Euphorbiasteroid Abrogates EGFR and Wnt/β-Catenin Signaling in Non-Small-Cell Lung Cancer Cells to Impart Anticancer Activity

EGFR and Wnt/β-catenin signaling pathways play a prominent role in tumor progression in various human cancers including non-small-cell lung carcinoma (NSCLC). Transactivation and crosstalk between the EGFR and Wnt/β-catenin pathways may contribute to the aggressiveness of cancers. Targeting these oncogenic pathways with small molecules is an attractive approach to counteract various types of cancers. In this study, we demonstrate the effect of euphorbiasteroid (EPBS) on the EGFR and Wnt/β-catenin pathways in NSCLC cells. EPBS induced preferential cytotoxicity toward A549 (wildtype EGFR-expressing) cells over PC-9 (mutant EGFR-expressing) cells. EPBS suppressed the expression of EGFR, Wnt3a, β-catenin, and FZD-1, and the reduction in β-catenin levels was found to be mediated through the activation of GSK-3β. EPBS reduced the phosphorylation of GSK-3β^S9 with a parallel increase in β-TrCP and phosphorylation of GSK-3β^Y216. Lithium chloride treatment increased the phosphorylation of GSK-3β^S9 and nuclear localization of β-catenin, whereas EPBS reverted these effects. Forced expression or depletion of EGFR in NSCLC cells increased or decreased the levels of Wnt3a, β-catenin, and FZD-1, respectively. Overall, EPBS abrogates EGFR and Wnt/β-catenin pathways to impart its anticancer activity in NSCLC cells.     

Recent Developments to Cope the Antibacterial Resistance via β-Lactamase Inhibition

Antibacterial resistance towards the β-lactam (BL) drugs is now ubiquitous, and there is a major global health concern associated with the emergence of new β-lactamases (BLAs) as the primary cause of resistance. In addition to the development of new antibacterial drugs, β-lactamase inhibition is an alternative modality that can be implemented to tackle this resistance channel. This strategy has successfully revitalized the efficacy of a number of otherwise obsolete BLs since the discovery of the first β-lactamase inhibitor (BLI), clavulanic acid. Over the years, β-lactamase inhibition research has grown, leading to the introduction of new synthetic inhibitors, and a few are currently in clinical trials. Of note, the 1, 6-diazabicyclo [3,2,1]octan-7-one (DBO) scaffold gained the attention of researchers around the world, which finally culminated in the approval of two BLIs, avibactam and relebactam, which can successfully inhibit Ambler class A, C, and D β-lactamases. Boronic acids have shown promise in coping with Ambler class B β-lactamases in recent research, in addition to classes A, C, and D with the clinical use of vaborbactam. This review focuses on the further developments in the synthetic strategies using DBO as well as boronic acid derivatives. In addition, various other potential serine- and metallo- β-lactamases inhibitors that have been developed in last few years are discussed briefly as well. Furthermore, binding interactions of the representative inhibitors have been discussed based on the crystal structure data of inhibitor-enzyme complex, published in the literature.     

Design and Synthesis of New Benzo[d]oxazole-Based Derivatives and Their Neuroprotective Effects on β-Amyloid-Induced PC12 Cells

A series of novel synthetic substituted benzo[d]oxazole-based derivatives (5a–5v) exerted neuroprotective effects on β-amyloid (Aβ)-induced PC12 cells as a potential approach for the treatment of Alzheimer’s disease (AD). In vitro studies show that most of the synthesized compounds were potent in reducing the neurotoxicity of Aβ_25-35-induced PC12 cells at 5 μg/mL. We found that compound 5c was non-neurotoxic at 30 μg/mL and significantly increased the viability of Aβ_25-35-induced PC12 cells at 1.25, 2.5 and 5 μg/mL. Western blot analysis showed that compound 5c promoted the phosphorylation of Akt and glycogen synthase kinase (GSK-3β) and decreased the expression of nuclear factor-κB (NF-κB) in Aβ_25-35-induced PC12 cells. In addition, our findings demonstrated that compound 5c protected PC12 cells from Aβ_25-35-induced apoptosis and reduced the hyperphosphorylation of tau protein, and decreased the expression of receptor for AGE (RAGE), β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), inducible nitric oxide synthase (iNOS) and Bcl-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) via Akt/GSK-3β/NF-κB signaling pathway. In vivo studies suggest that compound 5c shows less toxicity than donepezil in the heart and nervous system of zebrafish.     

