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.

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.

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.

Intravenous thrombolysis prior to endovascular treatment for acute ischemic stroke: a meta-analysis.

OBJECTIVES: Whether intravenous thrombolysis provides additional benefits before direct endovascular treatment (dEVT) in acute ischemic stroke remains unclear. We aimed to compare the functional and safety outcomes of dEVT to endovascular treatment with bridging using intravenous thrombolysis (BT) in acute ischemic stroke. METHODS: This meta-analysis included currently available eligible randomized clinical trials (RCTs) by searching in the PubMed, EMBASE, Cochrane Central Register, and the International Stroke Conference and European Stroke Organisation Conference posted abstracts. RESULTS: The six included RCTs yielded 2334 participants (mean age, 69.8 years [SD, 11.4]; women, 44.3%; 1164 in dEVT group and 1170 in BT group). We found not significantly different 90-day functional outcomes of modified Rankin scale (mRS 0 - 2, odds ratio [OR] 0.93, 95%CI 0.79 - 1.09; mRS 0 - 1, OR 0.99, 95%CI 0.82 - 1.18), mortality (OR 1.08, 95%CI 0.86 - 1.35), and symptomatic intracranial hemorrhage (OR 0.72, 95%CI 0.49 - 1.07) for patients in dEVT and BT group. Patients treated with dEVT were less likely to experience successful recanalization (OR 0.72, 95%CI 0.57 - 0.92, p = 0.009) and any intracranial hemorrhage (OR 0.81, 95%CI 0.68 - 0.97, p = 0.02). There were no significant differences regarding procedural complications between the two groups. CONCLUSION: This meta-analysis showed no significant differences in 90-day functional outcomes or mortality between dEVT and BT, but a lower possibility of successful recanalization and intracranial hemorrhage for dEVT.

H2S probe CPC inhibits autophagy and promotes apoptosis by inhibiting glutathionylation of Keap1 at Cys434.

H2S is actual an endogenous signaling gas molecule and involved in a range of cell physiological processes. However, the mechanism of endogenous H2S regulating autophagy and apoptosis has not been thoroughly investigated. Here, we try to address this issue by using a H2S probe, (E)-2-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)-piperazin-1-yl)-styryl)-1, 3, 3-trimethyl-3H-indol-1-ium iodide (CPC), which could react with endogenous H2S. Herein, we reported that CPC inhibited autophagy and decreased the expression and activity of NF-E2-related factor 2 (Nrf2), then induced cell apoptosis. CPC inhibited autophagy and promoted apoptosis by inhibiting Nrf2 activation, which was H2S dependent. Furthermore, we found that CPC inhibited Nrf2 nucleus translocation by inhibiting glutathionylation of Kelch-like ECH-associated protein 1 (Keap1) at the Cys434 residue. CPC also inhibited various cancer cell growth, but had no effect on normal cell growth in vitro, and inhibited A549 cancer growth, but did not affect normal angiogenesis in vivo. Therefore, we not only found a new inhibitor of autophagy and Nrf2, but also suggested a novel mechanism that endogenous H2S could regulate autophagy, apoptosis and Nrf2 activity through regulating glutathionylation of Keap1 at the Cys434 residue.

Promoting TTC4 and HSP70 interaction and translocation of annexin A7 to lysosome inhibits apoptosis in vascular endothelial cells.

We previously demonstrated that Tetraticopeptide 4 (TTC4) inhibited apoptosis in vascular endothelial cells (VEC) deprived of serum and fibroblast growth factor 2 (FGF-2). In this study, we aimed to resolve the mechanism of TTC4 inhibiting VEC apoptosis. TTC4, predicted as a HSP70 co-chaperone protein, may regulate the fate of cells by affecting the activity of HSP70, however, there is no experimental evidence showing the interaction of TTC4 and HSP70. Using Co-immunoprecipitation (Co-IP), we demonstrated that TTC4 interacted with HSP70. If HSP70 was knockdown, TTC4 no longer suppressed apoptosis. Furthermore, we found ABO, an inhibitor of annexin A7 (ANXA7) GTPase, could promote the interaction of TTC4 and HSP70 and the translocation of ANXA7 to lysosome. At the same time, ABO inhibited the interaction of HSP70 and ANXA7. Moreover, Akt, as a downstream effector of HSP70 was upregulated, and ANXA7 translocating to lysosome protected the stability of lysosomal membrane. Here, we discovered a special mechanism by which TTC4 inhibited apoptosis via HSP70 in VECs. On the one hand, increasing TTC4 and HSP70 interaction upregulated Akt that inhibited apoptosis. On the other hand, decreasing HSP70 and ANXA7 interaction promoted the translocation of ANXA7 to lysosome, which inhibited apoptosis through protecting the lysosomal membrane stability.

