An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2.

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

FOXL2 and FOXA1 cooperatively assemble on the TP53 promoter in alternative dimer configurations.

Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein-protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.

Activatable Fluorescent Probes Targeting Urokinase-Type Plasminogen Activator Receptor on the Cell Membrane.

Urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored protein located on the cell surface that is implicated in the promotion of metastasis. New fluorescent probes for the detection of uPAR expression that feature a rapid "turn-on" response are reported here. They consist of a donor-π-acceptor-based fluorophore conjugated with a uPAR-binding AE105 peptide. The resulting AE105-coupled uPAR-targeting probes are weakly emissive in aqueous buffer solutions; however, a fluorescence "turn-on" signal is instantly triggered upon specific binding to uPAR (KD =63.2 nM for P1 and 49.5 nM for P2), which restricts the rotational deactivation of the fluorophore. Applications of the probes were demonstrated in the imaging of uPAR overexpressed on the membrane of cancer cell and in a cell-based uPAR inhibitor assay.

Anticancer activity and metabolic profile alterations by ortho-topolin riboside in in vitro and in vivo models of non-small cell lung cancer.

Lung cancer has the highest incidence and mortality rates among all types of cancer worldwide, and 80%-85% of patients with lung cancer are diagnosed with non-small cell lung cancer (NSCLC), which has 5-year survival rate of only 5% at advanced stages. Development of new therapeutic agents and strategies is required to enhance the treatment efficiency in patients with NSCLC. Metabolic alterations and anticancer effects of plant hormones and their derivatives have not been investigated in NSCLC in vitro and in vivo. The present study investigated the cytotoxic effects of 11 plant hormones and their derivatives against NSCLC cell lines; ortho-topolin riboside (oTR) showed the highest cytotoxicity among all tested compounds against NSCLC cells. Alteration of metabolites and lipids was investigated using gas chromatography-mass spectrometry and nano electrospray ionization-mass spectrometry in oTR-treated NSCLC cells and a xenograft mouse model. oTR reduced amino acid and pyrimidine synthesis in NSCLC cells and xenograft tumors. Moreover, oTR reduced glycolytic function and decreased mitochondrial respiration function by inhibiting glutamine and fatty acid oxidation. Increased levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine species suggested that oTR might act as a fatty acid oxidation inhibitor. In addition, the increased level of phosphatidylserine species implied that phosphatidylserine-mediated apoptosis occurred in oTR-treated NSCLC cells and xenograft tumor. The antiproliferative and apoptotic effects of oTR were mediated by the reduced p-ERK and p-AKT levels and increased cleaved Caspase-3 levels, respectively. This is the first study to investigate the metabolic alterations and anticancer activity of oTR in in vitro and in vivo models of NSCLC. Our results provide basis for the development of oTR-based therapeutic agent for patients with NSCLC.

A low dose of bisphenol A stimulates estradiol production by regulating ß-catenin-FOXL2-CYP19A1 pathway in human ovarian granulosa cells.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical that interferes with normal steroid hormone production in various species. However, the underlying mechanism of the effect of BPA on steroid production in the human ovary is not well understood. In the present study, we found that BPA, at very low concentrations (10-11 to 10-8 M), significantly increased the expression of FOXL2, a transcriptional factor essential for proper ovarian development and function, in a human ovarian granulosa cell-derived cell line (KGN). Furthermore, BPA enhanced CYP19A1 (aromatase) expression levels and estradiol (E2) production, but these effects were not observed in FOXL2 knockout (KO) cells. In addition, we found that BPA upregulates ß-catenin (CTNNB1) and stimulates nuclear translocation of CTNNB1, leading to transcriptional activation of FOXL2 mRNA. Furthermore, BPA failed to induce CYP19A1 and E2 production in CTNNB1-silenced KGN cells. Thus, we reveal a comprehensive molecular signaling cascade encompassing BPA-CTNNB1-FOXL2-CYP19A1-E2 that contributes to the endocrine-disrupting activities of BPA in human ovarian granulosa cells.

Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins.

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.

BAX is an essential key mediator of AP5M1-induced apoptosis in cervical carcinoma cells.

The adaptor-related protein complex 5 subunit mu 1 (AP5M1) is an evolutionally conserved protein with ubiquitous expression in human tissues. However, the major function of AP5M1 in living organisms is unclear owing to few published studies. Here, we demonstrate that AP5M1 is a potent apoptosis-inducing molecule in cervical cancer cells. We also found that AP5M1 upregulated the level of BAX protein, a key pro-apoptotic B cell lymphoma (BCL)-2 family member regulating mitochondrial apoptotic cell death pathway. Moreover, AP5M1 completely lost its apoptotic activity in BAX-knockout or -knockdown cells, indicative of its functional dependence on BAX. Comparative analysis of cervical tissues from patients with cervical carcinoma and non-cancer control revealed a prominent downregulation in AP5M1 expression with a concomitant downregulation in BAX expression; AP5M1 and BAX mRNA expression levels in cervical tissues exhibited a strong positive correlation (r = 0.97). Thus, we identified AP5M1 as a previously unrecognized apoptotic protein that governs BAX expression and revealed the association between AP5M1 and malignancy.

