Novel fluorescence probe for ClO- in living cells: Based on FRET mechanism.

Hypochlorous acid (HClO) as a kind of reactive oxygen species (ROS) plays a vital role in many biological processes. Organic fluorescence probes have attracted great interests for the detection of HClO, due to their relatively high selectivity and sensitivity, satisfactory spatiotemporal resolution and good biocompatibility. Constructing fluorescence probes to detect HClO with advantages of large Stokes shift, wide emission gap, near infrared emission and good water solubility is still challenging. In this work, a new ratiometric fluorescence probe (named HCY) for HClO was developed. FRET-based HCY was constructed by bonding a coumarin and a flavone fluorophore. In absence of HClO, HCY exists FRET process, however, FRET is inhibited in the presence of HClO because the conjugated double bond broke. Due to the good match of the emission spectrum of the donor and the absorption spectrum of the acceptor, the FRET system appears favorable energy transfer efficiency. HCY showed high sensitivity and rapid response time. The linearity between the ratios of fluorescence intensity and concentration of HClO was established with a low limit of detection. What's more, HCY was also applied for fluorescence images of HClO in RAW264.7 cells.

A near-infrared fluorescent probe based FRET for ratiometric sensing of H2O2 and viscosity in live cells.

Hydrogen peroxide (H2O2) and viscosity play vital roles in the cellular environment as signaling molecule and microenvironment parameter, respectively, and are associated with many physiological and pathological processes in biological systems. We developed a near-infrared fluorescent probe, CQ, which performed colorimetric and ratiometric detection of H2O2 and viscosity based on the FRET mechanism, and was capable of monitoring changes in viscosity and H2O2 levels simultaneously through two different channels. Based on the specific reaction of H2O2 with borate ester, CQ exhibited a significant ratiometric response to H2O2 with a large Stokes shift of 221 nm, a detection limit of 0.87 µM, a near-infrared emission wavelength of 671 nm, a response time of 1 h, a wide detection ranges of 0.87-800 µM and a high energy transfer efficiency of 99.9 %. CQ could also recognize viscosity by the TICT mechanism, and efficiently detect viscosity changes caused by food thickeners. More importantly, CQ could successfully detect endogenous/exogenous H2O2 and viscosity in live HeLa cells, which was expected to be a practical tool for detecting H2O2 and viscosity in live cells.

Inhibition of annexin A7 suppresses senescence-associated heterochromatin foci formation and senescence through the AMPK/mTOR pathway in human dermal fibroblasts.

Senescence-associated heterochromatin foci (SAHF) is often used as a biological marker for senescent cells, but the regulation of its formation process is unclear. To find a new modulator of SAHF, we screened our chemical small molecules and found 7-amino-2,3,4,5-tetrahedrobenzo[b][1,4] oxazepin-3-ol (ABO) that was identified as an inhibitor of annexin A7 GTPase (ANXA7) dramatically suppressed the aggregation of heterochromatin protein (HP1γ), an indicator of SAHF. To understand its action mechanism, we first observed the changes in the karyoplasmic ratio of ANXA7 because HP1γ mainly located in the nucleus. The results showed that ABO elevated the protein level of ANXA7 in the nucleus. Therefore, we raised a hypothesis that ANXA7 interacted with HP1γ and regulated its phosphorylation, which is closely related to the formation of SAHF. The co-immunoprecipitation and Western blot experiment results showed that ANXA7 had no direct interaction with HP1γ, however, the phosphorylation of HP1γ was increased by ABO, which suggested that ANXA7 indirectly regulated HP1γ phosphorylation. Then, based on our previous discovery of ANXA7 interacting with AMP-activated protein kinase (AMPK), we investigated the effect of the AMPK/mammalian target of rapamycin (mTOR) signaling pathway on ABO-increased phosphorylation of HP1γ. We found that ABO decreased AMPK phosphorylation and increased the phosphorylation level and activity of mTOR. In the presence of an AMPK activator or mTOR inhibitor, ABO could not increase HP1γ phosphorylation. As a result, ABO inhibited the senescence of human dermal fibroblasts (HDFs). In this study, we found that ANXA7 was a new regulator of SAHF, it could regulate the formation of SAHF through the AMPK/mTOR pathway. The data suggested that ABO could be used as a powerful tool to inhibit the replicative senescence of HDFs.

