable clinical efficacy. However, resistance typically emerges in most melanoma patients. Studies have demonstrated that reactivation of MAPK signaling via CRAF overexpression and dysregulation is a mechanism for vemurafenib resistance in melanoma. Prohibitins (PHBs) are highly conserved proteins that are thought to control the cell cycle, senescence and tumor suppression. PHB1 is essential for CRAF-mediated ERK1/2 activation through direct binding to CRAF. We developed a CRAF-mediated model of vemurafenib resistance in melanoma cells to assess the importance of the interaction between CRAF and PHB1 in resistance to BRAF-targeting agents. We demonstrate that CRAF overexpression renders melanoma cells resistant to BRAF-targeting agents. Moreover, treatment with the natural compound rocaglamide A disrupts the interaction between PHB and CRAF in melanoma cells, thus reducing MEK1/2 and ERK1/2 signaling, inhibiting melanoma cell growth and inducing apoptosis. The efficacy of these compounds was also demonstrated in a human melanoma xenograft model. Taken together, these data suggest that PHB1 may serve as a novel, druggable target in CRAF-mediated vemurafenib resistance.

Topics: Animals; Benzofurans; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Indoles; MAP Kinase Signaling System; Melanoma; Mice; Prohibitins; Protein Binding; Proto-Oncogene Proteins c-raf; Repressor Proteins; Sulfonamides; Vemurafenib; Xenograft Model Antitumor Assays

Multiple myeloma (MM) is an incurable malignancy by the presently known therapies. TRAIL is a promising anticancer agent that virtually not shows any toxicity to normal cells. We have recently carried out clinical trials with a human circularly permuted TRAIL, CPT, against MM saw a partial response in approximate 20-30% of patients. In the current study, we investigated the cause of CPT resistance and revealed that the majority of the MM patients express elevated levels of c-FLIP. Knockdown of c-FLIP expression by siRNA alone was sufficient to increase CPT-mediated apoptosis in a CPT-resistant human MM cell line U266. To overcome CPT resistance, we investigated the combination of CPT with Rocaglamides(s) in MM which has been shown to inhibit c-FLIP expression in vitro. We show that Rocaglamide(s) overcomes CPT resistance in U266 in vitro and significant increases in anti-tumor efficacies of CPT in mice xenografted with U266. Similar results were also obtained in mice xenografted with the CPT-resistant human acute T-cell leukemia cell line Molt-4. Our study suggests that the combination of Rocaglamide(s) with CPT may provide a more efficient treatment against myeloma and leukemia.

Topics: Animals; Benzofurans; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Leukemia-Lymphoma, Adult T-Cell; Mice; Multiple Myeloma; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Xenograft Model Antitumor Assays

Rocaglamides are bioactive natural compounds which have a cyclopenta[b]benzofuran core structure. The total synthesis of a reported natural product, 3'-hydroxymethylrocaglate (5), was achieved using [3 + 2] cycloaddition between 3-hydroxyflavone and methyl cinnamate. We also describe the synthesis of rocaglamide heterocycle derivatives and evaluate their Wnt signal inhibitory activities. Compounds 4, 5, 22a, 22b, 22c and 23c showed potent Wnt signal inhibitory activity.

Topics: Benzofurans; Biological Products; Cinnamates; Crystallography, X-Ray; Cycloaddition Reaction; Flavonoids; HEK293 Cells; Humans; Models, Molecular; Wnt Proteins; Wnt Signaling Pathway

The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Azocines; Benzhydryl Compounds; Benzofurans; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; X-Linked Inhibitor of Apoptosis Protein

Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions.

Topics: 5' Untranslated Regions; Adenosine Triphosphate; Benzofurans; DEAD-box RNA Helicases; Eukaryotic Initiation Factor-4A; HEK293 Cells; Humans; Hydrolysis; Nucleic Acid Conformation; Nucleotide Motifs; Open Reading Frames; Peptide Chain Initiation, Translational; Protein Binding; Protein Biosynthesis; Protein Stability; Repressor Proteins; RNA-Binding Proteins; RNA, Messenger; Substrate Specificity; Thermodynamics

Chemotherapy is one of the pillars of anti-cancer therapy. Although chemotherapeutics cause regression of the primary tumor, many chemotherapeutics are often shown to induce or accelerate metastasis formation. Moreover, metastatic tumors are largely resistant against chemotherapy. As more than 90% of cancer patients die due to metastases and not due to primary tumor formation, novel drugs are needed to overcome these shortcomings. In this study, we identified the anticancer phytochemical Rocaglamide (Roc-A) to be an inhibitor of cancer cell migration, a crucial event in metastasis formation. We show that Roc-A inhibits cellular migration and invasion independently of its anti-proliferative and cytotoxic effects in different types of human cancer cells. Mechanistically, Roc-A treatment induces F-actin-based morphological changes in membrane protrusions. Further investigation of the molecular mechanisms revealed that Roc-A inhibits the activities of the small GTPases RhoA, Rac1 and Cdc42, the master regulators of cellular migration. Taken together, our results provide evidence that Roc-A may be a lead candidate for a new class of anticancer drugs that inhibit metastasis formation.

