Dataset of chicken-embryo blood cells exposed to quercetin, methyl methanesulfonate, or cadmium chloride.

Toxicological analysis of the effects of natural compounds is frequently mandated to assess their safety. In addition to more simple in vitro cellular systems, more complex biological systems can be used to evaluate toxicity. This dataset is comprised of bright-field microscopy images of chicken-embryo blood cells, a complex biological model that recapitulates several features found in human organisms, including circulation in blood stream and biodistribution to different organs. In the presented collection of blood smear images, cells were exposed to the flavonoid quercetin, and the two mutagens methyl methanesulfonate (MMS) and cadmium chloride (CdCl2). In ovo models offer a unique opportunity to investigate the effects of various substances, pathogens, or cancer treatments on developing embryos, providing valuable insights into potential risks and therapeutic strategies. In toxicology, in ovo models allow for early detection of harmful compounds and their impact on embryonic development, aiding in the assessment of environmental hazards. In immunology, these models offer a controlled system to explore the developing immune responses and the interaction between pathogens and host defenses. Additionally, in ovo models are instrumental in oncology research as they enable the study of tumor development and response to therapies in a dynamic, rapidly developing environment. Thus, these versatile models play a crucial role in advancing our understanding of complex biological processes and guiding the development of safer therapeutics and interventions. The data presented here can aid in understanding the potential toxic effects of these substances on hematopoiesis and the overall health of the developing organism. Moreover, the large dataset of blood smear images can serve as a resource for training machine learning algorithms to automatically detect and classify blood cells, provided that specific optimized conditions such as image magnification and background light are maintained for comparison. This can lead to the development of automated tools for blood cell analysis, which can be useful in research. Moreover, the data is amenable to the use as teaching and learning resource for histology and developmental biology.

Gossypetin Is a Novel Modulator of Inflammatory Cytokine Production and a Suppressor of Osteosarcoma Cell Growth.

Osteosarcoma (OS) is a common childhood sarcoma, and its treatment is hindered by adverse effects, chemoresistance, and recurrence. Interleukin (IL)-6 production by tumors plays a significant role in inflammation, carcinogenesis, and metastasis. This study aimed to investigate the antiproliferative potential of luteolin derivatives in OS and to evaluate interleukin production. MG-63, Saos-2, HOS, and 143B human OS cell lines were incubated with luteolin and eight derivatives containing hydroxy, chlorine, or alkyl substitutions. The cell viability and growth were evaluated in the presence of these compounds. Apoptosis was also examined through the analysis of the Bax expression and caspase-3 activity. Finally, the gossypetin effects were measured regarding the production of proinflammatory cytokines interleukin (IL)-6, IL-1ß, and IL-12p70. Our findings show that gossypetin was the most potent compound, with proliferation-suppressing activities that induced a series of critical events, including the inhibition of the cell viability and growth. Apoptosis was associated with enhanced caspase-3 activity and increased Bax expression, indicating the involvement of the intrinsic pathway of apoptosis. Moreover, pre-/co-treatment with gossypetin significantly reduced the autocrine production of proinflammatory cytokines. Further investigation is required; nevertheless, considering the link between inflammation, carcinogenesis, and metastasis in OS, our findings suggest that gossypetin exhibits anti-proliferative and anti-inflammatory properties that are potentially relevant in the clinical context.

The role of flavonoids in the regulation of epithelial-mesenchymal transition in cancer: A review on targeting signaling pathways and metastasis.

