Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

RNA polymerase common subunit ZmRPABC5b is transcriptionally activated by Opaque2 and essential for endosperm development in maize.

Maize (Zea mays) kernel size is an important factor determining grain yield; although numerous genes regulate kernel development, the roles of RNA polymerases in this process are largely unclear. Here, we characterized the defective kernel 701 (dek701) mutant that displays delayed endosperm development but normal vegetative growth and flowering transition, compared to its wild type. We cloned Dek701, which encoded ZmRPABC5b, a common subunit to RNA polymerases I, II and III. Loss-of-function mutation of Dek701 impaired the function of all three RNA polymerases and altered the transcription of genes related to RNA biosynthesis, phytohormone response and starch accumulation. Consistent with this observation, loss-of-function mutation of Dek701 affected cell proliferation and phytohormone homeostasis in maize endosperm. Dek701 was transcriptionally regulated in the endosperm by the transcription factor Opaque2 through binding to the GCN4 motif within the Dek701 promoter, which was subjected to strong artificial selection during maize domestication. Further investigation revealed that DEK701 interacts with the other common RNA polymerase subunit ZmRPABC2. The results of this study provide substantial insight into the Opaque2-ZmRPABC5b transcriptional regulatory network as a central hub for regulating endosperm development in maize.

A genome-wide association study uncovers a ZmRap2.7-ZCN9/ZCN10 module to regulate ABA signalling and seed vigour in maize.

Seed vigour, including rapid, uniform germination and robust seedling establishment under various field conditions, is becoming an increasingly essential agronomic trait for achieving high yield in crops. However, little is known about this important seed quality trait. In this study, we performed a genome-wide association study to identify a key transcription factor ZmRap2.7, which regulates seed vigour through transcriptionally repressing expressions of three ABA signalling genes ZmPYL3, ZmPP2C and ZmABI5 and two phosphatidylethanolamine-binding genes ZCN9 and ZCN10. In addition, ZCN9 and ZCN10 proteins could interact with ZmPYL3, ZmPP2C and ZmABI5 proteins, and loss-of-function of ZmRap2.7 and overexpression of ZCN9 and ZCN10 reduced ABA sensitivity and seed vigour, suggesting a complex regulatory network for regulation of ABA signalling mediated seed vigour. Finally, we showed that four SNPs in ZmRap2.7 coding region influenced its transcriptionally binding activity to the downstream gene promoters. Together with previously identified functional variants within and surrounding ZmRap2.7, we concluded that the distinct allelic variations of ZmRap2.7 were obtained independently during maize domestication and improvement, and responded separately for the diversities of seed vigour, flowering time and brace root development. These results provide novel genes, a new regulatory network and an evolutional mechanism for understanding the molecular mechanism of seed vigour.

Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

Genome-wide expression quantitative trait locus analysis reveals silk-preferential gene regulatory network in maize.

Silk of maize (Zea mays L.) contains diverse metabolites with complicated structures and functions, making it a great challenge to explore the mechanisms of metabolic regulation. Genome-wide identification of silk-preferential genes and investigation of their expression regulation provide an opportunity to reveal the regulatory networks of metabolism. Here, we applied the expression quantitative trait locus (eQTL) mapping on a maize natural population to explore the regulation of gene expression in unpollinated silk of maize. We obtained 3,985 silk-preferential genes that were specifically or preferentially expressed in silk using our population. Silk-preferential genes showed more obvious expression variations compared with broadly expressed genes that were ubiquitously expressed in most tissues. We found that trans-eQTL regulation played a more important role for silk-preferential genes compared to the broadly expressed genes. The relationship between 38 transcription factors and 85 target genes, including silk-preferential genes, were detected. Finally, we constructed a transcriptional regulatory network around the silk-preferential gene Bx10, which was proposed to be associated with response to abiotic stress and biotic stress. Taken together, this study deepened our understanding of transcriptome variation in maize silk and the expression regulation of silk-preferential genes, enhancing the investigation of regulatory networks on metabolic pathways.