Topics: Antineoplastic Agents; Benzofurans; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Humans; rho GTP-Binding Proteins

The complex flavagline, (-)-rocaglamide, possesses a synthetically intriguing tricyclic scaffold with five contiguous stereocenters and also exhibits potent anticancer, anti-inflammatory and insecticidal activity. This full account details distinct approaches to (±)- and (-)-rocaglamide utilizing Brønsted acid catalyzed and asymmetric Pd

Topics: Acids; Anti-Inflammatory Agents; Benzofurans; Catalysis; Cyclization; Palladium

The enhancement of apoptosis is a therapeutic strategy used in the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, hepatocellular carcinoma (HCC) cells exhibit marked resistance to the induction of cell death by TRAIL. The present study investigated whether rocaglamide, a naturally occurring product isolated from the genus Aglaia, is able to sensitize resistant HCC cells to TRAIL-mediated apoptosis. Two HCC cell lines, HepG2 and Huh-7, were treated with rocaglamide and/or TRAIL and the induction of apoptosis and effects on the TRAIL signaling pathway were investigated. The in vivo efficacy of rocaglamide was determined in TRAIL-resistant Huh-7-derived tumor xenografts. Rocaglamide significantly sensitized the TRAIL-resistant HCC cells to apoptosis by TRAIL, which resulted from the rocaglamide-mediated downregulation of cellular FLICE-like inhibitory protein and subsequent caspase-8 activation. Furthermore, rocaglamide markedly inhibited tumor growth from Huh-7 cells propagated in severe combined immunodeficient mice, suggesting that chemosentization also occurred in vivo. These data suggest that rocaglamide acted synergistically with TRAIL against the TRAIL-resistant HCC cells. Thus, it is concluded that rocaglamide as an adjuvant to TRAIL-based therapy may present a promising therapeutic approach for the treatment of HCC.

Topics: Animals; Apoptosis; Benzofurans; Carcinoma, Hepatocellular; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Line, Tumor; Disease Models, Animal; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Knockdown Techniques; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, SCID; TNF-Related Apoptosis-Inducing Ligand

We have previously reported asymmetric syntheses and absolute configuration assignments of the aglains (+)-ponapensin and (+)-elliptifoline and proposed a biosynthetic kinetic resolution process to produce enantiomeric rocaglamides and aglains. Herein, we report a biomimetic approach for the synthesis of enantiomerically enriched aglains and rocaglamides via kinetic resolution of a bridged ketone utilizing enantioselective transfer hydrogenation. The methodology has been employed to synthesize and confirm the absolute stereochemistries of the pyrimidone rocaglamides (+)-aglaiastatin and (-)-aglaroxin C. Additionally, the enantiomers and racemate of each metabolite were assayed for inhibition of the heat-shock response, cytotoxicity, and translation inhibition.

Topics: Benzofurans; Biological Products; Biomimetic Materials; Crystallography, X-Ray; Heterocyclic Compounds, 3-Ring; Hydrogenation; Ketones; Kinetics; Models, Molecular; Molecular Structure; Pyrroles; Quantum Theory; Stereoisomerism

A recently reported Pd(0)-catalyzed asymmetric Nazarov-type cyclization has been successfully applied in the key step of the first catalytic asymmetric total synthesis of (-)-rocaglamide (natural) and (+)-rocaglamide. The stereochemistry at the C3 position that controls the stereochemistry of all other stereocenters is determined in the cyclization step. This versatile and modular synthesis proceeds from simple reagents.

Topics: Benzofurans; Catalysis; Cyclization; Molecular Structure; Palladium; Stereoisomerism

Rheumatoid arthritis is a chronic inflammatory disease that leads to bone and cartilage erosion. The inhibition of osteoblast differentiation by the inflammatory factor TNF-α is critical for the pathogenesis of rheumatoid arthritis. To modulate TNF-α mediated inhibition of osteoblast differentiation is required to improve therapeutic efficacy of rheumatoid arthritis. Here, we explored the potential role of rocaglamide-A, a component of Aglaia plant, in osteoblast differentiation. Rocaglamide-A prevented TNF-α mediated inhibition of osteoblast differentiation, and promoted osteoblast differentiation directly, in both C2C12 and primary mesenchymal stromal cells. Mechanistically, Rocaglamide-A inhibited the phosphorylation of NF-κB component p65 protein and the accumulation of p65 in nucleus, which resulted in the diminished NF-κB responsible transcriptional activity. Oppositely, overexpression of p65 reversed rocaglamide-A's protective effects on osteoblast differentiation. Collectively, rocaglamide-A protected and stimulated osteoblast differentiation via blocking NF-κB pathway. It suggests that rocaglamide-A may be a good candidate to develop as therapeutic drug for rheumatoid arthritis associated bone loss diseases.