One of the hallmarks of cancer is metastasis, a process that entails the spread of cancer cells to distant regions in the body, culminating in tumor formation in secondary organs. Importantly, the proinflammatory environment surrounding cancer cells further contributes to cancer cell transformation and extracellular matrix destruction. During metastasis, front-rear polarity and emergence of migratory and invasive features are manifestations of epithelial-mesenchymal transition (EMT). A variety of transcription factors (TFs) are implicated in the execution of EMT, the most prominent belonging to the Snail Family Transcriptional Repressor (SNAI) and Zinc Finger E-Box Binding Homeobox (ZEB) families of TFs. These TFs are regulated by interaction with specific microRNAs (miRNAs), as miR34 and miR200. Among the several secondary metabolites produced in plants, flavonoids constitute a major group of bioactive molecules, with several described effects including antioxidant, antiinflammatory, antidiabetic, antiobesogenic, and anticancer effects. This review scrutinizes the modulatory role of flavonoids on the activity of SNAI/ZEB TFs and on their regulatory miRNAs, miR-34, and miR-200. The modulatory role of flavonoids can attenuate mesenchymal features and stimulate epithelial features, thereby inhibiting and reversing EMT. Moreover, this modulation is concomitant with the attenuation of signaling pathways involved in diverse processes as cell proliferation, cell growth, cell cycle progression, apoptosis inhibition, morphogenesis, cell fate, cell migration, cell polarity, and wound healing. The antimetastatic potential of these versatile compounds is emerging and represents an opportunity for the synthesis of more specific and potent agents.

Inhibitory activity of flavonoids against human sucrase-isomaltase (α-glucosidase) activity in a Caco-2/TC7 cellular model.

Type 2 diabetes (T2D) is the most common form of diabetes, and the number of people with this metabolic disease is steadily increasing worldwide. Among the available antidiabetic agents, α-glucosidase inhibitors are the most effective at reducing postprandial hyperglycaemia (PPHG), one of the main characteristics of T2D. However, most of the studies that have been performed have used the more readily available rat intestinal preparations or yeast α-glucosidase as the enzyme source, which despite being useful and cost effective, have a questionable physiological value. The present study evaluates the inhibitory activity of a selected group of flavonoids against human sucrase-isomaltase (SI), the α-glucosidase found in Caco-2/TC7 cells. A microassay using the physiological substrates sucrose and maltose, and a synthetic substrate, p-nitrophenyl-α-D-glucopyranoside (pNPG) was performed. The most active flavonoid was compound 4 (melanoxetin), presenting an IC50 value similar using the two natural substrates. In contrast, the tested flavonoids were not effective at inhibiting SI, when pNPG was used as a substrate. Hydroxylation of flavonoids at C-3 of the C ring, at C-3' and C-4' of the B ring, and at C-7 and C-8 of the A ring were the features that improved the inhibitory activity of flavonoids against human SI. These phenolic compounds deserve further exploration as alternatives to the currently available α-glucosidase inhibitors. The present study also demonstrates that the non-clinical in vitro studies conducted for the evaluation of α-glucosidase activity should use the human source rather than surrogate sources of α-glucosidase.

3',4'-Dihydroxyflavonol Modulates the Cell Cycle in Cancer Cells: Implication as a Potential Combination Drug in Osteosarcoma.

New agents are demanded to increase the therapeutic options for osteosarcoma (OS). Although OS is the most common bone cancer in children and adolescents, it is considered a rare disorder. Therefore, finding adjuvant drugs has potential to advance therapy for this disease. In this study, 3',4'-dihydroxyflavonol (DiOHF) was investigated to assess the effects in OS cellular models in combination with doxorubicin (Dox). MG-63 and U2OS human OS cells were exposed to DiOHF and Dox and tested for cell viability and growth. To elucidate the inhibitory effects of DiOHF, additional studies were conducted to assess apoptosis and cell cycle distribution, gene expression quantification of cell cycle regulators, and cytokinesis-block cytome assay to determine nuclear division rate. DiOHF decreased OS cell growth and viability in a concentration-dependent manner. Its combination with Dox enabled Dox dose reduction in both cell lines, with synergistic interactions in U2OS cells. Although no significant apoptotic effects were detected at low concentrations, cytostatic effects were demonstrated in both cell lines. Incubation with DiOHF altered cell cycle dynamics and resulted in differential cyclin and cyclin-dependent kinase expression. Overall, this study presents an antiproliferative action of DiOHF in OS combination therapy via modulation of the cell cycle and nuclear division.

