The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA.

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.

Rocaglamide promotes infiltration and differentiation of T cells and coordinates with PD-1 inhibitor to overcome checkpoint resistance in multiple tumor models.

Tumor-infiltrating lymphocyte (TIL) deficiency is the most conspicuous obstacle to limit the cancer immunotherapy. Immune checkpoint inhibitors (ICIs), such as anti-PD-1 antibody, have achieved great success in clinical practice. However, due to the limitation of response rates of ICIs, some patients fail to benefit from monotherapy. Thus, novel combination therapy that could improve the response rates emerges as new strategies for cancer treatment. Here, we reported that the natural product rocaglamide (RocA) increased tumor-infiltrating T cells and promoted Th17 differentiation of CD4+ TILs. Despite RocA monotherapy upregulated PD-1 expression of TILs, which was considered as the consequence of T cell activation, combining RocA with anti-PD-1 antibody significantly downregulated the expression of PD-1 and promoted proliferation of TILs. Taken together, these findings demonstrated that RocA could fuel the T cell anti-tumor immunity and revealed the remarkable potential of RocA as a therapeutic candidate when combining with the ICIs.

A subset of flavaglines inhibits KRAS nanoclustering and activation.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications.

Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells.

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.

Targeted intervention of eIF4A1 inhibits EMT and metastasis of pancreatic cancer cells via c-MYC/miR-9 signaling.

BACKGROUND: Owing to the lack of effective treatment options, early metastasis remains the major cause of pancreatic ductal adenocarcinoma (PDAC) recurrence and mortality. However, the molecular mechanism of early metastasis is largely unknown. We characterized the function of eukaryotic translation initiation factors (eIFs) in epithelial-mesenchymal-transition (EMT) and metastasis in pancreatic cancer cells to investigate whether eIFs and downstream c-MYC affect EMT and metastasis by joint interference. METHODS: We used The Cancer Genome Atlas (TCGA) and Genome Tissue Expression (GTEx) databases to analyze eIF4A1 expression in PDAC tissues and further validated the findings with a microarray containing 53 PDAC samples. Expression regulation and pharmacological inhibition of eIF4A1 and c-MYC were performed to determine their role in migration, invasion, and metastasis in pancreatic cancer cells in vitro and in vivo. RESULTS: Elevated eIF4A1 expression was positively correlated with lymph node infiltration, tumor size, and indicated a poor prognosis. eIF4A1 decreased E-cadherin expression through the c-MYC/miR-9 axis. Loss of eIF4A1 and c-MYC decreased the EMT and metastasis capabilities of pancreatic cancer cells, whereas upregulation of eIF4A1 attenuated the inhibition of EMT and metastasis induced by c-MYC downregulation. Treatment with the eIF4A1 inhibitor rocaglamide (RocA) or the c-MYC inhibitor Mycro3 either alone or in combination significantly decreased the expression level of EMT markers in pancreatic cancer cells in vitro. However, the efficiency and safety of RocA alone were not inferior to those of the combination treatment in vivo. CONCLUSION: Overexpression of eIF4A1 downregulated E-cadherin expression through the c-MYC/miR-9 axis, which promoted EMT and metastasis of pancreatic cancer cells. Despite the potential feedback loop between eIF4A1 and c-MYC, RocA monotherapy is a promising treatment inhibiting eIF4A1-induced PDAC metastasis.

Natural products as drugs and tools for influencing core processes of eukaryotic mRNA translation.