Excimer Emission-Based Fluorescent Probe Targeting Caspase-3.

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.

EGR2 is a gonadotropin-induced survival factor that controls the expression of IER3 in ovarian granulosa cells.

Pituitary gonadotropins are key hormones that orchestrate the growth and development of ovarian follicles. However, limited information is available on intra-ovarian factors that mediate the actions of gonadotropins. In this study, we identified that the early growth response 2 gene (EGR2) is a gonadotropin-inducible gene in granulosa cells of rats and humans. Analysis of consensus EGR-binding elements (EBEs) showed that the immediate early response 3 gene (IER3) is a novel transcriptional target gene of EGR2 as confirmed by the luciferase assay, electrophoretic mobility-shift assay (EMSA), chromatin immunoprecipitation (ChIP), and western blot analysis. Overexpression of EGR2 promoted survival of KGN human granulosa-derived cells in which IER3 acts as a mediator; knockdown of EGR2 induced death in KGN cells. Additionally, EGR2 was found to regulate the expression of myeloid cell leukemia 1 (MCL-1), which belongs to the BCL-2 family of proteins regulating cell survival. Thus, this study identified a novel signaling axis, comprised of gonadotropins-EGR2-IER3, which is important for the survival of granulosa cells during folliculogenesis.

Molecular dynamics simulations of acylpeptide hydrolase bound to chlorpyrifosmethyl oxon and dichlorvos.

Acylpeptide hydrolases (APHs) catalyze the removal of N-acylated amino acids from blocked peptides. Like other prolyloligopeptidase (POP) family members, APHs are believed to be important targets for drug design. To date, the binding pose of organophosphorus (OP) compounds of APH, as well as the different OP compounds binding and inducing conformational changes in two domains, namely, α/ß hydrolase and ß-propeller, remain poorly understood. We report a computational study of APH bound to chlorpyrifosmethyl oxon and dichlorvos. In our docking study, Val471 and Gly368 are important residues for chlorpyrifosmethyl oxon and dichlorvos binding. Molecular dynamics simulations were also performed to explore the conformational changes between the chlorpyrifosmethyl oxon and dichlorvos bound to APH, which indicated that the structural feature of chlorpyrifosmethyl oxon binding in APH permitted partial opening of the ß-propeller fold and allowed the chlorpyrifosmethyl oxon to easily enter the catalytic site. These results may facilitate the design of APH-targeting drugs with improved efficacy.

Cloning, Expression, and Characterization of a Thermophilic Endoglucanase, AcCel12B from Acidothermus cellulolyticus 11B.

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U·mg(-1), respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL(-1) and 131.75 U·mg(-1), respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future.

Molecular modeling and MM-PBSA free energy analysis of endo-1,4-ß-xylanase from Ruminococcus albus 8.

Endo-1,4-ß-xylanase (EC 3.2.1.8) is the enzyme from Ruminococcus albus 8 (R. albus 8) (Xyn10A), and catalyzes the degradation of arabinoxylan, which is a major cell wall non-starch polysaccharide of cereals. The crystallographic structure of Xyn10A is still unknown. For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme. In this study, the best similarity was found with the Clostridium thermocellum (C. thermocellum) N-terminal endo-1,4-ß-D-xylanase 10 b. Following the 100 ns molecular dynamics (MD) simulation, a reliable model was obtained for further studies. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods were used for the substrate xylotetraose having the reactive sugar, which was bound in the -1 subsite of Xyn10A in the 4C1 (chair) and 2SO (skew boat) ground state conformations. According to the simulations and free energy analysis, Xyn10A binds the substrate with the -1 sugar in the 2SO conformation 39.27 kcal·mol(-1) tighter than the substrate with the sugar in the 4C1 conformation. According to the Xyn10A-2SO Xylotetraose (X4(sb) interaction energies, the most important subsite for the substrate binding is subsite -1. The results of this study indicate that the substrate is bound in a skew boat conformation with Xyn10A and the -1 sugar subsite proceeds from the 4C1 conformation through 2SO to the transition state. MM-PBSA free energy analysis indicates that Asn187 and Trp344 in subsite -1 may an important residue for substrate binding. Our findings provide fundamental knowledge that may contribute to further enhancement of enzyme performance through molecular engineering.

Lutein inhibits glutamate-induced apoptosis in HT22 cells via the Nrf2/HO-1 signaling pathway.