A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway

As a neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.     

Schisandrin B Attenuates Hepatic Stellate Cell Activation and Promotes Apoptosis to Protect against Liver Fibrosis

The activation of hepatic stellate cells (HSC) plays a key role in the progression of hepatic fibrosis, it is essential to remove activated HSC through apoptosis to reverse hepatic fibrosis. Schisandrin B (Sch B) is the main chemical component of schisandrin lignan, and it has been reported to have good hepatoprotective effects. However, Schisandrin B on HSC apoptosis remains unclear. In our study, we stimulated the HSC-T6 and LX-2 cell lines with TGF-β1 to induce cell activation, and the proliferation and apoptosis of the activated HSC-T6 and LX-2 cells were detected after treatment with different doses of Schisandrin B. Flow cytometry results showed that Sch B significantly reduced the activity of activated HSC-T6 and LX-2 cells and significantly induced apoptosis. In addition, the cleaved-Caspase-3 levels were increased, the Bax activity was increased, and the Bcl-2 expression was decreased in HSC-T6 and LX-2 cells treated with Sch B. Our study showed that Sch B inhibited the TGF-β1-induced activity of hepatic stellate cells by promoting apoptosis.     

A Bioactive Compound from Sanguisorba officinalis L. Inhibits Cell Proliferation and Induces Cell Death in 5-Fluorouracil-Sensitive/Resistant Colorectal Cancer Cells

Colorectal cancer (CRC) is one of the most common cancer in the world. The first line chemotherapeutic agent, 5-fluorouracil (5-FU), plays a predominant role in the clinical treatment of CRC. However, with the wide use of 5-FU, more and more CRC patients have been obtaining drug resistance to 5-FU, which leads to a large amount of treatment failures. One of the effective strategies to overcome this obstacle is to find bioactive natural products from traditional medicine. In our previous work, Sanguisorba officinalis L. was found to exert a strong anti-proliferative activity against 5-FU-senstive/resistant CRC cells. Therefore, several compounds were isolated from this herb and screened for their anti-CRC effects to find promising compounds. Among them, a triterpenoid compound named 3β-[(α-l-arabinopyranosyl) oxy]-urs-12,18(19)-dien-28-oic acid β-d-glucopyranosyl ester (AGE), showed strong activity against both 5-FU-senstive and resistant CRC cells. In order to further study the mechanism of AGE on CRC cells, flow cytometer analysis, mitochondrial membrane potential (MMP) measurement, Western blotting, and RT-PCR assays were performed. Results demonstrated that AGE induced cell death by apoptosis pathway and autophagy, and inhibited cell proliferation via cell cycle arrest in G0-G1 phase mediated by Wnt signaling pathway. Therefore, AGE may be a potential bioactive compound for CRC treatment in clinic.     

Development and Validation of a Novel HPLC Method to Analyse Metabolic Reaction Products Catalysed by the CYP3A2 Isoform: In Vitro Inhibition of CYP3A2 Enzyme Activity by Aspirin (Drugs Often Used Together in COVID-19 Treatment)

