Fisetin Inhibits Autophagy in HepG2 Cells via PI3K/Akt/mTOR and AMPK Pathway.

The effect of fisetin on autophagy in hepatocellular carcinoma remains uncovered. HepG2 cells were exposed to different concentrations of fisetin (25, 50, and 100 µM) for 24 h. The cells were also treated with rapamycin and chloroquine alone or in combination with fisetin. Autophagic flux formation and ATP levels were determined. The changes in autophagic markers and AMPK signaling proteins were analyzed using qRT-PCR and Western blotting. Cyto-ID staining followed by flow cytometry showed that fisetin decreased autophagic flux formation in a dose-dependent manner. In gene expression analysis, the mRNA levels of mTOR, Atg5, Atg16L, and LC3A were elevated, whereas the mRNA levels of Atg7 and Beclin1 were downregulated in a dose-dependent manner compared to control. In the Western blotting analysis, fisetin treatment inhibited the expression of Atg7, Atg16L, mTOR, and pACC and elevated the expression of Atg5, AMPKα, AMPKß1/2, ACC and Akt. Taken together, the results revealed that fisetin inhibited autophagy by the activation of PI3K/Akt/mTOR and modulation of AMPK signaling pathways. Our findings indicate that suppression of autophagy by fisetin may serve as an effective therapeutic strategy against HCC.

Ferulic acid attenuates arsenic-induced cardiotoxicity in rats.

Arsenic (As), a potent environmental toxin, causes cardiac functional impairments. Ferulic acid (FA), a ubiquitous dietary hydroxycinnamate, exerts beneficial effects on human health. Hence, the present study investigated the effect of FA on myocardial oxidative stress parameters, ATP level, the status of cardiac cytoskeleton intermediate filaments-desmin and vimentin, and AMPK signaling proteins in As-intoxicated rats. Wistar rats were administered orally with FA-40 mg/kg and As-5 mg/kg alone and in combination for 30 days. Myocardial As content, serum cardiac marker enzyme activities including creatine kinase-isoenzyme, lactate dehydrogenase, and aspartate aminotransferase were increased in As-exposed rats. An accumulation of myocardial oxidants such as reactive oxygen species, lipid peroxidation, nitric oxide, protein carbonyl content, and histological aberrations was observed. A significant decrease of myocardial antioxidants comprises superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, and ascorbic acid and declined expression of desmin and vimentin was noted. Impaired energy signaling molecules AMPKα (Thr172), AMPKß1/2 (Ser108), ACC (Ser79), and intracellular myocardial ATP depletion were observed in As-intoxicated animals. FA attenuates As-induced cardiac dysfunction by restoring the expression of intermediate filaments and AMPK proteins. Based on the above findings, FA treatment could be used as a novel therapeutic against As-induced cardiac dysfunction.

Acute fluoride exposure alters myocardial redox and inflammatory markers in rats.

Acute fluoride (F-) exposure adversely impairs cardiac functions. We previously reported that acute F- toxicity causes modulation in oxidant and antioxidant systems, heat shock proteins, cytoskeletal proteins and AMPK signaling proteins in the myocardium of rats. With these findings, we hypothesized that acute F- intoxication may trigger an acute myocardial inflammatory response through the activation of NF-κB signaling and reduction of redox signaling regulatory system. To test this hypothesis, we treated male Wistar rats with single oral doses of 45 and 90 mg/kg of F- for 24 h. The myocardium of F- treated rats showed increased expression of pNF-κB, pIκKα/ß eventually leading to the increased expression of downstream target TNFα-a major proinflammatory cytokine secreted in the inflammatory process. F- intoxication decreased the mRNA expression of redox genes-Sirt1, Sirt3, Prdx2, Glrx1, Trx1, and Trx2. In addition, we observed decreased protein expression of Nrf2, GCLC, and NQO1 in the cardiac tissues of F- treated rats. This study reveals that F- toxicity triggers myocardial inflammatory response and depletes redox signaling molecules in the myocardium of rats. We conclude that NF-κB activation with decreased redox gene expression might be associated with the pathophysiology of F- induced cardiac dysfunction in rats. This finding provides new insights into the cardiovascular pathophysiology in acute F- toxicity.

Genome-wide identification and analysis of Nrf2 binding sites - Antioxidant response elements in zebrafish.

