Fluorescence-based amine-reactive dyes are highly valuable for the sensing of amines and the labeling of biomolecules. Although it would be highly desirable, large changes in emission spectra and ...intensity seldom accompany the conjugation of known amine-reactive dyes to their target molecules. On the contrary, amide bond formation between amines and the pentafluorophenyl (2-PFP) and succinimidyl (2-NHS) esters of meso-carboxyBODIPY results in significant changes in emission maxima (Δλ: 70–100 nm) and intensity (up to 3000-fold), enabling the fast (down to 5 min) and selective fluorogenic detection and labeling of amines, amino acids, and proteins. This approach further benefits from the demonstrated versatility and high reliability of activated ester chemistry, and background hydrolysis is negligible. The large “turn-on” response is a testament of the extreme sensitivity of meso-carboxyBODIPYs to the minimal changes in electronic properties that distinguish esters from amides. Applications to the detection of food spoilage, staining of proteins on electrophoretic gels or in living cells, and the expedited synthesis of organelle-specific fluorescence microscope imaging agents are further demonstrated.
A fluorescent probe based on an excimer-forming benzothiazolyl-cyanovinylene (CV) dye was developed to target the apoptotic protease caspase-3. Upon the action of caspase-3, the water-soluble ...fluorescent probe Ac-DEVD-NH-CV, which is weakly green emissive in aqueous solution, is converted to hydrophobic CV-NH2 , which spontaneously aggregates. Aggregation of CV-NH2 promotes excimer emission of the CV dye, which allows for the study of caspase-3 activity in vitro and for imaging the activity of the enzyme in living cells because of the large red shift and enhanced fluorescence signal of the probe.
The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) ...but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.
Malignant melanoma, characterized by its ability to metastasize to other organs, is responsible for 90% of skin cancer mortality. To investigate alterations in the cellular metabolome and lipidome ...related to melanoma metastasis, gas chromatography-mass spectrometry (GC-MS) and direct infusion-mass spectrometry (DI-MS)-based metabolic and lipidomic profiling were performed on extracts of normal human melanocyte (HEMn-LP), low metastatic melanoma (A375, G361), and highly metastatic melanoma (A2058, SK-MEL-28) cell lines. In this study, metabolomic analysis identified aminomalonic acid as a novel potential biomarker to discriminate between different stages of melanoma metastasis. Uptake and release of major metabolites as hallmarks of cancer were also measured between high and low metastatic melanoma cells. Lipid analysis showed a progressive increase in phosphatidylinositol (PI) species with saturated and monounsaturated fatty acyl chains, including 16:0/18:0, 16:0/18:1, 18:0/18:0, and 18:0/18:1, with increasing metastatic potential of melanoma cells, defining these lipids as possible biomarkers. In addition, a partial-least-squares projection to latent structure regression (PLSR) model for the prediction of metastatic properties of melanoma was established, and central metabolic and lipidomic pathways involved in the increased motility and metastatic potential of melanoma cells were identified as therapeutic targets. These results could be used to diagnose and control of melanoma metastasis.
Abstract
Interferon-inducible guanylate-binding proteins (GBPs) are well-known for mediating host-defense mechanisms against cellular pathogens. Emerging evidence suggests that GBPs are also ...implicated in tumorigenesis; however, their underlying molecular mechanism is still unknown. In this study, we identified that GBP1 and GBP2 interact with MCL-1, the key prosurvival member of the BCL-2 family, via its BH3 domain. GBPs induce caspase-dependent apoptosis in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells, where the proapoptotic BCL-2 member, BAK, is an indispensable mediator. In particular, GBP2 completely inhibited the MCL-1-mediated promotion of the survival of CML cells through competitive inhibition, resulting in BAK liberation from MCL-1. Concurrently, GBP2 dramatically upregulates BAK expression via its inhibition of the PI3K/AKT pathway. Moreover, paclitaxel upregulates GBP2 expression, and paclitaxel-induced apoptotic activity was distinctively compromised by knockout of
GBP2
in CML cells. Bioinformatics analyses of leukemia databases revealed that transcripts of
GBPs
were generally downregulated in leukemia patients and that GBPs were favorable prognosis markers. Thus, these findings provide molecular evidence of GBPs as apoptosis-inducing proteins of leukemia cells and suggest that GBPs are attractive targets for the development of chemotherapeutics.
