Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone from Syzygium nervosum Seeds on Antiproliferative,DNA Damage,Cell Cycle Arrest,and Apoptosis in Human Cervical Cancer Cell Lines

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a natural product derived from Syzygium nervosum A. Cunn. ex DC., was investigated for its inhibitory activities against various cancer cell lines. In this work, we investigated the effects of DMC and available anticervical cancer drugs (5-fluorouracil, cisplatin, and doxorubicin) on three human cervical cancer cell lines (C-33A, HeLa, and SiHa). DMC displayed antiproliferative cervical cancer activity in C-33A, HeLa, and SiHa cells, with IC₅₀ values of 15.76 ± 1.49, 10.05 ± 0.22, and 18.31 ± 3.10 µM, respectively. DMC presented higher antiproliferative cancer activity in HeLa cells; therefore, we further investigated DMC-induced apoptosis in this cell line, including DNA damage, cell cycle arrest, and apoptosis assays. As a potential anticancer agent, DMC treatment increased DNA damage in cancer cells, observed through fluorescence inverted microscopy and a comet assay. The cell cycle assay showed an increased number of cells in the G₀/G₁ phase following DMC treatment. Furthermore, DMC treatment-induced apoptosis cell death was approximately three- to four-fold higher compared to the untreated group. Here, DMC represented a compound-induced apoptosis for cell death in the HeLa cervical cancer cell line. Our findings suggest that DMC, a phytochemical agent, is a potential candidate for antiproliferative cervical cancer drug development.     

Molecular Determinant of DIDS Analogs Targeting RAD51 Activity

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.     

Development and Validation of an HPLC-UV Method for the Quantification of 4′-Hydroxydiclofenac Using Salicylic Acid: Future Applications for Measurement of In Vitro Drug–Drug Interaction in Rat Liver Microsomes

Salicylic acid is a key compound in nonsteroidal anti-inflammatory drugs that has been recently used for preventing the risk of hospitalization and death among COVID-19 patients and in preventing colorectal cancer (CRC) by suppressing two key proteins. Understanding drug–drug interaction pathways prevent the occurrence of adverse drug reactions in clinical trials. Drug–drug interactions can result in the variation of the pharmacodynamics and pharmacokinetic of the drug. Inhibition of the Cytochrome P450 enzyme activity leads to the withdrawal of the drug from the market. The aim of this paper was to develop and validate an HPLC-UV method for the quantification of 4′-hydroxydiclofenac as a CYP2C9 metabolite using salicylic acid as an inhibitor in rat liver microsomes. A CYP2C9 assay was developed and validated on the reversed phase C₁₈ column (SUPELCO 25 cm × 4.6 mm × 5 µm) using a low-pressure gradient elution programming at T = 30 °C, a wavelength of 282 nm, and a flow rate of 1 mL/min. 4′-hydroxydiclofenac demonstrated a good linearity (R² > 0.99), good reproducibility, low detection, and quantitation limit, and the inter and intra-day precision met the ICH guidelines (<15%). 4′-hydroxydiclofenac was stable for three days and showed an acceptable accuracy and recovery (80–120%) within the ICH guidelines in a rat liver microsome sample. This method will be beneficial for future applications of the in vitro inhibitory effect of salicylic acid on the CYP2C9 enzyme activity in rat microsomes and the in vivo administration of salicylic acid in clinical trials.     

Cytochrome P450 reaction phenotyping and inhibition and induction studies of pinostrobin in human liver microsomes and hepatocytes

Pinostrobin (PI, 5‐hydroxy‐7‐methoxyflavanone) is a natural flavonoid known for its rich pharmacological activities. The objective of this study was to identify the human liver cytochrome P450 (CYP450) isoenzymes involved in the metabolism of PI. A single hydoxylated metabolite was obtained from PI after an incubation with pooled human liver microsomes (HLMs). The relative contributions of different CYP450s were evaluated using CYP450‐selective inhibitors in HLMs and recombinant human CYP450 enzymes, and the results revealed the major involvement of CYP1A2, CYP2C9 and CYP2E1 in PI metabolism. We also evaluated the ability of PI to inhibit and induce human cytochrome P450 enzymes in vitro . High‐performance liquid chromatography and liquid chromatography–tandem mass spectrometry analytical techniques were used to estimate the enzymatic activities of seven drug‐metabolizing CYP450 isozymes in vitro . In HLMs, PI did not inhibit CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 (IC₅₀ > 100 μm ). In the induction studies, PI had minimal effects on CYP1A2, CYP2B6and CYP3A4 activity. Based on these results, PI would not be expected to cause clinically significant CYP450 inhibition or induction.     