Rimonabant potentiates the antifungal activity of amphotericin B by increasing cellular oxidative stress and cell membrane permeability.

Amphotericin B (AmB) is a very effective antifungal agent, and resistance in clinical isolates is rare. However, clinical treatment with AmB is often associated with severe side effects. Reducing the administration dose of AmB by combining it with other agents is a promising strategy to minimize this toxicity. In this study, we screened a small compound library and observed that the anti-obesity drug rimonabant exhibited synergistic antifungal action with AmB against Candida species and Cryptococcus neoformans. Moreover, the combination of AmB and rimonabant exhibited synergistic or additive effects against Candida albicans biofilm formation and cell viability in preformed biofilms. The effects of this combination were further confirmed in vivo using a murine systemic infection model. Exploration of the mechanism of synergy revealed that rimonabant enhances the fungicidal activity of AmB by increasing cellular oxidative stress and cell membrane permeability. These findings provide a foundation for the possible development of AmB-rimonabant polytherapies for fungal infections.

The Discovery and Biosynthesis of Nicotinic Myxochelins from an Archangium sp. SDU34.

Myxobacteria are a prolific source of structurally diverse natural products, and one of the best-studied myxobacterial products is the siderophore myxochelin. Herein, we report two new compounds, myxochelins N (1) and O (2), that are nicotinic paralogs of myxochelin A, from the terrestrial myxobacterium Archangium sp. SDU34; 2 is functionalized with a rare 2-oxazolidinone. A precursor-feeding experiment implied that the biosynthesis of 1 or 2 was due to altered substrate specificity of the loading module of MxcE, which likely accepts nicotinic acid and benzoic acid instead of more conventional 2,3-dihydroxybenzoic acid. We also employed a phylogenomic approach to map the evolutionary relationships of the myxochelin biosynthetic gene clusters (BGCs) in all the available myxobacterial genomes, to pave the way for the future discovery of potentially hidden myxochelin derivatives. Although the biological function of 1 and 2 is unclear yet, this work underpins that even extensively studied BGCs in myxobacteria can still produce new chemistry.

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.

Discovery of a fluorescigenic pyrazoline derivative targeting ubiquitin.

Despite significant process in ubiquitin modification by using traditional genetic methods, chemical small molecules that directly target and modify ubiquitin are little reported. Here, we find that a fluorescigenic pyrazoline derivative (FPD5) could do so effectively. Molecule docking revealed that lysine 11 of ubiquitin was the key contact residue. FPD5, with stronger fluorescence, elevated the ubiquitination of beclin 1 (BECN1) and promoted autophagy. This study highlights that targeting ubiquitin by chemical small molecules enables us to modulate ubiquitination and the downstream signaling in the ubiquitin system.

A mitochondria-targeted fluorescent probe for the detection of endogenous SO2 derivatives in living cells.

A unique fluorescent probe (ZACA) for the monitoring of SO2 derivatives was developed from coumarin and benzoindoles based on FRET and ICT. ZACA exhibited an active emission signal, large Stokes shift, wide emission window distance, and high photostability. It also possessed many advantages in the ratiometric detection of HSO3-/SO32- including low detection limit and high selectivity and sensitivity. Importantly, ZACA was successfully applied in the ratiometric detection of endogenous HSO3-/SO32- in living cells with excellent cellular imaging capability (1 µM) and mitochondria-targeting ability (co-localization coefficient: 0.91).

Low-concentration HCP1 inhibits apoptosis in vascular endothelial cells.

Vascular endothelial cell (VEC) apoptosis takes part in the development of various cardiovascular diseases. Heat shock protein 90 (HSP90) regulates apoptosis through various apoptosis associated client proteins. In previous study, we identified a novel HSP90 inhibitor HCP1 induced apoptosis in A549 human lung cancer cells. Here, we found that low-concentration HCP1 (1 µM, 2 µM) suppressed VEC apoptosis caused by serum and fibroblast growth factor 2 (FGF-2) deprivation. HCP1 directly bound to glucose-regulated protein 94 (Grp94), an isoform of HSP90 located in endoplasmic reticulum, and HCP1 selectively inhibited Grp94 activity via binding to site 3. Overexpression of Grp94 inhibited the anti-apoptotic effect of HCP1 in human umbilical vein endothelial cells. Therefore, we provided HCP1 as a new VEC apoptosis inhibitor which might be a potential compound in the treatment of VEC apoptosis related vascular diseases. And we provided new pieces of evidence to understand the role of Grp94 in VEC apoptosis.

Discovery of a new autophagy inducer for A549 lung cancer cells.

