Aniquinazoline B, a fungal natural product, activates the µ-opioid receptor.

The development of new µ-opioid receptor (MOR) agonists without the undesirable side effects, such as addiction or respiratory depression, has been a difficult challenge over the years. In the search for new compounds, we screened our chemical database of over 40.000 substances and further assessed the best 100 through molecular docking. We selected the top 10 compounds and evaluated them for their biological activity and potential to influence cyclic adenosine monophosphate (cAMP) levels. From the tested compounds, compound 7, called aniquinazoline B, belonging to the quinazolinone alkaloids class and isolated from the marine fungus Aspergillus nidulans, showed promising results, by inhibiting cAMP levels and in vitro binding to MOR, verified through microscale thermophoresis. Transcriptomic data investigation profiled the genes affected by compound 7 and discovered activation of different pathways compared to opioids. The western blot analysis revealed compound 7 as a balanced ligand, activating both p-ERK1/2 and ß-arrestin1/2 pathways, showing this is a favorable candidate to be further tested.

Jozimine A2, a Dimeric Naphthylisoquinoline (NIQ) Alkaloid, Shows In Vitro Cytotoxic Effects against Leukemia Cells through NF-κB Inhibition.

Naphthylisoquinoline (NIQ) alkaloids are rising as a promising class of secondary metabolites with pharmaceutical potential. NF-κB has already been recognized as a significant modulator of cancer proliferation and drug resistance. We have previously reported the mechanisms behind the cytotoxic effect of dioncophylline A, an NIQ monomer, in leukemia cells. In the current study, we have investigated the cytotoxic effect of jozimine A2, an NIQ dimer, on leukemia cells in comparison to a second, structurally unsymmetric dimer, michellamine B. To this end, molecular docking was applied to predict the binding affinity of the dimers towards NF-κB, which was then validated through microscale thermophoresis. Next, cytotoxicity assays were performed on CCRF-CEM cells and multidrug-resistant CEM/ADR5000 cells following treatment. Transcriptome analysis uncovered the molecular networks affected by jozimine A2 and identified the cell cycle as one of the major affected processes. Cell death modes were evaluated through flow cytometry, while angiogenesis was measured with the endothelial cell tube formation assay on human umbilical vein endothelial cells (HUVECs). The results indicated that jozimine A2 bound to NF-κB, inhibited its activity and prevented its translocation to the nucleus. In addition, jozimine A2 induced cell death through apoptosis and prevented angiogenesis. Our study describes the cytotoxic effect of jozimine A2 on leukemia cells and explains the interactions with the NF-κB signaling pathway and the anticancer activity.

Cytotoxicity of dioncophylline A and related naphthylisoquinolines in leukemia cells, mediated by NF-κB inhibition, angiogenesis suppression, G2/M cell cycle arrest, and autophagy induction.

BACKGROUND: Inhibition of NF-κB activity represents a strategy to treat acute myeloid leukemia, one of the most lethal leukemia types. Naphthylisoquinolines (NIQs) are cytotoxic alkaloids from lianas of the families Ancistrocladaceae and Dioncophyllaceae, which are indigenous to tropical rainforests. PURPOSE: Uncovering therapeutic possibilities and underlying molecular mechanisms of dioncophylline A and its derivatives towards NF-κB related cellular processes. METHODS: Resazurin-based cell viability assay was performed for dioncophylline A and three derivatives on wild-type CCRF-CEM and multidrug-resistant CEM/ADR5000 cells. Transcriptome analysis was executed to discover cellular functions and molecular networks associated with dioncophylline A treatment. Expression changes obtained by mRNA microarray hybridization were confirmed using qRT-PCR. Molecular docking was applied to predict the affinity of the NIQs with NF-κB. To validate the in silico approach, NF-κB reporter assays were conducted on HEK-Blue™ Null1 cells. Cell death mechanisms and cell cycle arrest were studied using flow cytometry. The potential activity on angiogenesis was evaluated with the endothelial cell tube formation assay on HUVECs using fluorescence microscopy. Intracellular NF-κB location in HEK-Blue™ Null1 cells was visualized with immunofluorescence. Finally, the anti-tumor activity of dioncophylline A was studied by a xenograft zebrafish model in vivo. RESULTS: Our study demonstrated that dioncophylline A and its derivatives exerted potent cytotoxicity on leukemia cells. Using Ingenuity Pathway Analysis, we identified the NF-κB network as the top network, and docking experiments predicted dioncophylline A and two of its derivatives sharing the same binding pocket with the positive control compound, triptolide. Dioncophylline A showed the best inhibitory activity in NF-κB reporter assays compared to its derivatives, caused autophagy rather than apoptosis, and induced G2/M arrest. It also prevented NF-κB translocation from the cytoplasm to the nucleus. Tube formation as an angiogenesis marker was significantly suppressed by dioncophylline A treatment. Finally, the remarkable anti-tumor activity of dioncophylline A was proven in zebrafish in vivo. CONCLUSION: Taken together, we report for the first time the molecular mechanism behind the cytotoxic effect of dioncophylline A on leukemia cells. Dioncophylline A showed strong cytotoxic activity, inhibited NF-κB translocation, significantly affected the NF-κB in silico and in vitro, subdued tube formation, induced autophagy, and exerted antitumor activity in vivo. Our findings enlighten both the cellular functions including the NF-κB signaling pathway and the cytotoxic mechanism affected by dioncophylline A.