Dual-response and lysosome-targeted fluorescent probe for viscosity and sulfur dioxide derivatives.

Viscosity and sulfur dioxide levels are important factors to evaluate the changes of cell micro-environment because a series of diseases usually occur when they are abnormal. At present, dual-response probes that can detect both viscosity and sulfur dioxide are rare. Therefore, we developed a novel fluorescent probe CBN for simultaneous detection of sulfur dioxide and viscosity. Besides, probe CBN could target lysosome of which normal function will be disrupted by the abnormality of viscosity. Therefore, probe CBN has the potential to be served as an effective biological tool to monitor the intracellular micro-environment.

FRET-based fluorescent probe with favorable water solubility for simultaneous detection of SO2 derivatives and viscosity.

The intracellular viscosity is an important parameter of the microenvironment and SO2 is a vital gas signal molecule. At present, some dual-response fluorescence probes for simultaneous measurements of viscosity and SO2 derivatives (HSO3-/SO32-) possessed poor water solubility. In this work, we developed a water-soluble fluorescence probe CIJ (0.0864 g/100 mL of water at 20 °C) for simultaneous measurements of viscosity and SO2 derivatives. CIJ exhibited a sensitive fluorescence enhancement to environmental viscosity from 0.97 to 28.04 cP based on a twisted intramolecular charge transfer mechanism and was applied to effective measurement of viscosity in vitro and in vivo. CIJ could also respond to SO2 derivatives with a low detection limit (44 nM) and a fast response time (5 min) based on the nucleophilic addition reaction. Furthermore, CIJ was applied to monitor SO2 derivatives in ratiometric response manner in living cells.

Esterase D interacts with metallothionein 2A and inhibits the migration of A549 lung cancer cells in vitro.

Esterase D (ESD) is a nonspecific esterase widely distributed in various organisms. ESD plays an important role in regulating cholesterol efflux, inhibiting viral replication and lung cancer growth. MT2A (metallothionein 2A) is the most important isoform of metallothionein (MTs) in human and high expression of MT2A in tumors represents poor prognosis and metastatic behavior. However, there are no reports about the molecular mechanism of ESD in the regulation of tumor metastasis. In this study, we found for the first time that activation ESD promoted its interaction with MT2A and decreased the protein level of MT2A, which resulting in the concentration of free zinc ions up-regulated, and inhibited the migration of A549 lung cancer cells in vitro.

Activation of GRP78 ATPase suppresses A549 lung cancer cell migration by promoting ITGB4 degradation.

Hypochlorous acid (HOCl) is an essential signal molecule in cancer cells. Activated GRP78 ATPase by a HOCl probe named ZBM-H inhibits lung cancer cell growth. However, the role and underlying mechanism of GRP78 ATPase in lung cancer cell migration have not been established. Here, we reported that activation of GRP78 ATPase by ZBM-H suppressed A549 cell migration and inhibited EMT process. Notably, ZBM-H time-dependently decreased the protein level of integrin ß4 (ITGB4) in A549 cells. Combinatorial treatment of 3BDO (an autophagy inhibitor) and ZBM-H partially rescued the protein level of ITGB4. Consistently, 3BDO partially reversed ZBM-H-inhibited cell migration. Furthermore, ZBM-H promoted the interaction between ANXA7 and Hsc70, which participated in the regulation of selective autophagy and degradation of ITGB4.

Identification of a new way to induce differentiation of dermal fibroblasts into vascular endothelial cells.

