Selenium-enriched Spirulina protects INS-1E pancreatic beta cells from human islet amyloid polypeptide-induced apoptosis through suppression of ROS-mediated mitochondrial dysfunction and PI3/AKT pathway.

PURPOSE: Human islet amyloid polypeptide (hIAPP) aggregation is linked to loss of pancreatic beta cells in type 2 diabetes, in part due to oxidative stress. Currently, little is known about the effects of selenium-enriched Spirulina on beta cells with the presence of hIAPP. In this study, INS-1E rat insulinoma cells were used as a model to evaluate in vitro protective effects of Se-enriched Spirulina extract (Se-SE) against hIAPP-induced cell death, as well as the underlying mechanisms. METHODS: Flow cytometric analysis was used to evaluate cell apoptosis, mitochondrial membrane potential (ΔΨm) and ROS generation. Caspase activity was measured using a fluorometric method. Western blotting was applied to detect protein expression. RESULTS: Our results showed that exposure of INS-1E cells to hIAPP resulted in cell viability loss, LDH release and appearance of sub-G peak. However, cytotoxicity of hIAPP was significantly attenuated by co-treatment with Se-SE. Se-SE also inhibited hIAPP-induced activation of caspase-3, -8 and -9. Additionally, hIAPP-induced accumulation of ROS and superoxide was suppressed by co-treatment with Se-SE. Moreover, Se-SE was able to prevent hIAPP-induced depletion of ΔΨm and intracellular ATP, reduction in mitochondrial mass, changes in the expression of Bcl-2 family members, release of mitochondrial apoptogenic factors. Furthermore, hIAPP-mediated AKT inhibition was restored by co-treatment with Se-SE. CONCLUSION: Our results showed that Se-SE protects INS-1E cells from hIAPP-induced cell death through preventing ROS overproduction, mitochondrial dysfunction and modulating PI3K/AKT pathway.

Desacetyluvaricin induces S phase arrest in SW480 colorectal cancer cells through superoxide overproduction.

Annonaceous acetogenins (ACGs) are a group of fatty acid-derivatives with potent anticancer effects. In the present study, we found desacetyluvaricin (Dau) exhibited notable in vitro antiproliferative effect on SW480 human colorectal carcinoma cells with IC50 value of 14 nM. The studies on the underlying mechanisms revealed that Dau inhibited the cancer cell growth through induction of S phase cell cycle arrest from 11.3% (control) to 33.2% (160 nM Dau), which was evidenced by the decreased protein expression of cyclin A Overproduction of superoxide, intracellular DNA damage, and inhibition of MEK/ERK signaling pathway, were also found involved in cells exposed to Dau. Moreover, pre-treatment of the cells with ascorbic acid significantly prevented the Dau-induced overproduction of superoxide, DNA damage and cell cycle arrest. Taken together, our results suggest that Dau induces S phase arrest in cancer cells by firstly superoxide overproduction and subsequently the involvement of various signaling pathways.

Synergistic induction of apoptosis by methylseleninic acid and cisplatin, the role of ROS-ERK/AKT-p53 pathway.

Cisplatin-based therapy is one of the most important chemotherapy treatments for cancers. However, its efficacy is greatly limited by drug resistance and undesirable side effects. Therefore, it is of great importance to develop chemosensitizing agents to cisplatin. In the present study, we demonstrated the strategy to use methylseleninic acid (MeSe) as a synergistic agent of cisplatin and elucidated their action mechanisms. The combination of MeSe and cisplatin exhibited synergistic anticancer efficacy and achieved greater selectivity between cancer cell and normal cell. By inducing intracellular oxidative stress, MeSe potentiated cisplatin-induced DNA damage and led to enhanced p53 phosphorylation, followed by increased activation of both mitochondrial and death receptor pathway. Down-regulation of phosphorylated AKT and ERK also played important roles in the synergistic effects of MeSe and cisplatin. Our results suggested that the strategy to apply MeSe as a synergistic agent to cisplatin could be a highly efficient way to achieve anticancer synergism by targeting the intracellular redox system. MeSe might be a candidate for clinical application as a chemosensitizer to cisplatin-based therapy for cancer treatments, especially for hepatocellular carcinoma.

Comparative proteomics analysis of selenium responses in selenium-enriched rice grains.