Topics: Aglaia; Animals; Arthritis, Rheumatoid; Benzofurans; Cell Differentiation; Cell Line; Mesenchymal Stem Cells; Mice; Osteoblasts; Plants, Medicinal; Primary Cell Culture; Signal Transduction; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Identification of novel drug targets and affordable therapeutic agents remains a high priority in the fight against chronic hepatitis C virus (HCV) infection. Here, we report that the cellular proteins prohibitin 1 (PHB1) and 2 (PHB2) are pan-genotypic HCV entry factors functioning at a post-binding step. While predominantly found in mitochondria, PHBs localize to the plasma membrane of hepatocytes through their transmembrane domains and interact with both EGFR and CRaf. Targeting PHB by rocaglamide (Roc-A), a natural product that binds PHB1 and 2, reduced cell surface PHB1 and 2, disrupted PHB-CRaf interaction, and inhibited HCV entry at low nanomolar concentrations. A structure-activity analysis of 32 synthetic Roc-A analogs indicated that the chiral, racemic version of aglaroxin C, a natural product biosynthetically related to Roc-A, displayed improved potency and therapeutic index against HCV infection. This study reveals a new class of HCV entry inhibitors that target the PHB1/2-CRaf pathway.

Topics: Antiviral Agents; Benzofurans; Cell Line; Drug Discovery; Hepacivirus; Hepatitis C; Humans; Prohibitins; Protein Binding; Proto-Oncogene Proteins c-raf; Repressor Proteins; Signal Transduction; Viral Envelope Proteins; Virus Internalization

Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over-expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide-A (Roc-A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1-S phase. Roc-A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc-A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway. However, Roc-A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc-A by a time-resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc-A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc-A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.

Topics: Antineoplastic Agents; Ataxia Telangiectasia Mutated Proteins; Benzofurans; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Checkpoint Kinase 2; DNA Damage; HCT116 Cells; Hep G2 Cells; HT29 Cells; Humans; Jurkat Cells; Leukemia; MCF-7 Cells; Phosphorylation; Plant Extracts; Protein Biosynthesis; Protein Kinases; RNA Interference; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; T-Lymphocytes

One of the main obstacles of conventional anticancer therapy is the toxicity of chemotherapeutics to normal tissues. So far, clinical approaches that aim to specifically reduce chemotherapy-mediated toxicities are rare. Recently, a number of studies have demonstrated that herbal extracts derived from traditional Chinese medicine (TCM) may reduce chemotherapy-induced side effects. Thus, we screened a panel of published cancer-inhibiting TCM compounds for their chemoprotective potential and identified the phytochemical Rocaglamide (Roc-A) as a candidate. We show that Roc-A significantly reduces apoptotic cell death induced by DNA-damaging anticancer drugs in primary human and murine cells. Investigation of the molecular mechanism of Roc-A-mediated protection revealed that Roc-A specifically blocks DNA damage-induced upregulation of the transcription factor p53 by inhibiting its protein synthesis. The essential role of p53 in Roc-A-mediated protection was confirmed by siRNA knockdown of p53 and by comparison of the effects of Roc-A on chemoprotection of splenocytes isolated from wild-type and p53-deficient mice. Importantly, Roc-A did not protect p53-deficient or -mutated cancer cells. Our data suggest that Roc-A may be used as an adjuvant to reduce the side effects of chemotherapy in patients with p53-deficient or -mutated tumors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzofurans; Cell Line, Tumor; DNA Damage; Down-Regulation; Drug Interactions; Drugs, Chinese Herbal; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Protective Agents; Tumor Suppressor Protein p53

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Topics: Animals; Antigens, CD34; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzofurans; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Leukemia; Leukocytes, Mononuclear; Mice; Mitochondria; Neoplastic Stem Cells; Phenotype; Reactive Oxygen Species; Sirolimus; Stem Cells; Triterpenes; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Phytochemical analysis of the leaves of different Aglaia species collected in Vietnam yielded eight rocaglamide derivatives, which are responsible for the strong insecticidal activity against Spodoptera littoralis, including rocaglamide A (1), rocaglamide 1 (2), rocaglamide W (3), rocaglamide AB (4), rocaglamide J (5), rocaglaol (6), rocaglamide S (7) and the new rocaglamide AY (8). The structures of these compounds were elucidated through extensive 1D and 2D NMR spectroscopy and analysis of their mass spectrometric (ESI-MS) and HRESIMS data.

Topics: Aglaia; Benzofurans; Insecticides; Molecular Structure; Plant Leaves; Vietnam

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.