Biological activities of a series of fluorescent compounds against human lung cancer cell line A549 were investigated. The results showed that (E)-1,3,3-trimethyl-2-(4-(piperidin-1-yl)styryl)-3H-indol-1-ium iodide (8) and (E)-2-(5,5-dimethyl-3-(4-(piperazin-1-yl)styryl)cyclohex-2-en-1-ylidene) malononitrile (11) could inhibit the growth of A549 cancer cells in a dose and time-dependent manner. Furthermore, compound 8 could trigger autophagy and apoptosis, but not obviously induce necrosis under the stimulatory condition. Therefore, 8 can be used as autophagy activator to investigate the regulatory mechanism of autophagy and may offer a new candidate for the treatment of lung cancer.

Acetyl-11-keto-ß-boswellic acid suppresses docetaxel-resistant prostate cancer cells in vitro and in vivo by blocking Akt and Stat3 signaling, thus suppressing chemoresistant stem cell-like properties.

Acquired docetaxel-resistance of prostate cancer (PCa) remains a clinical obstacle due to the lack of effective therapies. Acetyl-11-keto-ß-boswellic acid (AKBA) is a pentacyclic triterpenic acid isolated from the fragrant gum resin of the Boswellia serrata tree, which has shown intriguing antitumor activity against human cell lines established from PCa, colon cancer, malignant glioma, and leukemia. In this study, we examined the effects of AKBA against docetaxel-resistant PCa in vitro and in vivo as well as its anticancer mechanisms. We showed that AKBA dose-dependently inhibited cell proliferation and induced cell apoptosis in docetaxel-resistant PC3/Doc cells; its IC50 value in anti-proliferation was ∼17 µM. Furthermore, AKBA dose-dependently suppressed the chemoresistant stem cell-like properties of PC3/Doc cells, evidenced by significant decrease in the ability of mammosphere formation and down-regulated expression of a number of stemness-associated genes. The activation of Akt and Stat3 signaling pathways was remarkably enhanced in PC3/Doc cells, which contributed to their chemoresistant stem-like phenotype. AKBA (10-30 µM) dose-dependently suppressed the activation of Akt and Stat3 signaling pathways in PC3/Doc cells. In contrast, overexpression of Akt and Stat3 significantly attenuated the inhibition of AKBA on PC3/Doc cell proliferation. In docetaxel-resistant PCa homograft mice, treatment with AKBA significantly suppresses the growth of homograft RM-1/Doc, equivalent to its human PC3/Doc, but did not decrease their body weight. In summary, we demonstrate that AKBA inhibits the growth inhibition of docetaxel-resistant PCa cells in vitro and in vivo via blocking Akt and Stat3 signaling, thus suppressing their cancer stem cell-like properties.

Jungermannenone A and B induce ROS- and cell cycle-dependent apoptosis in prostate cancer cells in vitro.

AIM: Jungermannenone A and B (JA, JB) are new ent-kaurane diterpenoids isolated from Chinese liverwort Jungermannia fauriana, which show anti-proliferation activities in cancer cells. In this study we investigated the mechanisms underlying the anticancer action of JA and JB in PC3 human prostate cancer cells in vitro. METHODS: A panel of 9 human cancer cell lines was tested. Cell proliferation was assessed with a real-time cell analyzer and MTT assay. Cell apoptosis, cell cycle distribution and ROS levels were measured using cytometry. Mitochondrial damage was examined by transmission electron microscopy. DNA damage was detected with comet assay. Apoptotic, DNA damage- and cell cycle-related proteins were analyzed using Western blotting. The expression of DNA repair genes was measured with qRT-PCR. RESULTS: Both JA and JB exerted potent anti-proliferative action against the 9 cancer cell lines, and PC3 cells were more sensitive with IC50 values of 1.34±0.09 and 4.93±0.20 µmol/L, respectively. JA (1.5 µmol/L) and JB (5 µmol/L) induced PC3 cell apoptosis, which was attenuated by the caspase inhibitor Z-VAD. Furthermore, both JA and JB caused mitochondrial damage and ROS accumulation in PC3 cells, whereas vitamin C blocked the ROS accumulation and attenuated the cytotoxicity of JA and JB. Moreover, both JA and JB induced DNA damage, accompanied by downregulated DNA repair proteins Ku70/Ku80 and RDA51. JA induced marked cell cycle arrest at the G0/G1 phase, which was related to c-Myc suppression, whereas JB enforced the cell cycle blockade in the G2/M phase, which associated with activation of the JNK signaling. CONCLUSION: Both JA and JB induce prostate cancer apoptosis via ROS accumulation and induction of cell cycle arrest.

Impact of O6-methylguanine-DNA methyltransferase expression on the drug resistance of clear cell renal cell carcinoma.