Eukaryotic protein synthesis is the highly conserved, complex mechanism of translating genetic information into proteins. Although this process is essential for cellular homoeostasis, dysregulations are associated with cellular malfunctions and diseases including cancer and diabetes. In the challenging and ongoing search for adequate treatment possibilities, natural products represent excellent research tools and drug leads for new interactions with the translational machinery and for influencing mRNA translation. In this review, bacterial-, marine- and plant-derived natural compounds that interact with different steps of mRNA translation, comprising ribosomal assembly, translation initiation and elongation, are highlighted. Thereby, the exact binding and interacting partners are unveiled in order to accurately understand the mode of action of each natural product. The pharmacological relevance of these compounds is furthermore assessed by evaluating the observed biological activities in the light of translational inhibition and by enlightening potential obstacles and undesired side-effects, e.g. in clinical trials. As many of the natural products presented here possess the potential to serve as drug leads for synthetic derivatives, structural motifs, which are indispensable for both mode of action and biological activities, are discussed. Evaluating the natural products emphasises the strong diversity of their points of attack. Especially the fact that selected binding partners can be set in direct relation to different diseases emphasises the indispensability of natural products in the field of drug development. Discovery of new, unique and unusual interacting partners again renders them promising tools for future research in the field of eukaryotic mRNA translation.

The Evolution of the Total Synthesis of Rocaglamide.

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 Pd0 -catalyzed Nazarov chemistry developed in our laboratory, respectively. The successful asymmetric synthesis revealed unforeseen mechanistic complexity that required adjusting our strategy to overcome an unanticipated racemization process, an unusual reversible ring-cleavage step and a very facile trialkylsilyl group migration.

Molecular mechanisms and anti-cancer aspects of the medicinal phytochemicals rocaglamides (=flavaglines).

Rocaglamides (= flavaglines) are potent natural anti-cancer phytochemicals that inhibit cancer growth at nanomolar concentrations by the following mechanisms: (1) inhibition of translation initiation via inhibition of phosphorylation of the mRNA cap-binding eukaryotic translation initiation factor eIF4E and stabilization of RNA-binding of the translation initiation factor eIF4A in the eIF4F complex; (2) blocking cell cycle progression by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway; (3) inactivation of the heat shock factor 1 (HSF1) leading to up-regulation of thioredoxin-interacting protein (TXNIP) and consequent reduction of glucose uptake and (4) induction of apoptosis through activation of the MAPK p38 and JNK and inhibition of the Ras-CRaf-MEK-ERK signaling pathway. Besides the anti-cancer activities, rocaglamides are also shown to protect primary cells from chemotherapy-induced cell death and alleviate inflammation- and drug-induced injury in neuronal tissues. This review will focus on the recently discovered molecular mechanisms of the actions of rocaglamides and highlights the benefits of using rocaglamides in cancer treatment.

Rocaglamide regulates iron homeostasis by suppressing hepcidin expression.

The anemia of inflammation (AI) is characterized by the presence of inflammation and abnormal elevation of hepcidin. Accumulating evidence has proved that Rocaglamide (RocA) was involved in inflammation regulation. Nevertheless, the role of RocA in AI, especially in iron metabolism, has not been investigated, and its underlying mechanism remains elusive. Here, we demonstrated that RocA dramatically suppressed the elevation of hepcidin and ferritin in LPS-treated mice cell line RAW264.7 and peritoneal macrophages. In vivo study showed that RocA can restrain the depletion of serum iron (SI) and transferrin (Tf) saturation caused by LPS. Further investigation showed that RocA suppressed the upregulation of hepcidin mRNA and downregulation of Fpn1 protein expression in the spleen and liver of LPS-treated mice. Mechanistically, this effect was attributed to RocA's ability to inhibit the IL-6/STAT3 pathway, resulting in the suppression of hepcidin mRNA and subsequent increase in Fpn1 and TfR1 expression in LPS-treated macrophages. Moreover, RocA inhibited the elevation of the cellular labile iron pool (LIP) and reactive oxygen species (ROS) induced by LPS in RAW264.7 cells. These findings reveal a pivotal mechanism underlying the roles of RocA in modulating iron homeostasis and also provide a candidate natural product on alleviating AI.

Anti-tumor effects of the eIF4A inhibitor didesmethylrocaglamide and its derivatives in human and canine osteosarcomas.