Topics: Benzofurans; Binding Sites; Eukaryotic Initiation Factor-4F; Models, Biological; Saccharomyces cerevisiae; Triterpenes

The ribosome is centrally situated to sense metabolic states, but whether its activity, in turn, coherently rewires transcriptional responses is unknown. Here, through integrated chemical-genetic analyses, we found that a dominant transcriptional effect of blocking protein translation in cancer cells was inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for anabolic metabolism, cellular proliferation, and tumorigenesis. These analyses linked translational flux to the regulation of HSF1 transcriptional activity and to the modulation of energy metabolism. Targeting this link with translation initiation inhibitors such as rocaglates deprived cancer cells of their energy and chaperone armamentarium and selectively impaired the proliferation of both malignant and premalignant cells with early-stage oncogenic lesions.

Topics: Animals; Antineoplastic Agents; Benzofurans; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; DNA-Binding Proteins; Energy Metabolism; Gene Expression Regulation, Neoplastic; Heat Shock Transcription Factors; High-Throughput Screening Assays; Humans; Mice; Neoplasm Transplantation; Neoplasms; NIH 3T3 Cells; Protein Biosynthesis; Ribosomes; Transcription Factors

Although most of the patients with Hodgkin's lymphoma (HL) can be cured by the current regimen of high-dose multiagent chemotherapy, the treatment causes high risks of later toxicities including secondary malignancies. Therefore, new rational strategies are needed for HL treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its tumor selectivity and its lack of toxicity for normal cells. Unfortunately, many cancers remain resistant to TRAIL including HL. HL is characterized by enhanced expression of cellular caspase-8 (FLICE)-inhibitory protein (c-FLIP) and X-linked inhibitor of apoptosis (XIAP), which block receptor-mediated apoptosis by inhibiting caspase-8 and caspase-3, respectively. We have recently discovered the herbal compound Rocaglamide, which breaks TRAIL-resistance in acute T cell leukemia through inhibition of c-FLIP expression. We have also shown that small molecule XIAP inhibitors can sensitize TRAIL-mediated apoptosis in several resistant tumors. However, whether targeting XIAP or c-FLIP is also a suitable strategy to prime HL cells for TRAIL-induced apoptosis has not yet been investigated. In our study, we show that Rocaglamide suppresses c-FLIP expression in HL cells in a dose- and time-dependent manner. However, downregulation of c-FLIP alone was not sufficient to sensitize TRAIL-induced apoptosis in HL cells. Similarly, treatment of HL cells with a small molecule XIAP inhibitor resulted in a moderate induction of apoptosis. However, inhibition of XIAP alone was also not sufficient to enhance TRAIL-induced cell death. Synergistic increase in TRAIL-mediated killing of HL cells was only obtained by combination of Rocaglamide and XIAP inhibitors. Our study demonstrates that targeting both c-FLIP and XIAP are necessary for an efficient treatment of HL.

Topics: Apoptosis; Benzofurans; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Hodgkin Disease; Humans; TNF-Related Apoptosis-Inducing Ligand; X-Linked Inhibitor of Apoptosis Protein

The rocaglates/rocaglamides are a class of natural products known to display potent anticancer activity. One such derivative, silvestrol, has shown activity comparable to taxol in certain settings. Here, we report the synthesis of various rocaglamide analogues and identification of a hydroxamate derivative (-)-9 having activity similar to silvestrol in vitro and ex vivo for inhibition of protein synthesis. We also show that (-)-9 synergizes with doxorubicin in vivo to reduce Eμ-Myc driven lymphomas.

Topics: Animals; Antineoplastic Agents; Benzofurans; Cell Line, Tumor; Cell Survival; Doxorubicin; Drug Screening Assays, Antitumor; Drug Synergism; Eukaryotic Initiation Factor-4F; Humans; Hydroxamic Acids; Lymphoma; Mice; Mice, Inbred C57BL; Microsomes, Liver; Protein Subunits; Protein Synthesis Inhibitors; Stereoisomerism; Structure-Activity Relationship; Triterpenes

This article describes the evolution of a Nazarov cyclization-based synthetic strategy targeting the anticancer, antiinflammatory, and insecticidal natural product (±)-rocaglamide. Initial pursuit of a polarized heteroaromatic Nazarov cyclization to construct the congested cyclopentane core revealed an unanticipated electronic bias in the pentadienyl cation. This reactivity was harnessed in a successful second-generation approach using an oxidation-initiated Nazarov cyclization of a heteroaryl alkoxyallene. Full details of these two approaches are given, as well as the characterization of undesired reaction pathways available to the Nazarov cyclization product. A sequence of experiments that led to an understanding of the unexpected reactivity of this key intermediate is described, which culminated in the successful total synthesis of (+)-rocaglamide.