Identification of EDNRA as the Key Biomarker for Hypercholesterolemia and Colorectal Cancer.

Some studies have investigated the role of cholesterol in the progression of colorectal cancer (CRC). However, the underlying mechanism of action is not clear. In this study, we used bioinformatics tools to elucidate the molecular mechanisms involved. We initially obtained CRC datasets from the Gene Expression Omnibus (GEO) database and hypercholesterolemia data from GeneCards and DisGeNE. Common differentially expressed genes (DEGs) were determined by using Venn diagram web tools. Next, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The hub gene was identified through common expression pattern analysis and survival analysis. Finally, we conducted an immune regulatory point analysis and predicted target drugs based on the hub gene. The results of our analysis revealed 13 common DEGs, with endothelin receptor type A (EDNRA) identified as the hub gene linking hypercholesterolemia and CRC. The results of the GO analysis showed that the common DEGs were primarily associated with the G-protein coupled receptor signaling pathway, extracellular space, and receptor binding. The results of the KEGG pathway enrichment analysis indicated enrichment in pathways related to cancer and the phospholipase D signaling pathway. Additionally, we identified potential target drugs, including Podocarpus montanus, Diospyros kaki, Herba Salviae japoniae, sitaxentan, and ambrisentan. We found that EDNRA might be an underlying biomarker for both hypercholesterolemia and CRC. The predicted target drugs provide new strategies for treating CRC.

cis-Regulatory variation affecting gene expression contributes to the improvement of maize kernel size.

cis-Regulatory variations contribute to trait evolution and adaptation during crop domestication and improvement. As the most important harvested organ in maize (Zea mays L.), kernel size has undergone intensive selection for size. However, the associations between maize kernel size and cis-regulatory variations remain unclear. We chose two independent association populations to dissect the genetic architecture of maize kernel size together with transcriptomic and genotypic data. The resulting phenotypes reflected a strong influence of population structure on kernel size. Compared with genome-wide association studies (GWASs), which accounted for population structure and relatedness, GWAS based on a naïve or simple linear model revealed additional associated single-nucleotide polymorphisms significantly involved in the conserved pathways controlling seed size in plants. Regulation analyses through expression quantitative trait locus mapping revealed that cis-regulatory variations likely control kernel size by fine-tuning the expression of proximal genes, among which ZmKL1 (GRMZM2G098305) was transgenically validated. We also proved that the pyramiding of the favorable cis-regulatory variations has contributed to the improvement of maize kernel size. Collectively, our results demonstrate that cis-regulatory variations, together with their regulatory genes, provide excellent targets for future maize improvement.

Synthesis, characterization, anticancer efficacy evaluation of ruthenium(II) and iridium(III) polypyridyl complexes toward A549 cells.

A new ligand DFIP (2-(dibenzo[b,d]furan-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its two complexes iridium(III) [Ir(ppy)2(DFIP)](PF6) (ppy = 2-phenylpyridine, Ir1) and ruthenium(II) [Ru(bpy)2(DFIP)](PF6)2 (bpy = 2,2'-bipyridine, Ru1) were synthesized and characterized. The anticancer effects of the two complexes on A549, BEL-7402, HepG2, SGC-7901, HCT116 and normal LO2 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complex Ir1 shows high cytotoxic activity on A549, BEL-7402, SGC-7901 and HepG2, Ru1 exhibits moderate anticancer activity toward A549, BEL-7402 and SGC-7901 cells. The IC50 values of Ir1 and Ru1 toward A549 are 7.2 ± 0.1 and 22.6 ± 1.4 µM, respectively. The localization of complexes Ir1 and Ru1 in the mitochondrial, intracellular accumulation of reactive oxygen species (ROS) levels, and the changes of mitochondrial membrane potential (MMP) and cytochrome c (cyto-c) were investigated. Apoptosis and cell cycle were detected by flow cytometry. Immunogenic cell death (ICD) was used to detect the effects of Ir1 and Ru1 on the A549 using a confocal laser scanning microscope. The expression of apoptosis-related proteins was detected by western blotting. Ir1 and Ru1 can increase the intracellular ROS levels and release cyto-c, reduce the MMP, leading to the apoptosis of A549 cells and blocking the A549 cells at the G0/G1 phase. Additionally, the complexes caused a decrease of the expression of polyADP-ribose polymerase (PARP), caspase 3, Bcl-2 (B-cell lymphoma-2), PI3K (phosphoinositide-3 kinase) and upregulated the expression of Bax. All these findings indicated that the complexes exert anticancer efficacy to induce cell death through immunogenic cell death, apoptosis, and autophagy.