Modes of Action of a Novel c-MYC Inhibiting 1,2,4-Oxadiazole Derivative in Leukemia and Breast Cancer Cells.

The c-MYC oncogene regulates multiple cellular activities and is a potent driver of many highly aggressive human cancers, such as leukemia and triple-negative breast cancer. The oxadiazole class of compounds has gained increasing interest for its anticancer activities. The aim of this study was to investigate the molecular modes of action of a 1,2,4-oxadiazole derivative (ZINC15675948) as a c-MYC inhibitor. ZINC15675948 displayed profound cytotoxicity at the nanomolar range in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells. Multidrug-resistant sublines thereof (i.e., CEM/ADR5000 and MDA-MB-231-BCRP) were moderately cross-resistant to this compound (<10-fold). Molecular docking and microscale thermophoresis revealed a strong binding of ZINC15675948 to c-MYC by interacting close to the c-MYC/MAX interface. A c-MYC reporter assay demonstrated that ZINC15675948 inhibited c-MYC activity. Western blotting and qRT-PCR showed that c-MYC expression was downregulated by ZINC15675948. Applying microarray hybridization and signaling pathway analyses, ZINC15675948 affected signaling routes downstream of c-MYC in both leukemia and breast cancer cells as demonstrated by the induction of DNA damage using single cell gel electrophoresis (alkaline comet assay) and induction of apoptosis using flow cytometry. ZINC15675948 also caused G2/M phase and S phase arrest in CCRF-CEM cells and MDA-MB-231-pcDNA3 cells, respectively, accompanied by the downregulation of CDK1 and p-CDK2 expression using western blotting. Autophagy induction was observed in CCRF-CEM cells but not MDA-MB-231-pcDNA3 cells. Furthermore, microarray-based mRNA expression profiling indicated that ZINC15675948 may target c-MYC-regulated ubiquitination, since the novel ubiquitin ligase (ELL2) was upregulated in the absence of c-MYC expression. We propose that ZINC15675948 is a promising natural product-derived compound targeting c-MYC in c-MYC-driven cancers through DNA damage, cell cycle arrest, and apoptosis.

The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells.

Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and recently emerged in a new role in the treatment of cancer. ZINC253504760, a synthetic cardenolide that is structurally similar to well-known GCs, digitoxin and digoxin, has not been investigated yet. This study aims to investigate the cytotoxicity of ZINC253504760 on MDR cell lines and its molecular mode of action for cancer treatment. Four drug-resistant cell lines (P-glycoprotein-, ABCB5-, and EGFR-overexpressing cells, and TP53-knockout cells) did not show cross-resistance to ZINC253504760 except BCRP-overexpressing cells. Transcriptomic profiling indicated that cell death and survival as well as cell cycle (G2/M damage) were the top cellular functions affected by ZINC253504760 in CCRF-CEM cells, while CDK1 was linked with the downregulation of MEK and ERK. With flow cytometry, ZINC253504760 induced G2/M phase arrest. Interestingly, ZINC253504760 induced a novel state-of-the-art mode of cell death (parthanatos) through PARP and PAR overexpression as shown by western blotting, apoptosis-inducing factor (AIF) translocation by immunofluorescence, DNA damage by comet assay, and mitochondrial membrane potential collapse by flow cytometry. These results were ROS-independent. Furthermore, ZINC253504760 is an ATP-competitive MEK inhibitor evidenced by its interaction with the MEK phosphorylation site as shown by molecular docking in silico and binding to recombinant MEK by microscale thermophoresis in vitro. To the best of our knowledge, this is the first time to describe a cardenolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug resistance in cancer. A cardiac glycoside compound ZINC253504760 displayed cytotoxicity against different multidrug-resistant cell lines. ZINC253504760 exhibited cytotoxicity in CCRF-CEM leukemia cells by predominantly inducing a new mode of cell death (parthanatos). ZINC253504760 downregulated MEK1/2 phosphorylation and further affected ERK activation, which induced G2/M phase arrest.

Cynaropicrin disrupts tubulin and c-Myc-related signaling and induces parthanatos-type cell death in multiple myeloma.

The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM) cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line (IC50 = 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation, resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos. Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia (T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of hematopoietic tumor cells in vitro and in vivo.

The Novel Artemisinin Dimer Isoniazide ELI-XXIII-98-2 Induces c-MYC Inhibition, DNA Damage, and Autophagy in Leukemia Cells.

The proto-oncogenic transcription factor c-MYC plays a pivotal role in the development of tumorigenesis, cellular proliferation, and the control of cell death. Its expression is frequently altered in many cancer types, including hematological malignancies such as leukemia. The dimer isoniazide ELI-XXIII-98-2 is a derivative of the natural product artemisinin, with two artemisinin molecules and an isoniazide moiety as a linker in between them. In this study, we aimed to study the anticancer activity and the molecular mechanisms of this dimer molecule in drug-sensitive CCRF-CEM leukemia cells and their corresponding multidrug-resistant CEM/ADR5000 sub-line. The growth inhibitory activity was studied using the resazurin assay. To reveal the molecular mechanisms underlying the growth inhibitory activity, we performed in silico molecular docking, followed by several in vitro approaches such as the MYC reporter assay, microscale thermophoresis, microarray analyses, immunoblotting, qPCR, and comet assay. The artemisinin dimer isoniazide showed a potent growth inhibitory activity in CCRF-CEM but a 12-fold cross-resistance in multidrug-resistant CEM/ADR5000 cells. The molecular docking of artemisinin dimer isoniazide with c-MYC revealed a good binding (lowest binding energy of -9.84 ± 0.3 kcal/mol) and a predicted inhibition constant (pKi) of 66.46 ± 29.5 nM, which was confirmed by microscale thermophoresis and MYC reporter cell assays. Furthermore, c-MYC expression was downregulated by this compound in microarray hybridization and Western blotting analyses. Finally, the artemisinin dimer isoniazide modulated the expression of autophagy markers (LC3B and p62) and the DNA damage marker pH2AX, indicating the stimulation of both autophagy and DNA damage, respectively. Additionally, DNA double-strand breaks were observed in the alkaline comet assay. DNA damage, apoptosis, and autophagy induction could be attributed to the inhibition of c-MYC by ELI-XXIII-98-2.

Modulation of Intestinal Corticotropin-Releasing Hormone Signaling by the Herbal Preparation STW 5-II: Possible Mechanisms for Irritable Bowel Syndrome Management.

