G-quadruplex (G4) is a promising target for anti-cancer treatment. In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells. The molecular engineering of a fluorescent G4 ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), can change its major cellular localization from the nucleus to the mitochondria in cancer cells, while remaining primarily in the cytoplasm of normal cells. A number of BMVC derivatives with sufficient mitochondrial uptake can induce cancer cell death without damaging normal cells. Fluorescence studies of these anti-cancer agents in live cells and in isolated mitochondria from HeLa cells have demonstrated that their major target is mtDNA. In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells. Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression. This work underlines the importance of fluorescence in the monitoring of drug-target interactions in cells and illustrates the emerging development of drugs in which mtDNA G4 is the primary target.

DNA secondary structures and methylation are two well-known mechanisms that regulate gene expression. The catalytic subunit of telomerase, human telomerase reverse transcriptase (), is overexpressed in ∼90% of human cancers to maintain telomere length for cell immortalization. Binding of CCCTC-binding factor (CTCF) to the first exon of the gene can down-regulate its expression. However, DNA methylation in the first exon can prevent CTCF binding in most cancers, but the molecular mechanism is unknown. The NMR analysis showed that a stretch of guanine-rich sequence in the first exon of and located within the CTCF-binding region can form two secondary structures, a hairpin and a quadruplex. A key finding was that the methylation of cytosine at the specific CpG dinucleotides will participate in quartet formation, causing the shift of the equilibrium from the hairpin structure to the quadruplex structure. Of further importance was the finding that the quadruplex formation disrupts CTCF protein binding, which results in an increase in gene expression. Our results not only identify quadruplex formation in the first exon promoted by CpG dinucleotide methylation as a regulator of expression but also provide a possible mechanistic insight into the regulation of gene expression via secondary DNA structures.

Lipocalin 2 (LCN2) is an induced stressor that promotes the epithelial-mesenchymal transition (EMT). We previously demonstrated that the development of endometriosis in mice correlates with the secretion of LCN2 in the uterus. Here, we sought to clarify the relationship between LCN2 and EMT in endometrial epithelial cells and to determine whether LCN2 plays a role in endometriosis. Antibodies that functionally inhibit LCN2 slowed the growth of ectopic endometrial tissue in a mouse model of endometriosis, suggesting that LCN2 promotes the formation of endometriotic lesions. Using nutrient deprivation as a stressor, LCN2 expression was induced in cultured primary endometrial epithelial cells. As LCN2 levels increased, the cells transitioned from a round to a spindle-like morphology and dispersed. Immunochemical analyses revealed decreased levels of cytokeratin and increased levels of fibronectin in these endometrial cells, adhesive changes that correlate with induction of cell migration and invasion. Lcn2 knockdown also indicated that LCN2 promotes EMT and migration of endometrial epithelial cells. Our results suggest that stressful cellular microenvironments cause uterine tissues to secrete LCN2 and that this results in EMT of endometrial epithelial cells, which may correlate with the development of ectopic endometriosis. These findings shed light on the role of LCN2 in the pathology of endometrial disorders.

It is well known that mouse uterine 24p3 protein, is an acute phase protein, secreted from the L929 cell line, and that it will be induced by the dexamethasone stimulation of the cell. We investigated the possible effects of 24p3 protein on the L929 cell line, by observing its morphological change, ROS increase and viability decrease, by the process of culturing in a 24p3 protein-supplemented medium. Following the L929 cells′ exposure to the 24p3 protein supplement for a period of 72 hours, S-phase cells accumulated to a significant degree, suggesting that the entry into the G2/M phase from the S phase, in the cell cycle progression, was blocked. There was a significant decrease in cell numbers and increased DNA damage within the cells in the presence of 24p3 protein within the medium for 96 hours, implying that they have undergone pathway of cell death. After 96h incubation in low concentration of 24p3 protein, the result of PI/annexin V double staining showed cell death obviously. These results suggest that 24p3 protein-induced S phase arrest in the cell cycle, would cause DNA damage, followed by cell death in the L929 cells.

Previously, we have found that cancer cells survived from 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) have abnormal mitochondrial function and suppressed cellular invasiveness. Here we report that both the mRNA expression level and enzymatic activity of histone deacetylase (HDAC) were elevated in the PDT-derived variants with dysfunctional mitochondria. The activated HDAC deacetylated histone H3 and further resulted in the reduced migration and invasion, which correlated with the reduced expression of the invasion-related genes, matrix metalloproteinase 9 (MMP9), paternally expressed gene 1 (PEG1), and miR-355, the intronic miRNA. Using chromatin immunoprecipitation, we further demonstrate the reduced amount of acetylated histone H3 on the promoter regions of MMP9 and PEG1, supporting the down-regulation of these two genes in PDT-derived variants. These results indicate that HDAC activation induced by mitochondrial dysfunction could modulate the cellular invasiveness and its related gene expression. This argument was further verified in the 51-10 cybrid cells with the 4977 bp mtDNA deletion and A375 ρ0 cells with depleted mitochondria. These results indicate that mitochondrial dysfunction might suppress tumor invasion through modulating histone acetylation.