Search Results (22)

Leishmaniasis and Chagas disease are parasitic diseases considered to be among the most important neglected diseases, with implications for both developed and developing countries. Currently, there are no effective therapeutic treatments for these diseases due to challenges in drug administration, high toxicity, high costs, and drug resistance. In this study, a series of eleven thymol derivatives were designed, synthesized, and evaluated for their in vitro kinetoplastid activity against Leishmania amazonensis and Trypanosoma cruzi, as well as their cytotoxicity against a murine macrophage cell line. The most active compounds, thymol anysoate (9) and thymol picolinate (10), displayed the highest kinetoplastid activity with IC₅₀ values of 22.87 and 25.16 µM against L. amazonensis and T. cruzi, respectively. Notably, both compounds demonstrated an excellent selectivity index against the mammal cell line. Structure–activity relationship studies revealed that the ester group plays a crucial role in activity. The most promising derivatives, 9 and 10, activate autophagy and apoptosis-like processes in the treated cells. Atomic force microscopy observations showed that derivative 9 induces the formation of cytoplasmic vacuoles, indicating an autophagic process, and drug-likeness analysis revealed that it meets all the pharmacokinetic criteria. Overall, these results highlight derivative 9 as a potential lead compound for the development of new drugs for the treatment of Trypanosomatidae infections and warrants further studies to elucidate the cell death cascade involved. Full article

Background: This study investigated the combined effects of hesperidin (Hesp), a natural flavonoid, with bleomycin (BL), a commonly used chemotherapy agent, on A549 human lung cancer cells. Methods: Key parameters assessed included cell viability, colony formation, and cell migration, alongside the expression of apoptotic and autophagic markers (p53, p21, Bax, cleaved PARP, and Beclin-1), VEGF levels, and caspase-3 activity. Results: The findings revealed that the Hesp + BL combination significantly amplified antiproliferative, apoptotic, anti-angiogenic, and autophagic effects compared to either treatment alone. The combination therapy effectively inhibited colony formation and cell migration while markedly reducing VEGF levels, indicating strong anti-angiogenic properties. Apoptotic markers such as p53, p21, Bax, and cPARP were significantly upregulated, with caspase-3 activity confirming robust apoptosis induction. Furthermore, autophagy was notably enhanced, as reflected by increased Beclin-1 expression. Conclusions: Synergistic interactions between Hesp and BL, validated through combination index analysis, underscore the therapeutic potential of this combination. These findings underscore the therapeutic potential of the Hesp + BL combination as a promising strategy for lung cancer treatment, meriting further exploration in diverse lung cancer cell lines to validate and expand its applicability in developing novel therapeutic approaches. Full article

Despite significant efforts, cancer remains the second leading cause of mortality worldwide. The medicinal plant Nepeta nuda L. represents a valuable source of biologically active compounds with pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, and antiviral. This study aimed to assess the antiproliferative potential and mechanisms of action of aqueous extract from the leaves of wild-grown N. nuda. Cancer cell lines, MDA-MB-231, MCF7 (breast), HT29, Colon 26 (colon), and HepG2 (liver cancer), and a non-cancerous skin cell line, BJ, were assessed for antiproliferative activity by MTT assay and observation of cell morphological alterations. The cancer cell line that was most sensitive to the extract was further studied for apoptotic alterations by Annexin V/propidium iodide staining, colony-forming assay, and qRT-PCR analysis. The results revealed that the plant extract inhibited the proliferation of all investigated cancer cell lines with the strongest cytostatic effect on Colon 26 cells with a half maximal inhibitory concentration (IC₅₀) value of 380.2 μg/mL and a selectivity index (SI) of 3.5. The extract significantly inhibited the ability of cells to form colonies, exhibited considerable proapoptotic potential involving the participation of the CASP8 gene, and increased the expression levels of ATG3 and the BECN1 gene, which suggests a role of autophagic cell death in the antitumor action. Full article