Aspirin (also known as acetylsalicylic acid) is a drug intended to treat fever, pain, or inflammation. Treatment of moderate to severe cases of COVID-19 using aspirin along with dexamethasone has gained major attention globally in recent times. Thus, the purpose of this study was to use High-Performance Liquid Chromatography (HPLC) to evaluate the in vitro inhibition of CYP3A2 enzyme activity using aspirin in rat liver microsomes (RLMs). In this study, an efficient and sensitive HPLC method was developed using a reversed phase C18 column (X Bridge 4.6 mm × 150 mm, 3.5 µm) at 243 nm using acetonitrile and water (70:30 v/v). The linearity (r² > 0.999), precision (<15%), accuracy and recovery (80–120%), limit of detection (5.60 µM and 0.06 µM), limit of quantification (16.98 µM and 0.19 µM), and stability of the newly developed method were validated for dexamethasone and 6β-hydroxydexamethasone, respectively, following International Conference on Harmonization (ICH) guidelines. This method was applied in vitro to measure CYP3A2 activity. The results showed that aspirin competitively inhibits 6β-hydroxylation (CYP3A2 activity) with an inhibition constant (Ki) = 95.46 µM and the concentration of the inhibitor causing 50% inhibition of original enzyme activity (IC₅₀) = 190.92 µM. This indicated that there is a minimal risk of toxicity when dexamethasone and aspirin are co-administrated and a very low risk of toxicity and drug interaction with drugs that are a substrate for CYP3A2 in healthcare settings.     

Biological Activity of Extracts of Red and Yellow Fruits of Cornus mas L.—An In Vitro Evaluation of Antioxidant Activity,Inhibitory Activity against α-Glucosidase,Acetylcholinesterase, and Binding Capacity to Human Serum Albumin

Although extracts are broadly used in order to support the treatment of numerous diseases, only in a limited number of cases is the process of applying and establishing their mechanisms of action scientifically analyzed. Fruits of Cornelian cherry are an abundant source of iridoids, anthocyanins, flavonols and phenolic acids. The aim of the present study was to evaluate the in vitro bioactivity of red and yellow Cornelian cherry fruits’ extracts. The biological potential of extracts, in a broad sense, involved antioxidant activity in relation to phosphatidylcholine liposomes, inhibitory ability against α-glucosidase and acetylcholinesterase enzymes, as well as interactions with human serum albumin. Studies showed that both extracts were more effective in protecting liposome membranes against free radicals produced by AAPH in an aqueous environment due to the fact that they can be better eliminated by the hydrophilic components of the extracts than those produced by UVB radiation. Extracts exhibited inhibitory activity against acetylcholinesterase and α-glucosidase, wherein loganic acid extract showed noncompetitive inhibition of the enzyme. Moreover, extracts binded to albumin mainly through hydrogen bonds and van der Waals forces. Taken together, red and yellow cherry fruits’ extracts exhibit diverse biological properties and can be exploited as a source of natural therapeutic agents.     

Novel Sterically Crowded and Conformationally Constrained α-Aminophosphonates with a Near-Neutral pKa as Highly Accurate 31P NMR pH Probes. Application to Subtle pH Gradients Determination in Dictyostelium discoideum Cells

In order to discover new ³¹P NMR markers for probing subtle pH changes (<0.2 pH unit) in biological environments, fifteen new conformationally constrained or sterically hindered α-aminophosphonates derived from diethyl(2-methylpyrrolidin-2-yl)phosphonate were synthesized and tested for their pH reporting and cytotoxic properties in vitro. All compounds showed near-neutral pK_as (ranging 6.28–6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δδ_ab between the acidic and basic forms) ranging from 9.2–10.7 ppm, being fourfold larger than conventional endogenous markers such as inorganic phosphate. X-ray crystallographic studies combined with predictive empirical relationships and ab initio calculations addressed the inductive and stereochemical effects of substituents linked to the protonated amine function. Satisfactory correlations were established between pK_as and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δδ_ab. Finally, the hit ³¹P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing pK_a = 6.45 with Δδ_ab = 10.64 ppm, allowed the first clear-cut evidence of trans-sarcolemmal pH gradients in normoxic Dictyostelium discoideum cells with an accuracy of <0.05 pH units.