Astaxanthin Attenuates Early Acute Kidney Injury Following Severe Burns in Rats by Ameliorating Oxidative Stress and Mitochondrial-Related Apoptosis

Early acute kidney injury (AKI) is a devastating complication in critical burn patients, and it is associated with severe morbidity and mortality. The mechanism of AKI is multifactorial. Astaxanthin (ATX) is a natural compound that is widely distributed in marine organisms; it is a strong antioxidant and exhibits other biological effects that have been well studied in various traumatic injuries and diseases. Hence, we attempted to explore the potential protection of ATX against early post burn AKI and its possible mechanisms of action. The classic severe burn rat model was utilized for the histological and biochemical assessments of the therapeutic value and mechanisms of action of ATX. Upon ATX treatment, renal tubular injury and the levels of serum creatinine and neutrophil gelatinase-associated lipocalin were improved. Furthermore, relief of oxidative stress and tubular apoptosis in rat kidneys post burn was also observed. Additionally, ATX administration increased Akt and Bad phosphorylation and further down-regulated the expression of other downstream pro-apoptotic proteins (cytochrome c and caspase-3/9); these effects were reversed by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, the protective effect of ATX presents a dose-dependent enhancement. The data above suggested that ATX protects against early AKI following severe burns in rats, which was attributed to its ability to ameliorate oxidative stress and inhibit apoptosis by modulating the mitochondrial-apoptotic pathway, regarded as the Akt/Bad/Caspases signalling cascade.     

Actinomycin V Induces Apoptosis Associated with Mitochondrial and PI3K/AKT Pathways in Human CRC Cells

Actinomycin (Act) V, an analogue of Act D, presented stronger antitumor activity and less hepatorenal toxicity than Act D in our previous studies, which is worthy of further investigation. We hereby report that Act V induces apoptosis via mitochondrial and PI3K/AKT pathways in colorectal cancer (CRC) cells. Act V-induced apoptosis was characterized by mitochondrial dysfunction, with loss of mitochondria membrane potential (MMP) and cytochrome c release, which then activated cleaved caspase-9, cleaved caspase-3, and cleaved PARP, revealing that it was related to the mitochondrial pathway, and the apoptotic trendency can be reversed by caspase inhibitor Z-VAD-FMK. Furthermore, we proved that Act V significantly inhibited PI3K/AKT signalling in HCT-116 cells using cell experiments in vitro, and it also presented a potential targeted PI3Kα inhibition using computer docking models. Further elucidation revealed that it exhibited a 28-fold greater potency than the PI3K inhibitor LY294002 on PI3K inhibition efficacy. Taken together, Act V, as a superior potential replacement of Act D, is a potential candidate for inhibiting the PI3K/AKT pathway and is worthy of more pre-clinical studies in the therapy of CRC.     

Marine cyclotripeptide X-13 promotes angiogenesis in zebrafish and human endothelial cells via PI3K/Akt/eNOS signaling pathways

Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or L-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.     

Assessment of bioflocculant production by Bacillus sp. Gilbert, a marine bacterium isolated from the bottom sediment of Algoa Bay

The bioflocculant-producing potentials of a marine bacteria isolated from the bottom sediment of Algoa Bay was investigated using standard methods. The 16S rDNA sequence analysis revealed 98% similarity to that of Bacillus sp. HXG-C1 and the nucleotide sequence was deposited in GenBank as Bacillus sp. Gilbert with accession number {"type":"entrez-nucleotide","attrs":{"text":"HQ537128","term_id":"312064208","term_text":"HQ537128"}}HQ537128. Bioflocculant was optimally produced when sucrose (72% flocculating activity) and ammonium chloride (91% flocculating activity) were used as sole sources of carbon and nitrogen, respectively; an initial pH 6.2 of the production medium; and Mg²⁺ as cation. Chemical analysis of the purified bioflocculant revealed the compound to be a polysaccharide.     

Three New Asperentin Derivatives from the Algicolous Fungus Aspergillus sp. F00785

Three new asperentin-type compounds, 6-O-α-d-ribosylasperentin (1) and 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5–9), were isolated from a halotolerant Aspergillus sp. strain {"type":"entrez-nucleotide","attrs":{"text":"F00785","term_id":"707638","term_text":"F00785"}}F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.     

Canolol induces apoptosis in human gastric carcinoma cell through PI3K/Akt signaling pathway

Canolol is a natural polyphenolic compound in rapeseed oil with multiple biological activities.Anti-cancer potential of canolol has been proposed in some studies.However,the effect and underlying mechanism of canolol on human gastric carcinoma(AGS)cells have not been well understood.This study showed that canolol had significant inhibition in proliferation and strongly apoptotic effects in AGS cells,which were verified by the decrease of cell viability,high level of reactive oxygen species(ROS),and damage in mitochondria membrane potential(MMP).Treatment with canolol arrested cells in S phase,and increased expressions of Bax,cleaved caspase-9,and cleaved caspase-3 proteins.Meanwhile,low expression of Bcl-2 and cytochrome C(Cyt C)release were found.The expressions of PI3K(phosphoinositide 3-kinase)and p-AKT(phosphorylated serine/threonine kinase)were also downregulated.Overall,these results suggested that canolol inhibited proliferation and induced apoptosis in AGS cells by regulating PI3K/Akt pathway,demonstrating a potential in gastric cancer treatment.     

Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ ² P > 0.05), including only one common haplotype (GenBank access no. {"type":"entrez-nucleotide","attrs":{"text":"EF413075","term_id":"151936076","term_text":"EF413075"}}EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran     

Dieckol Ameliorates Aβ Production via PI3K/Akt/GSK-3β Regulated APP Processing in SweAPP N2a Cell

The proteolytic processing of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase releases amyloid-β peptide (Aβ), which deposits in amyloid plaques and contributes to the initial causative events of Alzheimer’s disease (AD). In the present study, the regulatory mechanism of APP processing of three phlorotannins was elucidated in Swedish mutant APP overexpressed N2a (SweAPP N2a) cells. Among the tested compounds, dieckol exhibited the highest inhibitory effect on both intra- and extracellular Aβ accumulation. In addition, dieckol regulated the APP processing enzymes, such as α-secretase (ADAM10), β-secretase, and γ-secretase, presenilin-1 (PS1), and their proteolytic products, sAPPα and sAPPβ, implying that the compound acts on both the amyloidogenic and non-amyloidogenic pathways. In addition, dieckol increased the phosphorylation of protein kinase B (Akt) at Ser473 and GSK-3β at Ser9, suggesting dieckol induced the activation of Akt, which phosphorylated GSK-3β. The specific phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 triggered GSK-3β activation and Aβ expression. In addition, co-treatment with LY294002 noticeably blocked the effect of dieckol on Aβ production, demonstrating that dieckol promoted the PI3K/Akt signaling pathway, which in turn inactivated GSK-3β, resulting in the reduction in Aβ levels.     

Induction of Apoptosis by Sinulariolide from Soft Coral through Mitochondrial-Related and p38MAPK Pathways on Human Bladder Carcinoma Cells

Sinulariolide, an isolated compound from the soft coral Sinularia flexibilis, possesses the anti-proliferative, anti-migratory and apoptosis-inducing activities against the TSGH bladder carcinoma cell. The anti-tumor effects of sinulariolide were determined by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell migration assay and flow cytometry, respectively. Sinulariolide inhibited the growth and migration of bladder carcinoma cells in a dose-dependent manner, as well as induced both early and late apoptosis as determined by the flow cytometer. Also, the sinulariolide-induced apoptosis is related to the mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome C, activation of caspase-3/-9, Bax and Bad, as well as suppression of Bcl-2/Bcl-xL/Mcl-1. Detection of the PARP-1 cleaved product suggested the partial involvement of caspase-independent pathways. Moreover, inhibition of p38MAPK activity leads to the rescue of the cell cytotoxicity of sinulariolide-treated TSGH cells, indicating that the p38MAPK pathway is also involved in the sinulariolide-induced cell apoptosis. Altogether, these results suggest that sinulariolide induces apoptosis against bladder cancer cells through mitochondrial-related and p38MAPK pathways.     

Sinularin Induces Apoptosis through Mitochondria Dysfunction and Inactivation of the pI3K/Akt/mTOR Pathway in Gastric Carcinoma Cells

Sinularin is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinularin on two human gastric cancer cell lines, AGS and NCI-N87. Our results demonstrated that sinularin suppressed the proliferation of gastric cancer cells in a dose-dependent manner and induced apoptosis. In addition, the loss of mitochondrial membrane potential, the release of cytochrome C, the activation of Bax, Bad and caspase-3/9, and the suppression of p-Bad, Bcl-xL and Bcl-2 were observed in the cells treated with sinularin. This finding suggests that sinularin-induced apoptosis is associated with mitochondria-mediated apoptosis and occurs through caspase-dependent pathways. Furthermore, sinularin inhibited the phosphoinositol 3-kinase/Akt/mechanistic target of the rapamycin signaling pathway. Taken together, our results show that sinularin-induced apoptosis is mediated by activation of the caspase cascade and mitochondrial dysfunction. Our findings suggest that sinularin merits further evaluation as a chemotherapeutic agent for human gastric cancer.     

Fucoidan and Derived Oligo-Fucoses: Structural Features with Relevance in Competitive Inhibition of Gastrointestinal Norovirus Binding

Norovirus infections belong to the most common causes of human gastroenteritis worldwide and epidemic outbreaks are responsible for hundreds of thousands of deaths annually. In humans, noroviruses are known to bind to gastrointestinal epithelia via recognition of blood-group active mucin-type O-glycans. Considering the involvement of l-α-fucose residues in these glycans, their high valency on epithelial surfaces far surpasses the low affinity, though specific interactions of monovalent milk oligosaccharides. Based on these findings, we attempted to identify polyfucoses (fucans) with the capacity to block binding of the currently most prevalent norovirus strain GII.4 (Sydney, 2012, {"type":"entrez-nucleotide","attrs":{"text":"JX459908","term_id":"409032931"}}JX459908) to human and animal gastrointestinal mucins. We provide evidence that inhibitory effects on capsid binding are exerted in a competitive manner by α-fucosyl residues on Fucus vesiculosus fucoidan, but also on the galacto-fucan from Undaria pinnatifida and their oligo-fucose processing products. Insight into novel structural aspects of fucoidan and derived oligosaccharides from low-mass Undaria pinnatifida were revealed by GCMS and MALDI mass spectrometry. In targeting noroviral spread attenuation, this study provides first steps towards a prophylactic food additive that is produced from algal species.     

