Antiproliferative Effect of Amaranth Proteins and Peptides on HT-29 Human Colon Tumor Cell Line

Antiproliferative effect of Amaranthus mantegazzianus proteins and peptides released after simulated gastrointestinal digestion (DH% 37.8?±?3.8) was investigated on human colon cancer cell line HT-29. Inhibition of proliferation of HT-29 cells was exhibited after a 24 h treatment with different concentrations of amaranth protein isolate (API) and the peptides released after digestion (DGS), presenting IC₅₀ values of 1.35?±?0.12 and 0.30?±?0.07 mg soluble protein/mL, respectively. Lactate dehydrogenase assay indicated that both samples caused the loss of membrane integrity and cell lysis over HT-29 cells, and DAPI fluorescence microscopies evidenced typical apoptotic features. Moreover, Annexin V-FITC flow cytometry showed a significant increase of early apoptotic and late apoptotic/necrotic HT-29 cells compared to untreated ones, and caspase-3 assay confirmed the apoptosis induction with a 43.0?±?10.3 and 65.8?±?12.7% increase of caspase-3 activity produced by a 2 mg/mL treatment of API and DGS, respectively. In conclusion, amaranth peptides successfully released after simulated gastrointestinal digestion would exert a potential antiproliferative activity over HT-29 tumor cells. This effect was linked to the induction of cell necrosis and apoptosis, supporting the idea of using amaranth proteins as a potential food alternative ingredient for functional foods.

     

Downregulation of Kinesin Spindle Protein Inhibits Proliferation,Induces Apoptosis and Increases Chemosensitivity in Hepatocellular Carcinoma Cells

Background: Kinesin spindle protein (KSP) plays a critical role in mitosis. Inhibition of KSP function leads to cell cycle arrest at mitosis and ultimately to cell death. The aim of this study was to suppress KSP expression by specific small-interfering RNA (siRNA) in Hep3B cells and evaluate its anti-tumor activity. Methods: Three siRNA targeting KSP (KSP-siRNA #1-3) and one mismatched-siRNA (Cont-siRNA) were transfected into cells. Subsequently, KSP mRNA and protein levels, cell proliferation, and apoptosis were examined in both Hep3B cells and THLE-3 cells. In addition, the chemosensitivity of KSP-siRNA-treated Hep3B cells with doxorubicin was also investigated using cell proliferation and clonogenic survival assays. Results: The expression of endogenous KSP at both mRNA and protein levels in Hep3B cells was higher than in THLE-3 cells. In Hep3B cells, KSP-siRNA #2 showed a further downregulation of KSP as compared to KSP-siRNA #1 or KSP-siRNA #3. It also exhibited greater suppression of cell proliferation and induction of apoptosis than KSP-siRNA #1 or KSP-siRNA #3; this could be explained by the significant downregulation of cyclin D1, Bcl-2, and survivin. In contrast, KSP-siRNAs had no or lower effects on KSP expression, cell proliferation and apoptosis in THLE-3 cells. We also noticed that KSP-siRNA transfection could increase chemosensitivity to doxorubicin in Hep3B cells, even at low doses compared to control. Conclusion: Reducing the expression level of KSP, combined with drug treatment, yields promising results for eradicating hepatocellular carcinoma (HCC) cells in vitro. This study opens a new direction for liver cancer treatment. Key Words: Apoptosis, Chemosensitivity, Doxorubicin, Hepatocellular carcinoma (HCC) cells, Kinesin spindle protein (KSP)     

Exposure to the Neurotoxic Dinoflagellate,Alexandrium catenella,Induces Apoptosis of the Hemocytes of the Oyster,Crassostrea gigas

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.     

Anti-Cancer and Anti-Inflammatory Activities of Three New Chromone Derivatives from the Marine-Derived Penicillium citrinum

Three new and uncommon chromone analogs, epiremisporine F (1), epiremisporine G (2), and epiremisporine H (3), were isolated from marine-origin Penicillium citrinum. Among the isolated compounds, compounds 2–3 remarkably suppressed fMLP-induced superoxide anion generation by human neutrophils, with IC₅₀ values of 31.68 ± 2.53, and 33.52 ± 0.42 μM, respectively. Compound 3 exhibited cytotoxic activities against human colon carcinoma (HT-29) and non-small lung cancer cell (A549) with IC₅₀ values of 21.17 ± 4.89 and 31.43 ± 3.01 μM, respectively, and Western blot assay confirmed that compound 3 obviously induced apoptosis of HT-29 cells, via Bcl-2, Bax, and caspase 3 signaling cascades.     