OBJECTIVE: The deoxyribonucleic acid-repair protein O(6)-methylguanine-deoxyribonucleic acid methyltransferase is a major determinant of resistance of cells to various alkylating drugs. Its expression profile is different in different cancer types. Here, we studied the expression and function of O(6)-methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma. METHODS: The expression of O(6)-methylguanine-deoxyribonucleic acid methyltransferase was evaluated in clear cell renal cell carcinoma tissues and cell lines by quantitative real-time polymerase chain reaction and immunohistochemistry. The relationship between O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression and clinicopathological characteristics was analyzed. To further investigate the function of O(6)-methylguanine-deoxyribonucleic acid methyltransferase in clear cell renal cell carcinoma resistance to alkylating agents, siRNA targeting O(6)-methylguanine-deoxyribonucleic acid methyltransferase were used to silence the O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression. RESULTS: We found that O(6)-methylguanine-deoxyribonucleic acid methyltransferase is over-expressed in clear cell renal cell carcinoma tissues and cell lines. O(6)-methylguanine-deoxyribonucleic acid methyltransferase expression is related with tumor progression in clear cell renal cell carcinoma patients. Up-regulation of O(6)-methylguanine-deoxyribonucleic acid methyltransferase plays a critical role in primary resistance to alkylating agents. CONCLUSIONS: The overexpression of O(6)-methylguanine-deoxyribonucleic acid methyltransferase contributes to resistance of clear cell renal cell carcinoma to standard chemotherapy. Our results have significance for understanding a new pathway of the development of drug resistance of clear cell renal cell carcinoma.

Marsupellins A-F, ent-longipinane-type sesquiterpenoids from the Chinese liverwort Marsupella alpine with acetylcholinesterase inhibitory activity.

Acetylcholinesterase (AChE) inhibitory activity-guided fractionation of the Chinese liverwort Marsupella alpine afforded six new [marsupellins A-F (1-6)] and three known (7-9) ent-longipinane-type sesquiterpenoids. The structures were determined from MS and NMR spectroscopic data, single-crystal X-ray diffraction, and electronic circular dichroism calculations. Compounds 1-9 exhibited moderate to weak AChE inhibitory activity.

Cembrane-type diterpenoids from the Chinese liverworts Chandonanthus hirtellus and C. birmensis.

Six new cembrane-type diterpenoids (1-6) were isolated from two species of Chandonanthus: Chandonanones A, B, and D-F (1, 2, and 4-6) were isolated from C. hirtellus, and chandonanones B, C, E, and F (2, 3, 5, and 6) from C. birmensis. Five known diterpenoids, (8E)-4α-acetoxy-12α,13α-epoxycembra-1(15),8-diene (7), isochandonanthone (8), chandonanthone (9), anadensin (10), and 2,10,14-triacetoxy-7,8,18,19-diepoxydolabell-3(E)-ene (11), were also obtained. The structures of the new metabolites were established by analyses of their spectroscopic data (1D NMR, 2D NMR, HRESIMS, and IR). The absolute configurations of compounds 1 and 2 were unequivocally confirmed using single-crystal X-ray diffraction analysis with Cu Kα radiation. Cytotoxicity tests of the isolated diterpenoids against seven cancer cell lines (DU145, PC3, A549, PC12, NCI-H292, NCI-H1299, and A172) revealed that some of the diterpenoids had weak activity.

Diterpenoids from the Chinese liverwort Heteroscyphus tener and their antiproliferative effects.

Four new ent-labdane diterpenoids, heteroscyphins A-D (1-4), and four known diterpenoids (5-8) were isolated from the Chinese liverwort Heteroscyphus tener (Steph.) Schiffn. The absolute configuration of compound 1 was defined by single-crystal X-ray diffraction using Cu Kα radiation. Cytotoxicity tests revealed that compounds 3 and 5 exhibited modest activity against seven cancer cell lines. Compound 5 showed inhibitory effects on prostate cancer (PCa) cell proliferation but with less inhibition on non-neoplastic prostate epithelial cells. Compound 5 markedly caused cell growth arrest at the G0/G1 phase and induced cellular apoptosis through ROS-mediated DNA damage in PCa cells.

Notolutesins A-J, dolabrane-type diterpenoids from the Chinese liverwort Notoscyphus lutescens.

Ten new dolabrane-type diterpenoids, notolutesins A-J (1-10), were isolated from the Chinese liverwort Notoscyphus lutescens, along with four known compounds. The structures of the new compounds were established on the basis of extensive spectroscopic data, and that of 1 was confirmed by single-crystal X-ray crystallography. The absolute configuration of 1 was determined by comparing its experimental and calculated electronic circular dichroism spectra. All of the isolates were evaluated for their cytotoxicity against a small panel of human cancer cell lines, and compound 1 exhibited an IC50 value of 6.2 µM against the PC3 human prostate cancer cell line.