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

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

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.

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.

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.

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.

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.

Strategy to enhance the therapeutic effect of doxorubicin in human hepatocellular carcinoma by selenocystine, a synergistic agent that regulates the ROS-mediated signaling.

Doxorubicin-based chemotherapy represents one of the most effective ways in combating human cancers. However, its clinical use is limited by severe side effects. Selenocystine (SeC) is a natural available selenoamino acid with novel anticancer efficacy. In this study, we used SeC to sensitize HepG2 human hepatocellular carcinoma (HCC) cells to DOX, and to achieve anticancer synergism in vitro and in vivo. Treatment with DOX dose-dependently reduced HepG2 cell viability through initiating cell apoptosis and strong G2/M phase cell cycle arrest. Mechanistic studies indicated that this sensitization of SeC to DOX was achieved by triggering inactivation of ERK and AKT and DNA damage through reactive oxygen species (ROS) overproduction. Pretreatment with inhibitors of ERK and AKT markedly enhanced combined treatment-induced cell killing, indicating that combined treatment-induced HCC cell killing with ERK- and AKT-dependent manner. Furthermore, inhibition of ROS effectively attenuated combined treatment-induced DNA damage and inactivation of ERK and AKT. Additionally, xenograft hepatocellular carcinoma growth was also effectively inhibited by combined treatment through induction of cell apoptosis in vivo. Taken together, our results suggest that the strategy to use SeC and DOX in combination could be a highly efficient way to achieve anticancer synergism against HCC.

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.

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.

Selenocystine potentiates cancer cell apoptosis induced by 5-fluorouracil by triggering reactive oxygen species-mediated DNA damage and inactivation of the ERK pathway.

5-Fluorouracil (5-FU)-based chemotherapy as a first-line treatment is quite limited, because of its inefficiency and clinical resistance to it. The search for chemosensitizers that could augment its efficiency and overcome the drug resistance to 5-FU has kindled great interest among scientists. Selenocystine (SeC), a naturally occurring selenoamino acid, displayed broad-spectrum anticancer activity in our previous studies. This study demonstrates that SeC acts as an effective enhancer of 5-FU-induced apoptosis in A375 human melanoma cells through induction of mitochondria-mediated apoptosis with the involvement of DNA damage-mediated p53 phosphorylation and ERK inactivation. Pretreatment of the cells with SeC significantly enhanced 5-FU-induced loss of mitochondrial membrane potential (∆ψm) by regulating the expression levels of Bcl-2 family proteins. SeC and 5-FU in combination also triggered cell oxidative stress through regulation of the intracellular redox system and led to DNA damage and inactivation of ERK and AKT. Moreover, inhibitors of ERK and AKT effectively enhanced the apoptotic cell death induced by the combined treatment. However, pretreatment of the cells with glutathione reversed the apoptosis induced by SeC and 5-FU and recovered the expression of ERK and AKT inactivation, which revealed the important role of reactive oxygen species in cell apoptosis and regulation of ERK and AKT pathways. Taken together, our results suggest that a strategy of using SeC and 5-FU in combination could be a highly efficient way to achieve anticancer synergism.

Generation of selenium-enriched rice with enhanced grain yield, selenium content and bioavailability through fertilisation with selenite.

To fulfill the natural human needs of selenium, selenium biofortification has been carried out in rice (Oryza sativa) in recent years. Despite some improvements have been made, the increase of selenium content in rice was still limited and a large amount of fertilisers are often required, which may cause environmental pullution. In this study, we further improved the selenium biofortification of rice by using less selenium fertilisers (10.5 g selenium/hectare) whereas, largely increasing selenium content in rice grains (up to 51 times vs. control). Furthermore, selenium speciation analysis, in vitro gastrointestinal digestion and antioxidant assays were performed to evaluate the selenium bioaccessibility and bioavailability in selenium-enriched rice grains. The major selenium species found were readily absorbable selenomethionine. Meanwhile, the selenium-enriched rice grains have significantly higher antioxidant bioactivities. In conclusion, this selenium-enriched rice has enormous potential for selenium supplementation in humans.

Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles.