Anti-Müllerian hormone (AMH) is required for proper sexual differentiation by regulating the regression of the Müllerian ducts in males. Recent studies indicate that AMH could be an important factor ...for maintaining the ovarian reserve. However, the mechanisms of AMH regulation in the ovary are largely unknown. Here, we provide evidence that AMH is an ovarian target gene of steroidogenic factor-1 (SF-1), an orphan nuclear receptor required for proper follicle development. FOXL2 is an evolutionally conserved transcription factor, and its mutations cause blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), wherein affected females display eyelid defects and premature ovarian failure (POF). Notably, we found that functional FOXL2 is essential for SF-1-induced AMH regulation, via protein-protein interactions between FOXL2 and SF-1. A BPES-inducing mutant of FOXL2 (290-291delCA) was unable to interact with SF-1 and failed to mediate the association between SF-1 and the AMH promoter. Therefore, this study identified a novel regulatory circuit for ovarian AMH production; specifically, through the coordinated interplay between FOXL2 and SF-1 that could control ovarian follicle development.
Melanoma is the most aggressive form of skin cancer, with metastatic melanoma being refractory to currently available conventional therapies. In this study, we evaluated the inhibitory effect of ...coronatine (COR) on the proliferation of metastatic melanoma cells. COR inhibited the proliferation of melanoma cells but negligibly affected the proliferation of normal melanocytes. Comparative metabolic and lipidomic profiling using gas chromatography-mass spectrometry and direct infusion-mass spectrometry was performed to investigate COR-induced metabolic changes. These analyses identified 33 metabolites and 82 lipids. Of these, the levels of lactic acid and glutamic acid, which are involved in energy metabolism, significantly decreased in COR-treated melanoma cells. Lipidomic profiling indicated that ceramide levels increased in COR-treated melanoma cells, suggesting that ceramides could function as a suppressor of cancer cell proliferation. In contrast, the levels of phosphatidylinositol (PI) species, including PI 16:0/18:0, 16:0/18:1, 18:0/18:0, and 18:0/18:1, which were found to be potential biomarkers of melanoma metastasis in our previous study, were lower in the COR-treated cells than in control cells. The findings of metabolomic and lipidomic profiling performed in the present study provide new insights on the anticancer mechanisms of COR and can be used to apply COR in cancer treatment.
The development of specifically targeted nanoparticles for subcellular organelles modified with a low-molecular-weight organic compound as drug nanocarriers can bring about wide applications in ...cancer therapy. However, their utility has been hampered by low selectivity, poor biodistribution, and limited efficiency. Herein, we report the aggregation behavior of a triphenylphosphonium-appended coumarin probe (TPP-C) in an aqueous solution and its applications as a mitochondria-targeting probe, and drug delivery carrier, which is a rare example for a low molecular-weight organic compound. The TPP-C formed homogeneous nanoparticles with small diameters in water as well as in mixtures of organic solvents and water. In pure water, the homogeneous nanoparticles induced J-aggregation, whereas in mixed solvents, the homogeneous nanoparticles induced H-aggregation. The luminescence intensities of nanoparticles originated from the aggregation-induced emission (AIE) effect in pure water and also in mixtures of organic solvents and water. These findings indicate that the AIE effect of TPP-C was dependent on the solvent. More interestingly, the TPP-C nanoparticles selectively accumulated in mitochondria. The TPP-C nanoparticles alone exhibited noncytotoxicity toward cancer cells. However, with the encapsulation of the anticancer drug doxorubicin (DOX) into the TPP-C nanoparticles, the DOX was efficiently delivered to the mitochondria. These results indicated that the proposed system demonstrates promise as a platform for future clinical medication, particularly for specific suborganelle-targeted drug delivery systems for cancer therapy.
DNA methylation is an epigenetic modification that regulates gene expression and plays an essential role in hematopoiesis. UHRF1 and DNMT1 are both crucial for regulating genome-wide maintenance of ...DNA methylation. Specifically, it is well known that hypermethylation is crucial characteristic of acute myeloid leukemia (AML). However, the mechanism underlying how DNA methylation regulates the differentiation of AML cells, including THP-1 is not fully elucidated. In this study, we report that UHRF1 or DNMT1 depletion enhances the phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP-1 cells. Transcriptome analysis and genome-wide methylation array results showed that depleting UHRF1 or DNMT1 induced changes that made THP-1 cells highly sensitive to PMA. Furthermore, knockdown of UHRF1 or DNMT1 impeded solid tumor formation in xenograft mouse model. These findings suggest that UHRF1 and DNMT1 play a pivotal role in regulating differentiation and proliferation of THP-1 cells and targeting these proteins may improve the efficiency of differentiation therapy in AML patients.
MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and ...characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.
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