Evaluation of the Anticancer Activity and Mechanism Studies of Glycyrrhetic Acid Derivatives toward HeLa Cells.

In this paper, a series of glycyrrhetic acid derivatives 3a-3f were synthesized via the esterification reaction. The cytotoxicity of these compounds against five tumor cells (SGC-7901, BEL-7402, A549, HeLa and B16) and normal LO2 cells was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The results showed that compound 3a exhibited high antiproliferative activity against HeLa cells (IC50 = 11.4 ± 0.2 µM). The anticancer activity was studied through apoptosis, cloning, and scratching; the levels of the intracellular ROS, GSH, and Ca2+; and the change in the mitochondrial membrane potential, cell cycle arrest and RNA sequencing. Furthermore, the effects of compound 3a on gene expression levels and metabolic pathways in HeLa cells were investigated via transcriptomics. The experimental results showed that this compound can block the cell cycle in the S phase and inhibit cell migration by downregulating Focal adhesion kinase (FAK) expression. Moreover, the compound can reduce the intracellular glutathione (GSH) content, increase the Ca2+ level and the intracellular ROS content, and induce a decrease in the mitochondrial membrane potential, further leading to cell death. In addition, it was also found that the mechanism of compounds inducing apoptosis was related to the regulation of the expression of mitochondria-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2-Associated X (Bax), and the activation of the caspase proteins. Taken together, this work provides a help for the development of glycyrrhetinic acid compounds as potential anticancer molecules.

Epigenetic Mutation in a Tubulin-Folding Cofactor B (ZmTFCB) Gene Arrests Kernel Development in Maize.

Epialleles, the heritable epigenetic variants that are not caused by changes in DNA sequences, can broaden genetic and phenotypic diversity and benefit to crop breeding, but very few epialleles related to agricultural traits have been identified in maize. Here, we cloned a small kernel mutant, smk-wl10, from maize, which encoded a tubulin-folding cofactor B (ZmTFCB) protein. Expression of the ZmTFCB gene decreased in the smk-wl10 mutant, which arrested embryo, endosperm and basal endosperm transfer layer developments. Overexpression of ZmTFCB could complement the defective phenotype of smk-wl10. No nucleotide sequence variation in ZmTFCB could be found between smk-wl10 and wild type (WT). Instead, we detected hypermethylation of nucleotide CHG (where H is A, C or T nucleotide) sequence contexts and increased level of histone H3K9me2 methylation in the upstream sequence of ZmTFCB in smk-wl10 compared with WT, which might respond to the attenuating transcription of ZmTFCB. In addition, yeast two-hybrid and bimolecular fluorescence complementation assays identified a strong interaction between ZmTFCB and its homolog ZmTFCE. Thus, our work identifies a novel epiallele of the maize ZmTFCB gene, which might represent a common phenomenon in the epigenetic regulation of important traits such as kernel development in maize.

Induction of apoptosis in SGC-7901 cells by iridium(III) complexes via endoplasmic reticulum stress-mitochondrial dysfunction pathway.