Introduction: Excessive glutamate levels induce oxidative stress, resulting in neuronal damage, and cell death. While natural antioxidants show promise for neuroprotection, their effectiveness in the central nervous system (CNS) is limited by the blood -brain barrier. Lutein, a neuroprotective carotenoid, has gained attention for its ability to traverse this barrier and accumulate in various brain regions. This study aimed to elucidate the mechanisms underlying the protective effects of lutein against glutamateinduced cell death in HT22 cells. Methods: HT22 cells were treated with lutein (1.25-20 µM) for 24 hours. Cell viability, ROS levels, apoptosis, and mitochondrial membrane potential were assessed following lutein pretreatment and glutamate exposure. Protein expression of apoptotic markers was analyzed using Western blotting. Results: Lutein effectively attenuated glutamate-induced apoptosis due to its antioxidant properties. Additionally, lutein inhibited glutamate-induced mitochondrial-mediated apoptosis. We observed that lutein modulated the nuclear translocation of nuclear factor erythroid 2 -related factor 2 (Nrf2) and upregulated the expression of heme oxygenase-1 (HO-1). Inhibition of HO-1 by tin protoporphyrin (SnPP), a synthetic inhibitor, weakened the protective effect of lutein. Furthermore, we demonstrated that lutein prevented the aberrant activation of MAPKs induced by glutamate, including ERK1/2, p38, and JNK, thereby conferring oxidative protection. Discussion: Our study highlights the potent antioxidant properties of lutein, which effectively safeguards against glutamate-induced mitochondrial apoptotic cell death through the Nrf2/HO-1 signaling pathway and inhibition of MAPK activation. These findings demonstrate that lutein exerts a neuroprotective effect against glutamate-induced neuronal cell damage.

Guanylate-binding proteins induce apoptosis of leukemia cells by regulating MCL-1 and BAK.

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.

Integrative Metabolomic and Lipidomic Profiling of Lung Squamous Cell Carcinoma for Characterization of Metabolites and Intact Lipid Species Related to the Metastatic Potential.

SQCC is a major type of NSCLC, which is a major cause of cancer-related deaths, and there were no reports regarding the prediction of metastatic potential of lung SQCC by metabolomic and lipidomic profiling. In this study, metabolomic and lipidomic profiling of lung SQCC were performed to predict its metastatic potential and to suggest potential therapeutic targets for the inhibition of lung SQCC metastasis. Human bronchial epithelial cells and four lung SQCC cell lines with different metastatic potentials were analyzed using gas chromatography-mass spectrometry and direct infusion-mass spectrometry. Based on the obtained metabolic and lipidomic profiles, we constructed models to predict the metastatic potential of lung SQCC; glycerol, putrescine, ß-alanine, hypoxanthine, inosine, myo-inositol, phosphatidylinositol (PI) 18:1/18:1, and PI 18:1/20:4 were suggested as characteristic metabolites and intact lipid species associated with lung SQCC metastatic potential. In this study, we established predictive models for the metastatic potential of lung SQCC; furthermore, we identified metabolites and intact lipid species relevant to lung SQCC metastatic potential that may serve as potential therapeutic targets for the inhibition of lung SQCC metastasis.

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku.

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.

Metabolic and lipidomic investigation of the antiproliferative effects of coronatine against human melanoma cells.

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.

LRIG2 is a growth suppressor of Hec-1A and Ishikawa endometrial adenocarcinoma cells by regulating PI3K/AKT- and EGFR-mediated apoptosis and cell-cycle.

Although endometrial cancer is the most common type of gynecological malignancy in developed countries, its molecular etiology is not well understood. Leucine-rich repeat and immunoglobulin-like domain 2 (LRIG2) is an evolutionarily conserved gene, but its functions in the endometrium are unknown. In this study, we found that LRIG2 is highly downregulated in endometrial adenocarcinoma patients and that it functions as a tumor suppressor. LRIG2 induced the mitochondrion-mediated apoptotic pathways by regulating stoichiometric balance among BCL-2 family proteins, whereby pro-survival members, MCL-1 and BCL-xL, were downregulated and pro-apoptotic BAK and BAX were upregulated. LRIG2 also inhibited proliferation of the Hec-1A and Ishikawa endometrial adenocarcinoma cells by upregulating p21. LRIG2 induced BAX- and BAK-dependent cell death that was efficiently prevented by MCL-1 overexpression. Furthermore, we found that LRIG2 unexpectedly phosphor-activates phosphoinositide 3-kinase (PI3K)/AKT and epidermal growth factor receptor (EGFR), which are conventionally accepted as survival signaling cues in diverse types of cancer. We observed that PI3K/AKT and EGFR serve as key kinases that have roles as growth suppressors of Hec-1A endometrial cancer cells by mediating the LRIG2-induced modulation of the BCL-2 family of proteins and p21. In vivo delivery of antisense DNAs against LRIG2 promoted the Hec-1A endometrial tumor growth in a xenograft mouse model, and immunoblotting of these tumor extracts showed consistent modulation of AKT, EGFR, the BCL-2 family members, and p21. Thus, our results demonstrated that LRIG2 is a growth suppressor of endometrial adenocarcinoma cells.