Cytostatic and cytotoxic effects of a hot water and methanol extract of Acokanthera oppositifolia in HepG2 hepatocarcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Herb-induced liver injury is poorly described for African herbal remedies, such as Acokanthera oppositifolia. Although a commonly used treatment for pain, snake bites and anthrax, it is also a well-known arrow poison, thus toxicity is to be expected. AIM OF THE STUDY: The cytotoxicity and preliminary mechanisms of toxicity in HepG2 hepatocarcinoma cells were assessed. MATERIALS AND METHODS: The effect of hot water and methanol extracts were on cell density, oxidative status, mitochondrial membrane potential, fatty acids, caspase-3/7 activity, adenosine triphosphate levels, cell cycling and viability was assessed. Phytochemicals were tentatively identified using ultra-performance liquid chromatography. RESULTS: The hot water extract displayed an IC50 of 24.26 µg/mL, and reduced proliferation (S- and G2/M-phase arrest) and viability (by 30.71%) as early as 24 h after incubation. The methanol extract had a comparable IC50 of 26.16 µg/mL, and arrested cells in the G2/M-phase (by 18.87%) and induced necrosis (by 13.21%). The hot water and methanol extracts depolarised the mitochondrial membrane (up to 0.84- and 0.74-fold), though did not generate reactive oxygen species. The hot water and methanol extracts decreased glutathione (0.42- and 0.62-fold) and adenosine triphosphate (0.08- and 0.26-fold) levels, while fatty acids (2.00- and 4.61-fold) and caspase-3/7 activity (1.98- and 5.82-fold) were increased. CONCLUSION: Extracts were both cytostatic and cytotoxic in HepG2 cells. Mitochondrial toxicity was evident and contributed to reducing adenosine triphosphate production and fatty acid accumulation. Altered redox status perturbed proliferation and promoted necrosis. Extracts of A. oppositifolia may thus promote necrotic cell death, which poses a risk for inflammatory hepatotoxicity with associated steatosis.

Comparative Evaluation of the Cytotoxicity of Doxorubicin in BT-20 Triple-Negative Breast Carcinoma Monolayer and Spheroid Cultures.

Three-dimensional cell culture models are increasingly adopted as preferred pre-clinical drug testing platforms, as they circumvent limitations associated with traditional monolayer cell cultures. However, many of these models are not fully characterized. This study aimed to characterize a BT-20 triple-negative breast carcinoma spheroid model and assess its susceptibility to doxorubicin in comparison to a monolayer model. Spheroids were developed using the liquid overlay method. Phenotypic attributes were analyzed by characterizing changes in size, gross morphology, protein content, metabolic activity, hypoxic status, and cell-cell junctions. The cytotoxic range of doxorubicin in monolayers was determined using the sulforhodamine B assay, and the comparative effect of toxic and sub-toxic concentrations was assessed in both spheroids and monolayers. Similar to the in vivo microenvironment, spheroids had a heterogeneous spatial cytoarchitecture, inherent hypoxia and strong adherens junctions. Doxorubicin induced dose-dependent cytotoxicity in monolayers (IC25: 130 nM, IC50: 320 nM and IC75: 1580 nM); however, these concentrations did not alter the spheroid size or acid phosphatase activity. Only concentrations ≥6 µM had any effect on spheroid integrity. In comparison to monolayers, the BT-20 spheroid model has decreased sensitivity to doxorubicin and could serve as a better model for susceptibility testing in triple-negative breast cancer.

Mechanisms facilitating the uptake of carboxyl-polythene glycol-functionalized gold nanoparticles into multicellular spheroids.

OBJECTIVES: Nanomedicines represent theragnostic alternatives to traditional candidate drugs, with increased targeting and delivery potential due to their size and functional tailorability. Biological activity typically relies on nanomaterials permeating into the intracellular environment, necessitating characterization of uptake and intracellular trafficking pathways. Spheroids' three-dimensional architecture and heterogenous cellular distribution offer an in-vivo-representative platform to assess the biological activity of nanoparticles (NPs). This study aimed to develop an A549 alveolar carcinoma spheroid model as a NP uptake assessment platform for carboxyl-polythene glycol-functionalized gold NPs affording further biological characterization opportunities in nanomedicine. METHODS: A549 spheroids were generated via the liquid overlay method, and their morphology and viability were assessed for 21 days. Cytotoxicity was assessed via lactate dehydrogenase release. NP uptake was elucidated using uptake pathway inhibition, combined with CytoViva hyperspectral imaging of sectioned spheroids to count internalized NPs. KEY FINDINGS: Cytotoxicity was absent for all exposure groups. Clathrin-mediated endocytosis was the primary endocytic mechanism (33.5-54.8% of uptake), which may precede lysosomal degradation. Lysosomal membrane permeabilization appears to be a potential downstream application. Low penetration into spheroids (4.5 µm) suggests the failure of NPs to traverse cellular layers in the spheroid. CONCLUSIONS: Although poor uptake was observed, a multicellular spheroid model of A549 alveolar carcinoma cells was established, allowing for similar future uptake assessment of various NPs.

