The GLP-1R Agonist Exendin-4 Attenuates Hyperglycemia-Induced Chemoresistance in Human Endometrial Cancer Cells Through ROS-Mediated Mitochondrial Pathway

This study aimed to assess the effects of the antidiabetic drug Exendin-4 (Exe-4), a GLP-1 receptor agonist, on the response of human endometrial cancer cells to chemotherapy under high glucose (HG) conditions. Cell viability was detected using a cell counting kit (CCK)-8. Cell apoptosis and reactive oxygen species (ROS) levels were measured by flow cytometry. Gene expression was evaluated by real-time PCR and immunoblotting. The chemotherapeutic drug cisplatin (DDP) dose-dependently inhibited both human endometrial adenocarcinoma Ishikawa and HEC1B cells, a response reversed by HG. Meanwhile, Exe-4 attenuated hyperglycemia’s effect by elevating intracellular lactate dehydrogenase (LDH) and ROS production. Similarly, DDP-induced elevation of intracellular rhodamine123 was attenuated by HG, and Exe-4 reversed HG’s impact. The chemoresistance genes multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (Pgp) were upregulated. At the same time, topoisomerase II (TOPO II) was downregulated under HG conditions, suggesting HG-induced chemoresistance. Exe-4 did not significantly influence the above genes. DDP downregulated Bcl-2 and Bcl-XL and upregulated Bax, cytosolic cytochrome c, and PARP under normal glucose (NG) versus HG conditions, and Exe-4 attenuated these effects. Upstream of Bax/Bcl, acetylated P53 was upregulated by DDP and downregulated by HG, whose effect was reversed by Exe-4. DPP treatment significantly induced apoptosis and cell cycle arrest in the S phase under NG, and HG reduced these effects. Prolonged exposure to HG induces DDP chemoresistance in human endometrial cancer cells but is alleviated by Exe-4.

A Unique Topoisomerase II Inhibitor with Dose-Affected Anticancer Mechanisms and Less Cardiotoxicity

Type II DNA topoisomerase (topo II) is an essential nuclear enzyme and a well-validated anticancer drug target. Previously, we have carried out several rounds of structural optimizations on our in-house topo II inhibitor E17, which was shown to have superior anticancer activity and less risk of multidrug resistance (MDR). Among the newly developed acridone derivatives, 6h displayed significant anticancer efficacy with unique mechanisms of action. At low concentrations, it arrested cancer cell cycles and triggered cell apoptosis, which is similar to the action of the well-known topo II inhibitor VP16. By contrast, 6h showed significant and long-term anti-proliferative activity at relatively high concentrations, with negligible influence on apoptosis. In addition, 6h exhibited no serious cardiotoxicity compared to doxorubicin (DOXO), a widely used topo II-targeting antineoplastic drug in clinic, but with damaging myocardial side effects. Collectively, our present work has supported the therapeutic value of 6h as a promising chemotherapy for cancers.

Potential Anti-Proliferative, Anti-Inflammatory and Anti-Viral Components from Sinopodophyllum Hexandrum Explored Using Bio-Affinity Ultrafiltration with Multiple Drug Targets

Background: Sinopodophyllum hexandrum (S. hexandrum) is a typical Chinese herbal medicine with numerous components and remarkable pharmacological activities. However, the specific phytochemicals responsible for its anti-proliferative, anti-inflammatory and anti-viral effects remain unexplored.Methods: The integrated analytical strategy combining bio-affinity ultrafiltration with multiple drug targets was developed to rapidly screen and identify bioactive ligands from S. hexandrum. The in vitro anti-proliferative and COX-2 inhibitory assays of bioactive ligands screened were further verified by sulforhodamine B (SRB) cell proliferation and cytotoxicity detection and COX-2 inhibitor screening kits, respectively. Molecular docking analysis was also implemented by the AutoDockTools 1.5.6 software.Results: 10, 7, 9 and 9 phytochemicals were screened out and identified as the potential Topo I, Topo II, COX-2 and ACE2 ligands, respectively. Hereinto, podophyllotoxin and quercetin with higher EF values displayed strong inhibitory effects on A549 and HT-29 cells comparable with etoposide and 5-FU. Furthermore, compared with indomethacin at 0.73 ± 0.07 mM, podophyllotoxin and kaempferol with higher EF values exerted stronger inhibitory effects with IC50 values at 0.36 ± 0.02 mM and 10.49 ± 0.61 mM, respectively. Additionally, the optimal binding sites and mode of action between bioactive ligands and multiple drug targets were determined by molecular docking. Wherein, isorhamnetin showed a stronger affinity to ACE2 with the binding energy of -5.72 kcal/mol and the IC50 value at 63.95 mM, lower than MLN-4760 (-4.27 kcal/mol and 738.62 mM). Conclusions: The integrative strategy combining multiple drug targets and bio-affinity ultrafiltration LC-MS in the present study showed very promising potential for the quick screening and identifying bioactive ligands in S. hexandrum for Topo I, Topo II, COX-2 and ACE2, and some bioactive compounds screened out from this work were verified with other in vitro assays, and even better than those positive drugs of interest. Based on these findings, we then first constructed an interacting network among multi-components and multi-targets. In this way, we showcased a quick and reliable experimental strategy for uncovering the underlying mechanism of the empirical traditional applications of S. hexandrum which could also provide valuable information for better understanding the therapeutic targets and therapeutic ligands of other herbal medicines.

Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome length and individualization

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.

Synthesis and anticancer evaluation of sulfur containing 9-anilinoacridines

Background: DNA topoisomerases are a class of enzymes that play a critical role in fundamental biological processes of replication, transcription, recombination, repair and chromatin remodeling. Amsacrine (m-AMSA), the best-known compound of 9-anilinoacridines series was one of the first DNA-intercalating agents to be considered as a Topoisomerase II inhibitor. Objective: A series of sulfur containing 9-anilinoacridines related to amsacrine were synthesized and evaluated for their anticancer activity. Methods: Cell viability was assessed by the MTT assay. The topoisomerase II inhibitory assay was performed using the Human topoisomerase II Assay kit and flow cytometry was used to evaluate the effects on cell cycle of K562 cells. Molecular docking was performed using Schrödinger Maestro program. Results: Compound 36 was found to be the most cytotoxic of the sulfide series against SW620, K562, and MCF-7. The limited SAR suggested the importance of the methansulfonamidoacetamide side chain functionality, the lipophilicity and relative metabolic stability of 36 in contributing to the cytotoxicity. Topoisomerase II α inhibitory activity appeared to be involved in the cytotoxicity of 36 through inhibition of decatenation of kinetoplast DNA (kDNA) in a concentration dependent manner. Cell cycle analysis further showed the Topo II inhibition through accumulation of K562 cells in G2/M phase of cell cycle. Docking of 36 into the Topo II α-DNA complex suggested that it may be an allosteric inhibitor of Topo II α. Conclusion: Compound 36 exhibits anticancer activity by inhibiting topoisomerase II and it could further be evaluated in in vivo models.

Combination Treatment of CI-994 With Etoposide Potentiates Anticancer Effects Through a Topoisomerase II-Dependent Mechanism in Atypical Teratoid/Rhabdoid Tumor (AT/RT)

PurposeAtypical teratoid/rhabdoid tumor (AT/RT) is arising typically in young children and is associated with a dismal prognosis which there is currently no curative chemotherapeutic regimen. Based on previous studies showing high histone deacetylase 1 (HDAC1) expression in AT/RT, the HDAC1 inhibitor CI-994 was used as a novel treatment strategy in this study. We assessed the anticancer effects of CI-994 and conventional drugs (etoposide, cisplatin or 4-HC) in AT/RT cells.MethodsAT/RT patient-derived primary cultured cells and cell lines were prepared. HDAC1 was estimated by real-time quantitative polymerase chain reaction (RT-qPCR). The interaction of the drugs was analyzed using isobologram analysis. Cell viability, apoptosis, HDAC enzyme activity and western blot assays were carried out.ResultsHDAC1 was overexpressed in AT/RT compared to medulloblastoma. The combination index (CI) of CI-994 with etoposide revealed a synergistic effect in all AT/RT cells, but no synergistic effect was observed between CI-994 and cisplatin or 4-HC. CI-994 effectively reduced not only Class I HDAC gene expression but also HDAC enzyme activity. The combination treatment of CI-994 with etoposide significantly increased apoptosis compared to the single treatment. The enhanced effect of apoptosis by this combination treatment is related to a signaling pathway which decreases topoisomerase (Topo) II and increases histone H3 acetylation (Ac-H3).ConclusionWe demonstrate that the combination treatment of CI-994 with etoposide exerts a synergistic anticancer effect against AT/RT by significantly inducing apoptosis through Topo II and Ac-H3 regulation.Clinical RelevanceThis combination treatment might be considered a viable therapeutic strategy for AT/RT patients.