This study was intended to evaluate the anticancer activity of three newly synthesized iridium(III) complexes [Ir(ppy)2(PEIP)](PF6) (1) (ppy = 2-phenylpyridine, PEIP = 2-phenethyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(SIP)](PF6) (2) (SIP = (E)-2-styryl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(PEYIP)](PF6) (3) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]phenanthroline). The cytotoxic activity in vitro against A549, SGC-7901, HepG2, HeLa and normal NIH3T3 cells was investigated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. We found that the complexes 1, 2 and 3 significantly inhibited cell proliferation, in particular, complexes 2 and 3 show high cytotoxic effect on SGC-7901 cells with an IC50 value of 5.8 ± 0.7 and 4.4 ± 0.1 µM. Moreover, cell cycle assay revealed that the complexes could block G2/M phase of the cell cycle. Apoptotic evaluation by Annexin V/PI staining indicated that complexes 1-3 can induce apoptosis in SGC-7901 cells. In addition, microscopy detection suggested that disruption of mitochondrial functions, characterized by increased generation of intracellular ROS and Ca2+ as well as decrease of mitochondrial membrane potential. Western blot analysis shows that the complexes upregulate the expression of pro-apoptotic Bax and downregulate the expression of anti-apoptotic Bcl-2, which further activates caspase-3 and prompts the cleavage of PARP. Taken together, these results demonstrated that complexes 1-3 exert a potent anticancer effect on SGC-7901 cells via ROS-mediated endoplasmic reticulum stress-mitochondrial apoptotic pathway and have a potential to be developed as novel chemotherapeutic agents for human gastric cancer. Three new iridium(III) complexes [Ir(ppy)2(PEIP)](PF6) (1) (ppy = 2-phenylpyridine, PEIP = 2-phenethyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(SIP)](PF6) (2) (SIP = 2-styryl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(PEYIP)](PF6) (3) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized. The anticancer activity in vitro was investigated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The results show that the complexes induce apoptosis via ROS-mediated endoplasmic reticulum stress-mitochondrial dysfunction pathway.

Construction of lipid-biomacromolecular compounds for loading and delivery of carotenoids: Preparation methods, structural properties, and absorption-enhancing mechanisms.

Due to the unstable chemical properties and poor water solubility of carotenoids, their processing adaptation and oral bioavailability are poor, limiting their application in hydrophilic food systems. Lipid-biomacromolecular compounds can be excellent carriers for carotenoid delivery by taking full advantage of the solubilization of lipids to non-polar nutrients and the water dispersion and gastrointestinal controlled release properties of biomacromolecules. This paper reviewed the research progress of lipid-biomacromolecular compounds as encapsulation and delivery carriers of carotenoids and summarized the material selection and preparation methods for biomacromolecular compounds. By considering the interaction between the two, this paper briefly discussed the effect of these compounds on carotenoid water solubility, stability, and bioavailability, emphasizing their delivery effect on carotenoids. Finally, various challenges and future trends of lipid-biomacromolecular compounds as carotenoid delivery carriers were discussed, providing new insight into efficient loading and delivery of carotenoids.

Synthesis, Characterization and Anticancer Efficacy Evaluation of Benzoxanthone Compounds toward Gastric Cancer SGC-7901.

Three benzoxanthone derivatives were synthesized through a new photochemical strategy. The in vitro cytotoxic activity of these compounds was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and their partition coefficients (logP) were measured by shake flask method. The pKa values of the compounds were detected by potentionmetric titration. The interaction of the compounds with calf thymus DNA (CT-DNA) was investigated by electronic absorption, luminescence spectra and viscosity. A molecular docking analysis was performed. The antitumor efficacy of the compounds was evaluated by cell apoptosis, cell cycle arrest, intracellular Ca2+ concentrations and reactive oxygen species (ROS) levels. The mitochondrial membrane potential was assayed using JC-1 (5,5',6,6'-tetrachloro-1,1,3',3'-tetraethyl-imidacarbocyanine iodide) as the fluorescence probe. The expression of Bcl-2 family protein, caspase 3 and poly ADP-ribose polymerase (PARP) was explored by western blot. The results showed that the compounds induced apoptosis through a ROS-mediated mitochondrial dysfunction pathway. This work provides an efficient approach to synthesize benzoxanthone derivatives, and is helpful for understanding the apoptotic mechanism.