By foliar fortification with selenite, selenium (Se)-enriched rice with a higher Se content and grain yield has been generated. However, the regulatory mechanisms of Se response in rice grains remain unknown; therefore, we carried out a comparative proteomics study in Se-enriched rice grains by using two approaches including two-dimensional gel electrophoresis (2-DE)-coupled MALDI-TOF/TOF MS and 1-DE/LC-FTICR-MS-coupled label-free quantification. By comparison between Se treatment and control, 62 and 250 abundance changed proteins were identified from 2-DE and 1-DE, respectively. By functional classification, proteins involved in metabolism, cell redox regulation, and seed nutritional storage were the most highly affected by Se accumulation. The up-regulation of late embryogenesis abundant proteins as well as proteins involved in sucrose synthesis and other metabolism pathways may contribute to the earlier maturation and higher yield of the Se-enriched rice. In addition, there have been six proteins identified to contain selenoamino acid modification, which is the first identification of selenoproteins in higher plants. In conclusion, our study provided novel insights into Se response in rice grains at the proteome level, which are expected to be highly useful for dissecting the Se response pathways in rice and for the production of Se-enriched rice in the future.

1,4-Diselenophene-1,4-diketone triggers caspase-dependent apoptosis in human melanoma A375 cells through induction of mitochondrial dysfunction.

Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound, 1,4-diselenophene-1,4-diketone (DSeD), has been synthesized by Double Friedel-Crafts reaction and identified as a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was relatively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initiation of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis pathway.

Ruthenium polypyridyl complexes that induce mitochondria-mediated apoptosis in cancer cells.

The limitations of cisplatin-based chemotherapy, including high toxicity, undesirable side effects, and drug resistance, have motivated extensive investigations into alternative metal-based cancer therapies. Ruthenium (Ru) possesses several favorable properties suited to rational anticancer drug design and biological applications. In the present study, we synthesized a series of ruthenium polypyridyl complexes containing N,N-chelating ligands, examined their anticancer activities, and elucidated the molecular mechanisms through which they caused the cancer cell death. The results demonstrated that [Ru(phen)(2)-p-MOPIP](PF(6))(2).2H(2)O (RuPOP), a complex with potent antiproliferative activity, is able to induce mitochondria-mediated and caspase-dependent apoptosis in human cancer cells. On the basis of these results, we suggest that RuPOP may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially for melanoma.

[Spectrometric investigation of the antioxidant activities of dangcong tea aqueous extracts against DPPH and ABTS free radicals].

The antioxidant activities of Dangcong tea aqueous extracts against DPPH and ABTS free radicals were evaluated using spectrometric methods. The results show that Dangcong tea aqueous extracts could effectively and rapidly inhibited the formation of ABTS and DPPH free radicals in a dose- and time-dependent manner, indicating the potent antioxidant activities of Dangcong tea aqueous extracts under both hydrophilic and hydrophobic conditions. In the optimized systems, the IC50 values of Baiye (I, II) and Fenghuang (III, IV) Dangcong teas against ABTS free radical were 26.9, 25.5, 28.0 and 31.7 microg x mL(-1), respectively, which were significantly lower than that of Trolox, the positive control (85.2 microg x mL(-1)), indicating the higher antioxidant activities of Dangcong teas. For DPPH free radical, the IC50 values for the Dangcong teas (I-IV) were 49.8, 41.6, 47.3 and 64.5 microg x mL(-1), respectively. Taken together, our results suggest the potential applications of Dangcong tea as functional food.

Mitochondria-mediated apoptosis in human breast carcinoma MCF-7 cells induced by a novel selenadiazole derivative.

The role of organoselenium compounds as potent cancer chemopreventive and chemotherapeutic agents has been supported by epidemiological, preclinical and clinical studies. In this study, a novel selenadiazole derivative, 1,2,5-selenadiazolo-[3,4-d]pyrimidine-5,7-(4H,6H)-dione (SPO), is identified as a potent antiproliferative agent against human breast adrenocarcinoma MCF-7 cells, human hepatoma HepG2 cells and human melanoma A375 cells. Induction of apoptosis in MCF-7 and A375 cells by SPO was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Further investigation on intracellular mechanisms found that SPO treatments induced activation of caspase-8 and caspase-9, overproduction of reactive oxygen species, and depletion of mitochondrial membrane potential (Delta Psi m) through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our findings suggest that SPO is a promising novel organoselenium compound with potential in the treatment of human cancers.

Selenium nanoparticles fabricated in Undaria pinnatifida polysaccharide solutions induce mitochondria-mediated apoptosis in A375 human melanoma cells.