BACKGROUND: Human dermal fibroblasts (HDFs) have the potential to differentiate into vascular endothelial cells (VECs), but their differentiation rate is low and the mechanism involved is not clear. The small molecule pathway controls the phenotype of fibroblasts by activating cellular signaling pathways, which is a more convenient method in the differentiation strategy of HDFs into VECs. METHODS: In this study, HDFs were treated with the different doses of CPP ((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl) piperazin-1-yl) styryl)-1-methylpyridin-1-ium iodide), and the mRNA and protein levels of HDFs were detected by qPCR, Western blot, flow cytometry and immunofluorescent staining. The matrigel assays, acetylated-LDL uptake and angiogenesis assays of chick embryo chorioallantoic membrane (CAM) and hindlimb ischemia model of nude mice were performed to evaluate the functions of VECs derived from HDFs. RESULTS: Here, we report that the small chemical molecule, CPP, can effectively induce HDFs to differentiate into VECs. First, we observed the morphological changes of HDFS treated with CPP. Flow cytometry, Western blot and qRT-PCR analyses showed that CPP effectively decreased the level of the HDFs-marker Vimentin and increased levels of the VEC-markers CD31, CD133, TEK, ERG, vWF, KDR and CDH5. Detection of the percentage of CD31-positive cells by immunofluorescent staining confirmed that CPP can effectively induce HDFs to differentiate into VECs. The results of Matrigel assays, DiI-ac-LDL uptake, angiogenesis assays on CAM and hindlimb ischemia model of nude mice showed that CPP-induced HDFs have the functions of VECs in vitro and in vivo. Western blot and qRT-PCR analysis showed that CPP induces HDFs to differentiate into VECs by promoting the expression of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB). CONCLUSIONS: Our data suggest that the small chemical molecule CPP efficiently induces the differentiation of HDFs into VECs. Simultaneously, this new inducer provides a potential to develop new approaches to restore vascular function for the treatment of ischemic vascular diseases.

HOCI Probe CPP Induces the Differentiation of Human Dermal Fibroblasts into Vascular Endothelial Cells through PHD2/HIF-1α/HEY1 Signaling Pathway.

Human dermal fibroblasts (HDFs) have the potential to differentiate into endothelial cells (VECs). In our previous research, we reported that a hypochlorous acid (HOCl) probe CPP efficiently induced the differentiation of HDFs into VECs, however, the mechanism of differentiation was not clear. As an HOCI probe, CPP binds HOCI to modulate its effects. In this study, through Western blotting, qPCR, and PHD2 enzyme activity assay, we found that CPP inhibited the enzyme activity of prolyl-4-hydroxylase 2 (PHD2), thereby stabilizing HIF-1α. To further clarify the mechanism by which CPP inhibits PHD2 enzyme activity, we constructed plasmids, and found that CPP inhibited PHD2 activity to increase the HIF-1α level through the modulation of PHD2 at Cys302 by HOCl in HDFs. Furthermore, RNA-seq experiments showed that CPP could induce the expression of HEY1, which is not only a target gene regulated by HIF1α, but also a key transcription factor for VECs. We used siRNA transfection and in vivo experiments to confirm that CPP could induce HDFs to differentiate into VECs by HEY1. In summary, we identified a new inhibitor of PHD2, demonstrated the new role of HOCl in cell differentiation, and elucidated the mechanism by which HOCl probe CPP induced the differentiation of HDFs into VECs.

Grp94 Inhibitor HCP1 Inhibits Human Dermal Fibroblast Senescence.

Researchers are paying more and more attention to aging, especially skin aging. Therefore, it is urgent to find an effective way to inhibit aging. Here, we report a small chemical molecule, HCP1, that inhibited the senescence of human dermal fibroblasts (HDFs). First, we performed morphological experiment and found that HCP1-treated HDFs were no longer elongated and flat compared to DMSO-treated groups. Next, we found that the number of ß-gal positive cells decreased compared to DMSO-treated groups. Through flow cytometry, western blot, and immunofluorescence, we found that HCP1 could inhibit the senescence of HDFs. In the study of the mechanism, we found that HCP1 could regulate the AMPK/mTOR signal pathway through glucose-regulated protein 94 (Grp94). In addition, we found that HCP1 could promote the interaction between Grp94 and lysosomes, which led to an increase in the activity of lysosomes and inhibited the senescence of HDFs. At the same time, we found that HCP1 decreased the concentration of Ca2+ in mitochondria, inhibiting the senescence of HCP1. Therefore, we propose that HCP1 is a potential aging-inhibiting compound, and provide a new idea for the development of senescence-inhibiting drugs.