Nuclear-Encoded Maturase Protein 3 Is Required for the Splicing of Various Group II Introns in Mitochondria during Maize (Zea mays L.) Seed Development.

Splicing of plant organellar group II introns from precursor-RNA transcripts requires the assistance of nuclear-encoded splicing factors. Maturase (nMAT) is one such factor, as its three homologs (nMAT1, 2 and 4) have been identified as being required for the splicing of various mitochondrial introns in Arabidopsis. However, the function of nMAT in maize (Zea mays L.) is unknown. In this study, we identified a seed development mutant, empty pericarp 2441 (emp2441) from maize, which showed severely arrested embryogenesis and endosperm development. Positional cloning and transgenic complementation assays revealed that Emp2441 encodes a maturase-related protein, ZmnMAT3. ZmnMAT3 is highly expressed during seed development and its protein locates to the mitochondria. The loss of function of ZmnMAT3 resulted in the reduced splicing efficiency of various mitochondrial group II introns, particularly of the trans-splicing of nad1 introns 1, 3 and 4, which consequently abolished the transcript of nad1 and severely impaired the assembly and activity of mitochondrial complex I. Moreover, the Zmnmat3 mutant showed defective mitochondrial structure and exhibited expression and activity of alternative oxidases. These results indicate that ZmnMAT3 is essential for mitochondrial complex I assembly during kernel development in maize.

Small kernel 501 (smk501) encodes the RUBylation activating enzyme E1 subunit ECR1 (E1 C-TERMINAL RELATED 1) and is essential for multiple aspects of cellular events during kernel development in maize.

RUBylation plays essential roles in plant growth and development through regulating Cullin-RING ubiquitin E3 ligase (CRL) activities and the CRL-mediated protein degradations. However, the function of RUBylation in regulating kernel development remains unclear. Through genetic and molecular analyses of a small kernel 501 (smk501) mutant in maize (Zea mays), we cloned the smk501 gene, revealed its molecular function, and defined its roles in RUBylation pathway and seed development. Smk501 encodes a RUBylation activating enzyme E1 subunit ZmECR1 (E1 C-TERMINAL RELATED 1) protein. Destruction in RUBylation by smk501 mutation resulted in less embryo and endosperm cell number and smaller kernel size. The transcriptome and proteome profiling, hormone evaluation and cell proliferation observation revealed that disturbing ZmECR1 expression mainly affects pathways on hormone signal transduction, cell cycle progression and starch accumulation during kernel development. In addition, mutant in zmaxr1 (Auxin resistant 1), another RUB E1 subunit, also showed similar defects in kernel development. Double mutation of zmecr1 and zmaxr1 lead to empty pericarp kernel phenotype. RUBylation is a novel regulatory pathway affecting maize kernel development, majorly through its functions in modifying multiple cellular progresses.

The pentatricopeptide repeat protein EMP603 is required for the splicing of mitochondrial Nad1 intron 2 and seed development in maize.

Intron splicing is an essential event in post-transcriptional RNA processing in plant mitochondria, which requires the participation of diverse nuclear-encoded splicing factors. However, it is presently unclear how these proteins cooperatively take part in the splicing of specific introns. In this study, we characterized a nuclear-encoded mitochondrial P-type pentatricopeptide repeat (PPR) protein named EMP603. This protein is essential for splicing of intron 2 in the Nad1 gene and interacts with the mitochondria-localized DEAD-box RNA helicase PMH2-5140, the RAD52-like proteins ODB1-0814 and ODB1-5061, and the CRM domain-containing protein Zm-mCSF1. Further study revealed that the N-terminal region of EMP603 interacts with the DEAD-box of PMH2-5140, the CRM domain of Zm-mCSF1, and OBD1-5061, but not with OBD1-0814, whereas the PPR domain of EMP603 can interact with ODB1-0814, ODB1-5061, and PMH2-5140, but not with Zm-mCSF1. Defects in EMP603 severely disrupt the assembly and activity of mitochondrial complex I, leading to impaired mitochondrial function, and delayed seed development. The interactions revealed between EMP603 and PMH2-5140, ODB1-0814, ODB1-5061, and Zm-mCSF1 indicate a possible involvement of a dynamic 'spliceosome-like' complex in intron splicing, and may accelerate the elucidation of the intron splicing mechanism in plant mitochondria.