Selenium nanoparticle (Nano-Se) is a novel Se species with novel biological activities and low toxicity. In the present study, we demonstrated a simple method for synthesis of size-controlled Nano-Se by adding Undaria pinnatifida polysaccharides to the redox system of selenite and ascorbic acid. A panel of four human cancer cell lines was shown to be susceptible to Nano-Se, with IC(50) values ranging from 3.0 to 14.1 microM. Treatment of A375 human melanoma cells with the Nano-Se resulted in dose-dependent cell apoptosis as indicated by DNA fragmentation and phosphatidylserine translocation. Further investigation on intracellular mechanisms found that Nano-Se treatment triggered apoptotic cell death in A375 cells with the involvement of oxidative stress and mitochondrial dysfunction. Our results suggest that Nano-Se may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially melanoma cancer.

Selenium-induced changes in activities of antioxidant enzymes and content of photosynthetic pigments in Spirulina platensis.

Spirulina platensis exposed to various selenium (Se) concentrations (0, 10, 20, 40, 80, 150, 175, 200, 250 mg/L) accumulated high amounts of Se in a dose- and time-dependent manner. Under low Se concentrations (or= 175 mg/L) led to higher Se accumulation and increases in activities of GPX, SOD, CAT and POD, but also induced lipid peroxidation (LPO) coupled with potassium leakage and decreases in biomass concentration and contents of photosynthetic pigment. The results indicate that increases in activities of the antioxidant enzymes were not sufficient to protect cell membranes against Se stress. Time-dependent variations in the activities of antioxidant enzymes, contents of chlorophyll a and carotenoid and the LPO level were also investigated under representative Se concentrations of 40 and 200 mg/L. Opposite variation trends between SOD-CAT activities, and GPX-POD-APX activities were observed during the growth cycles. The results showed that the prevention of damage to cell membranes of S. platensis cells could be achieved by cooperative effects of SOD-CAT and GPX-POD-APX enzymes. This study concludes that S. platensis possessed tolerance to Se and could protect itself from phytotoxicity induced by Se by altering various metabolic processes.

Apoptosis induced by 9,11­dehydroergosterol peroxide from Ganoderma Lucidum mycelium in human malignant melanoma cells is Mcl­1 dependent.

9,11-Dehydroergosterol peroxide [9(11)-DHEP] is an important steroid from medicinal mushroom, which has been reported to exert antitumor activity in several tumor types. However, the role of 9(11)­DHEP toward the malignant melanoma cells has not been investigated. In the present study, the steroid from Ganoderma lucidum was purified on a submerged culture, and its antitumor mechanisms on A375 human malignant melanoma cells was investigated by MTT, flow cytometry and western blotting. The studies demonstrated that apoptotic mechanisms of the steroid were caspase­dependent and mediated via the mitochondrial pathway. The steroid did not cause significant changes in the expression levels of B­cell lymphoma 2 (Bcl­2) family proteins, Bcl­2­like protein 11, p53 upregulated modulator of apoptosis, Bcl­2­associated X protein, BH3 interacting­domain death agonist, Bcl­2­associated death promoter and Bcl­2, but it significantly downregulated induced myeloid leukemia cell differentiation protein Mcl­1 (Mcl­1) in melanoma cells, suggesting the key role of Mcl­1 in regulating apoptosis of melanoma cells induced by the steroid. These properties of 9(11)­DHEP advocate its usage as supplements in human malignant melanoma chemoprevention. The present study also suggests that mycelium of G. lucidum has a potential for producing bioactive substances and extracts with applications in medicine.

Correction: Chiral ruthenium polypyridyl complexes as mitochondria-targeted apoptosis inducers.

[This corrects the article DOI: 10.1039/C0MD00060D.].

Fatty acid synthase inhibitors cerulenin and C75 retard growth and induce caspase-dependent apoptosis in human melanoma A-375 cells.