The Biological Fate of a Novel Anticancer Drug Candidate TNBG-5602: Metabolic Profile,Interaction with CYP450, and Pharmacokinetics in Rats

TNBG-5602, a novel anticancer drug candidate, may induce the expression of PPARγ, causing targeted lipotoxicity in cancer tissues. In this study, the in vivo metabolism in rats, in vitro metabolism in recombinant cytochromes, molecular docking for the CYP binding site, and pharmacokinetics in rats were explored to better understand TNBG-5602′s in vivo fate and behavior. Thirteen metabolites were identified using a high-resolution mass spectrometry method, and metabolizing pathways of TNBG-5602 were proposed. Results suggest that TNBG-5602 could be metabolized by CYP450s, while CYP2D6 may play an important role in its in vivo metabolism. The main metabolizing sites of TNBG-5602 are the amino group on the side chain and rings A and E in the molecule. TNBG-5602 is a potent CYP2D6 inhibitor, with an IC₅₀ value of 2.52 μM. An interaction responsible for its metabolism is formed by the NH on the side chain bonding with the ASP301 on the CYP2D6. The pharmacokinetics in rats after a single intravenous administration were fitted to a two-compartment model. The clearance was 0.022 L min⁻¹, and the elimination half-life was 710.9 min. The distribution volume of the peripheral compartment was 1.88-fold that of the central compartment, while the K₁₂ was 1.5-fold that of K₂₁. In conclusion, these studies have not only revealed the metabolizing pathways of TNBG-5602 using in vivo and in vitro methodology, but they have also provided the pharmacokinetic characteristics of TNBG-5602 in rats. The results suggest that TNBG-5602 has good drug developability in terms of pharmacokinetic behaviors.     

Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5′,5′-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging – they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands – folic acid, biotin, glucose, and cholesterol – to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure–activity relationship (SAR) study using model fluorescent probes and cap–ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Ligand assisted cellular delivery of negatively charged dinucleotides, which are potential antagonists of the protooncogenic protein eIF4E.     

New Polymethoxyflavones from Hottonia palustris Evoke DNA Biosynthesis-Inhibitory Activity in An Oral Squamous Carcinoma (SCC-25) Cell Line

Four new compounds, 5-hydroxy-2′,6′-dimethoxyflavone (4), 5-hydroxy-2′,3′,6′-trimethoxyflavone (5), 5-dihydroxy-6-methoxyflavone (6), and 5,6′-dihydroxy-2′,3′-dimethoxyflavone (7), and three known compounds, 1,3-diphenylpropane-1,3-dione (1), 5-hydroxyflavone (2), and 5-hydroxy-2′-methoxyflavone (3), were isolated from the aerial parts of Hottonia palustris. Their chemical structures were determined through the use of spectral, spectroscopic and crystallographic methods. The quantitative analysis of the compounds (1–7) and the zapotin (ZAP) in methanol (HP1), petroleum (HP6), and two chloroform extracts (HP7 and HP8) were also determined using HPLC-PDA. The biological activity of these compounds and extracts on the oral squamous carcinoma cell (SCC-25) line was investigated by considering their cytotoxic effects using the MTT assay. Subsequently, the most active compounds and extracts were assessed for their effect on DNA biosynthesis. It was found that all tested samples during 48 h treatment of SCC-25 cells induced the DNA biosynthesis-inhibitory activity: compound 1 (IC₅₀, 29.10 ± 1.45 µM), compound 7 (IC₅₀, 40.60 ± 1.65 µM) and extracts ZAP (IC₅₀, 20.33 ± 1.01 µM), HP6 (IC₅₀, 14.90 ± 0.74 µg), HP7 (IC₅₀, 16.70 ± 0.83 µg), and HP1 (IC₅₀, 30.30 ± 1.15 µg). The data suggest that the novel polymethoxyflavones isolated from Hottonia palustris evoke potent DNA biosynthesis inhibitory activity that may be considered in further studies on experimental pharmacotherapy of oral squamous cell carcinoma.     

