Search Results (17)

Targeted cancer therapy aims to disrupt the functions of proteins that regulate cancer progression, mainly by using small molecule inhibitors (SMIs). SMIs exert their effect by modulating signalling pathways, organelle integrity, chromatin components, and several biosynthetic processes essential for cell division and survival. Antiapoptotic protein BCL2 is highly upregulated in many cancers compared with normal cells, making it an ideal target for cancer therapy. Around 75% of primary breast cancers overexpress BCL2, providing an opportunity to explore BCL2 inhibitors as a therapeutic option. Disarib is an SMI that has been developed as a selective BCL2 inhibitor. Disarib works by disrupting BCL2-BAK interaction and activating intrinsic apoptotic pathways in leukemic cells while sparing normal cells. We investigated the effects of Disarib, a BCL2 specific inhibitor, on breast cancer cells and xenografts. Cytotoxicity and fluorometric assays revealed that Disarib induced cell death by increasing reactive oxygen species and activating intrinsic apoptotic pathways in Triple-Negative Breast Cancer cells (MDA-MB-231 and MDA-MB-468). Disarib also affected the colony-forming properties of these cells. MDA-MB-231- and MDA-MB-468-derived xenografts showed a significant reduction in tumours upon Disarib treatment. Through the transcriptomics approach, we also explored the influence of BCL2 inhibitors on energy metabolism, mitochondrial dynamics, and epithelial-to-mesenchymal transition (EMT). Mitochondrial dynamics and glucose metabolism mainly regulate energy metabolism. The change in energetics regulates tumour growth through epithelial–mesenchymal transition, and angiogenesis. RNA sequencing (RNAseq) analysis revealed that BCL2 inhibitors ABT-199 and Disarib maintain Oxphos levels in MDA-MB-231. However, key glycolytic genes were significantly downregulated. Mitochondrial fission genes were seen to be downregulated both in RNAseq data and semi quantitative real time polymerase chain reaction (qRTPCR) in Disarib-treated TNBC cells and xenografts. Lastly, Disarib inhibited wound healing and epithelial-to-mesenchymal transition. This study showed that Disarib disrupts mitochondrial function, activates the intrinsic apoptotic pathway in breast cancer, and inhibits epithelial-to-mesenchymal transition both in vitro and in vivo. These findings highlight Disarib’s potential as a multifaceted therapeutic strategy for patients with Triple-Negative Breast Cancer. Full article

Millet, a gluten-free cereal, has received attention for its environmental friendliness and higher protein content than other grains. It represents a staple food in many African countries, where fermentation is traditionally used for preserving food products and preparing different cereal-based products. This study aimed to assess the impact of sourdough fermentation on bioactive compounds and antioxidant and anti-inflammatory properties of pearl millet from Burkina Faso. Phenolic compounds were investigated spectrophotometrically and by HPLC-DAD. The antioxidant activity of unfermented (MF) and fermented (FeMF) millet was evaluated in vitro by spectrophotometric and fluorometric assays and ex vivo on oxidized human erythrocytes for hemolysis inhibition. Finally, the potential anti-inflammatory effect of FeMF and MF was evaluated on human adenocarcinoma cell line (HT-29) exposed to TNF-α inflammatory stimulus. Results revealed significantly higher levels of polyphenols, flavonoids, and in vitro antioxidant activity following millet fermentation. Notable differences in phenolic composition between FeMF and MF are observed, with fermentation facilitating the release of bioactive compounds such as gallic acid, quercetin, and rutin. A dose-dependent protection against oxidative hemolysis was observed in both FeMF- and MF-pretreated erythrocytes. Similarly, pretreatment with FeMF significantly reduced the levels of inflammatory markers in TNF-α-treated cells, with effects comparable to those of MF. Fermentation with sourdough represents a simple and low-cost method to improve the bioactive compounds content and in vitro antioxidant activity of millet flour with promising nutraceutical potential. Full article