Fatty acid synthase (FAS) has been shown previously to be highly expressed in breast and prostate carcinomas, but has low expression level in normal tissues. We also found in this study that FAS was expressed in a number of cancer cell lines of different histotypes. The growth-inhibitory effects of FAS inhibitors cerulenin and C75 were then investigated on these cancer cell lines, particularly the human melanoma A-375. MTT assay revealed that the cancer cell proliferation and viability was reduced dose- and time-dependently by 20.8%-87.1% of the control levels after 24 and 48 h of treatment with 20-160 microM of the inhibitor. Immunoblotting studies showed that both cerulenin and C75 induced poly(ADP-ribose) polymerase (PARP) cleavage in the melanoma cells dose-dependently. Procaspase-3 was also found to be processed into the active and smaller 17 and 19 kDa subunits, and administration of pan-caspase inhibitor Z-VAD-FMK completely rescued the cells from PARP cleavage. This indicated that the cerulenin- and C75-induced apoptosis involved caspase activation. The proapoptotic effects of the FAS inhibitors were further confirmed using confocal microscopy with annexin-V FITC and propidium iodide staining. DNA flow cytometric studies demonstrated that the FAS inhibitors accumulated G2/M cells preceding the elevation of sub G1 or apoptotic cells with fragmented DNA. The induced cell cycle arrest and apoptosis were associated with elevation of p21 and depletion of Bcl-xL and Mcl-1, respectively. Results from this study suggest that FAS inhibitors retard growth of melanoma A-375 cells, involving activation of caspase-dependent apoptosis.

A rapid and efficient method to study the function of crop plant transporters in Arabidopsis.

Iron (Fe) is an essential micronutrient for humans. Fe deficiency disease is widespread and has led to extensive studies on the mechanisms of Fe uptake and storage, especially in staple food crops such as rice. However, studies of functionally related genes in rice and other crops are often time and space demanding. Here, we demonstrate that transgenic Arabidopsis suspension culture cells and Arabidopsis plants can be used as an efficient expression system for gain-of-function study of selected transporters, using Fe transporters as a proof-of-principle. The vacuolar membrane transporters OsVIT1 and OsVIT2 have been described to be important for iron sequestration, and disruption of these two genes leads to Fe accumulation in rice seeds. In this study, we have taken advantage of the fluorescent-tagged protein GFP-OsVIT1, which functionally complements the Fe hypersensitivity of ccc1 yeast mutant, to generate transgenic Arabidopsis suspension cell lines and plants. GFP-OsVIT1 was shown to localize on the vacuolar membrane using confocal microscopy and immunogold EM. More importantly, the Fe concentration, as well as the concentration of Zn, in the transgenic cell lines and plants were significantly increased compared to that in the WT. Taken together, our study shows that the heterologous expression of rice vacuolar membrane transporter OsVIT1 in Arabidopsis system is functional and effectively enhances iron accumulation, indicating an useful approach for studying other putative transporters of crop plants in this system.

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.

Accumulation of selenium in mixotrophic culture of Spirulina platensis on glucose.

Accumulation of Se in mixotrophic culture of Spirulina platensis was investigated in this study. Results indicated that glucose was better than acetate as an organic carbon source for mixotrophic culture of S. platensis. Supplementation of glucose (2 gL(-1)) significantly enhanced the biomass concentration (2.57 gL(-1)) and the production of phycocyanin (0.279 gL(-1)) and allophycocyanin (0.126 gL(-1)) in S. platensis, which were much higher than those of photoautotrophic culture (1.08 gL(-1), 0.119 gL(-1) and 0.042 gL(-1), respectively). Stepwise addition of Se during the growth phase avoided the inhibitory effect of high Se concentration on the growth of S. platensis. The Se enrichment favored the production of phycocyanin and allophycocyanin in the algal cells. The highest Se yield (1033 microgL(-1)) was obtained at an accumulative Se concentration of 250 mgL(-1), with organic Se percentage, biomass concentration, phycocyanin and allophycocyanin yields of 92.3%, 2.55 gL(-1), 0.295 gL(-1) and 0.153 gL(-1), respectively. These results indicated that the application of mixotrophic culture S. platensis with stepwise addition of Se to the medium could offer an effective and economical way for the production of high Se-enriched algal products.

Identification of flavonoids in Hakmeitau beans (Vigna sinensis) by high-performance liquid chromatography-electrospray mass spectrometry (LC-ESI/MS).