Topics: Alkadienes; Anti-Inflammatory Agents; Antineoplastic Agents, Phytogenic; Benzofurans; Cyclization; Cyclopentanes; Insecticides; Ketones; Magnetic Resonance Spectroscopy; Molecular Structure; Oxidation-Reduction; Stereoisomerism

The RNA helicases eIF4AI and eIF4AII play key roles in recruiting ribosomes to mRNA templates during eukaryotic translation initiation. Small molecule inhibitors of eIF4AI and eIF4AII have been useful for chemically dissecting their role in translation in vitro and in vivo. Here, we describe a screen performed on a small focused library of kinase inhibitors to identify a novel helicase inhibitor. We describe assays that have been critical for characterizing novel RNA helicase inhibitors.

Topics: Animals; Anthracenes; Benzofurans; Epoxy Compounds; Eukaryotic Initiation Factor-4A; Humans; Macrolides; Perylene; Protein Kinase Inhibitors; Sterols; Thiazoles

Rocaglamides are potent natural anticancer products that inhibit proliferation of various cancer cells at nanomolar concentrations. We have recently shown that these compounds prevent tumor growth and sensitize resistant cancer cells to apoptosis by blocking the MEK-ERK-eIF4 pathway. However, their direct molecular target(s) remain(s) unknown. In this study, using an affinity chromatography approach we discovered that prohibitin (PHB) 1 and 2 are the direct targets of rocaglamides. Binding of rocaglamides to PHB prevents interaction between PHB and CRaf and, thereby, inhibits CRaf activation and subsequently CRaf-MEK-ERK signaling. Moreover, knockdown of PHB mimicked the effects of rocaglamides on the CRaf-MEK-ERK pathway and cell cycle progression. Thus, our finding suggests that rocaglamides are a new type of anticancer agent and that they may serve as a small-molecular tool for studying PHB-mediated cellular processes.

Topics: Aglaia; Antineoplastic Agents, Phytogenic; Benzofurans; Cell Cycle; Cell Proliferation; Cells, Cultured; Drug Screening Assays, Antitumor; Extracellular Signal-Regulated MAP Kinases; HEK293 Cells; HeLa Cells; Humans; Jurkat Cells; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Prohibitins; Proto-Oncogene Proteins c-raf; Repressor Proteins; Structure-Activity Relationship

The human T-cell leukemia virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATL) is incurable by currently known therapies. ATL samples and cell lines derived from ATL patients show restricted sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L). We have recently shown that HTLV-1-infected cells express elevated levels of cellular caspase-8 FLICE-inhibitory protein (c-FLIP) conferring resistance to receptor-mediated apoptosis. This finding underscores the demand to develop new strategies for treatment of ATL. In this study, we show that the naturally occurring herbal compound Rocaglamide (Roc) sensitizes CD95L- and TRAIL-induced apoptosis in HTLV-1-infected cells by downregulation of c-FLIP expression. Investigation of the molecular mechanism of Roc-mediated downregulation of c-FLIP revealed that it inhibits phosphorylation of the translation initiation factor 4E (eIF4E), a key factor that controls the rate-limiting step of translation, through inhibition of the MEK-ERK-MNK1 signaling pathway. This event prevents de novo synthesis of short-lived proteins such as c-FLIP in HTLV-1-infected cells. Our data suggest that Roc may serve as an adjuvant for TRAIL-based anticancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Benzofurans; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line, Tumor; Down-Regulation; Eukaryotic Initiation Factor-4E; Extracellular Signal-Regulated MAP Kinases; Fas Ligand Protein; Human T-lymphotropic virus 1; Humans; Intracellular Signaling Peptides and Proteins; Leukemia-Lymphoma, Adult T-Cell; MAP Kinase Kinase Kinases; Phosphorylation; Protein Biosynthesis; Protein Serine-Threonine Kinases; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

Drugs with tumor selectivity may have an important benefit in chemotherapies. We have previously shown that Rocaglamide(s), derived from the medicinal plant Aglaia, kills various leukemic cells through the mitochondrial apoptosis pathway with only minor toxicities to normal lymphocytes. Here, we show further that Rocaglamide preferentially promotes activation-induced cell death in malignant T cells by differential regulation of c-FLIP and CD95L expression. Rocaglamide enhances and also prolongs activation-induced JNK activation in malignant T cells leading to downregulation of c-FLIP but upregulation of CD95L expression. We also show that malignant T cells express a significantly higher amount of Bid - the molecular linker that bridges the receptor-mediated to the mitochondria-mediated apoptosis pathway. Conversely, a substantially lower amount of c-FLIP in response to T-cell stimulation compared to normal T cells is observed. This difference may provide a therapeutic window for cancer treatment. The effect of Rocaglamide on sensitization of activation-induced cell death in malignant T cells was further demonstrated in vivo in a mouse model. Our study demonstrates that Rocaglamide may be a potential anticancer drug that simultaneously targets both c-FLIP and CD95L expressions in tumor cells. This study may also provide a new clue to design a more efficient chemotherapy by using a combination of stimuli that engage the receptor-mediated and the mitochondria-mediated death pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzofurans; BH3 Interacting Domain Death Agonist Protein; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Line; Fas Ligand Protein; Humans; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukemia, T-Cell; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Signal Transduction; T-Lymphocytes; Transplantation, Homologous

The total syntheses of aglafolin, rocagloic acid, and rocaglamide using Nazarov cyclization are described. Generation of the necessary oxyallyl cation intermediate was accomplished via peracid oxidation of an allenol ether to generate an unusual oxycarbenium ion species that undergoes cyclization. The synthesis is efficient, highly diastereoselective, and strategically distinct from previous syntheses of rocaglamide.