In the post-genomic era, deciphering the Nrf2 binding sites - antioxidant response elements (AREs) is an essential task that underlies and governs the Keap1-Nrf2-ARE pathway - a cell survival response pathway to environmental stresses in the vertebrate model system. AREs regulate the transcription of a repertoire of phase II detoxifying and/or oxidative-stress responsive genes, offering protection against toxic chemicals, carcinogens, and xenobiotics. In order to identify and analyze AREs in zebrafish, a pattern search algorithm was developed to identify AREs and computational tools available online were utilized to analyze the identified AREs in zebrafish. This study identified the AREs within 30 kb upstream from the transcription start site of antioxidant genes and mitochondrial genes. We report for the first time the AREs of all the known protein coding genes in the zebrafish genome. Western blotting, RT2 profiler array PCR, and qRT-PCR were performed to test whether AREs influence the Nrf2 target genes expression in the zebrafish larvae using sulforaphane. This study reveals unique AREs that have not been previously reported in the cytoprotective genes. Nine TGAG/CNNNTC and six TGAG/CNNNGC AREs were observed significantly. Our findings suggest that AREs drive the dynamic transcriptional events of Nrf2 target genes in the zebrafish larvae on exposure to sulforaphane. The identified abundant putative AREs will define the Keap1-Nrf2-ARE network and elucidate the precise regulation of Nrf2-ARE pathway in not only diseases but also in embryonic development, inflammation, and aerobic respiration. Our results help to understand the dynamic complexity of the Nrf2-ARE system in zebrafish.

Iron oxide nanoparticles modulate heat shock proteins and organ specific markers expression in mice male accessory organs.

With increased industrial utilization of iron oxide nanoparticles (Fe2O3-NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe2O3-NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe2O3-NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe2O3-NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe2O3-NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe2O3-NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe2O3-NPs could be an environmental risk factor for reproductive disease.

The Wnt-pathway corepressor TLE3 interacts with the histone methyltransferase KMT1A to inhibit differentiation in Rhabdomyosarcoma.

Rhabdomyosarcoma tumor cells resemble differentiating skeletal muscle cells, which unlike normal muscle cells, fail to undergo terminal differentiation, underlying their proliferative and metastatic properties. We identify the corepressor TLE3 as a key regulator of rhabdomyosarcoma tumorigenesis by inhibiting the Wnt-pathway. Loss of TLE3 function leads to Wnt-pathway activation, reduced proliferation, decreased migration, and enhanced differentiation in rhabdomyosarcoma cells. Muscle-specific TLE3-knockout results in enhanced expression of terminal myogenic differentiation markers during normal mouse development. TLE3-knockout rhabdomyosarcoma cell xenografts result in significantly smaller tumors characterized by reduced proliferation, increased apoptosis and enhanced differentiation. We demonstrate that TLE3 interacts with and recruits the histone methyltransferase KMT1A, leading to repression of target gene activation and inhibition of differentiation in rhabdomyosarcoma. A combination drug therapy regime to promote Wnt-pathway activation by the small molecule BIO and inhibit KMT1A by the drug chaetocin led to significantly reduced tumor volume, decreased proliferation, increased expression of differentiation markers and increased survival in rhabdomyosarcoma tumor-bearing mice. Thus, TLE3, the Wnt-pathway and KMT1A are excellent drug targets which can be exploited for treating rhabdomyosarcoma tumors.

Fisetin, a phytopolyphenol, targets apoptotic and necroptotic cell death in HepG2 cells.

Fisetin (3,7,3',4'-tetrahydroxyflavone), a bioactive dietary flavonoid, intrigued scientists for its anticancer potential against various cancer types. We investigated the fisetin-induced inhibition of growth and survival of human hepatocellular carcinoma. Fisetin decreased cell viability and proliferation of HepG2 cells as revealed from MTT and clonogenicity assays. Cell cycle arrest in the G2/M phase was observed. Annexin V/propidium iodide (PI) staining followed by flow cytometry revealed that fisetin induced both apoptosis and necroptosis in HepG2 cells. Apoptotic cells were significantly increased on fisetin treatment as observed in morphological evaluations and 4',6-diamidino-2-phenylindole and Acridine orange staining. Flow cytometry, fluorescence imaging, and 2', 7'-dichlorofluorescein diacetate analyses showed an increase in reactive oxygen species (ROS) generation on fisetin treatment. Pretreatment with N-acetyl cysteine inhibited ROS production and also rescued mitochondrial membrane potential in HepG2 cells. The underlying mechanisms of apoptosis and necroptosis were determined by analysis of their respective signaling molecules using qRT-PCR and Western blotting. Fisetin showed a marked increase in the expression of TNFα and IKκB with a decrease in NF-κB, pNF-κB and pIKκB expression. Fisetin reduced the expression of Bcl2, and elevated levels of Bax, caspase-3, and PARP and thus induced apoptosis in HepG2 cells. zVAD suppressed the fisetin-induced expression of caspase-8, RIPK1, RIPK3, and MLKL as opposed to fisetin treatment. Nec-1 + fisetin could not completely block necroptosis, which warrants further investigation. Taken together, our findings demonstrate that the fisetin exhibited anti-proliferative effects on HepG2 cells through apoptosis and necroptosis via multiple signaling pathways. Fiestin has potential as a therapeutic agent against hepatocellular carcinoma.