Safrole oxide induced 5-HT neuron-like cell differentiation of bone marrow mesenchymal stem cells by elevating G9a.

In our previous study, we found that safrole oxide (SFO) could induce bone marrow mesenchymal stem cell differentiation into neuron-like cells. However, which kind of neuron cells was induced by SFO was unknown. Here, we found that SFO could induce BMSC differentiation into 5-hydroxytryptamine (5-HT) neuron-like cells. Microarray analysis of BMSCs treated with SFO for 6 h revealed a total of 35 genes changed more than twice. We selected G9a, a histone methyltransferase for further study. The upregulation of G9a was confirmed by RT-PCR and Western blot analysis. Small interfering RNA knockdown of G9a blocked SFO-induced BMSC differentiation. These results demonstrated that G9a was the pivotal factor in SFO-medicated 5-HT neuronal differentiation of BMSCs. Our findings provide a new clue for further investigating the gene control of BMSC differentiation into 5-HT neuron-like cells and provide a putative strategy for depression diseases therapies.

Activation of Esterase D by FPD5 Inhibits Growth of A549 Lung Cancer Cells via JAB1/p53 Pathway.

Esterase D (ESD) is widely distributed in mammals, and it plays an important role in drug metabolism, detoxification, and biomarkers and is closely related to the development of tumors. In our previous work, we found that a chemical small-molecule fluorescent pyrazoline derivative, FPD5, an ESD activator, could inhibit tumor growth by activating ESD, but its molecular mechanism is still unclear. Here, by using RNA interference (RNAi), andco-immunoprecipitation techniques, we found that ESD suppressed the nucleus exportation of p53 through reducing the interaction between p53 and JAB1. The protein level of p53 in the nucleus was upregulated and the downstream targets of p53 were found by Human Gene Expression Array. p53 inhibited the expression of CDCA8 and CDC20. Lastly, the cell cycle of A549 cells was arrested at the G0/G1 phase. Together, our data suggest that ESD inhibited the cancer cell growth by arresting the cell cycle of A549 cells via the JAB1/p53 signaling pathway. Our findings provide a new insight into how to inhibit the growth of lung cancer with the activation of ESD by FPD5.

A quinoline-coumarin near-infrared ratiometric fluorescent probe for detection of sulfur dioxide derivatives.

Sulfur dioxide derivatives (HSO3- and SO32-) play an important role in food preservative, antibacterial, antioxidant and other aspects, so it is urgent for us to develop more efficient detection methods to broaden their application in biochemical research and related disease diagnosis. Fluorescent probes are of particular interest because of their simplicity and high temporal and spatial resolution. Herein, we constructed a new near-infrared (NIR) fluorescence probe, CQC, composed of coumarin fluorophore and quinoline fluorophore, for detecting SO2 derivatives. The near-infrared emission probe CQC with a large Stokes shift (260 nm) not only kept the distance between the two emission peaks large enough (165 nm), but also had a particularly high energy transfer efficiency (99.5%), and was particularly sensitive to the detection of HSO3-/SO32- (LOD: 0.1 µM). The powerful probe CQC succeeded in real-time visualizing endogenous HSO3-/SO32- in living cells.

ZBM-H-induced activation of GRP78 ATPase promotes apoptosis via annexin A7 in A549 lung cancer cells.

Hypochlorous acid (HOCl) is an essential signal for the regulation of cancer cell fate, including autophagy and apoptosis. HOCl regulated autophagy by affecting the oxidation modification of glucose-regulated protein 78 (GRP78) and the activity of GRP78 ATPase. The mechanism of GRP78 ATPase in cell apoptosis has however not yet been clarified. Here we reported that ZBM-H, as a probe of HOCl, was able to directly bind to GRP78 in the presence or absence of ATP. Following ZBM-H treatment, the interaction between GRP78 and annexin A7 (ANXA7) was promoted, and this was accompanied by increased phosphorylation of integrin ß4 (ITGB4). In addition, ZBM-H enhanced the phosphorylation of ANXA7. ABO, an inhibitor of ANXA7, inhibited ZBM-H-induced ITGB4 phosphorylation and apoptosis, while ANXA7 activator SEC had opposite effect. Collectively, these data provide new evidence for the mechanism by which ZBM-H-induced activation of GRP78 ATPase regulates apoptosis of A549 lung cancer cells.