Topics: Benzofurans; Cyclization; Epoxy Compounds; Oxidation-Reduction; Stereoisomerism

The tricyclic core of the cyclopentabenzofurans has been prepared in an efficient and stereoselective manner utilizing an intramolecular silyl vinylketene formation/[4 + 1] annulation sequence. This novel approach affords the ABC ring system where the adjacent phenyl and aryl substituents of the C ring have the required cis relationship.

Topics: Animals; Benzofurans; Ethylenes; Ketones; Mice; Silanes; Stereoisomerism; Substrate Specificity; Vinyl Compounds

Two new compounds, a cyclopenta[bc]benzopyran, ponapensin (1), and an aglaialactone, 5,6-desmethylenedioxy-5-methoxy-aglalactone (2), together with nine known compounds were isolated from the CHCl(3) soluble extract of the leaves and twigs of Aglaia ponapensis. Their structures were established by spectroscopic data interpretation. Ponapensin (1) exhibited significant NF-kappaB inhibitory activity in an Elisa assay, and was found to be more potent than the positive control rocaglamide. All of the compounds isolated were also tested in a panel of human cancer cell lines, with the known sterol E-volkendousin (3) and methyl rocaglate (aglafoline) found to be the only active substances.

Topics: Aglaia; Benzofurans; Benzopyrans; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Heterocyclic Compounds, 3-Ring; Humans; Molecular Structure; NF-kappa B; Plant Leaves

With an increasing cancer rate worldwide, there is an urgent quest for the improvement of anticancer drugs. One of the main problems of present chemotherapy in treatment of tumor patients is the toxicity of drugs. Most of the existent anticancer drugs, unfortunately, attack also proliferating normal cells. In recent years, traditional Chinese herbal remedies have gradually gained considerable attention as a new source of anticancer drugs. Although their healing mechanisms are still largely unknown, some of the drugs have been used to help cancer patients fight their disease at reduced side effects compared to other treatments. In our study, we show that Rocaglamide (Roc), derived from the traditional Chinese medicinal plants Aglaia, induces apoptosis through the intrinsic death pathway in various human leukemia cell lines and in acute lymphoblastic leukemia, chronic myeloid leukemia and acute myeloid leukemia cells freshly isolated from patients. Investigation of the molecular mechanisms by which Roc kills tumors revealed that it induces a consistent activation of the stress-response mitogen-activated protein kinase (MAPK) p38 accompanied with a long-term suppression of the survival MAPK extracellular signal-regulated kinase. These events affect proapoptotic Bcl-2 family proteins leading to depolarization of the mitochondrial membrane potential and trigger caspase-mediated apoptosis involving caspase-9, -8, -3 and -2. Importantly, Roc shows no effects on MAPKs in normal lymphocytes and therefore has no or very low toxicity on healthy cells. Up to now, more than 50 different Roc derivatives have been isolated from Aglaia. Our study suggests that Roc derivatives may be promising candidates for the development of new drugs against hematologic malignancies.

Topics: Acute Disease; Antineoplastic Agents; Apoptosis; Benzofurans; Cell Line, Tumor; Drugs, Chinese Herbal; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tubulin

Enantioselective syntheses of methyl rocaglate and the related natural products rocaglamide and rocaglaol are outlined. The approach involves enantioselective [3 + 2] photocycloaddition promoted by chiral Brønsted acids (TADDOLs) to afford an aglain precursor followed by a ketol shift/reduction sequence to the rocaglate core.

Topics: Acids; Benzofurans; Meliaceae; Models, Chemical; Molecular Structure; Photochemistry; Plants, Medicinal; Spectrum Analysis; Stereoisomerism

Two new rocaglamide derivatives, 1-O-formylrocagloic acid (1) and 3'-hydroxy rocagloic acid (2), together with five known compounds, rocaglaol (3), rocagloic acid (4), 3'-hydroxymethylrocaglate (5), 1-O-formylmethyl rocaglate (6), and methylrocaglate (7), were isolated from the fruits of Amoora cucullata. Their structures were elucidated by spectroscopic methods. Compounds 1-3, 6, and 7 exhibited potent cytotoxicity against KB, BC, and NCI-H187 cell lines, whereas 4 and 5 showed selective cytotoxicity against NCI-H187 cell line.