Selenium nanoparticles (SeNPs) have garnered a great deal of attention as potential cancer therapeutic payloads. However, the in vivo targeting drug delivery has been challenging. Herein, we describe the synthesis of tansferrin (Tf)-conjugated SeNPs and its use as a cancer-targeted drug delivery system to achieve enhanced cellular uptake and anticancer efficacy. Tf as targeting ligand significantly enhances the cellular uptake of doxorubicin (DOX)-loaded SeNPs through clathrin-mediated and caveolae/lipid raft-mediated endocytosis in cancer cells overexpressing transferrin receptor, and increases their selectivity between cancer and normal cells. DOX-loaded and Tf-conjugated SeNPs (Tf-SeNPs) exhibits unprecedented enhanced cytotoxicity toward cancer cells through induction of apoptosis with the involvement of intrinsic and extrinsic pathways. Internalized Tf-SeNPs triggers intracellular ROS overproduction, thus activates p53 and MAPKs pathways to promote cell apoptosis. In the nude mice xenograft experiment, Tf-SeNPs significantly inhibits the tumor growth via induction of p53-mediated apoptosis. This cancer-targeted design of SeNPs opens a new path for synergistic treating of cancer with higher efficacy and decreased side effects.

Natural borneol, a monoterpenoid compound, potentiates selenocystine-induced apoptosis in human hepatocellular carcinoma cells by enhancement of cellular uptake and activation of ROS-mediated DNA damage.

Selenocystine (SeC) has been identified as a novel compound with broad-spectrum anticancer activities. Natural borneol (NB) is a monoterpenoid compound that has been used as a promoter of drug absorption. In the present study, we demonstrated that NB significantly enhanced the cellular uptake of SeC and potentiated its antiproliferative activity on HepG2 cells by induction of apoptosis. NB effectively synergized with SeC to reduce cancer cell growth through the triggering apoptotic cell death. Further mechanistic studies by Western blotting showed that treatment of the cells with NB and SeC activated the intrinsic apoptotic pathway by regulation of pro-survival and pro-apoptotic Bcl-2 family proteins. Treatment of the cells with NB and SeC induced the activation of p38MAPK and inactivation of Akt and ERK. NB also potentiated SeC to trigger intracellular ROS generation and DNA strand breaks as examined by Comet assay. Moreover, the thiol-reducing antioxidants effectively blocked the occurrence of cell apoptosis, which confirmed the important role of ROS in cell apoptosis. Taken together, these results reveal that NB strongly potentiates SeC-induced apoptosis in cancer cells by enhancement of cellular uptake and activation of ROS-mediated DNA damage. NB could be further developed as a chemosensitizer of SeC in treatment of human cancers.

Enhancement of auranofin-induced apoptosis in MCF-7 human breast cells by selenocystine, a synergistic inhibitor of thioredoxin reductase.

Thioredoxin system plays an important role in regulation of intracellular redox balance and various signaling pathways. Thioredoxin reductase (TrxR) is overexpressed in many cancer cells and has been identified as a potential target of anticancer drugs. Auranofin (AF) is potent TrxR inhibitor with novel in vitro and in vivo anticancer activities. Selenocystine (SeC) is a nutritionally available selenoamino acid with selective anticancer effects through induction of apoptosis. In the present study, we demonstrated the synergistic effects and the underlying molecular mechanisms of SeC in combination with AF on MCF-7 human breast cancer cells. The results showed that SeC and AF synergistically inhibited the cancer cell growth through induction of ROS-dependent apoptosis with the involvement of mitochondrial dysfunction. DNA damage-mediated p53 phosphorylation and down-regulation of phosphorylated AKT and ERK also contributed to cell apoptosis. Moreover, we demonstrated the important role of TrxR activity in the synergistic action of SeC and AF. Taken together, our results suggest the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism by targeting TrxR.

Cyanidin reverses cisplatin-induced apoptosis in HK-2 proximal tubular cells through inhibition of ROS-mediated DNA damage and modulation of the ERK and AKT pathways.

Cyanidin is an anthocyanin widely distributed in food diet with novel antioxidant activity. Herein, we investigated the protective effects of cyanidin on HK-2 proximal tubular cells against cisplatin-induced apoptosis and elucidated the underlying mechanisms. The results showed that cisplatin-induced apoptosis in HK-2 cells was significantly attenuated by cyanidin. The cleavage of caspases and PARP, activation of p53 and mitochondrial-mediated apoptosis pathways induced by cisplatin were effectively blocked by cyanidin. Moreover, cyanidin significantly suppressed the overproduction of ROS, and activation of ERK and AKT pathways triggered by cisplatin. Our results indicate that cyanidin exhibits therapeutic potential in prevention of cisplatin-induced nephrotoxicity.