Identification and Expression of Two Novel Cytochrome P450 Genes,CYP6CV1 and CYP9A38, in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Cnaphalocrocis medinalis Güenée can cause severe losses in rice. Cytochrome P450s play crucial roles in the metabolism of allelochemicals in herbivorous insects. Two novel P450 cDNAs, CYP6CV1 and CYP9A38, were cloned from the midgut of C. medinalis. CYP6CV1 encodes a protein of 500 amino acid residues, while CYP9A38-predicted protein has 531 amino acid residues. Both cDNA-predicted proteins contain the conserved functional domains for all P450s. Phylogenetic analyses showed that CYP6CV1 is grouped in the cluster containing CYP6B members, while CYP9A38 is in the cluster including CYP9 members. However, both clusters are contained in the same higher lineage. Homologous analysis revealed that CYP6CV1 is most similar to CYP6B8, CYP6B7, CYP6B6, CYP6B2, and CYP6B4 with the highest amino acid identity of 41%. CYP9A38 is closest to CYP9A17, CYP9A21, CYP9A20, and CYP9A19 with the highest amino acid identity of 66%. Studies of temporal expression profiles revealed that CYP9A38 showed a steady increase in mRNA level during the five instar stages, but a low-expression level in pupae, and then presented at a high-expression level again in adults. Similar expression patterns were obtained with CYP6CV1. In the fifth instar larvae, CYP6CV1 was mainly expressed in midgut and fat bodies, whereas CYP9A38 was mainly expressed in midgut. Expression studies also revealed a 3.20-fold over-expression of CYP6CV1 and 3.54-fold over-expression of CYP9A38 after larval exposure to host rice resistance. Our results suggest that both CYP6CV1 and CYP9A38 may be involved in detoxification of rice phytochemicals.     

Tackling the Cytotoxic Effect of a Marine Polycyclic Quinone-Type Metabolite: Halenaquinone Induces Molt 4 Cells Apoptosis via Oxidative Stress Combined with the Inhibition of HDAC and Topoisomerase Activities

A marine polycyclic quinone-type metabolite, halenaquinone (HQ), was found to inhibit the proliferation of Molt 4, K562, MDA-MB-231 and DLD-1 cancer cell lines, with IC₅₀ of 0.48, 0.18, 8.0 and 6.76 μg/mL, respectively. It exhibited the most potent activity against leukemia Molt 4 cells. Accumulating evidence showed that HQ may act as a potent protein kinase inhibitor in cancer therapy. To fully understand the mechanism of HQ, we further explored the precise molecular targets in leukemia Molt 4 cells. We found that the use of HQ increased apoptosis by 26.23%–70.27% and caused disruption of mitochondrial membrane potential (MMP) by 17.15%–53.25% in a dose-dependent manner, as demonstrated by Annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of Molt 4 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by HQ, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of HQ. The results of a cell-free system assay indicated that HQ could act as an HDAC and topoisomerase catalytic inhibitor through the inhibition of pan-HDAC and topoisomerase IIα expression, respectively. On the protein level, the expression of the anti-apoptotic proteins p-Akt, NFκB, HDAC and Bcl-2, as well as hexokinase II was inhibited by the use of HQ. On the other hand, the expression of the pro-apoptotic protein Bax, PARP cleavage, caspase activation and cytochrome c release were increased after HQ treatment. Taken together, our results suggested that the antileukemic effect of HQ is ROS-mediated mitochondrial apoptosis combined with the inhibitory effect on HDAC and topoisomerase activities.     