Topics: Benzofurans; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Magnetic Resonance Spectroscopy; Meliaceae; Molecular Conformation; Reference Standards; Seeds; Sensitivity and Specificity; Stereoisomerism; Structure-Activity Relationship

Aglaia (family Meliaceae) plants are used in traditional medicine (e.g., in Vietnam) for the treatment of inflammatory skin diseases and allergic inflammatory disorders such as asthma. Inflammatory diseases arise from inappropriate activation of the immune system, leading to abnormal expression of genes encoding inflammatory cytokines and tissue-destructive enzymes. The active compounds isolated from these plants are derivatives of rocaglamide. In this study we show that rocaglamides are potent immunosuppressive phytochemicals that suppress IFN-gamma, TNF-alpha, IL-2, and IL-4 production in peripheral blood T cells at nanomolar concentrations. We demonstrate that rocaglamides inhibit cytokine gene expression at the transcriptional level. At the doses that inhibit cytokine production, they selectively block NF-AT activity without impairing NF-kappaB and AP-1. We also show that inhibition of NF-AT activation by rocaglamide is mediated by strong activation of JNK and p38 kinases. Our study suggests that rocaglamide derivatives may serve as a new source of NF-AT-specific inhibitors for the treatment of certain inflammatory diseases.

Topics: Aglaia; Benzofurans; Cell Nucleus; Cytokines; DNA-Binding Proteins; Gene Expression Regulation; Humans; Immunosuppressive Agents; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; MAP Kinase Signaling System; NF-kappa B; NFATC Transcription Factors; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-jun; RNA, Messenger; T-Lymphocytes; Transcription Factors

A unified biomimetic approach to the aglain-forbaglin-rocaglamide classes of natural products is reported. The approach involves photogeneration of oxidopyryliums via excited-state intramolecular proton transfer (ESIPT) of 3-hydroxyflavones followed by [3+2] dipolar cycloaddition to the aglain core. An alpha-ketol (acyloin) rearrangement was employed to transform the aglain core to the rocaglamide framework. This approach was successfully used for the synthesis of the natural product (+/-)-methyl rocaglate.

Topics: Benzofurans; Biomimetic Materials; Flavonoids; Photochemistry; Pyridinium Compounds

Two rocaglamide-related natural products, the previously known compound 6-demethoxy-10-hydroxy-11-methoxy-6,7-methylendioxyrocaglamide (3), and cyclorocaglamide (4), its 8b,10-anhydro analogue, have been isolated from the tropical plant Aglaia oligophylla. Compound 4 is the first bridged cyclopentatetrahydrobenzofuran natural product, and it exhibited a CD spectrum virtually opposite that of all the other rocaglamide natural products known so far, but it still has the same absolute configuration at all stereogenic centers of the basic molecular framework. This was shown unequivocally by quantum chemical CD calculations (here based on molecular dynamics-weighted force field structures) and was finally confirmed experimentally, by a "biomimetic-type" cyclization of 3 to give 4, with the expected "inversion" of the CD spectrum. The opposite chiroptical properties of 3 and 4, despite their homochiral character, underline the necessity of handling chiroptical data with the greatest care, e.g., by simulating them by quantum chemical CD calculations. Compound 3 exhibited an LC(50) of 2.5 ppm when evaluated against neonate larvae of Spodoptera littoralis, while 4 was inactive in this assay up to 100 ppm.

Topics: Animals; Benzofurans; Bridged-Ring Compounds; Chromatography, High Pressure Liquid; Circular Dichroism; Cyclopentanes; Feeding Behavior; Insecticides; Larva; Meliaceae; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Spodoptera; Stereoisomerism

Crude extracts from different Aglaia species are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-kappaB transcription factors represent key regulators of genes involved in immune and inflammatory responses, we supposed that the anti-inflammatory effects of Aglaia extracts are mediated by the inhibition of NF-kappaB activity. Purified compounds of Aglaia species, namely 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one aglain congener were tested for their ability to inhibit NF-kappaB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB-dependent reporter gene activity in Jurkat T cells with IC(50) values in the nanomolar range. Some derivatives are less effective, and others are completely inactive. Rocaglamides are able to suppress the PMA-induced expression of NF-kappaB target genes and sensitize leukemic T cells to apoptosis induced by TNFalpha, cisplatin, and gamma-irradiation. The suppression of NF-kappaB activation correlated with the inhibition of induced IkappaB(alpha) degradation and IkappaB(alpha) kinase activation. The level of interference was determined and found to be localized upstream of the IkappaB kinase complex but downstream of the TNF receptor-associated protein 2. Our data suggest that rocaglamide derivatives could serve as lead structures in the development of anti-inflammatory and tumoricidal drugs.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Benzofurans; Cell Line; Dose-Response Relationship, Drug; Gene Expression Regulation; Genes, Reporter; Humans; I-kappa B Kinase; I-kappa B Proteins; Leukemia, T-Cell; Mice; Molecular Structure; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Serine-Threonine Kinases; Rats; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Organic-soluble extracts of the twigs of Aglaia oligophylla collected in Vietnam yielded four insecticidal cyclopentatetrahydrobenzofurans of the rocaglamide type including one new natural product (compound 4). Moreover, two cyclopentatetrahydrobenzopyran derivatives, belonging to the aglain and aglaforbesin types, respectively, were also isolated. The aglaforbesin derivative 6 proved likewise to be a new natural product. All isolated rocaglamide, aglain, and aglaforbesin derivatives have a characteristic methylenedioxy substituent linked to C-6 and C-7 or to C-7 and C-8, respectively. Structure elucidation of the new natural products and the determination of the absolute configuration of compound 1 by calculation of its CD spectrum with molecular dynamics simulation are described. All isolated rocaglamide derivatives exhibited strong insecticidal activity toward neonate larvae of the polyphageous pest insect Spodoptera littoralis when incorporated into an artificial diet, with LC(50) values varying between 2.15 and 6.52 ppm.