Inhibition of translation initiation using eIF4A inhibitors like (-)-didesmethylrocaglamide [(-)-DDR] and (-)-rocaglamide [(-)-Roc] is a potential cancer treatment strategy as they simultaneously diminish multiple oncogenic drivers. We showed that human and dog osteosarcoma cells expressed higher levels of eIF4A1/2 compared with mesenchymal stem cells. Genetic depletion of eIF4A1 and/or 2 slowed osteosarcoma cell growth. To advance preclinical development of eIF4A inhibitors, we demonstrated the importance of (-)-chirality in DDR for growth-inhibitory activity. Bromination of DDR at carbon-5 abolished growth-inhibitory activity, while acetylating DDR at carbon-1 was tolerated. Like (-)-DDR, (±)-DDR, and (-)-Roc, (±)-DDR-acetate increased γH2A.X levels and induced G2/M arrest and apoptosis. Consistent with translation inhibition, these rocaglates decreased the levels of several mitogenic kinases, the STAT3 transcription factor, and the stress-activated protein kinase p38. However, phosphorylated p38 was greatly enhanced in treated cells, suggesting activation of stress response pathways. RNA sequencing identified RHOB as a top upregulated gene in both (-)-DDR- and (-)-Roc-treated osteosarcoma cells, but the Rho inhibitor Rhosin did not enhance the growth-inhibitory activity of (-)-DDR or (-)-Roc. Nonetheless, these rocaglates potently suppressed tumor growth in a canine osteosarcoma patient-derived xenograft model. These results suggest that these eIF4A inhibitors can be leveraged to treat both human and dog osteosarcomas.

Anti-tumor Effects of the eIF4A Inhibitor Didesmethylrocaglamide and Its Derivatives in Human and Canine Osteosarcomas.

Inhibition of translation initiation using eIF4A inhibitors like (-)-didesmethylrocaglamide [(-)-DDR] and (-)-rocaglamide [(-)-Roc] is a potential cancer treatment strategy as they simultaneously diminish multiple oncogenic drivers. We showed that human and dog osteosarcoma cells expressed high levels of eIF4A1/2, particularly eIF4A2. Genetic depletion of eIF4A1 and/or 2 slowed osteosarcoma cell growth. To advance preclinical development of eIF4A inhibitors, we demonstrated the importance of (-)-chirality in DDR for growth-inhibitory activity. Bromination of DDR at carbon-5 abolished growth-inhibitory activity, while acetylating DDR at carbon-1 was tolerated. Like DDR and Roc, DDR-acetate increased the γH2A.X levels and induced G2/M arrest and apoptosis. Consistent with translation inhibition, these rocaglates decreased the levels of several mitogenic kinases, the STAT3 transcription factor, and the stress-activated protein kinase p38. However, phosphorylated p38 was greatly enhanced in treated cells, suggesting activation of stress response pathways. RNA sequencing identified RHOB as a top upregulated gene in both DDR- and Roc-treated osteosarcoma cells, but the Rho inhibitor Rhosin did not enhance the growth-inhibitory activity of (-)-DDR or (-)-Roc. Nonetheless, these rocaglates potently suppressed tumor growth in a canine osteosarcoma patient-derived xenograft model. These results suggest that these eIF4A inhibitors can be leveraged to treat both human and dog osteosarcomas.

Characterization of Plasmodium falciparum prohibitins as novel targets to block infection in humans by impairing the growth and transmission of the parasite.

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes ofPlasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated hese proteins as mitochondrial residents as they were found to be localized as branched structures. We further validated PfPHBs as organellar proteins residing in Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte and oocyst growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

Mitomycin C enhanced the antitumor efficacy of Rocaglamide in colorectal cancer.