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. It is important to discover new therapeutic regimens for treating CRC. Depression is known to be an important complication of cancer diseases. Repurposing antidepressants into anticancer drugs and exploring the combinational efficacy of antidepressants and chemotherapy are potentially good options for developing CRC treatment regimens. In this study, sertraline, an antidepressant drug, and paclitaxel, an anticancer drug, were chosen to study their antitumor effects in the treatment of colorectal cancer, alone or in combination, and to explore their underlying mechanisms. The data showed that sertraline exerted a dose-dependent cytotoxic effect on MC38 and CT26 colorectal cancer cell lines with IC₅₀ values of 10.53 μM and 7.47 μM, respectively. Furthermore, sertraline synergistically sensitized chemotherapeutic agent paclitaxel efficacy in CRC cells with combination index (CI) values at various concentrations consistently lower than 1. Sertraline remarkably augmented paclitaxel-induced autophagy by increasing autophagosome formation indicated by elevated LC3-II/I ratio and promoting autophagic flux by degrading autophagy cargo receptor SQSTM1/p62, which may explain the synergistically cytotoxic effect of sertraline and paclitaxel combination therapy on CRC cells. This study provides important evidence to support repurposing sertraline as an anticancer agent and suggests a novel combinational regimen for effectively treating CRC as well as in the simultaneous treatment of CRC and depression. Full article

Mycotoxins are produced by more than one hundred fungi and produce secondary metabolites that contaminate various agricultural commodities, especially rice and corn. Their presence in the food chain is considered a serious problem worldwide. In recent years, a link between exposure to mycotoxins and impaired fertility has been suggested. Consequently, it has become vital to investigate the interactive effects of these mycotoxins on ovarian function. In this study, we investigated the intergenerational effects of the mycotoxin fumonisin B1 (FB1) on ovarian structure and function. Virgin Wistar albino female rats were separated into control and FB1 treatment groups and examined from day 6 of pregnancy until delivery (20 and 50 mg/kg b.w./day). The obtained female rats of the first (F1) and second generations (F2) were euthanized at 4 weeks of age, and ovary samples were collected. We found that the ovary weight index increased with the high dose of the treatment (50 mg/kg b.w./day) among both F1 and F2, in a manner similar to that observed in polycystic ovary syndrome. As expected, FB1 at a high dose (50 mg/kg b.w.) reduced the number of primordial follicles in F1 and F2, leading to an accelerated age-related decline in reproductive capacity. Moreover, it reduced the fertility rate among the F1 female rats by affecting follicle growth and development, as the number of secondary and tertiary follicles decreased. Histopathological changes were evidenced by the altered structures of most of the growing follicle oocytes, as revealed by a thinning irregular zona pellucida and pyknosis in granulosa cells. These findings are concomitant with steroidogenesis- and folliculogenesis-related gene expression, as evidenced by the decrease in CYP19 activity and estrogen receptor beta (ESR2) gene expression. Additionally, GDF-9 mRNA levels were significantly decreased, and IGF-1 mRNA levels were significantly increased. However, the results from the ovaries of the F2 treatment groups were different and unexpected. While there was no significant variation in CYP19 activity compared to the control, the ESR2 significantly increased, leading to stereological and histopathological changes similar to those of the control, except for some altered follicles. The hallmark histological feature was the appearance of vacuolar structures within the oocyte and between granulosa cell layers. Interestingly, the autophagic marker LC3 was significantly increased in the F2 offspring, whereas this protein was significantly decreased in the F1 offspring. Therefore, we suggest that the promotion of autophagy in the ovaries of the F2 offspring may be considered a recovery mechanism from the effect of prenatal FB1 exposure. Thus, autophagy corrected the effect of FB1 during the early life of the F1 female rats, leading to F2 offspring with ovarian structure and function similar to those of the control. However, the offspring, treated female rats may experience early ovarian aging because their ovarian pool was affected. Full article