Corticotropin-releasing factor (CRF) mediates stress responses and alters the gut-brain axis, contributing to the pathogenesis of irritable bowel syndrome (IBS), which is recognized by abdominal pain accompanied by bowel habit disturbance. STW 5-II, a mixture of six herbal extracts, is clinically effective in functional dyspepsia and IBS. Here we aimed to establish an organoid-based stress-induced IBS-like model to investigate the mechanisms of action of STW 5-II. STW 5-II (10, 20, and 30 g/mL) was applied to intestinal organoids for 24 h before being treated with CRF (100 nM) for 48 h. The effects of STW 5-II on CRF signaling were investigated using several in vitro and in silico approaches. STW 5-II activities were further explored by in silico PyRx screening followed by molecular docking of the main 52 identified compounds in STW 5-II with both CRF receptors CRFR1 and CRFR2. CRF exposure stimulated inflammation and increased proinflammatory mediators, while STW 5-II dose-dependently counteracted these effects. STW 5-II inhibited CRF-induced claudin-2 overexpression and serotonin release. Docking of the STW 5-II constituents oleanolic acid and licorice saponin G2 to CRFR1 and CRFR2, respectively, showed a good affinity. These multi-target activities support and elucidate the clinically proven efficacy of STW 5-II in disorders of gut-brain interaction.

Disruption of Lipid Raft Microdomains, Regulation of CD38, TP53, and MYC Signaling, and Induction of Apoptosis by Lomitapide in Multiple Myeloma Cells.

BACKGROUND/AIM: Multiple myeloma (MM) is characterized by accumulation of a malignant clone of plasma cells in the bone marrow. Curative treatments are not yet available. Therefore, we undertook a drug repurposing approach to identify possible candidates from a chemical library of 1,230 FDA-approved drugs by virtual drug screening. As a target, we have chosen the non-receptor Bruton's tyrosine kinase (BTK) which is one of the main regulators of the MM biomarker CD38. MATERIALS AND METHODS: In silico virtual screening was performed by using PyRx. Flow cytometry was applied for cell cycle and apoptosis analysis. Furthermore, protein and gene expression was determined by western blotting and microarray hybridization. Lipid raft staining was observed by confocal microscopy. RESULTS: The in silico identified lipid-lowering lomitapide presented with the strongest cytotoxicity among the top 10 drug candidates. This drug arrested the cell cycle in the G2/M phase and induced apoptosis in MM cells. Western blot analyses revealed that treatment with lomitapide induced cleavage of the apoptosis regulator PARP and reduced the expression of CD38, an integral part of lipid rafts. Using confocal microscopy, we further observed that lipid raft microdomain formation in MM cells was inhibited by lomitapide. In four MM cell lines (KMS-12-BM, NCI-H929, RPMI-8226, and MOLP-8) treated with lomitapide, microarray analyses showed not only that the expression of CD38 and BTK was down-regulated, but also that the tumor suppressor gene TP53 and the oncogene c-MYC were among the top deregulated genes. Further analysis of these data by Ingenuity pathway analysis (IPA) suggested that lomitapide interferes with the cross-talk of CD38 and BTK and apoptosis-regulating genes via TP53 and c-MYC. CONCLUSION: Lomitapide treatment led to disruption of lipid raft domains and induction of pro-apoptotic factors and might, therefore, be considered as a potential therapeutic agent in MM.

Novel artemisinin derivative FO8643 with anti-angiogenic activity inhibits growth and migration of cancer cells via VEGFR2 signaling.

The vascular endothelial growth factor receptor 2 (VEGFR2) is widely recognized as a key effector in angiogenesis and cancer progression and has been considered a critical target for the development of anti-cancer drugs. Artemisinin (ARS) and its derivatives exert profound efficacy in treating not only malaria but also cancer. As a novel ARS-type compound, FO8643 caused significant suppression of the growth of a panel of cancer cells, including both solid and hematologic malignancies. In CCRF-CEM leukemia cells, FO8643 dramatically inhibited cell proliferation coupled with increased apoptosis and cell cycle arrest. Additionally, FO8643 restrained cell migration in the 2D wound healing assay as well as in a 3D spheroid model of human hepatocellular carcinoma HUH-7 cells. Importantly, SwissTargetPrediction predicted VEGFR2 as an underlying target for FO8643. Molecular docking simulation further indicated that FO8643 formed hydrogen bonds and hydrophobic interactions within the VEGFR2 kinase domain. Moreover, FO8643 directly inhibited VEGFR2 kinase activity and its downstream action including MAPK and PI3K/Akt signaling pathways in HUH-7 cells. Encouragingly, FO8643 decreased angiogenesis in the chorioallantoic membrane assay in vivo. Collectively, FO8643 is a novel ARS-type compound exerting potential VEGFR2 inhibition. FO8643 may be a viable drug candidate in cancer therapy.