Differential expression of myocardial heat shock proteins in rats acutely exposed to fluoride.

Acute fluoride (F-) toxicity is known to cause severe cardiac complications and leads to sudden heart failure. Previously, we reported that increased myocardial oxidative damage, apoptosis, altered cytoskeleton and AMPK signaling proteins associated with energy deprivation in acute F- induced cardiac dysfunction. The present study was aimed to decipher the status of myocardial heat shock proteins (Hsps-Hsp27, Hsp32, Hsp40, Hsp60, Hsp70, Hsp90) and heat shock transcription factor 1 (Hsf1) in acute F--intoxicated rats. In order to study the expression of myocardial Hsps, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F- for 24 h. The expression levels of myocardial Hsps were determined using RT-PCR, western blotting, and immunohistochemical studies. Acute F--intoxicated rats showed elevated levels of both the transcripts and protein expression of Hsf1, Hsp27, Hsp32, Hsp60, and Hsp70 when compared to control. In addition, the expression levels of Hsp40 and Hsp90 were significantly declined in a dose-dependent fashion in F--treated animals. Our result suggests that differential expression of Hsps in the rat myocardium could serve as a balance between pro-survival and death signal during acute F--induced heart failure.

Acute fluoride poisoning alters myocardial cytoskeletal and AMPK signaling proteins in rats.

BACKGROUND: Our previous findings revealed that increased oxidative stress, apoptosis and necrosis were implicated in acute fluoride (F-) induced cardiac dysfunction apart from hypocalcemia and hyperkalemia. Cardiac intermediate filaments (desmin and vimentin) and cytoskeleton linker molecule vinculin plays an imperative role in maintaining the architecture of cardiac cytoskeleton. In addition, AMPK is a stress activated kinase that regulates the energy homeostasis during stressed state. The present study was aimed to examine the role of cytoskeletal proteins and AMPK signaling molecules in acute F- induced cardiotoxicity in rats. METHODS: In order to study this, male Wistar rats were treated with single oral doses of 45 and 90mg/kgF- for 24h. RESULTS: Acute F- intoxicated rats showed declined cytoskeletal protein expression of desmin, vimentin and vinculin in a dose dependent manner compared to control. A significant increase in phosphorylation of AMPKα (Thr172), AMPKß1 (Ser108) and Acetyl-coA carboxylase (ACC) (Ser79) in the myocardium and associated ATP deprivation were found in acute F- intoxicated rats. Further, ultra-structural studies confirmed myofibril lysis with interruption of Z lines, dilated sarcoplasmic reticulum and damaged mitochondrion were observed in both the groups of F- intoxicated rats. CONCLUSION: Taken together, these findings reveal that acute F- exposure causes sudden heart failure by altering the expression of cytoskeletal proteins and AMPK signaling molecules.

Recurrent exposure to ferric oxide nanoparticles alters myocardial oxidative stress, apoptosis and necrotic markers in male mice.

The cardiotoxicity of iron oxide nanoparticles (Fe2O3-NPs) in mice was investigated. The mice were intraperitoneally administered with Fe2O3-NPs at the dose of 25 and 50 mg/kg bw for 30 days at seven days interval. In vivo MRI analysis reveals the Fe2O3-NPs accumulation in the cardiac system. Also, serum iron estimation and Prussian blue staining confirms the iron deposition in circulatory system. Cardiac dysfunction was assessed by ECG analysis and further validated by evaluating the functional markers such as cardiac Troponin-1 (cTnI) expression, AChE activity and levels of LDH and CK-MB in cardiac tissue. Fe2O3-NPs exposure disturbs the balance between the oxidants and antioxidants resulting in oxidative myocardial damages. In consequence, damaged mitochondria, diminished ATP level and NOX4 over expression were observed in the intoxicated groups indicating the role of Fe2O3-NPs in oxidative stress. A dose dependant increase in oxidative stress mediates apoptosis through upregulation of Bax, cytochrome c and cleaved caspase 3 in the 25 mg/kg treated group. Sustained oxidative stress suggest the occurrence of necrosis in addition to apoptosis in 50 mg/kg treated group evidenced by altered expression pattern of cleaved PARP, cytochrome c, Bax and cleaved caspase 3. In addition, triphenyl tetrazolium chloride (TTC) staining confirms cardiac necrosis in 50 mg/kg Fe2O3-NPs treated group.