Botulinum neurotoxin type-A (BoNT) injections are commonly used as spasticity treatment in cerebral palsy (CP). Despite improved clinical outcomes, concerns regarding harmful effects on muscle morphology have been raised, and the BoNT effect on muscle stem cells remains not well defined. This study aims at clarifying the impact of BoNT on growing muscles (1) by analyzing the in vitro effect of BoNT on satellite cell (SC)-derived myoblasts and fibroblasts obtained from medial gastrocnemius microbiopsies collected in young BoNT-naïve children (t0) compared to age ranged typically developing children; (2) by following the effect of in vivo BoNT administration on these cells obtained from the same children with CP at 3 (t1) and 6 (t2) months post BoNT; (3) by determining the direct effect of a single and repeated in vitro BoNT treatment on neuromuscular junctions (NMJs) differentiated from hiPSCs. In vitro BoNT did not affect myogenic differentiation or collagen production. The fusion index significantly decreased in CP at t2 compared to t0. In NMJ cocultures, BoNT treatment caused axonal swelling and fragmentation. Repeated treatments impaired the autophagic–lysosomal system. Further studies are warranted to understand the long-term and collateral effects of BoNT in the muscles of children with CP. Full article

Chagas disease (CD) caused by the protozoan Trypanosoma cruzi affects more than six million people worldwide. Treatment is restricted to benznidazole (Bz) and nifurtimox (Nf) that display low activity in the later chronic stage besides triggering toxic events that result in treatment abandonment. Therefore, new therapeutic options are necessary. In this scenario, natural products emerge as promising alternatives to treat CD. In the family Plumbaginaceae, Plumbago sp. exhibits a broad spectrum of biological and pharmacological activities. Thus, our main objective was to evaluate, in vitro and in silico, the biological effect of crude extracts of root and of aerial parts of P. auriculata, as well as its naphthoquinone Plumbagin (Pb) against T. cruzi. The phenotypic assays revealed potent activity of the root extract against different forms (trypomastigote and intracellular forms) and strains (Y and Tulahuen), with a compound concentration that reduced 50% of the number of the parasite (EC₅₀) values ranging from 1.9 to 3.9 µg/mL. In silico analysis showed that Pb is predicted to have good oral absorption and permeability in Caco2 cells, besides excellent probability of absorption by human intestinal cells, without toxic or mutagenic potential effects, not being predicted as a substrate or inhibitor of P-glycoprotein. Pb was as potent as Bz against intracellular forms and displayed a superior trypanosomicidal effect (about 10-fold) in bloodstream forms (EC₅₀ = 0.8 µM) as compared to the reference drug (8.5 µM). The cellular targets of Pb on T. cruzi were evaluated using electron microscopy assays and the findings on bloodstream trypomastigotes showed several cellular insults related to the autophagic process. Regarding toxicity in mammalian cells, the root extracts and the naphthoquinone present a moderate toxic profile on fibroblasts and cardiac cell lines. Then, aiming to reduce host toxicity, the root extract and Pb were tested in combination with Bz, and the data showed additive profiles with the sum of the fractional inhibitory concentration indexes (ΣFICIs) being 1.45 and 0.87, respectively. Thus, our work reveals the promising antiparasitic activity of Plumbago auriculata crude extracts and its purified naphthoquinone Plumbagin against different forms and strains of Trypanosoma cruzi in vitro. Full article

In recent years, lycopene has been highlighted due to its antioxidant and anti-inflammatory properties, associated with a beneficial effect on human health. The aim of this study was to advance the studies of antioxidant and anti-inflammatory mechanisms on human keratinocytes cells (HaCaT) of a self-emulsifying drug delivery system (SEDDS) loaded with lycopene purified from red guava (nanoLPG). The characteristics of nanoLPG were a hydrodynamic diameter of 205 nm, a polydispersity index of 0.21 and a zeta potential of −20.57, providing physical stability for the nanosystem. NanoLPG demonstrated antioxidant capacity, as shown using the ORAC methodology, and prevented DNA degradation (DNA agarose). Proinflammatory activity was evaluated by quantifying the cytokines TNF-α, IL-6 and IL-8, with only IL-8 showing a significant increase (p < 0.0001). NanoLPG showed greater inhibition of the tyrosinase and elastase enzymes, involved in the skin aging process, compared to purified lycopene (LPG). In vitro treatment for 24 h with 5.0 µg/mL of nanoLPG did not affect the viability of HaCaT cells. The ultrastructure of HaCaT cells demonstrated the maintenance of morphology. This contrasts with endoplasmic reticulum stresses and autophagic vacuoles when treated with LPG after stimulation or not with LPS. Therefore, the use of lycopene in a nanoemulsion may be beneficial in strategies and products associated with skin health. Full article