Alpha-Glucosidase Inhibitory Assay-Screened Isolation and Molecular Docking Model from Bauhinia pulla Active Compounds

The aim of this research was to establish the constituents of Bauhinia pulla as anti-diabetic agents. A phytochemistry analysis was conducted by chromatographic and spectroscopic techniques. The alpha-glucosidase inhibitory assay screening resulted in the isolation of eight known compounds of quercetin, quercitrin, luteolin, 5-deoxyluteolin, 4-methyl ether isoliquiritigenin, 3,2′,4′-trihydroxy-4-methoxychalcone, stigmasterol and β-sitosterol. Ethanol leaf extracts showed potential effects, which led to a strong inhibitory activity of isolated quercetin at 138.95 µg/mL and 5.41 µg/mL of IC₅₀, respectively. The docking confirmed that flavonoids and chalcones had the same potential binding sites and responsibilities for their activity. This study was the first report of Bauhinia pulla chemical constituents and its alpha-glucosidase inhibition.     

Development of Bicyclo[3.1.0]hexane-Based A3 Receptor Ligands: Closing the Gaps in the Structure–Affinity Relationships

The adenosine A₃ receptor is a promising target for treating and diagnosing inflammation and cancer. In this paper, a series of bicyclo[3.1.0]hexane-based nucleosides was synthesized and evaluated for their P1 receptor affinities in radioligand binding studies. The study focused on modifications at 1-, 2-, and 6-positions of the purine ring and variations of the 5′-position at the bicyclo[3.1.0]hexane moiety, closing existing gaps in the structure–affinity relationships. The most potent derivative 30 displayed moderate A₃AR affinity (Ki of 0.38 μM) and high A₃R selectivity. A subset of compounds varied at 5′-position was further evaluated in functional P2Y₁R assays, displaying no off-target activity.     

Potential Effects of Ibuprofen,Remdesivir and Omeprazole on Dexamethasone Metabolism in Control Sprague Dawley Male Rat Liver Microsomes (Drugs Often Used Together Alongside COVID-19 Treatment)

The role of individual cytochrome P450 (CYPs) responsible for the drug metabolism can be determined through their chemical inhibition. During the pandemic, dexamethasone and remdesivir with omeprazole were used for the treatment of COVID-19, while Ibuprofen was taken to treat the symptoms of fever and headache. This study aimed to examine the potency of ibuprofen remdesivir, and omeprazole as inhibitors of cytochrome P450s using rat liver microsomes in vitro. Dexamethasone a corticosteroid, sometimes used to reduce the body’s immune response in the treatment of COVID-19, was used as a probe substrate and the three inhibitors were added to the incubation system at different concentrations and analysed by a validated High Performance Liquid Chromatography (HPLC) method. The CYP3A2 isoenzyme is responsible for dexamethasone metabolism in vitro. The results showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with K_i = 224.981 ± 1.854 µM and IC₅₀ = 230.552 ± 2.020 µM, although remdesivir showed a mixed inhibition pattern with a K_i = 22.504 ± 0.008 µM and IC₅₀ = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a K_i = 39.175 ± 0.230 µM and IC₅₀ = 78.351 ± 0.460 µM. These results suggest that the tested inhibitors would not exert a significant effect on the CYP3A2 isoenzyme responsible for the co-administered dexamethasone drug’s metabolism in vivo.     