Pyrrolizidine alkaloids cause cell cycle and DNA damage repair defects as analyzed by transcriptomics in cytochrome P450 3A4-overexpressing HepG2 clone 9 cells.

Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.

Repurposing of the ALK Inhibitor Crizotinib for Acute Leukemia and Multiple Myeloma Cells.

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib's ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

Cytotoxicity of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide in multidrug-resistant cancer cells through activation of PERK/eIF2α/ATF4 pathway.

After decades of research, multidrug resistance (MDR) remains a huge challenge in cancer treatment. In this study, the cytotoxic of 4-hydroxy-N-(naphthalen-1-yl)-2-oxo-2H-chromene-3-carboxamide (MCC1734) has been investigated towards multidrug-resistant cancer cell lines. MCC1734 exerted cytotoxicity on cell lines expressing different mechanisms of drug resistance (P-glycoprotein, BCRP, ABCB5, EGFR, p53 knockout) to a different extent. Interestingly, sensitive CCRF-CEM cells and multidrug-resistant P-gp-overexpressing CEM/ADR5000 cells represented similar sensitivity towards MCC1734, indicating MCC1734 can bypass P-gp-mediated resistance. Microarray-based mRNA expression revealed that MCC1734 affected cells by multiple pathways, including cell cycle regulation, mitochondrial dysfunction, apoptosis signaling, and EIF2 signaling. MCC1734 stimulated the generation of excessive reactive oxygen species and the collapse of mitochondria membrane potential in CCRF-CEM cells, companied by the arrest of the cell cycle in the G2M phase and apoptosis induction as determined by flow cytometry. In addition, our immunoblotting analysis highlighted that MCC1734 triggered endoplasmic reticulum (ER) stress, evidenced by the activation of p-PERK, p-eIF2α, ATF4 and CHOP. The anti-cancer effects of MCC1734 were further observed in vivo using human xenograft tumors transplanted to zebrafish, providing further support for MCC1734 as a promising new candidate for cancer drug development.

A novel moniliformin derivative as pan-inhibitor of histone deacetylases triggering apoptosis of leukemia cells.

New and potent agents that evade multidrug resistance (MDR) and inhibit epigenetic modifications are of great interest in cancer drug development. Here, we describe that a moniliformin derivative (IUPAC name: 3-(naphthalen-2-ylsulfanyl)-4-{[(2Z)-1,3,3-trimethyl-2,3-dihydro-1H-indol-2-ylidene]methyl}cyclobut-3-ene-1,2-dione; code: MCC1381) bypasses P-gp-mediated MDR. Using transcriptomics, we identified a large number of genes significantly regulated in response to MCC1381, which affected the cell cycle and disturbed cellular death and survival. The potential targets of MCC1381 might be histone deacetylases (HDACs) as predicted by SwissTargetPrediction. In silico studies confirmed that MCC1381 presented comparable affinity with HDAC1, 2, 3, 6, 8 and 11. Besides, the inhibition activity of HDACs was dose-dependently inhibited by MCC1381. Particularly, a strong binding affinity was observed between MCC1381 and HDAC6 by microscale thermophoresis analysis. MCC1381 decreased the expression of HDAC6, inversely correlated with the increase of acetylated HDAC6 substrates, acetylation p53 and α-tubulin. Furthermore, MCC1381 arrested the cell cycle at the G2/M phase, induced the generation of reactive oxygen species and collapse of the mitochondrial membrane potential. MCC1381 exhibited in vivo anti-cancer activity in xenografted zebrafish. Collectively, MCC1381 extended cytotoxicity towards P-gp-resistant leukemia cancer cells and may act as a pan-HDACs inhibitor, indicating that MCC1381 is a novel candidate for cancer therapy.

Anti-inflammatory and tight junction protective activity of the herbal preparation STW 5-II on mouse intestinal organoids.