Liquid chromatography coupled with electrospray mass spectrometry (LC-ESI/MS) with positive and negative ion detection was used for the identification of flavonoids in Hakmeitau beans, a black seed cultivar of cowpea (Vigna sinensis). Gradient elution with water and acetonitrile, both containing 2% formic acid, was employed in chromatographic separation. The peaks were identified by comparison of the retention times and the UV-vis spectroscopic and mass spectrometric data with authentic standards and/or literature data. The identified flavonoids included six anthocyanins (cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, malvidin 3-O-glucoside, peonidin 3-O-glucoside, and petunidin 3-O-glucoside) and four flavonol/flavonol glycosides (kaempferol 3-O-glucoside, quercetin, quercetin 3-O-glucoside, and quercetin 3-O-6' '-acetylglucoside). The tentatively identified flavonoids included two anthocyanins (malvidin 3-O-acetylglucoside and peonidin 3-O-malonylglucoside) and three flavonol glycosides (myricetin-3-O-glucoside, quercetin 7-O-glucoside, and quercetin-3-O-diglucoside). These flavonoids are present in seed coats, and the contents of anthocyanins and flavonol glycosides were 20.7 and 2.0 mg/g, respectively.

Synergistic apoptosis-inducing effects on A375 human melanoma cells of natural borneol and curcumin.

This study was to investigate the synergistic effect of NB/Cur on growth and apoptosis in A375 human melanoma cell line by MTT assay, flow cytometry and Western blotting. Our results demonstrated that NB effectively synergized with Cur to enhance its antiproliferative activity on A375 human melanoma cells by induction of apoptosis, as evidenced by an increase in sub-G1 cell population, DNA fragmentation, PARP cleavage and caspase activation. Further mechanistic studies by Western blotting showed that after treatment of the cells with NB/Cur, up-regulation of the expression level of phosphorylated JNK and down-regulation of the expression level of phosphorylated ERK and Akt contributed to A375 cells apoptosis. Moreover, NB also potentiated Cur to trigger intracellular ROS overproduction and the DNA damage with up-regulation of the expression level of phosphorylated ATM, phosphorylated Brca1 and phosphorylated p53. The results indicate the combinational application potential of NB and Cur in treatments of cancers.

Selenocysteine derivative overcomes TRAIL resistance in melanoma cells: evidence for ROS-dependent synergism and signaling crosstalk.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as one of the most promising targeted drug for new cancer therapeutics, is limited in clinical application by the evolution of resistance in many cancer cell lines, especially in malignant melanoma. Thus, it is urgently needed to identify chemosensitizers to enhance the apoptotic inducing efficacy of TRAIL and overcome resistance of malignant melanoma cells. Herein, we reported that 3,3'-diselenodipropionic acid (DSeA), a Selenocysteine derivative, could synergistically enhance the growth inhibitory effect of TRAIL on A375 melanoma cells though induction of ROS-dependent apoptosis with involvement of PTEN-mediated Akt inactivation and DNA damage-mediated p53 phosphorylation, which subsequently activated mitochondrial and death receptor apoptotic pathways. Moreover, silencing of p53 down-regulated the expression levels of p53-inducible genes, and effectively blocked the cell apoptosis. Suppression of PI3K significantly increased the apoptotic cell death. In contrast, antioxidants effectively reversed the cell apoptosis through regulation of Akt and p53 signaling pathways. Taken together, the combination of DSeA and TRAIL could be a novel strategy to overcome TRAIL resistance in malignant melanoma, and DSeA may be candidates for further evaluation as a chemosensitizer in clinical trails.

pH-responsive cancer-targeted selenium nanoparticles: a transformable drug carrier with enhanced theranostic effects.

Selenium nanoparticles (SeNPs) have been widely used in various biomedical applications, including cancer therapy, diagnosis and drug delivery. Herein, we fabricated a novel type of structure-transformable capsules by decoration of SeNPs with folate-chitosan to form smart-shell nanocapsules (FAC@CurP-SeNPs). The shrink particles could target cancer cells over expressing folate receptor and enter the cells via folate receptor-mediated endocytosis. FAC@CurP-SeNPs were expanded to snowflake particles under acidifying stimulus (pH 5.3), which led to enhanced drug-release over prolonged periods. Treatment with FAC@CurP-SeNPs significantly inhibited the growth of MCF-7 human breast carcinoma cells through induction of apoptosis, which was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. The contribution of extrinsic and intrinsic apoptotic pathways to the cell apoptosis was confirmed by activation of caspase-9 and caspase-8. Internalized FAC@CurP-SeNPs triggers intracellular ROS overproduction, thus activates p53, MAPKs pathways and inhibits NFκB and to promote cell apoptosis. Our results suggest that FAC@CurP-SeNPs may be a candidate for further evaluation as a agent for human cancers, and the strategy to use transformable nanocapsules could be a highly efficient way to enhance controlled drug release and anticancer efficacy.