Phytochemical Investigation and Biological Activities of Lantana rhodesiensis

Lantana rhodesiensis Moldenke is a plant widely used to treat diseases, such as rheumatism, diabetes, and malaria in traditional medicine. To better understand the traditional uses of this plant, a phytochemical study was undertaken, revealing a higher proportion of polyphenols, including flavonoids in L. rhodesiensis leaf extract and moderate proportion in stem and root extracts. The antioxidant activity of the extracts was also determined using three different assays: the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, the FRAP method (Ferric-reducing antioxidant power) and the β-carotene bleaching test. The anti-malarial activity of each extract was also evaluated using asexual erythrocyte stages of Plasmodium falciparum, chloroquine-sensitive strain 3D7. The results showed that the leaf extract exhibited higher antioxidant and anti-malarial activities in comparison with the stem and root extracts, probably due to the presence of higher quantities of polyphenols including flavonoids in the leaves. A positive linear correlation was established between the phenolic compound content (total polyphenols including flavonoids and tannins; and total flavonoids) and the antioxidant activity of all extracts. Furthermore, four flavones were isolated from leaf dichloromethane and ethyl acetate fractions: a new flavone named rhodescine (5,6,3′,5′-tetrahydroxy-7,4′-dimethoxyflavone) (1), 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (2), 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (3), and 5,6,3′-trihydroxy-7,4′-dimethoxyflavone (4). Their structures were elucidated by ¹H, ¹³CNMR, COSY, HSQC, HMBC, and MS-EI spectral methods. Aside from compound 2, all other molecules were described for the first time in this plant species.     

Concentration of Bioactive Phenolic Compounds in Olive Mill Wastewater by Direct Contact Membrane Distillation

Olive mill wastewater (OMW), generated as a by-product of olive oil production, is considered one of the most polluting effluents produced by the agro-food industry, due to its high concentration of organic matter and nutrients. However, OMW is rich in several polyphenols, representing compounds with remarkable biological properties. This study aimed to analyze the chemical profile as well as the antioxidant and anti-obesity properties of concentrated fractions obtained from microfiltered OMW treated by direct contact membrane distillation (DCMD). Ultra-high performance liquid chromatography (UHPLC) analyses were applied to quantify some phenols selected as phytochemical markers. Moreover, α-Amylase, α-glucosidase, and lipase inhibitory activity were investigated together with the antioxidant activity by means of assays, namely β-carotene bleaching, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS) diammonium salts, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and Ferric Reducing Activity Power (FRAP) tests. MD retentate—which has content of about five times greater of hydroxytyrosol and verbascoside and about 7 times greater of oleuropein than the feed—was more active as an antioxidant in all applied assays. Of interest is the result obtained in the DPPH test (an inhibitory concentration 50% (IC₅₀) of 9.8 μg/mL in comparison to the feed (IC₅₀ of 97.2 μg/mL)) and in the ABTS assay (an IC₅₀ of 0.4 μg/mL in comparison to the feed (IC₅₀ of 1.2 μg/mL)).     

Using the Method of “Optical Biopsy” of Prostatic Tissue to Diagnose Prostate Cancer

Simple SummaryAnalytical discrimination models of Raman spectra of prostate cancer tissue were constructed by using the projections onto latent structures data analysis (PLS-DA) method for different wavelengths of exciting radiation—532 and 785 nm. These models allowed us to divide the Raman spectra of prostate cancer and the spectra of hyperplasia sites for validation datasets with the accuracy of 70–80%, depending on the specificity value. Meanwhile, for the calibration datasets, the accuracy values reached 100% for the excitation of a laser with a wavelength of 785 nm. Due to the registration of Raman “fingerprints”, the main features of cellular metabolism occurring in the tissue of a malignant prostate tumor were confirmed, namely the absence of aerobic glycolysis, over-expression of markers, and a strong increase in the concentration of cholesterol and its esters, as well as fatty acids and glutamic acid.AbstractThe possibilities of using optical spectroscopy methods in the differential diagnosis of prostate cancer were investigated. Analytical discrimination models of Raman spectra of prostate tissue were constructed by using the projections onto latent structures data analysis(PLS-DA) method for different wavelengths of exciting radiation—532 and 785 nm. These models allowed us to divide the Raman spectra of prostate cancer and the spectra of hyperplasia sites for validation datasets with the accuracy of 70–80%, depending on the specificity value. Meanwhile, for the calibration datasets, the accuracy values reached 100% for the excitation of a laser with a wavelength of 785 nm. Due to the registration of Raman “fingerprints”, the main features of cellular metabolism occurring in the tissue of a malignant prostate tumor were confirmed, namely the absence of aerobic glycolysis, over-expression of markers (FASN, SREBP1, stearoyl-CoA desaturase, etc.), and a strong increase in the concentration of cholesterol and its esters, as well as fatty acids and glutamic acid. The presence of an ensemble of Raman peaks with increased intensity, inherent in fatty acid, beta-glucose, glutamic acid, and cholesterol, is a fundamental factor for the identification of prostate cancer.     