BACKGROUND: Irritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS. AIM: This study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II. METHODS: Intestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1ß, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II. RESULTS: An organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS. CONCLUSION: STW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.

Selection of safe artemisinin derivatives using a machine learning-based cardiotoxicity platform and in vitro and in vivo validation.

The majority of drug candidates fails the approval phase due to unwanted toxicities and side effects. Establishment of an effective toxicity prediction platform is of utmost importance, to increase the efficiency of the drug discovery process. For this purpose, we developed a toxicity prediction platform with machine-learning strategies. Cardiotoxicity prediction was performed by establishing a model with five parameters (arrhythmia, cardiac failure, heart block, hypertension, myocardial infarction) and additional toxicity predictions such as hepatotoxicity, reproductive toxicity, mutagenicity, and tumorigenicity are performed by using Data Warrior and Pro-Tox-II software. As a case study, we selected artemisinin derivatives to evaluate the platform and to provide a list of safe artemisinin derivatives. Artemisinin from Artemisia annua was described first as an anti-malarial compound and later its anticancer properties were discovered. Here, random forest feature selection algorithm was used for the establishment of cardiotoxicity models. High AUC scores above 0.830 were achieved for all five cardiotoxicity indications. Using a chemical library of 374 artemisinin derivatives as a case study, 7 compounds (deoxydihydro-artemisinin, 3-hydroxy-deoxy-dihydroartemisinin, 3-desoxy-dihydroartemisinin, dihydroartemisinin-furano acetate-d3, deoxyartemisinin, artemisinin G, artemisinin B) passed the toxicity filtering process for hepatotoxicity, mutagenicity, tumorigenicity, and reproductive toxicity in addition to cardiotoxicity. Experimental validation with the cardiomyocyte cell line AC16 supported the findings from the in silico cardiotoxicity model predictions. Transcriptomic profiling of AC16 cells upon artemisinin B treatment revealed a similar gene expression profile as that of the control compound, dexrazoxane. In vivo experiments with a Zebrafish model further substantiated the in silico and in vitro data, as only slight cardiotoxicity in picomolar range was observed. In conclusion, our machine-learning approach combined with in vitro and in vivo experimentation represents a suitable method to predict cardiotoxicity of drug candidates.

miR-449a Repression Leads to Enhanced NOTCH Signaling in TMPRSS2:ERG Fusion Positive Prostate Cancer Cells.

About 50% of prostate cancer (PCa) tumors are TMPRSS2:ERG (T2E) fusion-positive (T2E+), but the role of T2E in PCa progression is not fully understood. We were interested in investigating epigenomic alterations associated with T2E+ PCa. Using different sequencing cohorts, we found several transcripts of the miR-449 cluster to be repressed in T2E+ PCa. This repression correlated strongly with enhanced expression of NOTCH and several of its target genes in TCGA and ICGC PCa RNA-seq data. We corroborated these findings using a cellular model with inducible T2E expression. Overexpression of miR-449a in vitro led to silencing of genes associated with NOTCH signaling (NOTCH1, HES1) and HDAC1. Interestingly, HDAC1 overexpression led to the repression of HES6, a negative regulator of the transcription factor HES1, the primary effector of NOTCH signaling, and promoted cell proliferation by repressing the cell cycle inhibitor p21. Inhibition of NOTCH as well as knockdown of HES1 reduced the oncogenic properties of PCa cell lines. Using tissue microarray analysis encompassing 533 human PCa cores, ERG-positive areas exhibited significantly increased HES1 expression. Taken together, our data suggest that an epigenomic regulatory network enhances NOTCH signaling and thereby contributes to the oncogenic properties of T2E+ PCa.

DNA Promoter Methylation and ERG Regulate the Expression of CD24 in Prostate Cancer.