Recent studies have demonstrated the association of APP and Aβ with cancer, suggesting that BACE1 may play an important role in carcinogenesis. In the present study, we assessed BACE1’s usefulness as a therapeutic target in prostate cancer (PCa). BACE1 expression was observed in human PCa tissue samples, patient-derived xenografts (PDX), human PCa xenograft tissue in nude mice, and transgenic adenocarcinoma of the mouse prostate (TRAMP) tissues by immunohistochemistry (IHC) analysis. Additionally, the downstream product of BACE1 activity, i.e., Aβ1-42 expression, was also observed in these PCa tissues by IHC as well as by PET imaging in TRAMP mice. Furthermore, BACE1 gene expression and activity was confirmed in several established PCa cell lines (LNCaP, C4-2B-enzalutamide sensitive [S], C4-2B-enzalutamide resistant [R], 22Rv1-S, 22Rv1-R, PC3, DU145, and TRAMP-C1) by real-time PCR and fluorometric assay, respectively. Treatment with a pharmacological inhibitor of BACE1 (MK-8931) strongly reduced the proliferation of PCa cells in in vitro and in vivo models, analyzed by multiple assays (MTT, clonogenic, and trypan blue exclusion assays and IHC). Cell cycle analyses revealed an increase in the sub-G1 population and a significant modulation in other cell cycle stages (G1/S/G2/M) following MK-8931 treatment. Most importantly, in vivo administration of MK-8931 intraperitoneal (30 mg/kg) strongly inhibited TRAMP-C1 allograft growth in immunocompetent C57BL/6 mice (approximately 81% decrease, p = 0.019). Furthermore, analysis of tumor tissue using the prostate cancer-specific pathway array revealed the alteration of several genes involved in PCa growth and progression including Forkhead O1 (FOXO1). All together, these findings suggest BACE1 as a novel therapeutic target in advanced PCa. Full article

Porphyromonas gingivalis is a periodontopathogenic bacterium that can adhere to and colonize periodontal tissues, leading to an inflammatory process, and, consequently, tissue destruction. New therapies using flavonoids, such as hesperidin, are being studied, and their promising properties have been highlighted. The aim of this study was to evaluate the effect of hesperidin on the epithelial barrier function, reactive oxygen species (ROS) production, and on the inflammatory response caused by P. gingivalis in in vitro models. The integrity of the epithelial tight junctions challenged by P. gingivalis was determined by monitoring the transepithelial electrical resistance (TER). P. gingivalis adherence to a gingival keratinocyte monolayer and a basement membrane model were evaluated by a fluorescence assay. A fluorometric assay was used to determine the ROS production in gingival keratinocytes. The level of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) secretion was evaluated by ELISA; to assess NF-κB activation, the U937-3xjB-LUC monocyte cell line transfected with a luciferase reporter gene was used. Hesperidin protected against gingival epithelial barrier dysfunction caused by P. gingivalis and reduced the adherence of P. gingivalis to the basement membrane model. Hesperidin dose-dependently inhibited P. gingivalis-mediated ROS production by oral epithelial cells as well as the secretion of IL-1β, TNF-α, IL-8, MMP-2, and MMP-9 by macrophages challenged with P. gingivalis. Additionally, it was able to attenuate NF-κB activation in macrophages stimulated with P. gingivalis. These findings suggest that hesperidin has a protective effect on the epithelial barrier function, in addition to reducing ROS production and attenuating the inflammatory response associated with periodontal disease. Full article

Quarantine pathogens require the investigation of new tools for effective plant protection. In particular, research on sustainable agrochemicals is the actual challenge. Plant extracts, essential oils, and gels are natural sources of efficient biocides, such as aromatic secondary metabolites. Thymol is the major phenolic constituent of thyme and oregano essential oils, and it can inhibit many pathogenic microbes. Thymol nanoparticles were obtained through adsorption on CaCO₃ nanocrystals, exploiting their carrier action. High loading efficiency and capability were reached as verified through UV and TGA measurements. We report the first study of thymol effect on Xylella fastidiosa, conducing both fluorometric assay and in vitro inhibition assay. The first test confirmed the great antibacterial effect of this compound. Finally, an in vitro test revealed an interesting synergistic action of thymol and nanocarriers, suggesting the potential application of thymol-nanoparticles as effective biocides to control Xylella fastidiosa infection. Full article