ß-Carotene and its physiological metabolites: Effects on oxidative status regulation and genotoxicity in in vitro models.

Carotenoids are ubiquitously distributed in nature, ß-carotene being the most frequently found carotenoid in the human diet. In the human body, ß-carotene is absorbed, distributed and metabolized by enzymatic and/or non-enzymatic oxidant cleavage into several metabolites. Despite the broadly accepted biological value of ß-carotene, it has also been considered a double-edged sword, mainly due to its potential antioxidant versus pro-oxidant behaviour. In this sense, the aim of this work was to scrutinize the antioxidant or pro-oxidant potential of ß-carotene and its metabolites, namely trans-ß-apo-8'-carotenal and ß-ionone. Several parameters were evaluated in this study, viz. their effects on reactive species production, both in human whole blood and neutrophils; their effects on lipid peroxidation, in the absence and presence of peroxynitrite anion (ONOO-) or hydrogen peroxide (H2O2), using a synaptosomal model; and finally, their putative genotoxic effects in the human hepatic HepG2 cell line. In general, depending on the cellular model and conditions tested, ß-carotene and its metabolites revealed antioxidant effects to varying degrees without significant pro-oxidant or genotoxic effects.

Therapeutic potential of hesperidin and its aglycone hesperetin: Cell cycle regulation and apoptosis induction in cancer models.

BACKGROUND: The ability of cancer cells to divide without restriction and to escape programmed cell death is a feature of the proliferative state. Citrus flavanones are flavonoids with potential multiple anticancer actions, from antioxidant and chemopreventive, to anti-inflammatory, anti-angiogenic, cytostatic and cytotoxic in different cancer models. PURPOSE: This review aims to summarize the current knowledge on the antiproliferative actions of the citrus flavanones hesperidin (HSD) and hesperetin (HST), with emphasis on cell cycle arrest and apoptosis. METHODS: Cochrane Library, Scopus, Pubmed and Web of Science collection databases were queried for publications reporting antiproliferative effects of HSD and HST in cancer models. RESULTS: HSD and HST have been proven to delay cell proliferation in several cancer models. Depending on the compound, dose and cell line studied, different effects have been reported. Cell cycle arrest associated with cytostatic effects has been reported in cells with increased levels of p53 and also cyclin-dependent kinase inhibitors, as well as decreased levels of specific cyclins and cyclin-dependent kinases. Moreover, apoptotic effects have been found to be associated with altered ratios of pro-/antiapoptotic proteins, caspase activation, c-Jun N-terminal kinase (JNK) pathway activation and caspase-independent pathways. CONCLUSION: Available scientific literature data indicate complex effects, dependent on cell lines and exposure conditions, suggesting that HSD and HST doses need to be optimized according to the cellular and organismal context. The establishment of the main antiproliferative mechanisms is of utmost importance for a possible therapeutic benefit of citrus flavanones in the context of cancer.

Antioxidant mechanisms to counteract TiO2-nanoparticles toxicity in wheat leaves and roots are organ dependent.

Nanoparticles (NP) bioactivity is under deep scrutiny. In this work, the antioxidant response to TiO2-NP in wheat (Triticum aestivum) was determined. For that, enzymatic and the non-enzymatic antioxidants were evaluated in plants exposed to the P25 anatase:rutile material composed of TiO2-NP and under environmentally realistic doses (0; 5; 50; 150 mg/L for 20 days). Shoot but not root growth was reduced. In leaves, thiol metabolism and ascorbate accumulation were the preferred route whereas in roots the pre-existing antioxidant capacity was preferentially utilized. Both leaves and roots showed increased glutathione reductase and dehydroascorbate reductase activities and decreased ascorbate peroxidase activity. Roots, nevertheless, presented higher enzymatic basal levels than leaves. On the other hand, when examining non-enzymatic antioxidants, the ratio of reduced-to-oxidized glutathione (GSH/GSSG) increased in leaves and decreased in roots. Exposed leaves also presented higher total ascorbate accumulation compared to roots. TiO2-NP exposure down regulated, with more prominence in roots, antioxidant enzyme genes encoding catalase, ascorbate peroxidase, monodehydroascorbate reductase and dehydroascorbate reductase. In leaves, superoxide dismutase gene expression was increased. All data pinpoint to TiO2-NP toxicity above 5 mg/L, with aerial parts being more susceptible, which draws concerns on the safety doses for the use of these NPs in agricultural practices.