Novel N-benzylpiperidine carboxamide derivatives as potential cholinesterase inhibitors for the treatment of Alzheimer's disease.

A series of fifteen acetylcholinesterase inhibitors were designed and synthesised based upon the previously identified lead compound 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-benzylpiperidine-4-carboxylate (5) which showed good inhibitory activity (IC50 0.03 ±â€¯0.07 µM) against acetylcholinesterase. A series of compounds were prepared wherein the ester linker in the original lead compound was exchanged for a more metabolically stable amide linker and the indanone moiety was exchanged for a range of aryl and aromatic heterocycles. The two most active analogues 1-benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide (28) and 1-benzyl-N-(1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide (20) afforded in vitro IC50 values of 0.41 ±â€¯1.25 and 5.94 ±â€¯1.08 µM, respectively. In silico screening predicts that 20 will be a blood brain-barrier permeant, and molecular dynamic simulations are indicative of a close correlation between the binding of 20 and the Food and Drug Administration-approved cholinesterase inhibitor donepezil (1).

Solamargine, a bioactive steroidal alkaloid isolated from Solanum aculeastrum induces non-selective cytotoxicity and P-glycoprotein inhibition.

BACKGROUND: Solanum aculeastrum fruits are used by some cancer sufferers as a form of alternative treatment. Scientific literature is scarce concerning its anticancer activity, and thus the aim of the study was to assess the in vitro anticancer and P-glycoprotein inhibitory potential of extracts of S. aculeastrum fruits. Furthermore, assessment of the combinational effect with doxorubicin was also done. METHODS: The crude extract was prepared by ultrasonic maceration. Liquid-liquid extraction yielded one aqueous and two organic fractions. Bioactive constituents were isolated from the aqueous fraction by means of column chromatography, solid phase extraction and preparative thin-layer chromatography. Confirmation of bioactive constituent identity was done by nuclear magnetic resonance and ultra-performance liquid chromatography mass spectrometry. The crude extract and fractions were assessed for cytotoxicity and P-glycoprotein inhibition in both cancerous and non-cancerous cell lines using the sulforhodamine B and rhodamine-123 assays, respectively. RESULTS: Both the crude extract and aqueous fraction was cytotoxic to all cell lines, with the SH-SY5Y neuroblastoma cell line being most susceptible to exposure (IC50 = 10.72 µg/mL [crude], 17.21 µg/mL [aqueous]). Dose-dependent P-glycoprotein inhibition was observed for the crude extract (5.9 to 18.9-fold at 100 µg/mL) and aqueous fraction (2.9 to 21.2 at 100 µg/mL). The steroidal alkaloids solamargine and solanine were identified. While solanine was not bioactive, solamargine displayed an IC50 of 15.62 µg/mL, and 9.1-fold P-glycoprotein inhibition at 100 µg/mL against the SH-SY5Y cell line. Additive effects were noted for combinations of doxorubicin against the SH-SY5Y cell line. CONCLUSIONS: The crude extract and aqueous fraction displayed potent non-selective cytotoxicity and noteworthy P-glycoprotein inhibition. These effects were attributed to solamargine. P-glycoprotein inhibitory activity was only present at concentrations higher than those inducing cytotoxicity, and thus does not appear to be the likely mechanism for the enhancement of doxorubicin's cytotoxicity. Preliminary results suggest that non-selective cytotoxicity may hinder drug development, however, further assessment of the mode of cell death is necessary to determine the route forward.

Targeting Alzheimer's disease by investigating previously unexplored chemical space surrounding the cholinesterase inhibitor donepezil.

A series of twenty seven acetylcholinesterase inhibitors, as potential agents for the treatment of Alzheimer's disease, were designed and synthesised based upon previously unexplored chemical space surrounding the molecular skeleton of the drug donepezil, which is currently used for the management of mild to severe Alzheimer's disease. Two series of analogues were prepared, the first looking at the replacement of the piperidine ring in donepezil with different sized saturated N-containing ring systems and the second looking at the introduction of different linkers between the indanone and piperidine rings in donepezil. The most active analogue 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-benzylpiperidine-4-carboxylate (67) afforded an in vitro IC50 value of 0.03 ± 0.07 µM against acetylcholinesterase with no cytotoxicity observed (IC50 of >100 µM, SH-SY5Y cell line). In comparison donepezil had an IC50 of 0.05 ± 0.06 µM and an observed cytotoxicity IC50 of 15.54 ± 1.12 µM. Molecular modelling showed a strong correlation between activity and in silico binding in the active site of acetylcholinesterase.