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD. Full article

Stevia species (Asteraceae) have been a rich source of terpenoid compounds, mainly sesquiterpene lactones, several of which show antiprotozoal activity. In the search for new trypanocidal compounds, S. satureiifolia var. satureiifolia and S. alpina were studied. Two sesquiterpene lactones, santhemoidin C and 2-oxo-8-deoxyligustrin, respectively, were isolated. These compounds were assessed in vitro against Trypanosoma cruzi stages, showing IC₅₀ values of 11.80 and 4.98 on epimastigotes, 56.08 and 26.19 on trypomastigotes and 4.88 and 20.20 µM on amastigotes, respectively. Cytotoxicity was evaluated on Vero cells by the MTT assay. The effect of the compounds on trypanothyone reductase (TcTR), Trans-sialidase (TcTS) and the prolyl oligopeptidase of 80 kDa (Tc80) as potential molecular targets of T. cruzi was investigated. Santhemoidin C inhibited oligopeptidase activity when tested against recombinant Tc80 using a fluorometric assay, reaching an IC₅₀ of 34.9 µM. Molecular docking was performed to study the interaction between santhemoidin C and the Tc80 protein, reaching high docking energy levels. Plasma membrane shedding and cytoplasmic vacuoles, resembling autophagosomes, were detected by transmission microscopy in parasites treated with santhemoidin C. Based on these results, santhemoidin C represents a promising candidate for further studies in the search for new molecules for the development of trypanocidal drugs. Full article

2-Styrylchromones (2-SC) are heterocyclic compounds with a structure of at least 17 carbons and a styryl group attached to a benzoannelated γ-pyrone ring. Although the anti-inflammatory potential of 2-SC has become a subject of interest, their effects in inflammatory pathways are still unexplored. Therefore, to better understand the mechanisms of anti-inflammatory action of 2-SC, this study investigated the influence of 10 hydroxylated and methoxylated 2-SC on the inhibitory activity of cyclooxygenase (COX-2), through an in vitro non-cellular assay and an ex vivo assay in human whole blood, which were based on the fluorometric detection of prostaglandin (PG) G₂ and colorimetric detection of PGE₂, respectively. A 2-SC hydroxylated at C-7 and C-8 on the A-ring and C-3′ and C-4′ on the B-ring was the most active in the direct inhibition of COX-2 activity, whereas a 2-SC methoxylated at C-4′ on the B-ring was the most active in the ex vivo inhibition of PGE₂ production. The obtained results suggest that the presence of OH groups, especially at C-8 on the A-ring, favor the direct inhibition of COX-2. Conversely, for inhibition of PGE₂ production in a more complex matrix, human blood, it is the presence of an OCH₃ at C-4′ on the B-ring that seems to be important. Full article

Angiotensin-converting enzyme (ACE) inhibitors are anti-hypertensive drugs associated with several side effects. Natural compounds, namely bioactive peptides from brewing by-products—brewer’s spent grain (BSG) and yeast (BSY)—are promising alternatives, as they can inhibit ACE in vitro and are less likely to cause severe side effects, while maintaining therapeutic efficacy. However, the impact of oral administration on peptides’ bioavailability has not been assessed so far. Thus, the aim of this study was to understand in vitro the impact of the oral route on the effectiveness of BSG/BSY peptides as ACE inhibitors. Extracted BSG/BSY proteins were hydrolysed and sequentially subjected to simulated gastrointestinal digestion (INFOGEST), intestinal absorption and liver metabolism (co-culture of Caco-2 and HepG2 cells). MTT assay was used to assess BSG/BSY peptides’ safeness. The ACE-inhibitory potential of initial and final products (BSY, BSG and a mixture 50:50—MIX) at an identical concentration (0.857mg/mL) was measured (fluorometric assay) and compared with Captopril (1 µM, a clinically used ACE-inhibitory drug). Simulation of oral administration increased brewing peptides’ ACE-inhibitory capacity. When comparing the final peptides with captopril, BSY demonstrated identical potency, while BSG showed 22% greater efficacy; the new tested product MIX presented 30% higher inhibition. In conclusion, the current study shows that BSG, BSY and MIX natural peptides derived from the brewing industry enhance their bioactive properties as ACE-inhibitors after oral administration, validating the usefulness of these peptides to reduce the risk of, ameliorate or treat primary hypertension. Full article