Topics: Animals; Benzofurans; Circular Dichroism; Insecticides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Molecular; Molecular Structure; Rosales; Spodoptera; Stereoisomerism

A phytochemical analysis of the leaves of Aglaia dasyclada collected in Yunnan Province (People's Republic of China) yielded five cyclopentabenzofurans (1-5) of the rocaglamide family that are common secondary metabolites of Aglaia species as well as four biogenetically related compounds of the aglain (7), aglaforbesin (8) and forbaglin (9, 10) types. In addition, the cinnamic acid amide dasyclamide (6), which is a putative biogenetic precursor of these compounds (7-10), was isolated. The structures of the new compounds (6-10) were assigned unambiguously from the combined use of 1D and 2D NMR spectroscopy and mass spectrometry.

Topics: Animals; Benzofurans; China; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Circular Dichroism; Dose-Response Relationship, Drug; Feeding Behavior; Glycosides; Insecticides; Larva; Magnetic Resonance Spectroscopy; Mass Spectrometry; Meliaceae; Molecular Structure; Plant Leaves; Plants, Medicinal; Putrescine; Spodoptera; Stereoisomerism

Bark of Aglaia spectabilis collected on the island of Phu Quoc (Vietnam) yielded insecticidal cyclopentatetrahydrobenzofurans of the rocaglamide type including four new natural products. Structure elucidation of the new compounds is described. All rocaglamide derivatives isolated exhibited strong insecticidal activity towards neonate larvae of the polyphagous pest insect Spodoptera littoralis when incorporated into an artificial diet. LC50 values varied from 0.8 to 80 ppm. The most active compounds isolated, methylrocaglate and C-3' hydroxylmethylrocaglate, were similar with regard to their insecticidal activity to the well-known natural insecticide azadirachtin.

Topics: Benzofurans; Insecticides; Molecular Structure; Rosales; Spectrum Analysis

Thirteen naturally occurring 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one naturally occurring aglain congener all of them isolated from three Aglaia species (Aglaia duperreana, A. oligophylla and A. spectabilis) collected in Vietnam were studied for their antiproliferative effects using the human monocytic leukemia cell lines MONO-MAC-1 and MONO-MAC-6. Only rocaglamide type compounds showed significant inhibition of [3H-]thymidine incorporation and the most active compound didesmethylrocaglamide inhibited cell growth in a similar concentration range as the well-known anticancer drug vinblastine sulfate. Detailed structure-activity analysis indicated that the OH-group at C-8b which is a common structural feature of most naturally occurring rocaglamide compounds is essential for the described antiproliferative activity since replacement of this group by methylation led to a complete loss of the inhibitory activity for the resulting derivative. Rocaglamide derivatives rapidly inhibited DNA as well as protein biosynthesis of MONO-MAC-6 cells at concentrations well below those of actinomycin D or cycloheximide which were used as positive controls in the respective experiments. Didesmethylrocaglamide was furthermore able to induce growth arrest of MONO-MAC-1 cells in the G2/M and probably G0/G1-phase of the cell cycle with no morphological indication of cellular damage. Our data suggests that 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type act primarily by a cytostatic mechanism.

Topics: Antineoplastic Agents, Phytogenic; Benzofurans; Cell Cycle; Cell Division; Humans; Leukemia; Lignans; Plants; Tumor Cells, Cultured

Two related 1H-2,3,3a,8b-tetrahydrocyclopenta[b]benzofurans, aglafolin (1a) and rocaglamide (2), isolated from the stems of Aglaia elliptifolia, showed significant cytotoxicity in six cancer cell lines. Aglafolin (1a) was also found to completely block platelet aggregation caused by arachidonic acid and platelet-activating factor at 100 microM and 2 ng/mL, respectively.

Topics: Animals; Antineoplastic Agents, Phytogenic; Benzofurans; Drug Screening Assays, Antitumor; Humans; In Vitro Techniques; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mice; Plant Extracts; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet; Tumor Cells, Cultured