CD24 is overexpressed in many human cancers and is a driver of tumor progression. Herein, molecular mechanisms leading to up-regulation of CD24 in prostate cancer were studied. DNA methylation of the CD24 gene promoter at four loci using quantitative methylation-specific PCR was evaluated. Expression of CD24 in tumor tissues was studied by immunohistochemistry. To corroborate the results in vitro, ERG-inducible LNCaP TMPRSS2:ERG (T2E) cells and luciferase promoter assays were used. DNA methylation of the CD24 promoter was significantly higher in tumors than in benign tissue and was associated with biochemical recurrence-free survival, tumor grade, and stage. CD24 mRNA and protein expression were significantly higher in T2E-positive, ERG-overexpressing, and/or PTEN-deficient cases. Higher levels of CD24 protein expression conferred shorter biochemical recurrence-free survival, and these observations were confirmed using The Cancer Genome Atlas prostate adenocarcinoma data. In silico analysis of the CD24 promoter revealed an ERG binding site in between the DNA methylation sites. ERG overexpression led to a strong induction of CD24 mRNA and protein expression. Luciferase promoter assays using the wild-type and mutated ERG binding site within the CD24 promoter showed ERG-dependent activation. Collectively, our results suggest that promoter DNA methylation of the CD24 gene and T2E fusion status are factors involved in the up-regulation of CD24 in patients with prostate cancer.

2α-Hydroxyalantolactone from Pulicaria undulata: activity against multidrug-resistant tumor cells and modes of action.

BACKGROUND: Sesquiterpene lactones having α-methylene-γ-lactone moiety are promising natural metabolites showing various biological activity. One of the major metabolites isolated from Pulicaria undulata, 2α-hydroxyalantolactone (PU-1), has not been investigated in detail yet. Multidrug resistance (MDR) represents a major obstacle for cancer chemotherapy and the capability of novel natural products to overcoming MDR is of great interest. PURPOSE: Exploring the molecular modes of action for potent natural product metabolites. METHODS: The resazurin reduction assay was employed to evaluate the cytotoxicity of PU-1 on sensitive and their corresponding drug-resistant cell lines (overexpressing P-glycoprotein, BCRP, ABCB5, ΔEGFR, or TP53 knockout). Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human CCRF-CEM leukemic cells after treatment with PU-1. The top significantly up- or down-regulated genes were identified by Chipster program and analyzed using Ingenuity Pathway Analysis (IPA) software. Finally, flow cytometry and Western blotting were performed for cell cycle analyses and apoptosis detection. RESULTS: The sesquiterpene lactone, PU-1, showed potent cytotoxicity towards the drug-sensitive and -resistant cell lines. Transcriptome-wide mRNA expression profiling and pathway analysis pointed to genes involved in DNA damage response and G2/M cell cycle arrest. G2/M arrest was verified by flow cytometry and further confirmed by the upregulation of p21 and downregulation of p-CDC25C expression in Western blotting. Moreover, the suggested DNA damage checkpoint regulation was confirmed by immunofluorescence and Western blotting by upregulation of pS345 Chk1, p-H3 and γ-H2AX. Furthermore, PU-1 inhibited PI3K/AKT pathway, which is involved in signaling DNA damage and G2/M arrest. Cells ultimately induced apoptosis upon PU-1 treatment. CONCLUSIONS: PU-1 is a potent natural product inhibiting otherwise drug-resistant human tumor cell growth through DNA damage, G2/M cell cycle arrest and apoptosis.

Multiple modes of cell death in neuroendocrine tumors induced by artesunate.

BACKGROUND: The paucity of effective treatment in neuroendocrine tumors (NETs) encouraged us to investigate the therapeutic value of artesunate (ART) promised by its inhibitory effect against various tumors and broad safety profile. METHODS: We evaluated the impact of ART on three NET cell lines, BON-1, QGP-1 and NCI-H727 on cellular and molecular levels. RESULTS: Our results showed that ART induced endoplasmic reticulum (ER) stress through phosphorylation of eIF2α, which further gave rise to autophagy in all three NET cell lines. Specifically, apoptosis and ferroptosis were also observed in BON-1 cells, which made BON-1 cell line more vulnerable upon ART treatment. The different sensitivities presented on the three cell lines also associated with a differential regulation of p21 on the long run. Co-treatment with p21 inhibitor UC2288 showed an additive effect on QGP-1 and NCI-H727 cell lines indicating p21 upregulation in these two cell lines might confer resistance towards ART treatment. CONCLUSIONS: It is possible to include ART in the treatment of NETs in the future.