Attenuation of oxidative stress in U937 cells by polyphenolic-rich bark fractions of Burkea africana and Syzygium cordatum.

BACKGROUND: Oxidative stress has been implicated in the progression of various diseases, which may result in the depletion of endogenous antioxidants. Exogenous supplementation with antioxidants could result in increased protection against oxidative stress. As concerns have been raised regarding synthetic antioxidant usage, the identification of alternative treatments is justified. The aim of the present study was to determine the antioxidant efficacy of Burkea africana and Syzygium cordatum bark extracts in an in vitro oxidative stress model. METHODS: Cytotoxicity of crude aqueous and methanolic extracts, as well as polyphenolic-rich fractions, was determined in C2C12 myoblasts, 3T3-L1 pre-adipocytes, normal human dermal fibroblasts and U937 macrophage-like cells using the neutral red uptake assay. Polyphenolic content was determined using the Folin-Ciocalteau and aluminium trichloride assays, and antioxidant activity using the Trolox Equivalence Antioxidant Capacity and DPPH assays. The extracts efficacy against oxidative stress in AAPH-exposed U937 cells was assessed with regards to reactive oxygen species generation, cytotoxicity, apoptosis, lipid peroxidation and reduced glutathione depletion. RESULTS: B. africana and S. cordatum showed enrichment of polyphenols from the aqueous extract, to methanolic extract, to polyphenolic-rich fractions. Antioxidant activity followed the same trend, which correlated well with the increased concentration of polyphenols, and was between two- to three-fold stronger than the Trolox antioxidant control. Both plants had superior activity compared to ascorbic acid in the DPPH assay. Polyphenolic-rich fractions were most toxic to the 3T3-L1 (IC50's between 13 and 21 µg/ml) and C2C12 (IC50's approximately 25 µg/ml) cell lines, but were not cytotoxic in the U937 and normal human dermal fibroblasts cultures. Free radical-induced generation of reactive oxygen species (up to 80%), cytotoxicity (up to 20%), lipid peroxidation (up to 200%) and apoptosis (up to 60%) was successfully reduced by crude extracts of B. africana and the polyphenolic-rich fractions of both plants. The crude extracts of S. cordatum were not as effective in reducing cytotoxic parameters. CONCLUSION: Although oxidative stress was attenuated in U937 cells, cytotoxicity was observed in the 3T3-L1 and C2C12 cell lines. Further isolation and purification of polyphenolic-fractions could increase the potential use of these extracts as supplements by decreasing cytotoxicity and maintaining antioxidant quality.

Effects of selected South African plant extracts on haemolysis and coagulation.

The use of herbal preparations for staunching blood flow and reducing the risk of vascular thrombosis is common worldwide. In this study, aqueous and methanolic extracts of plants used to treat blood-associated complaints were investigated to determine their effects on red blood cell haemolysis and coagulation. The extent of haemolysis was determined spectrophotometrically. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) as indicators of coagulation rate were determined using a coagulatometer. All of the plant extracts tested had a significant effect on coagulation time, prolonging the aPTT. Cassia petersiana had the greatest prolonging effect on PT compared to the control, phosphate buffered saline (PBS). As all of the herbal extracts tested had a delaying effect on coagulation, patients using herbal/plant therapies should be cautioned to stop their medication before surgery.

Herbal remedies affecting coagulation: a review.

CONTEXT: Herbal remedies are used to treat a large variety of diseases, including blood-related disorders. However, a number of herbal preparations have been reported to cause variations in clotting time, this is mainly by disruption of the coagulation cascade. OBJECTIVE: The compiling of plants investigated for effects on the coagulation cascade. METHODS: Information was withdrawn from Google Scholar and the journal databases Scopus and PubMed. RESULTS: Sixty-five herbal remedies were identified with antiplatelet, anticoagulant, or coagulating ability. Bioactive compounds included polyphenols, taxanes, coumarins, saponins, fucoidans, and polysaccharides. CONCLUSION: Although research has been conducted on the effect of herbal remedies on coagulation, most information relies on in vitro assays. Contradictory evidence is present on bleeding risks with herbal uses, though herb-drug interactions pose a threat. As the safety of many herbals has not been proven, nor their effect on blood parameters determined, the use of herbal preparations before undergoing any surgical procedure should discontinued.