Responses of olive plants exposed to different irrigation treatments in combination with heat shock: physiological and molecular mechanisms during exposure and recovery.

MAIN CONCLUSION: A water-deficit period, leading to stomatal control and overexpression of protective proteins (sHSP and DHN), contributes to olive´s tolerance to later imposed stress episodes. Aquaporins modulation is important in olive recovery. Olive is traditionally cultivated in dry farming or in high water demanding irrigated orchards. The impact of climate change on these orchards remains to unveil, as heat and drought episodes are increasing in the Mediterranean region. To understand how young plants face such stress episodes, olive plants growing in pots were exposed to well-irrigated and non-irrigated treatments. Subsequently, plants from each treatment were either exposed to 40 °C for 2 h or remained under control temperature. After treatments, all plants were allowed to grow under well-irrigated conditions (recovery). Leaves were compared for photosynthesis, relative water content, mineral status, pigments, carbohydrates, cell membrane permeability, lipid peroxidation and expression of the protective proteins' dehydrin (OeDHN1), heat-shock proteins (OeHSP18.3), and aquaporins (OePIP1.1 and OePIP2.1). Non-irrigation, whilst increasing carbohydrates, reduced some photosynthetic parameters to values below the ones of the well-irrigated plants. However, when both groups of plants were exposed to heat, well-irrigated plants suffered more drastic decreases of net CO2 assimilation rate and chlorophyll b than non-irrigated plants. Overall, OeDHN1 and OeHSP18.3 expression, which was increased in non-irrigated treatment, was potentiated by heat, possibly to counteract the increase of lipid peroxidation and loss of membrane integrity. Plants recovered similarly from both irrigation and temperature treatments, and recovery was associated with increased aquaporin expression in plants exposed to one type of stress (drought or heat). These data represent an important contribution for further understanding how dry-farming olive will cope with drought and heat episodes.

Biochemical and transcriptional analyses of cadmium-induced mitochondrial dysfunction and oxidative stress in human osteoblasts.

Cadmium (Cd) accumulation is known to occur predominantly in kidney and liver; however, low-level long-term exposure to Cd may also result in bone damage. Few studies have addressed Cd-induced toxicity in osteoblasts, particularly upon cell mitochondrial energy processing and putative associations with oxidative stress in bone. To assess the influence of Cd treatment on mitochondrial function and oxidative status in osteoblast cells, human MG-63 cells were treated with Cd (up to 65 µM) for 24 or 48 h. Intracellular reactive oxygen species (ROS), lipid and protein oxidation and antioxidant defense mechanisms such as total antioxidant activity (TAA) and gene expression of antioxidant enzymes were analyzed. In addition, Cd-induced effects on mitochondrial function were assessed by analyzing the activity of enzymes involved in mitochondrial respiration, membrane potential (ΔΨm), mitochondrial morphology and adenylate energy charge. Treatment with Cd increased oxidative stress, concomitantly with lipid and protein oxidation. Real-time polymerase chain reaction (qRT-PCR) analyses of antioxidant genes catalase (CAT), glutathione peroxidase 1 (GPX1), glutathione S-reductase (GSR), and superoxide dismutase (SOD1 and SOD2) exhibited a trend toward decrease in transcripts in Cd-stressed cells, particularly a downregulation of GSR. Longer treatment with Cd (48 h) resulted in energy charge states significantly below those commonly observed in living cells. Mitochondrial function was affected by ΔΨm reduction. Inhibition of mitochondrial respiratory chain enzymes and citrate synthase also occurred following Cd treatment. In conclusion, Cd induced mitochondrial dysfunction which appeared to be associated with oxidative stress in human osteoblasts.