Rocaglamide (ROC), a natural phytochemical isolated from Aglaia species, is a translational inhibitor of de novo c-FLIP synthesis, which relieves the inhibition of c-FLIP dimerization with procasoase-8 and downstream activation. Unfortunately, a lot of cancer cells, especially colorectal cancer cells (CRC), exhibit marked resistance to Rocaglamide-induced cell death. Research has demonstrated that mitomycin C (MMC) has broad-spectrum anti-tumor activity that it can synergize with a wide range of clinical drugs to inhibit tumor growth. The current study investigated whether MMC combined with ROC could sensitize CRC cells with different ROC resistance to apoptosis. HCT116 and HT29, two different CRC cells, were treated with ROC and/or MMC, and the induction of apoptosis, inhibition of cell migration and invasion, arrest of cell cycle, induction of reactive oxygen species, and effects on Bcl-2 family signaling pathway were investigated. The results showed that low concentration of MMC combined with ROC significantly promoted HCT116 and HT29 cell apoptosis and inhibited cell proliferation by downregulating the expression of Bcl-2 and c-FLIP, upregulating the expression of Bax, activating the caspase cascade (involving the mitochondrial apoptosis pathway), arresting cell cycle in G1 phase, and increasing the level of reactive oxygen species (ROS). In addition, the viability and morphology of MRC-5 cells were not significantly affected by the combined treatment with ROC and MMC, indicating its safety. Therefore, it is concluded that the combination treatment of ROC and MMC is a highly effective tumor therapy and may offer a promising therapeutic strategy for the treatment of CRC.

Hierarchical Virtual Screening Based on Rocaglamide Derivatives to Discover New Potential Anti-Skin Cancer Agents.

Skin Cancer (SC) is among the most common type of cancers worldwide. The search for SC therapeutics using molecular modeling strategies as well as considering natural plant-derived products seems to be a promising strategy. The phytochemical Rocaglamide A (Roc-A) and its derivatives rise as an interesting set of reference compounds due to their in vitro cytotoxic activity with SC cell lines. In view of this, we performed a hierarchical virtual screening study considering Roc-A and its derivatives, with the aim to find new chemical entities with potential activity against SC. For this, we selected 15 molecules (Roc-A and 14 derivatives) and initially used them in docking studies to predict their interactions with Checkpoint kinase 1 (Chk1) as a target for SC. This allowed us to compile and use them as a training set to build robust pharmacophore models, validated by Pearson's correlation (p) values and hierarchical cluster analysis (HCA), subsequentially submitted to prospective virtual screening using the Molport® database. Outputted compounds were then selected considering their similarities to Roc-A, followed by analyses of predicted toxicity and pharmacokinetic properties as well as of consensus molecular docking using three software. 10 promising compounds were selected and analyzed in terms of their properties and structural features and, also, considering their previous reports in literature. In this way, the 10 promising virtual hits found in this work may represent potential anti-SC agents and further investigations concerning their biological tests shall be conducted.

Inhibitory effects of Rocaglamide-A on PPARγ-driven adipogenesis through regulation of mitotic clonal expansion involving the JAK2/STAT3 pathway.

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.

Rocaglamide promotes the infiltration and antitumor immunity of NK cells by activating cGAS-STING signaling in non-small cell lung cancer.

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition. Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism. Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA. Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.

Known Inhibitors of RNA Helicases and Their Therapeutic Potential.

RNA helicases are proteins found in all kingdoms of life, and they are associated with all processes involving RNA from transcription to decay. They use NTP binding and hydrolysis to unwind duplexes, to remodel RNA structures and protein-RNA complexes, and to facilitate the unidirectional metabolism of biological processes. Viral, bacterial, and eukaryotic parasites have an intimate need for RNA helicases in their reproduction. Moreover, various disorders, like cancers, are often associated with a perturbation of the host's helicase activity. Thus, RNA helicases provide a rich source of targets for the development of therapeutic or prophylactic drugs. In this review, we provide an overview of the different targeting strategies against helicases, the different types of compounds explored, the proposed inhibitory mechanisms of the compounds on the proteins, and the therapeutic potential of these compounds in the treatment of various disorders.

Neurofibromatosis: Molecular Pathogenesis and Natural Compounds as Potential Treatments.