The treatment of hypertension is of major importance to reduce the risk of cardiovascular disease, the leading cause of death worldwide. Angiotensin-converting enzyme (ACE) inhibitors are anti-hypertensive drugs associated with several side effects. Natural products, namely bioactive peptides from brewing by-products, brewers’ spent grain (BSG), and yeast (BSY), are promising alternatives since they can inhibit ACE in vitro. However, the oral intake of these peptides may modify their expected inhibitory effect owing to possible changes in active peptides’ bioavailability, which have not been assessed so far. The goal of this study was to simulate oral administration to evaluate BSG/BSY peptides’ effectiveness by submitting protein hydrolysates sequentially to simulated gastrointestinal digestion, intestinal absorption (Caco-2 cells), and liver metabolism (HepG2 cells). MTT assay was used to assess BSG/BSY protein hydrolysates safeness. The ACE-inhibitory potential of initial and final protein hydrolysates (BSY, BSG, and a new product, MIX) were tested using a fluorometric assay and compared with captopril (1 µM, an ACE-inhibitory drug). Simulation of oral administration greatly increased BSY and MIX protein hydrolysates’ ACE-inhibitory capacity, though final MIX and BSG revealed greater ACE-inhibitory potential than captopril. Notwithstanding, all final protein hydrolysates presented ACE-inhibitory capacity, thus being promising compounds to manage hypertension. Full article

Pueraria lobata leaves contain a variety of phytoestrogens, including flavonoids, isoflavonoids, and coumestan derivatives. In this study, we aimed to identify the active ingredients of P. lobata leaves and to elucidate their function in monoamine oxidase (MAO) activation and Aβ self-aggregation using in vitro and in silico approaches. To the best of our knowledge, this is the first study to elucidate coumestrol as a selective and competitive MAO-A inhibitor. We identified that coumestrol, a coumestan-derivative, exhibited a selective inhibitory effect against MAO-A (IC₅₀ = 1.99 ± 0.68 µM), a key target protein for depression. In a kinetics analysis with 0.5 µg MAO-A, 40–160 µM substrate, and 25 °C reaction conditions, coumestrol acts as a competitive MAO-A inhibitor with an inhibition constant of 1.32 µM. During an in silico molecular docking analysis, coumestrol formed hydrogen bonds with FAD and pi–pi bonds with hydrophobic residues at the active site of the enzyme. Moreover, based on thioflavin-T-based fluorometric assays, we elucidated that coumestrol effectively prevented self-aggregation of amyloid beta (Aβ), which induces an inflammatory response in the central nervous system (CNS) and is a major cause of Alzheimer’s disease (AD). Therefore, coumestrol could be used as a CNS drug to prevent diseases such as depression and AD by the inhibition of MAO-A and Aβ self-aggregation. Full article

Many organophosphorus compounds (OPs), especially various α-aminophosphonates, exhibit anti-cancer activities. They act, among others, as inhibitors of the proteases implicated in cancerogenesis. Thesetypes of inhibitors weredescribed, e.g., for neutral endopeptidase (NEP) expressed in different cancer cells, including osteosarcoma (OS). The aim of the present study isto evaluate new borane-protected derivatives of phosphonous acid (compounds 1–7) in terms of their drug-likeness properties, anti-osteosarcoma activities in vitro (against HOS and Saos-2 cells), and use as potential NEP inhibitors. The results revealed that all tested compounds exhibited the physicochemical and ADME properties typical for small-molecule drugs. However, compound 4 did not show capability of blood–brain barrier penetration (Lipiński and Veber rules;SwissAdme tool). Moreover, the α-aminophosphonite-boranes (compounds 4–7) exhibited stronger anti-proliferative activity against OS cells than the other phosphonous acid-borane derivatives (compounds 1–3),especially regarding HOS cells (MTT assay). The most promising compounds 4 and 6 induced apoptosis through the activation of caspase 3 and/or cell cycle arrest at the G₂ phase (flow cytometry). Compound 4 inhibited the migration and invasiveness of highly aggressive HOS cells (wound/transwell and BME-coated transwell assays, respectively). Additionally, compound 4 and, to a lesser extent, compound 6 inhibited NEP activity (fluorometric assay). This activity of compound 4 was involved in its anti-proliferative potential (BrdU assay). The present study shows that compound 4 can be considered a potential anti-osteosarcoma agent and a scaffold for the development of new NEP inhibitors. Full article