Our previous studies have confirmed that cadmium (Cd) exposure causes hepatotoxicity; it also induces autophagy and blocks the autophagy flux. Therefore, we hypothesized that Cd hepatotoxicity could be alleviated through nutritional intervention. Taurine (Tau) has various biological functions such as acting as an antioxidant, acting as an anti-inflammatory, and stabilizing cell membranes. In order to explore the protective effect and internal mechanism of Tau on Cd-induced hepatotoxicity, normal rat liver cell line BRL3A cells were treated with Cd alone or in combination with Tau to detect cell injury and autophagy-related indexes in this study. We found that Tau can alleviate Cd-induced cell-proliferation decline and morphological changes in the cell. In addition, Tau activates autophagy and alleviates the blockage of Cd-induced autophagy flux. In this process, lysosome acidification and degradation were enhanced, and autophagosomes were further fused with lysosomes. Then, we found that Tau alleviated autophagic flux block by promoting the transfer of membrane fusion proteins STX17 and SNAP29 to autophagosomes and the translocation of VAMP8 to lysosomes, which in turn attenuated the hepatocyte injury induced by Cd exposure. This will further reveal the hepatotoxicity mechanism of Cd and provide the theoretical basis for the prevention and treatment of Cd poisoning. Full article

We previously demonstrated that SAOS human osteosarcoma cells, incubated with carotenoid-enriched nanoemulsions (CEN), activated a nonprotective form of autophagy and delayed cell proliferation. The present work focuses on the biological effects of CEN on a derivative of SAOS cells named SAOS400, recently described for their radiation resistance and higher expression of therapy-induced senescence (TIS) markers. SAOS400 cells, incubated with CEN, activated a “cytostatic” form of autophagy confirmed by cell cycle arrest in the G2/M phase and increased expression of autophagic proteins. Treatment of SAOS400 cells with CEN also resulted in decreased expression of the senescence marker p16^INK4. However, when SAOS400 cells were γ-irradiated in combination with CEN, the threshold for cell death was reached (>60% after 96 h). We showed that this type of cell death corresponded to ‘cytotoxic’ or ‘lethal’ autophagy and that the combined treatment of CEN plus γ-rays was synergistic, with the combination index < 1. Since CEN contained β-carotene, the pure compound was used in SAOS400 cells at the same concentration present in CEN and up to 10 times higher. However, no radio-sensitizing effect of β-carotene was observed, suggesting that the biological effect of CEN was due to less abundant but more bioactive molecules, or to the synergistic activity of multiple components present in the extracts, confirming the functional pleiotropy of natural extracts enriched in bioactive molecules. Full article

A major cause of cancer cell resistance to chemotherapeutics is the blocking of apoptosis and induction of autophagy in the context of cell adaptation and survival. Therefore, new compounds are being sought, also among drugs that are commonly used in other therapies. Due to the involvement of histamine in the regulation of processes occurring during the development of many types of cancer, antihistamines are now receiving special attention. Our study concerned the identification of new mechanisms of action of azelastine hydrochloride, used in antiallergic treatment. The study was performed on HeLa cells treated with different concentrations of azelastine (15–90 µM). Cell cycle, level of autophagy (LC3 protein activity) and apoptosis (annexin V assay), activity of caspase 3/7, anti-apoptotic protein of Bcl-2 family, ROS concentration, measurement of mitochondrial membrane potential (Δψm), and level of phosphorylated H2A.X in response to DSB were evaluated by cytometric method. Cellular changes were also demonstrated at the level of transmission electron microscopy and optical and fluorescence microscopy. Lysosomal enzyme activities-cathepsin D and L and cell viability (MTT assay) were assessed spectrophotometrically. Results: Azelastine in concentrations of 15–25 µM induced degradation processes, vacuolization, increase in cathepsin D and L activity, and LC3 protein activation. By increasing ROS, it also caused DNA damage and blocked cells in the S phase of the cell cycle. At the concentrations of 45–90 µM, azelastine clearly promoted apoptosis by activation of caspase 3/7 and inactivation of Bcl-2 protein. Fragmentation of cell nucleus was confirmed by DAPI staining. Changes were also found in the endoplasmic reticulum and mitochondria, whose damage was confirmed by staining with rhodamine 123 and in the MTT test. Azelastine decreased the mitotic index and induced mitotic catastrophe. Studies demonstrated the multidirectional effects of azelastine on HeLa cells, including anti-proliferative, cytotoxic, autophagic, and apoptotic properties, which were the predominant mechanism of death. The revealed novel properties of azelastine may be practically used in anti-cancer therapy in the future. Full article