A ratiometric fluorescent probe for selective detection of thiophenol derivatives.

Though a number of on-off or off-on fluorescent probes have been developed for the detection of thiophenol by using its unique recognition groups, such as 2, 4-dinitrophenyl ether, 2, 4-dinitrophenyl sulfonamide, and 2, 4-dinitrophenyl sulfonate, up to now, there are few probes that can detect thiophenol by the proportional fluorescence signal. We developed a ratiometric fluorescent probe with coumarin pyridine derivative as fluorophore and 2, 4-dinitrophenyl ether moiety as the sensing unit which could be used to detect thiophenol derivatives by the aromatic nucleophilic substitution reaction. This probe (CPBPN) displayed significant change in fluorescence ratio (256 fold) to result in a more reliable analysis by self-calibration and a relatively low detection limit of 24 nM toward 4-methylthiophenol (MTP) within 30 min to achieve more sensitivity. Besides, the probe was also applied to detect the presence of thiophenol derivatives in actual water samples and fluorescence imaging in living cells. The present work is of great importance for monitoring environmental pollutants and studying their biological function.

Two simple but effective turn-on benzothiazole-based fluorescent probes for detecting hydrogen sulfide in real water samples and HeLa cells.

Two simple turn-on fluorescent probes, containing a benzothiazole and the 2,4-dinitrobenzenesulfonyl group, were designed for detecting H2S. Two probes exhibited good selectivity and high sensitivity, which were applied to detect the H2S in real water samples. Probe P2 with a positive charge had better solubility than probe P1 in water; therefore, probe P2 was successfully applied to detect both the endogenous and exogenous H2S in lysosomes of living HeLa cells.

A novel sulfur dioxide probe inhibits high glucose-induced endothelial cell senescence.

Sulfur dioxide (SO2) is an important gas signal molecule produced in the cardiovascular system, so it has an important regulatory effect on human umbilical vascular endothelial cells (HUVECs). Studies have shown that high glucose (HG) has become the main cause of endothelial dysfunction and aging. However, the mechanism by which SO2 regulates the senescence of vascular endothelial cells induced by HG has not yet been clarified, so it is necessary to find effective tools to elucidate the effect of SO2 on senescence of HUVECs. In this paper, we identified a novel sulfur dioxide probe (2-(4-(dimethylamino)styryl)-1,1,3-trimethyl-1H-benzo [e]indol-3-ium, DLC) that inhibited the senescence of HUVECs. Our results suggested that DLC facilitated lipid droplets (LDs) translocation to lysosomes and triggered upregulation of LAMP1 protein levels by targeting LDs. Further study elucidated that DLC inhibited HG-induced HUVECs senescence by promoting the decomposition of LDs and protecting the proton channel of V-ATPase on lysosomes. In conclusion, our study revealed the regulatory effect of lipid droplet-targeted sulfur dioxide probes DLC on HG-induced HUVECs senescence. At the same time, it provided the new experimental evidence for elucidating the regulatory mechanism of intracellular gas signaling molecule sulfur dioxide on vascular endothelial fate.

An Inhibitor of Grp94 Inhibits OxLDL-Induced Autophagy and Apoptosis in VECs and Stabilized Atherosclerotic Plaques.