Monascin and monascinol, azaphilonoid pigments from Mortierella polycephala AM1: in silico and in vitro targeting of the angiogenic VEGFR2 kinase

The fungus, Mortierella polycephala is one of the most productive sources of anticancer bioactive compounds namely those of pigment nature. During our investigation of the produced bioactive metabolites by the terrestrial M. polycephala AM1 isolated from Egyptian poultry feather waste, two main azaphilonoid pigments, monascin (1) and monascinol (2) were obtained as major products; their structures were identified by 1D (1H&13C) and 2D (1H–1H COSY, HMBC) NMR and HRESI-MS spectroscopic data. Biologically, cytotoxic activities of these compounds were broadly studied compared with the fungal extract. To predict the biological target for the presumed antitumor activity, an in silico study was run toward three proteins, topoisomerase IIα, topoisomerase IIβ, and VEGFR2 kinase. Monascinol (2) was expected to be moderately active against VEGFR2 kinase without any anticipated inhibition toward topo II isoforms. The in vitro study confirmed the docked investigation consistently and introduced monascinol (2) rather than its counterpart (1) as a potent inhibitor to the tested VEGFR2 kinase. Taxonomically, the fungus was identified using morphological and genetic assessments.

Guiding functions of the C-terminal domain of topoisomerase IIα advance mitotic chromosome assembly

Topoisomerase II (topo II) is one of the six proteins essential for mitotic chromatid reconstitution in vitro. It is not fully understood, however, mechanistically how this enzyme regulates this process. In an attempt to further refine the reconstitution assay, we have found that chromosomal binding of Xenopus laevis topo IIα is sensitive to buffer conditions and depends on its C-terminal domain (CTD). Enzymological assays using circular DNA substrates supports the idea that topo IIα first resolves inter-chromatid entanglements to drive individualization and then generates intra-chromatid entanglements to promote thickening. Importantly, only the latter process requires the CTD. By using frog egg extracts, we also show that the CTD contributes to proper formation of nucleosome-depleted chromatids by competing with a linker histone for non-nucleosomal DNA. Our results demonstrate that topo IIα utilizes its CTD to deliver the enzymatic core to crowded environments created during mitotic chromatid assembly, thereby fine-tuning this process.

Benzoquinoline Derivatives: A Straightforward and Efficient Route to Antibacterial and Antifungal Agents

We report here the design, synthesis, experimental and in silico evaluation of the antibacterial and antifungal activity of some new benzo[f]quinoline derivatives. Two classes of benzo[f]quinolinium derivatives—(benzo[f]quinolinium salts (BQS) and pyrrolobenzo[f]quinolinium cycloadducts (PBQC)—were designed and obtained in two steps via a direct and facile procedure: quaternization followed by a cycloaddition reaction. The synthesized compounds were characterized by elemental and spectral analysis (FT-IR, 1H-NMR, 13C-NMR). The antimicrobial assay reveals that the BQS salts have an excellent quasi-nonselective antifungal activity against the fungus Candida albicans (some of them higher that the control drug nystatin) and very good antibacterial activity against the Gram positive bacterium Staphylococcus aureus. The PBQC compounds are inactive. Analysis of the biological data reveals interesting SAR correlations in the benzo[f]quinolinium series of compounds. The in silico studies furnished important data concerning the pharmacodynamics, pharmacokinetics and ADMET parameters of the BQS salts. Studies of the interaction of each BQS salt 3a–o with ATP synthase in the formed complex, reveal that salts 3j, 3i, and 3n have the best fit in a complex with ATP synthase. Study of the interaction of each BQS salt 3a-o with TOPO II in the formed complex reveals that salts 3j and 3n have the best-fit in complex with TOPO II. The in silico ADMET studies reveal that the BQS salts have excellent drug-like properties, including a low toxicity profile. Overall, the experimental and in silico studies indicate that compounds 3e and 3f (from the aliphatic series), respectively, and 3i, 3j and 3n (from the aromatic series), are promising leading drug candidates.