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.

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.

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.

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.

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.

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.

Synaptic, transcriptional and chromatin genes disrupted in autism.

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

Common functional variants of the glutamatergic system in Autism spectrum disorder with high and low intellectual abilities.

The genetic architecture underlying Autism spectrum disorder (ASD) has been suggested to differ between individuals with lower (IQ ≤ 70; LIQ) and higher intellectual abilities (IQ > 70; HIQ). Among the identified pathomechanisms, the glutamatergic signalling pathway is of specific interest in ASD. We investigated 187 common functional variants of this neurotransmitter system for association with ASD and with symptom severity in two independent samples, a German (German-ALL: N = 583 families) and the Autism Genome Project cohort (AGP-ALL: N = 2001 families), split into HIQ, and LIQ subgroups. We did not identify any association withstanding correction for multiple testing. However, we report a replicated nominal significant under-transmission (OR < 0.79, p < 0.04) of the AKAP13 rs745191-T allele in both LIQ cohorts, but not in the much larger HIQ cohorts. At the phenotypic level, we nominally replicated associations of CAMK2A-rs2241694 with non-verbal communication in both combined LIQ and HIQ ASD cohorts. Variants PLD1-rs2124147 and ADCY1-rs2461127 were nominally associated with impaired non-verbal abilities and AKAP2-rs3739456 with repetitive behaviour in both LIQ cohorts. All four LIQ-associated genes are involved in G-protein coupled signal transduction, a downstream pathway of metabotropic glutamate receptor activation. We conclude that functional common variants of glutamatergic genes do not have a strong impact on ASD, but seem to moderately affect ASD risk and phenotypic expression. Since most of our nominally replicated hits were identified in the LIQ cohort, further investigation of the glutamatergic system in this subpopulation might be warranted.

Anticancer activity of cryptotanshinone on acute lymphoblastic leukemia cells.

Cryptotanshinone, a well-known diterpene quinone from a widely used traditional Chinese herb named Salvia miltiorrhiza, has been reported for its therapeutical potentials on diverse activities. In this study, pharmacological effects of cryptotanshinone on acute lymphoblastic leukemia cells were investigated. IC50 values of 5.0 and 4.8 were obtained in CEM/ADR5000 and CCRF-CEM. Microarray-based mRNA expression revealed that cryptotanshinone regulated genes associated with cell cycle, DNA damage, reactive oxygen species (ROS), NFκB signaling and cellular movement. The involvement of these pathways in the mode of action of cryptotanshinone was subsequently validated by additional independent in vitro studies. Cryptotanshinone stimulated ROS generation and induced DNA damage. It arrested cells in G2/M phase of the cell cycle and induced apoptosis as measured by annexin V-FITC-conjugating fluorescence. The induction of the intrinsic apoptotic pathway by cryptotanshinone was proved by loss of mitochondrial membrane potential and increased cleavage of caspase 3/7, caspase 9 and poly ADP ribose polymerase (PARP). DNA-binding motif analysis of the microarray-retrieved deregulated genes in the promoter region revealed NFκB as potential transcription factor involved in cryptotanshinone's mode of action. Molecular docking and Western blotting provided supportive evidence, suggesting that cryptotanshinone binds to IKK-ß and inhibits the translocation of p65 from the cytosol to the nucleus. In addition, cryptotanshinone inhibited cellular movement as shown by a fibronectin-based cellular adhesion assay, indicating that this compound exerts anti-invasive features. In conclusion, cryptotanshinone exerts profound cytotoxicity, which is caused by multispecific modes of actions, including G2/M arrest, apoptosis and inhibition of cellular movement. The inhibitory activities of this compound may be explained by inhibition of NFκB, which orchestrates all these mechanisms.

A Novel Mutation in RPL10 (Ribosomal Protein L10) Causes X-Linked Intellectual Disability, Cerebellar Hypoplasia, and Spondylo-Epiphyseal Dysplasia.

RPL10 encodes ribosomal protein L10 (uL16), a highly conserved multifunctional component of the large ribosomal subunit, involved in ribosome biogenesis and function. Using X-exome resequencing, we identified a novel missense mutation (c.191C>T; p.(A64V)) in the N-terminal domain of the protein, in a family with two affected cousins presenting with X-linked intellectual disability, cerebellar hypoplasia, and spondylo-epiphyseal dysplasia (SED). We assessed the impact of the mutation on the translational capacity of the cell using yeast as model system. The mutation generates a functional ribosomal protein, able to complement the translational defects of a conditional lethal mutation of yeast rpl10. However, unlike previously reported mutations, this novel RPL10 missense mutation results in an increase in the actively translating ribosome population. Our results expand the mutational and clinical spectrum of RPL10 identifying a new genetic cause of SED and highlight the emerging role of ribosomal proteins in the pathogenesis of neurodevelopmental disorders.