Tacrine-Coumarin Derivatives as Topoisomerase Inhibitors with Antitumor Effects on A549 Human Lung Carcinoma Cancer Cell Lines

A549 human lung carcinoma cell lines were treated with a series of new drugs with both tacrine and coumarin pharmacophores (derivatives 1a–2c) in order to test the compounds’ ability to inhibit both cancer cell growth and topoisomerase I and II activity. The ability of human topoisomerase I (hTOPI) and II to relax supercoiled plasmid DNA in the presence of various concentrations of the tacrine-coumarin hybrid molecules was studied with agarose gel electrophoresis. The biological activities of the derivatives were studied using MTT assays, clonogenic assays, cell cycle analysis and quantification of cell number and viability. The content and localization of the derivatives in the cells were analysed using flow cytometry and confocal microscopy. All of the studied compounds were found to have inhibited topoisomerase I activity completely. The effect of the tacrine-coumarin hybrid compounds on cancer cells is likely to be dependent on the length of the chain between the tacrine and coumarin moieties (1c, 1d = tacrine-(CH₂)_8–9-coumarin). The most active of the tested compounds, derivatives 1c and 1d, both display longer chains.     

β-Carotene: Preventive Role for Type 2 Diabetes Mellitus and Obesity: A Review

Carotenoids are vital antioxidants for plants and animals. They protect cells from oxidative events and act against the inflammatory process and carcinogenesis. Among the most abundant carotenoids in human and foods is β-carotene. This carotenoid has the highest level of provitamin A activity, as it splits into two molecules of retinol through the actions of the cytosolic enzymes: β-carotene-15,15′-monooxygenase (β-carotene-15,15′-oxygenase 1) and β-carotene-9′,10′-dioxygenase (β-carotene-9′,10′-oxygenase 2). The literature supports the idea that β-carotene acts against type 2 diabetes mellitus, cardiovascular diseases, obesity, and metabolic syndrome. Due to the many processes involved in β-carotene biosynthesis and metabolic function, little is known about such components, since many mechanisms have not yet been fully elucidated. Therefore, our study concisely described the relationships between the consumption of carotenoids, with emphasis on β-carotene, and obesity and type 2 diabetes mellitus and its associated parameters in order to understand the preventive role of carotenoids better and encourage their consumption.     

Determination of Flavonoids in Selected Scleranthus Species and Their Anti-Collagenase and Antioxidant Potential

A new 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-arabinopyranoside-2″-O-(4‴-acetoxy)-glucoside (6) and three known flavone C-glycosides—5,7,3′,4′-tetrahydroxyflavone-6-C-xyloside-8-C-β-d-glucoside (lucenin-1) (7), 5,7,3′-trihydroxyflavone-6-C-glucoside-8-C-β-d-glucoside (vicenin-2) (8), and 5,7,4′-trihydroxy-3′-methoxyflavone-6-C-β-d-glucopyranoside-8-C-α-arabinopyranoside (chrysoeriol-6-C-β-d-glucopyranoside-8-C-α-arabinopyranoside) (9)—were isolated from aerial parts of Scleranthus perennis L. (Caryophyllaceae). Their structures were determined through the use of comprehensive spectroscopic and spectrometric methods, and a method for the quantification of the major constituents of S. perennis and S. annuus L. was developed. Furthermore, the anti-collagenase and antioxidant activities of all isolated compounds obtained from extracts and fractions from both Scleranthus species were evaluated. The highest percentage of collagenase inhibition (at 400 µg/mL) was distinguished for methanolic extracts (22.06%, 32.04%) and ethyl acetate fractions (16.59%, 14.40%) from S. annuus and S. perennis. Compounds 6–9 displayed moderate inhibitory activity, with IC₅₀ values ranging from 39.59–73.86 µM.     

Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC₅₀ = 2.5 ± 0.6 µM, K_i = 2.4 ± 1.0 µM, k_inact = 0.07 ± 0.01 min⁻¹, k_inact/K_i = 0.03 min⁻¹ µM⁻¹. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.