Studies of anticancer activity in vivo and in vitro behaviors of liposomes encapsulated iridium(III) complex.

Iridium(III) complexes have gained great attention in cancer treatment in recent years. In this paper, we designed and synthesized a new iridium(III) complex [Ir(piq)2(DQTT)](PF6) Ir1 (piq = 1-phenylisoquinoline, DQTT = 12-(1,4-dihydroquinoxalin-6-yl)-4,5,9,14-tetraazabenzo[b]triphenylene). The Ir1-loaded PEGylated liposomes (Lipo-Ir1) were prepared using the ethanol injection method. The anticancer activity of the complex and Lipo-Ir1 against SGC-7901 (human gastric adenocarcinoma), A549 (human lung carcinoma), HeLa (human cervical carcinoma), HepG2 (human hepatocellular carcinoma), BEL-7402 (human hepatocellular carcinoma), and normal NIH3T3 (mouse embryonic fibroblasts) was tested by the MTT method. The complex Ir1 shows moderate or low cytotoxicity against the selected cancer cells, whereas the Lipo-Ir1 exhibits high anticancer activity toward the same cancer cells. The apoptosis induced by Lipo-Ir1 was assayed by flow cytometry and Lipo-Ir1 induced apoptosis through increasing intracellular reactive-oxygen species levels, decreasing mitochondrial membrane potential, further promoting cytochrome c release and causing the increase of level of intracellular Ca2+. Western blot was used to detect the changes in Bcl-2 family protein and PI3K/AKT pathway proteins. The cloning experiments demonstrated that the Lipo-Ir1 can effectively inhibit cell proliferation. In vivo experiments, Lipo-Ir1 inhibited tumor growth in xenograft nude mice, and the percentage of tumor growth inhibition in vivo was 75.70%. Overall, the liposomes Lipo-Ir1 exhibits higher anticancer activity than Ir1 under the same conditions. These results indicated that Lipo-Ir1 may be a valuable resource for cancer therapy.

Synthesis and evaluation of iridium(III) complexes on antineoplastic activity against human gastric carcinoma SGC-7901 cells.

The study was intended to determine the antineoplastic effects of two new iridium(III) complexes [Ir(ppy)2(PTTP)](PF6) (1) (ppy = 2-phenylpyridine) and [Ir(piq)2(PTTP)](PF6) (2) (piq = 1-phenylisoquinoline, PTTP = 2-phenoxy-1,4,8,9-tetraazatriphenylene). In MTT assay, the ligand PTTP displayed ineffective inhibition on cell growth in SGC-7901, BEL-7402, HepG2 as well as NIH3T3 cell lines, while complexes 1 and 2 showed high cytotoxic activity on SGC-7901 cells with an IC50 value of 0.5 ± 0.1 µM and 4.4 ± 0.6 µM, respectively. Cellular uptake, cell cloning experiments, wound healing assay and cell cycle arrest indicated that the two complexes can inhibit the cell proliferation in SGC-7901 and induce cell cycle arrest at G0/G1 phase. Additionally, reactive oxygen species (ROS) and mitochondrial membrane potential suggested that the two complexes induced cell apoptosis through disrupting mitochondrial functions. Further, western blot analysis illustrated that the two complexes caused apoptosis via regulating expression levels of Bcl-2 family proteins. Moreover, complex 1 could suppress tumor growth in vivo with an inhibitory rate of 49.41%. Altogether, these results demonstrated that complexes 1 and 2 exert a potent anticancer effect against SGC-7901 cells via mitochondrial apoptotic pathway and have a potential to be developed as antineoplastic drug candidates for human gastric cancer.