In silico prediction of microRNAs on fluoride induced sperm toxicity in mice.

Fluorosis is an endemic global problem causing male reproductive impairment. F mediates male reproductive toxicity in mice down-regulating 63 genes involved in diverse biological processes - apoptosis, cell cycle, cell signaling, chemotaxis, electron transport, glycolysis, oxidative stress, sperm capacitation and spermatogenesis. We predicted the miRNAs down-regulating these 63 genes using TargetScan, DIANA and MicroCosm. The prediction tools identified 3059 miRNAs targeting 63 genes. Of the predicted interactions, 11 miRNAs (mmu-miR-103, -107, -122, -188a, -199a-5p, -205, -340-5p, -345-3p, -452-5p, -499, -878-3p) were commonly found in the three tools utilized and seven miRNAs (miR-9-5p, miR-511-3p, miR-7b-5p, miR-30e-5p, miR-17-5p, miR-122-5p and miR-541-5p) targeting six genes (Traf3, Rock2, Rgs8, Atp1b2, Cacna2d1 and Aldoa) were already validated experimentally in mice. The miRNA-mRNA network of the predicted miRNAs with its respective targets revealed the complex interaction within a biological process leading to sperm dysfunction on exposure to F. Our findings not only suggest that the predicted miRs furnish evidence, but also have the potential to serve as non-invasive biomarkers on F-induced sperm dysfunction. Our data contribute towards elucidating the function of miRNAs in the fluoride induced infertility. miRNA molecular pathways in F intoxication will open new avenues on the use of antagomirs in recovering fertility.

Tamarind seed coat ameliorates fluoride induced cytotoxicity, oxidative stress, mitochondrial dysfunction and apoptosis in A549 cells.

Fluoride (F) is an environmental contaminant and industrial pollutant. Molecular mechanisms remain unclear in F induced pulmonary toxicity even after numerous studies. Tamarind fruits act as defluoridating agents, but no study was conducted in in vitro systems. Hence, we aimed to assess the ameliorative impact of the tamarind seed coat extract (TSCE) against F toxicity utilizing lung epithelial cells, A549. Cells were exposed to sodium fluoride (NaF-5 mM) alone and in combination with TSCE (750 ng/ml) or Vitamin C (positive control) for 24 h and analyzed for F content, intracellular calcium ([Ca(2+)]i) level, oxidative stress, mitochondrial integrity and apoptotic markers. TSCE treatment prevented the F induced alterations in [Ca(2+)]i overload, F content, oxidant (reactive oxygen species generation, lipid peroxidation, protein carbonyl content and nitric oxide) and antioxidant (superoxide dismutase, catalase, glutathione peroxidase and glutathione) parameters. Further, TSCE modulates F activated changes in mitochondrial membrane potential, permeability transition pore opening, cytochrome-C release, Bax/Bcl-2 ratio, caspase-3 and PARP-1 expressions. In conclusion, our study demonstrated that TSCE as a potential protective agent against F toxicity, which can be utilized as a neutraceutical.

Single oral acute fluoride exposure causes changes in cardiac expression of oxidant and antioxidant enzymes, apoptotic and necrotic markers in male rats.