Cytotoxic and Apoptosis-Inducing Activity of Triterpene Glycosides from Holothuria scabra and Cucumaria frondosa against HepG2 Cells

The cytotoxic effects of thirteen triterpene glycosides from Holothuria scabra Jaeger and Cucumaria frondosa Gunnerus (Holothuroidea) against four human cell lines were detected and their cytotoxicity-structure relationships were established. The apoptosis-inducing activity of a more potent glycoside echinoside A (1) in HepG2 cells was further investigated by determining its effect on the morphology, mitochondrial transmembrane potential (Δψ_m) and mRNA expression levels of the apoptosis-related genes. The results showed that the number of glycosyl residues in sugar chains and the side chain of aglycone could affect their cytotoxicity towards tumor cells and selective cytotoxicity. 1 significantly inhibited cell viability and induced apoptosis in HepG2 cells. 1 also markedly decreased the Δψ_m and Bcl-2/Bax mRNA express ratio, and up-regulated the mRNA expression levels of Caspase-3, Caspase-8 and Caspase-9 in HepG2 cells. Therefore, 1 induced apoptosis in HepG2 cells through both intrinsic and extrinsic pathway. These findings could potentially promote the usage of these glycosides as leading compounds for developing new antitumor drugs.     

Flaxseed (Linum usitatissimum L.) and Its Total Non-digestible Fraction Influence the Expression of Genes Involved in Azoxymethane-induced Colon Cancer in Rats

The influence of flaxseed (Linum usitatissimum L.) and its total non-digestible fraction (TNDF) on the expression of genes involved in azoxymethane (AOM)-induced colon cancer in Sprague Dawley rats was analyzed. The dose used in the animal model was two tablespoons of flaxseed per day, which is the dose recommended for humans. Flaxseed significantly decreased the crypt multiplicity (10.50?±?3.5) compared with the AOM treatment (34.00?±?11.0), which suggests that flaxseed exhibits a preventive effect against colon cancer. Both treatments (flaxseed and TNDF) influence the overexpression of genes involved in cell cycle arrest and mitochondrial apoptosis: p53, p21, bcl-2, bax and caspase-3. Flaxseed induced the expression of p53 and p21, whereas TNDF triggered the p21-independent expression of p53. This finding suggests that both of these treatments induced cell cycle arrest. In addition, TNDF induced mitochondrial apoptosis because the TNDF + AOM group exhibited the expression of caspase-3, decreased bcl-2 expression and increased bax expression. These results suggest that the expression of the analyzed genes is associated with the presence of dietary antioxidants linked to the cell wall of flaxseed.     

The Marine Fungal Metabolite,Dicitrinone B,Induces A375 Cell Apoptosis through the ROS-Related Caspase Pathway

Dicitrinone B, a rare carbon-bridged citrinin dimer, was isolated from the marine-derived fungus, Penicillium citrinum. It was reported to have antitumor effects on tumor cells previously; however, the details of the mechanism remain unclear. In this study, we found that dicitrinone B inhibited the proliferation of multiple tumor types. Among them, the human malignant melanoma cell, A375, was confirmed to be the most sensitive. Morphologic evaluation, cell cycle arrest and apoptosis rate analysis results showed that dicitrinone B significantly induced A375 cell apoptosis. Subsequent observation of reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) reduction revealed that the apoptosis induced by dicitrinone B may be triggered by over-producing ROS. Further studies indicated that the apoptosis was associated with both intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. Caspase-9, caspase-8 and caspase-3 were activated during the process, leading to PARP cleavage. The pan-caspase inhibitor, Z-VAD-FMK, could reverse dicitrinone B-induced apoptosis, suggesting that it is a caspase-dependent pathway. Our data for the first time showed that dicitrinone B inhibits the proliferation of tumor cells by inducing cell apoptosis. Moreover, compared with the first-line chemotherapy drug, 5-fluorouracil (5-Fu), dicitrinone B showed much more potent anticancer efficacy, suggesting that it might serve as a potential antitumor agent.     

Ecklonia cava Attenuates PM2.5-Induced Cognitive Decline through Mitochondrial Activation and Anti-Inflammatory Effect