Inorganic Hg toxicity in plants: A comparison of different genotoxic parameters.

Inorganic Mercury (Hg) contamination persists an environmental problem, but its cyto- and genotoxicity in plants remains yet unquantified. To determine the extent of Hg-induced cyto- and genotoxicity, and assess most sensitive endpoints in plants, Pisum sativum L. seedlings were exposed for 14 days to different HgCl2 concentrations up to 100 µM. Shoots and roots from hydroponic exposure presented growth impairment and/or morphological disorders for doses >1 µM, being the roots more sensitive. Plant growth, ploidy changes, clastogenicity (HPCV), cell cycle dynamics (G1-S-G2), Comet-tail moment (TM), Comet-TD, Mitotic-index (MI) and cell proliferation index (CPI) were used to evaluate Hg-induced cyto/genotoxicity. Both leaf and root DNA-ploidy levels, assessed by flow cytometry (FCM), remained unaltered after exposure. Root cell cycle impairment occurred at lower doses (≥1 µM) than structural DNA damages (≥10 µM). Cytostatic effects depended on the Hg concentration, with delays during S-phase at lower doses, and arrests at G1 at higher ones. This arrest was paralleled with decreases of both mitotic index (MI) and cell proliferation index (CPI). DNA fragmentation, assessed by the Comet assay parameters of TD and TM, could be visualized for conditions ≥10 µM, while FCM-clastogenic parameter (FPCV) and micronuclei (MNC) were only altered in roots exposed to 100 µM. We demonstrate that inorganic-Hg induced cytostaticity is detectable even at 1 µM (a value found in contaminated sites), while structural DNA breaks/damage are only visualized in plants at concentrations ≥10 µM. We also demonstrate that among the different techniques tested for cyto- and genotoxicity, TD and TM Comet endpoints were more sensitive than FPCV or MNC. Regarding cytostatic effects, cell cycle analysis by FCM, including the difference in % cell cycle phases and CPI were more sensitive than MI or MNC frequency. Our data contribute to better understand Hg cyto- and genotoxicity in plants and to understand the information and sensitivity provided by each of the genotoxic techniques used.

Combination of etoposide and fisetin results in anti-cancer efficiency against osteosarcoma cell models.

Osteosarcoma chemotherapy is often limited by chemoresistance, resulting in poor prognosis. Combined chemotherapy could, therefore, be used to prevent resistance to chemotherapeutics. Here, the effects of fisetin on osteosarcoma cells were investigated, as well as cytostatic potential in combination with the anti-cancer drug etoposide. For this, different osteosarcoma cell lines were treated with fisetin, with etoposide and with respective combinations. Fisetin was associated with decrease in colony formation in Saos-2 and in U2OS cells but not in MG-63 cells. Notwithstanding, upon evaluation of cellular growth by crystal violet assay, MG-63 and Saos-2 cells showed decreased cell proliferation at 40 and 20 µM fisetin, respectively. Depending on the relative concentrations, fisetin:etoposide combinations showed negative-to-positive interactions on the inhibition of cell proliferation. In addition, fisetin treatment up to 50 µM for 48 h resulted in G2-phase cell cycle arrest. Regardless of the combination, fisetin:etoposide increased % cells in G2-phase and decreased % cells in G1-phase. In addition, mixtures with more positive combined effects induced increased % cells in S-phase. Compared to etoposide treatment, these combinations resulted in decreased levels of cyclins B1 and E1, pointing to the role of these regulators in fisetin-induced cell cycle arrest. In conclusion, these results show that the combination of fisetin with etoposide has higher anti-proliferative effects in osteosarcoma associated with cell cycle arrest, allowing the use of lower doses of the chemotherapeutic agent, which has important implications for osteosarcoma treatment.