Several studies have shown that acute fluoride (F(-)) exposure impairs cardiac function, but the molecular mechanism is not clear. In order to study this, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F(-) for 24 h. A significant accumulation of F(-) was found in the serum and myocardium of experimental rats. F(-) treatment causes myocardial necrosis as evident from increased levels of myocardial troponin I, creatine kinase, lactate dehydrogenase and aspartate transaminase. In addition, F(-) induces myocardial oxidative stress via increased reactive oxygen species, lipid peroxidation, protein carbonyl content and nitrate levels along with decreased in the levels of enzymatic (superoxide dismutase 2, catalase, glutathione peroxidase and glutathione s transferase pi class) and non-enzymatic (reduced glutathione) antioxidants. Notably, F(-) triggers myocardial apoptosis through altered Bax/Bcl2 ratio and increased cytochrome c, caspase 3p20 and terminal deoxynucleotidyl transferase dUTP nick end labeled positive cells. An increased cardiac expression of Nox4 and p38α MAPK in F(-) treated rats indicates the oxidative and apoptotic damage. Moreover, ultra-structural changes, histopathological and luxol fast blue staining demonstrates the degree of myocardial damage at subcellular level. Taken together, these findings reveal that acute F(-) exposure causes cardiac impairment by altering the expression of oxidative stress, apoptosis and necrotic markers.

Caffeic acid, a phyto polyphenol mitigates fluoride induced hepatotoxicity in rats: A possible mechanism.

Fluoride induced hepatotoxicity has been extensively studied in both humans and animals. However, the mechanism underlying in the hepatotoxicity of experimental fluorosis remains obscure. The severity of fluoride toxicity was reduced by oral administration of certain plant derived antioxidants. Caffeic acid (CA) a polyphenolic compound has diverse range of pharmacological activity in the biological system. Therefore, the present study was aimed to investigate the protective mechanism of CA, against fluoride induced hepatotoxicity in rats. The rats were treated with 300 ppm of NaF via drinking water ad libitum alone and in combination with CA at a dose of 50 mg/kg daily for 30 days by oral intubation. CA treatment significantly prevented fluoride induced hepatic damage as evident from the histopathological studies and declined levels of serum fluoride and liver marker enzymes. A significant decrease in the levels of enzymatic (SOD2, CAT, GPx, and GSTpi class) and nonenzymatic (GSH and Vitamin C) antioxidants along with increased ROS, lipid peroxidation, protein carbonyl content, and nitrate levels by fluoride were also prevented in these groups. In addition, CA inhibits fluoride induced apoptosis by altering the Bax and caspase-3p20 expressions. Further, fluoride induced upregulation of Nox4, p38α MAPK, Hsp60, and downregulation of Hsp27 are the indicators for the detection of oxidative damage, apoptosis, and mitochondrial stress was also modulated by CA. These findings reveal that CA supplementation has a protective effect against fluoride induced hepatotoxicity in rats.

Ferulic acid modulates fluoride-induced oxidative hepatotoxicity in male Wistar rats.

The present study is aimed to evaluate the protective effect of ferulic acid (FA) on fluoride-induced oxidative hepatotoxicity in male Wistar rats. Fluoride (25 mg/L) was given orally to induce hepatotoxicity for 12 weeks. Hepatic damage were assessed using status of pathophysiological markers like serum marker enzymes like aspartate transaminase, alanine transaminase, alkaline phosphatase, acid phosphatase, gamma glutamyl transferase, lactate dehydrogenase, bilirubin, lipid profile, total protein content levels, and histopathological studies. Treatment with FA significantly reduced the degree of histological aberrations and rescued lipid peroxidation, as observed from reduced levels of lipid hydroperoxides, nitric oxide, restored levels of enzymic and non-enzymic antioxidants, and total protein content, with a concomitant decline in the levels of marker enzymes and lipid profile in fluoride-induced rats. These results suggest that ferulic acid has the ability to protect fluoride-induced hepatic damage.

A brief review on experimental fluorosis.

Fluoride (F) is a naturally occurring contaminant in the water. F is essential for normal maintenance of teeth and bones. However, prolonged exposure to high concentration of F is found to be deleterious to teeth, bones and other organs. Besides drinking water, F can enter the body through food, dental products, drugs and industrial emission. People living in areas where F contamination is much higher than the expected level, are found to suffer from not only teeth and bone problem but also other systems, including brain and its functions. Since animals respond to the toxic effects of F like human beings, the deleterious effects of F have been produced experimentally in animals in order to determine the mechanism involved in the action of F. The reports indicating the chronic harmful effects of F in teeth, bones, heart, liver, kidneys, gastrointestinal tract, lungs, brain, blood, hormones and biochemical parameters of experimental animals and in in vitro studies have been reviewed in this article. The neurotoxic action of F that produces chiefly learning and memory impairment has also been included. The review also points out the harmful effects of F on reproduction, its teratogenic action and in inducing premature ageing. Finally, the reports indicating a reversal of certain toxicities of F in experimental animals after withdrawal of its exposure has been included.