To evaluate the effects of Ecklonia cava (E. cava) on ambient-pollution-induced neurotoxicity, we used a mouse model exposed to particulate matter smaller than 2.5 µm in aerodynamic diameter (PM_2.5). The intake of water extract from E. cava (WEE) effectively prevented the learning and memory decline. After a behavioral test, the toll-like receptor (TLR)-4-initiated inflammatory response was confirmed by PM_2.5 exposure in the lung and brain tissues, and the WEE was regulated through the inhibition of nuclear factor-kappa B (NF-κB)/inflammasome formation signaling pathway and pro-inflammatory cytokines (IL-6 and IFN-γ). The WEE also effectively improved the PM_2.5-induced oxidative damage of the lungs and brain through the inhibition of malondialdehyde (MDA) production and the activation of mitochondrial activity (mitochondrial ROS content, mitochondria membrane potential (MMP), adenosine triphosphate (ATP) content, and mitochondria-mediated apoptotic molecules). In particular, the WEE regulated the cognition-related proteins (a decreased amyloid precursor protein (APP) and p-Tau, and an increased brain-derived neurotrophic factor (BDNF)) associated with PM_2.5-induced cognitive dysfunction. Additionally, the WEE prevented the inactivation of acetylcholine (ACh) synthesis and release as a neurotransmitter by regulating the acetylcholinesterase (AChE) activity, choline acetyltransferase (ChAT), and ACh receptor (AChR)-α3 in the brain tissue. The bioactive compounds of the WEE were detected as the polysaccharide (average Mw; 160.13 kDa) and phenolic compounds including 2′-phloroeckol.     

Fish Oil Increases Diet-Induced Thermogenesis in Mice

Increasing energy expenditure (EE) is beneficial for preventing obesity. Diet-induced thermogenesis (DIT) is one of the components of total EE. Therefore, increasing DIT is effective against obesity. We examined how much fish oil (FO) increased DIT by measuring absolute values of DIT in mice. C57BL/6J male mice were given diets of 30 energy% fat consisting of FO or safflower oil plus butter as control oil (Con). After administration for 9 days, respiration in mice was monitored, and then the data were used to calculate DIT and EE. DIT increased significantly by 1.2-fold in the FO-fed mice compared with the Con-fed mice. Body weight gain was significantly lower in the FO-fed mice. FO increased the levels of uncoupling protein 1 (Ucp1) mRNA and UCP1 protein in brown adipose tissue (BAT) by 1.5- and 1.2-fold, respectively. In subcutaneous white adipose tissue (subWAT), the levels of Ucp1 mRNA and UCP1 protein were increased by 6.3- and 2.7-fold, respectively, by FO administration. FO also significantly increased the expression of markers of browning in subWAT such as fibroblast growth factor 21 and cell death-inducing DNA fragmentation factor α-like effector a. Thus, dietary FO seems to increase DIT in mice via the increased expressions of Ucp1 in BAT and induced browning of subWAT. FO might be a promising dietary fat in the prevention of obesity by upregulation of energy metabolism.     

Polyhydroxy p-Terphenyls from a Mangrove Endophytic Fungus Aspergillus candidus LDJ-5

Six undescribed polyhydroxy p-terphenyls, namely asperterphenyllins A–F, were isolated from an endophytic fungus Aspergillus candidus LDJ-5. Their structures were determined by NMR and MS data. Differing from the previously reported p-terphenyls, asperterphenyllin A represents the first p-terphenyl dimer connected by a C-C bond. Asperterphenyllin A displayed anti-influenza virus A (H1N1) activity and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC₅₀ values of 53 μM and 21 μM, respectively. The anti-influenza virus A (H1N1) activity and protein tyrosine phosphatase 1B (PTP1B) inhibitory activity of p-terphenyls are reported for the first time. Asperterphenyllin G exhibited cytotoxicity against nine cell lines with IC₅₀ values ranging from 0.4 to 1.7 μM. Asperterphenyllin C showed antimicrobial activity against Proteus species with a MIC value of 19 μg/mL.     

Semi-Synthesis,Cytotoxic Evaluation,and Structure—Activity Relationships of Brefeldin A Derivatives with Antileukemia Activity

Brefeldin A (1), a potent cytotoxic natural macrolactone, was produced by the marine fungus Penicillium sp. (HS-N-29) from the medicinal mangrove Acanthus ilicifolius. Series of its ester derivatives 2–16 were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure–activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7-O-2-chloro-4,5-difluorobenzoate (7) exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC₅₀ value of 0.84 µM. Further evaluations indicated that 7 induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after 7-treatment in a dose-dependent manner. Furthermore, molecular modeling of 7 docked into 1 binding site of an ARF1–GDP-GEF complex represented well-tolerance. Taken together, 7 had the potential to be served as an effective antileukemia agent or lead compound for further exploration.