Cytotoxic effect of the serotonergic drug 1-(1-Naphthyl)piperazine against melanoma cells.

1-(1-Naphthyl)piperazine (1-NPZ) is a serotonergic derivative of quipazine acting both as antagonist and agonist of different serotonin receptors, with promising results for the management of skin cancer. In this work, we studied the effect of 1-NPZ on human MNT-1 melanoma cells by evaluating its effects on cell viability, ability to form colonies, cell cycle dynamics, reactive oxygen species (ROS) production and apoptosis. Treatment of MNT-1 cells with 1-NPZ for 24h decreased cell viability and induced apoptosis in a dose-dependent manner. Activity against melanoma was confirmed with a different melanoma cell line, SK-MEL-28. Simultaneously, 1-NPZ affected cell cycle progression by mediating a S-phase delay. Higher levels of ROS were also detected in MNT-1 cells after treatment with 1-NPZ. Furthermore, 1-NPZ significantly increased the expression of cyclooxygenase-2 in MNT-1 cells. These findings suggest that 1-NPZ pretreatment is able to induce oxidative stress, and consequently apoptotic cell death in melanoma cells. In conclusion, this study demonstrates the cytotoxic and genotoxic potential of 1-NPZ against melanoma cells.

Hesperetin-etoposide combinations induce cytotoxicity in U2OS cells: Implications on therapeutic developments for osteosarcoma.

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma.

Insights into the impact of silver nanoparticles on human keratinocytes metabolism through NMR metabolomics.

Due to their antimicrobial properties, silver nanoparticles (AgNPs) are increasingly incorporated into consumer goods and medical products. Their potential toxicity to human cells is however a major concern, and there is a need for improved understanding of their effects on cell metabolism and function. Here, Nuclear Magnetic Resonance (NMR) metabolomics was used to investigate the metabolic profile of human epidermis keratinocytes (HaCaT cell line) exposed for 48 h to 30 nm citrate-stabilized spherical AgNPs (10 and 40 µg/mL). Intracellular aqueous extracts, organic extracts and extracellular culture medium were analysed to provide an integrated view of the cellular metabolic response. The specific metabolite variations, highlighted through multivariate analysis and confirmed by spectral integration, suggested that HaCaT cells exposed to AgNPs displayed upregulated glutathione-based antioxidant protection, increased glutaminolysis, downregulated tricarboxylic acid (TCA) cycle activity, energy depletion and cell membrane modification. Importantly, most metabolic changes were apparent in cells exposed to a concentration of AgNPs which did not affect cell viability at significant levels, thus underlying the sensitivity of NMR metabolomics to detect early biochemical events, even in the absence of a clear cytotoxic response. It can be concluded that NMR metabolomics is an important new tool in the field of in vitro nanotoxicology.

Cadmium-induced genotoxicity in human osteoblast-like cells.