Background: Oxidized low-density lipoprotein (oxLDL) induces vascular endothelial cell (VEC) injury and atherosclerosis through activating endoplasmic reticulum stress. Expression of glucose-regulated protein 94 (Grp94) is induced by endoplasmic reticulum stress and Grp94 is involved in cardiovascular diseases. This study aimed to determine the role of Grp94 in oxLDL-induced vascular endothelial cell injury and atherosclerosis. Methods and Results: An inhibitor of Grp94, HCP1, was used to investigate the role of Grp94 in oxLDL-induced VEC injury in human umbilical vein endothelial cells and atherosclerosis in apolipoprotein E-/- mice. Results showed that HCP1 inhibited autophagy and apoptosis induced by oxLDL in VECs. And we found that Grp94 might interact with adenosine monophosphate-activated protein kinase (AMPK) and activate its activity. HCP1 inhibited AMPK activity and overexpression of Grp94 blocked the effect of HCP1. Besides, HCP1 activated the activity of mechanistic target of rapamycin complex 1 (mTORC1), co-treatment with AMPK activator acadesine eliminated the effect of HCP1 on mTORC1 activity as well as autophagy. In apolipoprotein E-/- mice, HCP1 suppressed autophagy and apoptosis of atherosclerotic plaque endothelium. In addition, HCP1 increased the content of collagen, smooth muscle cells, and anti-inflammatory macrophages while reducing the activity of MMP-2/9 and pro-inflammatory macrophages in the atherosclerotic lesion. Conclusion: HCP1 inhibited oxLDL-induced VEC injury and promoted the stabilization of atherosclerotic plaque in apoE-/- mice. Grp94 might be a potential therapeutic target in the clinical treatment of atherosclerosis.

Esterase D stabilizes FKBP25 to suppress mTORC1.

BACKGROUND: Esterase D (ESD) is a nonspecific esterase that detoxifies formaldehyde. Many reports have stated that ESD activity is associated with a variety of physiological and pathological processes. However, the detailed signaling pathway of ESD remains poorly understood. METHODS: Considering the advantages of the small chemical molecule, our recent work demonstrated that 4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro-1H-pyrazol-3-yl) phenol (FPD5) activates ESD, and will be a good tool for studying ESD further. Firstly, we determined the interaction between ESD and FK506 binding protein 25 (FKBP25) by yeast two-hybrid assay and co-immunoprecipitation (CO-IP) and analyzed the phosphorylation levels of mTORC1, P70S6K and 4EBP1 by western blot. Furthermore, we used the sulforhodamine B (SRB) and chick chorioallantoic membrane (CAM) assay to analyze cell viability in vitro and in vivo after treatment with ESD activator FPD5. RESULTS: We screened FKBP25 as a candidate protein to interact with ESD by yeast two-hybrid assay. Then we verified the interaction between ESD and endogenous FKBP25 or ectopically expressed GFP-FKBP25 by CO-IP. Moreover, the N-terminus (1-90 aa) domain of FKBP25 served as the crucial element for their interaction. More importantly, ESD reduced the K48-linked poly-ubiquitin chains of FKBP25 and thus stabilized cytoplasmic FKBP25. ESD also promoted FKBP25 to bind more mTORC1, suppressing the activity of mTORC1. In addition, ESD suppressed tumor cell growth in vitro and in vivo through autophagy. CONCLUSIONS: These findings provide novel evidence for elucidating the molecular mechanism of ESD and ubiquitination of FKBP25 to regulate autophagy and cancer cell growth. The ESD/FKBP25/mTORC1 signaling pathway is involved in inhibiting tumor cell growth via regulating autophagy.

Identification of a new autophagy inhibitor targeting lipid droplets in vascular endothelial cells.

Autophagy of vascular endothelial cells (VECs) plays an important role in maintaining vascular homeostasis. Lipid droplets (LDs) are organelles that can be formed in response to various stimuli, including excessive lipid or various stresses. LDs sequester toxic lipids, thereby preventing lipotoxic cell damage and have a complex relationship with autophagy. In the previous study, we identified a novel Grp94 inhibitor HCP1 inhibited apoptosis in VECs. Here we found that HCP1 targeted LDs and promoted the accumulation of LDs in VECs. Our results showed that HCP1 upregulated the protein levels of autophagy-related proteins. We demonstrated that HCP1 upregulated the number of LDs and suppressed autophagy by inhibiting Grp94. Therefore, we provided HCP1 as a new VECs autophagy inhibitor targeting LDs, which might be a potential compound in the treatment of VECs autophagy related vascular diseases.