Genetic and functional analyses of SHANK2 mutations suggest a multiple hit model of autism spectrum disorders.

Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23-4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11-q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the "multiple hit model" for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.

Common variants in genes of the postsynaptic FMRP signalling pathway are risk factors for autism spectrum disorders.

Autism spectrum disorders (ASD) are heterogeneous disorders with a high heritability and complex genetic architecture. Due to the central role of the fragile X mental retardation gene 1 protein (FMRP) pathway in ASD we investigated common functional variants of ASD risk genes regulating FMRP. We genotyped ten SNPs in two German patient sets (N = 192 and N = 254 families, respectively) and report association for rs7170637 (CYFIP1; set 1 and combined sets), rs6923492 (GRM1; combined sets), and rs25925 (CAMK4; combined sets). An additional risk score based on variants with an odds ratio (OR) >1.25 in set 1 and weighted by their respective log transmitted/untransmitted ratio revealed a significant effect (OR 1.30, 95 % CI 1.11-1.53; P = 0.0013) in the combined German sample. A subsequent meta-analysis including the two German samples, the "Strict/European" ASD subsample of the Autism Genome Project (1,466 families) and a French case/control (541/366) cohort showed again association of rs7170637-A (OR 0.85, 95 % CI 0.75-0.96; P = 0.007) and rs25925-G (OR 1.31, 95 % CI 1.04-1.64; P = 0.021) with ASD. Functional analyses revealed that these minor alleles predicted to alter splicing factor binding sites significantly increase levels of an alternative mRNA isoform of the respective gene while keeping the overall expression of the gene constant. These findings underpin the role of ASD candidate genes in postsynaptic FMRP regulation suggesting that an imbalance of specific isoforms of CYFIP1, an FMRP interaction partner, and CAMK4, a transcriptional regulator of the FMRP gene, modulates ASD risk. Both gene products are related to neuronal regulation of synaptic plasticity, a pathomechanism underlying ASD and may thus present future targets for pharmacological therapies in ASD.

Enhancing cisplatin drug sensitivity through PARP3 inhibition: The influence on PDGF and G-coupled signal pathways in cancer.

Drug resistance poses a significant challenge in cancer treatment despite the clinical efficacy of cisplatin. Identifying and targeting biomarkers open new ways to improve therapeutic outcomes. In this study, comprehensive bioinformatic analyses were employed, including a comparative analysis of multiple datasets, to evaluate overall survival and mutation hotspots in 27 base excision repair (BER) genes of more than 7,500 tumors across 23 cancer types. By using various parameters influencing patient survival, revealing that the overexpression of 15 distinct BER genes, particularly PARP3, NEIL3, and TDG, consistently correlated with poorer survival across multiple factors such as race, gender, and metastasis. Single nucleotide polymorphism (SNP) analyses within protein-coding regions highlighted the potential deleterious effects of mutations on protein structure and function. The investigation of mutation hotspots in BER proteins identified PARP3 due to its high mutation frequency. Moving from bioinformatics to wet lab experiments, cytotoxic experiments demonstrated that the absence of PARP3 by CRISPR/Cas9-mediated knockdown in MDA-MB-231 breast cancer cells increased drug activity towards cisplatin, carboplatin, and doxorubicin. Pathway analyses indicated the impact of PARP3 absence on the platelet-derived growth factor (PDGF) and G-coupled signal pathways on cisplatin exposure. PDGF, a critical regulator of various cellular functions, was downregulated in the absence of PARP3, suggesting a role in cancer progression. Moreover, the influence of PARP3 knockdown on G protein-coupled receptors (GPCRs) affects their function in the presence of cisplatin. In conclusion, the study demonstrated a synthetic lethal interaction between GPCRs, PDGF signaling pathways, and PARP3 gene silencing. PARP3 emerged as a promising target.

Inhibition of MSH6 augments the antineoplastic efficacy of cisplatin in non-small cell lung cancer as autophagy modulator.