13-Acetoxysarcocrassolide Induces Apoptosis on Human Gastric Carcinoma Cells Through Mitochondria-Related Apoptotic Pathways: p38/JNK Activation and PI3K/AKT Suppression

13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ΔΨm, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways.     

Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number {"type":"entrez-nucleotide","attrs":{"text":"AY729671","term_id":"56718401","term_text":"AY729671"}}AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.     

In Vitro Antitumor Activity of Stellettin B,a Triterpene from Marine Sponge Jaspis stellifera,on Human Glioblastoma Cancer SF295 Cells

Stellettin B was isolated from marine sponge Jaspis stellifera. In vitro antitumor activities were investigated on 39 human cancer cell lines. Stellettin B exhibited highly potent inhibition against the growth of a human glioblastoma cell line SF295, with a GI50 of 0.01 μM. In contrast, stellettin B showed very weak inhibitory activity on normal cell lines including HMEC, RPTEC, NHBE and PrEC, with GI50s higher than 10 μM, suggesting its relatively selective cytotoxicity against human cancer cells compared to normal human cell lines. We then focused on the antitumor activity of this compound on SF295 cells. Flow cytometric analysis indicated that stellettin B induced apoptosis in SF295 cells in a concentration-dependent manner. Further study indicated that stellettin B increased the production of ROS, the activity of caspase 3/7, as well as the cleavage of PARP, each of which is known to be involved in apoptosis. To investigate the molecular mechanism for cell proliferation inhibition and apoptosis induction, effect on the phosphorylation of several signal proteins of PI3K/Akt and RAS/MAPK pathways was examined. Stellettin B inhibited the phosphorylation of Akt potently, with no activity on p-ERK and p-p38, suggesting that inhibition of PI3K/Akt pathway might be involved in the antiproliferative and apoptosis-inducing effect. However, homogenous time-resolved fluorescence (HTRF) assay indicated that stellettin B did not inhibit PI3K activity, suggesting that the direct target might be signal protein upstream of Akt pathway other than PI3K.     

The Inhibition Effect of the Seaweed Polyphenol, 7-Phloro-Eckol from Ecklonia Cava on Alcohol-Induced Oxidative Stress in HepG2/CYP2E1 Cells

The liver is vulnerable to oxidative stress-induced damage, which leads to many diseases, including alcoholic liver disease (ALD). Liver disease endanger people’s health, and the incidence of ALD is increasing; therefore, prevention is very important. 7-phloro-eckol (7PE) is a seaweed polyphenol, which was isolated from Ecklonia cava in a previous study. In this study, the antioxidative stress effect of 7PE on HepG2/CYP2E1 cells was evaluated by alcohol-induced cytotoxicity, DNA damage, and expression of related inflammation and apoptosis proteins. The results showed that 7PE caused alcohol-induced cytotoxicity to abate, reduced the amount of reactive oxygen species (ROS) and nitric oxide (NO), and effectively inhibited DNA damage in HepG2/CYP2E1 cells. Additionally, the expression levels of glutathione (GSH), superoxide dismutase (SOD), B cell lymphoma 2 (Bcl-2), and Akt increased, while γ-glutamyltransferase (GGT), Bcl-2 related x (Bax), cleaved caspase-3, cleaved caspase-9, nuclear factor-κB (NF-κB), and JNK decreased. Finally, molecular docking proved that 7PE could bind to BCL-2 and GSH protein. These results indicate that 7PE can alleviate the alcohol-induced oxidative stress injury of HepG2 cells and that 7PE may have a potential application prospect in the future development of antioxidants.     

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.     

Protective Role of Oleuropein against Acute Deltamethrin-Induced Neurotoxicity in Rat Brain

Background:

Deltamethrin (DM) is a synthetic pyrethroid insecticide that can elicit neurotoxicity, leading to apoptosis. There is accumulating evidence that oleuropein (OE) has anti-apoptotic effect. The purpose of this study was to determine the anti-apoptotic effect of OE pretreatment in the neuronal cells of cerebral cortex.

Methods:

Rats were randomly divided into four groups each containing five rats: DM-treated group (12.5 mg/kg, a single dose), OE-treated group (20 mg/kg per day), DM + OE-treated group, and vehicle group. Sections of the brain were obtained 24 hours after DM injection and studied for histopathological and immunohistochemistry assessment.

Results:

The histopathological assessments showed lesser characteristics of neural degeneration in DM + OE group compared with DM group. Greater Bcl-2 and attenuated Bax expression could be detected in the DM + OE treated-mice compared with DM group.

Conclusion:

The results suggested that DM-induced neurotoxicity can be subsided by OE.Key Words: Deltamethrin, Oleuropein, Apoptosis