Cadmium (Cd) is a widespread heavy metal used in numerous industrial processes. Cd exerts toxicological effects mostly in kidney and liver. Bone is also an important target of Cd, however, the cellular mechanisms of Cd toxicological effects in the bone cells are still poorly understood. Therefore, the present work aimed to investigate the putative cytotoxic and genotoxic effects of Cd to human bone cells. For that, the osteoblast-like MG-63 cells were exposed to 20 and 50µM Cd for 24 and 48h. Results showed a dose-dependent increase in Cd accumulation in cells and a decrease in cell viability, especially after 48h. Cell cycle analysis showed a delay at S phase concomitant with a decrease in cells at G0/G1 phase. After 24h, Cd treatment downregulated the expression of CHEK1, CHEK2 and CDK2 genes and upregulated the expression of CCNE1 gene. After 48h, the expression of ATM and CCNB1 genes were downregulated. Also, a 3.3 fold increase on the expression of gene CCNE1 was detected. Both Cd doses induced DNA fragmentation at 48h, while an increase in micronuclei (MN) and nucleoplasmic bridges (NPBs) together with an increase in the percentage of apoptotic/necrotic cells was detected for both time periods. Overall, our results demonstrate the cytotoxicity and genotoxicity of Cd in human bone cells. Also, the cytokinesis-block micronucleus (CBMN) assay parameters (MN, NPBs and the percentage of cells under apoptosis or necrosis) together with the cell cycle appear as the most sensitive to Cd cyto- and genotoxicity, being early affected even with the lowest Cd dose. Therefore, these cyto-/genotoxic techniques may be selected for early detection of Cd-induced toxicity.

Sulforaphane induces oxidative stress and death by p53-independent mechanism: implication of impaired glutathione recycling.

Sulforaphane (SFN) is a naturally-occurring isothiocyanate best known for its role as an indirect antioxidant. Notwithstanding, in different cancer cell lines, SFN may promote the accumulation of reactive oxygen species (ROS) and cause cell death e.g. by apoptosis. Osteosarcoma often becomes chemoresistant, and new molecular targets to prevent drug resistance are needed. Here, we aimed to determine the effect of SFN on ROS levels and to identify key biomarkers leading to ROS unbalance and apoptosis in the p53-null MG-63 osteosarcoma cell line. MG-63 cells were exposed to SFN for up to 48 h. At 10 µM concentration or higher, SFN decreased cell viability, increased the%early apoptotic cells and increased caspase 3 activity. At these higher doses, SFN increased ROS levels, which correlated with apoptotic endpoints and cell viability decline. In exposed cells, gene expression analysis revealed only partial induction of phase-2 detoxification genes. More importantly, SFN inhibited ROS-scavenging enzymes and impaired glutathione recycling, as evidenced by inhibition of glutathione reductase (GR) activity and combined inhibition of glutathione peroxidase (GPx) gene expression and enzyme activity. In conclusion, SFN induced oxidative stress and apoptosis via a p53-independent mechanism. GPx expression and activity were found associated with ROS accumulation in MG-63 cells and are potential biomarkers for the efficacy of ROS-inducing agents e.g. as co-adjuvant drugs in osteosarcoma.

Sulforaphane induces DNA damage and mitotic abnormalities in human osteosarcoma MG-63 cells: correlation with cell cycle arrest and apoptosis.

Osteosarcoma is a recalcitrant bone malignancy with poor responsiveness to treatments; therefore, new chemotherapeutic compounds are needed. Sulforaphane (SFN) has been considered a promising chemotherapeutic compound for several types of tumors by inducing apoptosis and cytostasis, but its effects (e.g., genotoxicity) in osteosarcoma cells remains exploratory. In this work, the MG-63 osteosarcoma cell line was exposed to SFN up to 20 µM for 24 and 48 h. SFN induced G2/M phase arrest and decreased nuclear division index, associated with disruption of cytoskeletal organization. Noteworthy, SFN induced a transcriptome response supportive of G2/M phase arrest, namely a decrease in Chk1- and Cdc25C-encoding transcripts, and an increase in Cdk1-encoding transcripts. After 48-h exposure, SFN at a dietary concentration (5 µM) contributed to genomic instability in the MG-63 cells as confirmed by increased number of DNA breaks, clastogenicity, and nuclear and mitotic abnormalities. The increased formation of nucleoplasmic bridges, micronuclei, and apoptotic cells positively correlated with loss of viability. These results suggest that genotoxic damage is an important step for SFN-induced cytotoxicity in MG-63 cells. In conclusion, SFN shows potential to induce genotoxic damage at low concentrations and such potential deserves further investigation in other tumor cell types.

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction.

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.