The signaling pathways involved in age-related inflammation are increasingly recognized as targets for the development of preventive and therapeutic strategies. Our previous study elucidated the structure–activity relationship of monoterpene compounds derived from p-menthane as potential anti-inflammatory drugs and identified (S)-(+)-carvone as the most potent among the compounds tested. This study aims at identifying the molecular mechanism underlying the anti-inflammatory properties of (S)-(+)-carvone. The murine macrophage cell line, Raw 264.7, was stimulated with bacterial lipopolysaccharide (LPS) to simulate inflammation. Western blot was used to assess protein levels and post-translational modifications. The subcellular localization of NF-κB/p65 was visualized by immunocytochemistry. An in vitro fluorometric assay was used to measure Sirtuin-1 (SIRT1) activity. (S)-(+)-carvone inhibited LPS-induced JNK1 phosphorylation, but not that of p38 and ERK1/2 and also did not affect the phosphorylation and degradation of the NF-κB inhibitor, IκB-α. Accordingly, (S)-(+)-carvone did not affect LPS-induced phosphorylation of NF-κB/p65 on Ser536 and its nuclear translocation, but it significantly decreased LPS-induced IκB-α resynthesis, a NF-κB-dependent process, and NF-κB/p65 acetylation on lysine (Lys) 310. Deacetylation of that Lys residue is dependent on the activity of SIRT1, which was found to be increased by (S)-(+)-carvone, while its protein levels were unaffected. Taken together, these results show that (S)-(+)-carvone is a new SIRT1 activator with the potential to counteract the chronic low-grade inflammation characteristic of age-related diseases. Full article

Evidence suggests that histone deacetylases (HDACs) inhibitors could be used as an effective treatment for some psychiatric and neurological conditions such as depression, anxiety and age-related cognitive decline. However, non-specific HDAC inhibiting compounds have a clear disadvantage regarding their efficacy and safety, thus the need to develop more selective ones. The present study evaluated the toxicity, the capacity to inhibit HDAC activity and antidepressant-like activity of three recently described class I HDAC inhibitors IN01, IN04 and IN14, using A. salina toxicity test, in vitro fluorometric HDAC activity assay and forced-swimming test, respectively. Our data show that IN14 possesses a better profile than the other two. Therefore, the pro-cognitive and antidepressant effects of IN14 were evaluated. In the forced-swimming test model of depression, intraperitoneal administration of IN14 (100 mg/Kg/day) for five days decreased immobility, a putative marker of behavioral despair, significantly more than tricyclic antidepressant desipramine, while also increasing climbing behavior, a putative marker of motivational behavior. On the other hand, IN14 left the retention latency in the elevated T-maze unaltered. These results suggest that novel HDAC class I inhibitor IN14 may represent a promising new antidepressant with low toxicity and encourages further studies on this compound. Full article

Chemoresistance is a major obstacle that limits the benefits of cisplatin-based chemotherapy in various cancers, including hepatocellular carcinoma. De-regulation of the poly(ADP-ribose) polymerase 1 (PARP1)/high-mobility group box 1 (HMGB1) signaling pathway has been proposed as an important mechanism involved in cisplatin-resistance. In this study, we investigated therapeutic potential of a natural flavonoid Morin hydrate against cisplatin-induced toxicity using the HepG2^DR multi-drug resistant cell line, which is derived from the HepG2 human hepatocellular carcinoma cell line. HepG2^DR cells were exposed to cisplatin and Morin hydrate alone or together after which autophagy and apoptotic signaling pathways were monitored by fluorometric assay and Western blot analysis. Xenograft mouse models were performed to confirm the in vitro effect of Morin hydrate. PARP1 was hyper activated in cisplatin-resistant HepG2^DR cells. Cisplatin-induced PARP1 activation resulted in chemoresistance via increased autophagy. The cisplatin/Morin hydrate combination was effective in the reversal of the HepG2^DR cell resistance via suppression of PARP1-mediated autophagy by regulating the HMGB1 and microtubule-associated protein 1A/1B light chain 3B (LC3) I/II. Moreover, PARP1 inhibition by 4-amino-1,8-naphthalimide or autophagy inhibition by a knockdown of the autophagy-related 5 (ATG5) gene resulted in sensitizing the HepG2^DR cells to cisplatin (CP) through activation of the c-Jun N-terminal kinase (JNK) pathway. In a mouse xenograft model, the treatment of cisplatin with Morin hydrate reversed the increased expression of PARP and HMGB1 and significantly suppressed tumor growth. These findings indicate dysregulated expression of PARP1 confers cisplatin-resistance via autophagy activation in HepG2^DR cells. Morin hydrate inhibits cisplatin-mediated autophagy induction, resulting in increased susceptibility of HepG2^DR cells to cisplatin cytotoxicity. The combination of Morin hydrate with cisplatin may be a promising therapeutic strategy to enhance the efficacy of conventional chemotherapeutic drugs. Full article