The altered response to chemotherapeutic agents predominantly stems from heightened single-point mutations within coding regions and dysregulated expression levels of genes implicated in drug resistance mechanisms. The identification of biomarkers based on mutation profiles and expression levels is pivotal for elucidating the underlying mechanisms of altered drug responses and for refining combinatorial therapeutic strategies in the field of oncology. Utilizing comprehensive bioinformatic analyses, we investigated the impact of eight mismatch repair (MMR) genes on overall survival across 23 cancer types, encompassing more than 7500 tumors, by integrating their mutation profiles. Among these genes, MSH6 emerged as the most predictive biomarker, characterized by a pronounced mutation frequency and elevated expression levels, which correlated with poorer patient survival outcomes. The wet lab experiments disclosed the impact of MSH6 in mediating altered drug responses. Cytotoxic assays conducted revealed that the depletion of MSH6 in H460 non-small lung cancer cells augmented the efficacy of cisplatin, carboplatin, and gemcitabine. Pathway analyses further delineated the involvement of MSH6 as a modulator, influencing the delicate equilibrium between the pro-survival and pro-death functions of autophagy. Our study elucidates the intricate interplay between MSH6, autophagy, and cisplatin efficacy, highlighting MSH6 as a potential therapeutic target to overcome cisplatin resistance. By revealing the modulation of autophagy pathways by MSH6 inhibition, our findings offer insights into novel approaches for enhancing the efficacy of cisplatin-based cancer therapy through targeted interventions.

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.

Identification of Cytisine Derivatives as Agonists of the Human Delta Opioid Receptor by Supercomputer-Based Virtual Drug Screening and Transcriptomics.

Delta opioid receptors (DORs) are rising as therapeutic targets, not only for the treatment of pain but also other neurological disorders (e.g., Parkinson's disease). The advantage of DOR agonists compared to µ-opioid receptor agonists is that they have fewer side effects and a lower potential to induce tolerance. However, although multiple candidates have been tested in the past few decades, none have been approved for clinical use. The current study focused on searching for new DOR agonists by screening a chemical library containing 40,000 natural and natural-derived products. The functional activity of the top molecules was evaluated in vitro through the cyclic adenosine monophosphate accumulation assay. Compound 3 showed promising results, and its activity was further investigated through transcriptomic methods. Compound 3 inhibited the expression of TNF-α, prevented NF-κB translocation to the nucleus, and activated the G-protein-mediated ERK1/2 pathway. Additionally, compound 3 is structurally different from known DOR agonists, making it a valuable candidate for further investigation for its anti-inflammatory and analgesic potential.

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.

The cytotoxic activities of the major diterpene extracted from Salvia multicaulis (Bardakosh) are mediated by the regulation of heat-shock response and fatty acid metabolism pathways in human leukemia cells.

BACKGROUND: Leukemia is one of the most lethal cancers worldwide and represents the sixth-leading cause of cancer deaths. The results of leukemia treatment have not been as positive as desired, and recurrence is common. PURPOSE: Thus, there is an urgent requirement for the development of new therapeutic drugs. Salvia multicaulis (Bardakosh) is a widespread species that contains multiple phytochemical components with anti-cancer activities. METHODS: We isolated and characterized the major diterpene candesalvone B methyl ester from S. multicaulis and investigated its action as a cytotoxic agent towards sensitive and drug-resistant leukemia cells by the resazurin reduction assay. Additionally, the targeted genes and the affected molecular mechanisms attributed to the potent cytotoxic activities were discovered by transcriptome-wide mRNA expression profiling. The targets predicted to be regulated by candesalvone B methyl ester in each cell line were confirmed by qRT-PCR, molecular docking, microscale thermophoresis, and western blotting. Moreover, cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: Candesalvone B methyl ester was cytotoxic with IC50 values of 20.95 ± 0.15 µM against CCRF-CEM cells and 4.13 ± 0.10 µM against multidrug-resistant CEM/ADR5000 leukemia cells. The pathway enrichment analysis disclosed that candesalvone B methyl ester could regulate the heat-shock response signaling pathway via targeting heat shock factor 1 (HSF1) in CCRF-CEM cells and ELOVL fatty acid elongase 5 (ELOVL5) controls the fatty acid metabolism pathway in CEM/ADR5000 cells. Microscale thermophoresis showed the binding of candesalvone B methyl ester with HSF1 and ELOVL5, confirming the results of molecular docking analysis. Down-regulation of both HSF1 and ELOVL5 by candesalvone B methyl ester as detected by both western blotting and RT-qPCR was related to the reversal of drug resistance in the leukemia cells. Furthermore, candesalvone B methyl ester increased the arrest in the sub-G1 phase of the cell cycle in a dose-dependent manner from 1.3 % to 32.3 % with concomitant induction of apoptosis up to 29.0 % in CCRF-CEM leukemic cells upon inhibition of HSF1. CONCLUSION: Candesalvone B methyl ester isolated from S. multicaulis exerted cytotoxicity by affecting apoptosis, cell division, and modulation of expression levels of genes contributing to the heat stress signaling and fatty acid metabolism pathways that could relieve drug resistance of leukemia cells.

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.