The research has demonstrated a synergistic anticancer effect of Seabuckthorn pulp oil (SBO) and the standard chemotherapy regimen Docetaxel (DTX) against two non-small cell lung cancer (NSCLC) cell lines: A549 and H23. The synergizing effect of an SBO and DTX combination was detected utilizing SRB assay and combination index (CI) approaches. Flow cytometry was carried out using fluorescent probes to measure cell cycle analysis by DNA content and reactive oxygen species (ROS) generation. Further, we demonstrated that the synergistic anticancer activity of SBO merged with DTX was achieved by caspase-independent autophagy and senescence induction. These changes were concomitant with increased generation of ROS production and microtubule-associated protein 1 light chain 3 (LC3) protein expression, G1-phase arrest, and enhanced senescence-associated β-galactosidase staining activity. Our data also demonstrated that SBO or DTX treatment groups solely upregulated the phosphorylation of ERK, which coincided with the induction of autophagy vacuoles and was functionally associated with ROS activation. Moreover, endogenous LC3 puncta staining was performed and monitored by confocal microscopy. Overall, these results suggest new mechanisms for the antitumor activity of SBO co-treated with DTX through triggering autophagic cell death and senescence against cancer cells as a result of sustained ERK phosphorylation and intracellular ROS production in NSCLC. In addition, we also highlight SBO as an alternative therapeutic option or adjunct therapeutic strategy in combination with chemotherapeutic agents in lung cancer therapy management. Full article

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients’ PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors’ PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14⁻ (PBLs) and CD14⁺ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4⁺, CD8⁺ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients’ serum IL-18 concentrations occurred. CD4⁺ and CD8⁺ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE. Full article

(1) Background: Growth differentiation factor-15 (GDF-15) is associated with cardiovascular diseases and autophagy in human macrophages (MΦ). Thus, we are interested in investigating autophagic mechanisms with special respect to the role of GDF-15. (2) Methods: Recombinant (r)GDF-15 and siRNA GDF-15 were used to investigate the effects of GDF-15 on autophagic and lysosomal activity, as well as autophagosome formation by transmission electron microscopy (TEM) in MΦ. To ascertain the effects of GDF-15^−/− on the progression of atherosclerotic lesions, we used GDF-15^−/−/ApoE^−/− and ApoE^−/− mice under a cholesterol-enriched diet (CED). Body weight, body mass index (BMI), blood lipid levels and lumen stenosis in the brachiocephalic trunk (BT) were analyzed. Identification of different cell types and localization of autophagy-relevant proteins in atherosclerotic plaques were performed by immunofluorescence. (3) Results: siGDF-15 reduced and, conversely, rGDF-15 increased the autophagic activity in MΦ, whereas lysosomal activity was unaffected. Autophagic degradation after starvation and rGDF-15 treatment was observed by TEM. GDF-15^−/−/ApoE^−/− mice, after CED, showed reduced lumen stenosis in the BT, while body weight, BMI and triglycerides were increased compared with ApoE^−/− mice. GDF-15^−/− decreased p62-accumulation in atherosclerotic lesions, especially in endothelial cells (ECs). (4) Conclusion: GDF-15 seems to be an important factor in the regulation of autophagy, especially in ECs of atherosclerotic lesions, indicating its crucial pathophysiological function during atherosclerosis development. Full article

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC₅₀ and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug. Full article