Anticancer effect evaluation in vitro and in vivo of iridium(III) polypyridyl complexes targeting DNA and mitochondria.

To investigate the antitumor effect of iridium complexes, three iridium (III) complexes [Ir(ppy)2(dcdppz)]PF6 (ppy = 2-phenylpyridine, dcdppz = 11,12-dichlorodipyrido[3,2-a:2',3'-c]phenazine) (Ir1), [Ir(bzq)2(dcdppz)]PF6 (bzq = benzo[h]quinoline) (Ir2) and [Ir(piq)2(dcdppz)]PF6 (piq = 1-phenylisoquinoline) (Ir3) were synthesized and characterized. Geometry optimization, molecular dynamics simulation and docking studies have been performed to further explore the antitumor mechanism. The cytotoxicity of Ir1-3 toward cancer cells was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The localization of complexes Ir1-3 in the mitochondria, intracellular accumulation of reactive oxygen species (ROS) levels, the changes of mitochondrial membrane potential and morphological changes in apoptosis were investigated. Flow cytometry was applied to quantify fluorescence intensity and determine cell cycle distribution. Western blotting was used to detect the expression of apoptosis-related proteins. The anti-tumor effect of Ir1 in vivo was evaluated. The results showed that Ir1-3 had high cytotoxicity to most tumor cells, especially to SGC-7901 cells with a low IC50 value. Ir1-3 can increase the intracellular ROS levels, reduce the mitochondrial membrane potential. Additionally, the complexes induce an increase of apoptosis-related protein expression, enhance the percentage of apoptosis. The complexes inhibit the cell proliferation at G0/G1 phase. The results obtained from antitumor in vivo indicate that Ir1 can significantly inhibit the growth of tumors with an inhibitory rate of 54.08%. The docking studies show that complexes Ir1-3 interact with DNA through minor-groove intercalation, which increases the distance of DNA base pairs, leading to a change of DNA helix structure. These experimental and theoretical findings indicate that complexes Ir1-3 can induce apoptosis in SGC-7901 cells through the mitochondrial dysfunction and DNA damage pathways, and then exerting anti-tumor activity in vitro and vivo.

Fucoxanthin Pretreatment Ameliorates Visible Light-Induced Phagocytosis Disruption of RPE Cells under a Lipid-Rich Environment via the Nrf2 Pathway.

Fucoxanthin, a special xanthophyll derived from marine algae, has increasingly attracted attention due to its diverse biological functions. However, reports on its ocular benefits are still limited. In this work, the ameliorative effect of fucoxanthin on visible light and lipid peroxidation-induced phagocytosis disruption in retinal pigment epithelium (RPE) cells was investigated in vitro. Marked oxidative stress, inflammation, and phagocytosis disruption were evident in differentiated RPE cells following their exposure to visible light under a docosahexaenoic acid (DHA)-rich environment. Following pretreatment with fucoxanthin, however, the activated nuclear factor erythroid-derived-2-like 2 (Nrf2) signaling pathway was observed and, furthermore, when the fucoxanthin -pretreated RPE cells were irradiated with visible light, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) levels and inflammation were obviously suppressed, while phagocytosis was significantly improved. However, following the addition of Nrf2 inhibitor ML385, the fucoxanthin exhibited no ameliorative effects on the oxidative stress, inflammation, and phagocytosis disruption in the RPE cells, thus indicating that the ameliorative effect of fucoxanthin on the phagocytosis of RPE cells is closely related to the Nrf2 signaling pathway. In conclusion, these results suggest that fucoxanthin supplementation might be beneficial to the prevention of visible light-induced retinal injury.