The neurofibromatosis syndromes, including NF1, NF2, and schwannomatosis, are tumor suppressor syndromes characterized by multiple nervous system tumors, particularly Schwann cell neoplasms. NF-related tumors are mainly treated by surgery, and some of them have been treated by but are refractory to conventional chemotherapy. Recent advances in molecular genetics and genomics alongside the development of multiple animal models have provided a better understanding of NF tumor biology and facilitated target identification and therapeutic evaluation. Many targeted therapies have been evaluated in preclinical models and patients with limited success. One major advance is the FDA approval of the MEK inhibitor selumetinib for the treatment of NF1-associated plexiform neurofibroma. Due to their anti-neoplastic, antioxidant, and anti-inflammatory properties, selected natural compounds could be useful as a primary therapy or as an adjuvant therapy prior to or following surgery and/or radiation for patients with tumor predisposition syndromes, as patients often take them as dietary supplements and for health enhancement purposes. Here we review the natural compounds that have been evaluated in NF models. Some have demonstrated potent anti-tumor effects and may become viable treatments in the future.

Identification of Cardiac Glycosides as Novel Inhibitors of eIF4A1-Mediated Translation in Triple-Negative Breast Cancer Cells.

The eukaryotic translation initiation factor 4F complex (eIF4F) is a potential chemotherapeutic target in triple-negative breast cancer (TNBC). This complex regulates cap-dependent translational initiation and consists of three core proteins: eIF4E, eIF4G, and eIF4A1. In this study, we focus on repositioning compounds as novel inhibitors of eIF4A1-mediated translation. In order to accomplish this goal, a modified synthetic reporter assay was established. More specifically, a (CGG)4 motif, which confers eIF4A dependency, was incorporated into the 5'-leader region of a luciferase-tdTomato lentiviral reporter construct. The Prestwick Chemical Library was then screened in multiple TNBC cell lines by measuring the tdTomato fluorescent intensity. We identified several cardiac glycosides as potential inhibitors of eIF4A1-mediated translation. Based on our studies, we find that cardiac glycosides inhibit the expression of eIF4A1. To identify a potential mechanism by which this was occurring, we utilized the Integrative Library of Integrated Network-Based Cellular Signatures (iLINCS). Our pursuits led us to the discovery that cardiac glycosides also decrease levels of c-MYC. Quantitative PCR confirmed that decreases in c-MYC and eIF4A were occurring at the transcriptional level. As such, disruption of the eIF4A1-c-MYC axis may be a viable approach in the treatment of TNBC. The novel combination of rocaglamide A and digoxin exhibited synergistic anti-cancer activity against TNBC cells in vitro. The findings in this study and others are important for formulating potential combination chemotherapies against eIF4A1 in vivo. Thus, drug repositioning may be one classical approach to successfully target eIF4A1 in TNBC patients.

PINK1/Parkin-Mediated Mitophagy Regulation by Reactive Oxygen Species Alleviates Rocaglamide A-Induced Apoptosis in Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the most lethal diseases, and effective treatment of PC patients remains an enormous challenge. Rocaglamide A (Roc-A), a bioactive molecule extracted from the plant Aglaia elliptifolia, has aroused considerable attention as a therapeutic choice for numerous cancer treatments. Nevertheless, the effects and underlying mechanism of Roc-A in PC are still poorly understood. Here, we found that Roc-A inhibited growth and stimulated apoptosis by induction of mitochondria dysfunction in PC. Moreover, Roc-A accelerated autophagosome synthesis and triggered mitophagy involving the PTEN-induced putative kinase 1 (PINK1)/Parkin signal pathway. We also demonstrated that inhibition of autophagy/mitophagy can sensitize PC cells to Roc-A. Finally, Roc-A treatment results in an obvious accumulation of reactive oxygen species (ROS), and pretreatment of cells with the reactive oxygen species scavenger N-acetylcysteine reversed the apoptosis and autophagy/mitophagy induced by Roc-A. Together, our results elucidate the potential mechanisms of action of Roc-A. Our findings indicate Roc-A as a potential therapeutic agent against PC and suggest that combination inhibition of autophagy/mitophagy may be a promising therapeutic strategy in PC.