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.

PEG-nanolized ultrasmall selenium nanoparticles overcome drug resistance in hepatocellular carcinoma HepG2 cells through induction of mitochondria dysfunction.

Gray selenium (Se) is one of the most widely used Se sources with very limited biocompatibility and bioactivity. In the present study, a simple method for the preparation of ultrasmall selenium nanoparticles (SeNPs) through direct nanolization of gray selenium by polyethylene glycol (PEG) was demonstrated. Monodisperse and homogeneous PEG-SeNPs with ultrasmall diameters were successfully prepared under optimized conditions. The products were characterized using various microscopic and spectroscopic methods, and the results suggest that the amphoteric properties of PEG and the coordination between oxygen and selenium atoms contributed to the formation of ultrasmall nanoparticles. PEG-SeNPs exhibited stronger growth inhibition on drug-resistant hepatocellular carcinoma (R-HepG2) cells than on normal HepG2 cells. Dose-dependent apoptosis was induced by PEG-SeNPs in R-HepG2 cells, as evidenced by an increase in the sub-G1 cell population. Further investigation on the underlying molecular mechanisms revealed that depletion of mitochondrial membrane potential and generation of superoxide anions contributed to PEG-SeNPs-induced apoptotic cell death in R-HepG2 cells. Our results suggest that PEG-SeNPs may be a candidate for further evaluation as a chemotherapeutic agent for drug-resistant liver cancer, and the strategy to use PEG200 as a surface decorator could be a highly efficient way to enhance the anticancer efficacy of nanomaterials.

Selenium nanoparticles as a carrier of 5-fluorouracil to achieve anticancer synergism.

A simple method for preparing 5-fluorouracil surface-functionalized selenium nanoparticles (5FU-SeNPs) with enhanced anticancer activity has been demonstrated in the present study. Spherical SeNPs were capped with 5FU through formation of Se-O and Se-N bonds and physical adsorption, leading to the stable structure of the conjugates. 5FU surface decoration significantly enhanced the cellular uptake of SeNPs through endocytosis. A panel of five human cancer cell lines was shown to be susceptible to 5FU-SeNPs, with IC(50) values ranging from 6.2 to 14.4 µM. Despite this potency, 5FU-SeNP possesses great selectivity between cancer and normal cells. Induction of apoptosis in A375 human melanoma cells by 5FU-SeNPs was evidenced by accumulation of sub-G1 cell population, DNA fragmentation, and nuclear condensation. The contribution of the intrinsic apoptotic pathway to the cell apoptosis was confirmed by activation of caspase-9 and depletion of mitochondrial membrane potential. Pretreatment of cells with a general caspase inhibitor z-VAD-fmk significantly prevented 5FU-SeNP-induced apoptosis, indicating that 5FU-SeNP induced caspase-dependent apoptosis in A375 cells. Furthermore, 5FU-SeNP-induced apoptosis was found dependent on ROS generation. Our results suggest that the strategy to use SeNPs as a carrier of 5FU could be a highly efficient way to achieve anticancer synergism. 5FU-SeNPs may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers, especially melanoma.

Proteomics analysis reveals multiple regulatory mechanisms in response to selenium in rice.

Selenium (Se) shows both beneficial and toxic effects on plant growth. Rice (Oryza sativa L.) seedlings cultivated under lower concentrations of sodium selenite showed enhanced growth, whereas higher concentrations of sodium selenite repressed seedling growth. To acquire detailed regulatory mechanisms underlying these effects, a comparative proteomics study using 2-dimensional gel electrophoresis and MALDI-TOF/TOF MS was performed. By comparison of gel images between Se treatments and control, 66 and 97 differentially expressed proteins were identified in shoot and root, respectively under at least one of the Se treatment concentrations. Gene Ontology and Clustering analysis reveal primary metabolism, photosynthesis and redox homeostasis are the most highly affected biological processes by Se treatments. Lower Se treatments (2 and 6 mg/L sodium selenite) activated antioxidative system, enhanced photosynthesis and primary metabolism. However, higher Se treatment (10mg/L sodium selenite) damaged photosynthesis apparatus, inhibited photosynthesis and primary metabolism. Protein ubiquitination and phosphorylation may also play important roles in Se response in rice. In conclusion, our study provided novel insights into Se response in rice at the proteome level, which are expected to be highly useful for dissecting the Se response pathways in higher plants and for producing Se enriched rice cultivars in the future.