Disulfide Cleavage in a Dimeric Epipolythiodioxopiperazine Natural Product Diminishes Its Apoptosis-Inducing Effect but Enhances Autophagy in Tumor Cells.

Gliocladicillin C (3) is a cytotoxic epipolythiodioxopiperazine (ETP) isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana. Although the disulfides/polysulfides in ETPs are believed to account for their cytotoxicity, and 11'-deoxyverticillin A was demonstrated to induce apoptosis and autophagy, how they mediate apoptosis and autophagy remained unknown. Here, we revealed that 3 activated caspase-dependent apoptosis and autophagy in human tumor cells, while the prepared disulfide-cleavage product failed to induce reactive oxygen species production and PARP cleavage, but further enhanced the autophagic flux compared to 3. Gliocladicillin C and its derivative also increased the phosphorylation of AMP-activated protein kinase and stimulated autophagy by affecting the glycolytic pathway. These results demonstrated that the disulfides played an essential role in inducing apoptosis, but not autophagy.

Using optical coherence tomography and intravascular ultrasound imaging to quantify coronary plaque cap thickness and vulnerability: a pilot study.

BACKGROUND: Detecting coronary vulnerable plaques in vivo and assessing their vulnerability have been great challenges for clinicians and the research community. Intravascular ultrasound (IVUS) is commonly used in clinical practice for diagnosis and treatment decisions. However, due to IVUS limited resolution (about 150-200 µm), it is not sufficient to detect vulnerable plaques with a threshold cap thickness of 65 µm. Optical Coherence Tomography (OCT) has a resolution of 15-20 µm and can measure fibrous cap thickness more accurately. The aim of this study was to use OCT as the benchmark to obtain patient-specific coronary plaque cap thickness and evaluate the differences between OCT and IVUS fibrous cap quantifications. A cap index with integer values 0-4 was also introduced as a quantitative measure of plaque vulnerability to study plaque vulnerability. METHODS: Data from 10 patients (mean age: 70.4; m: 6; f: 4) with coronary heart disease who underwent IVUS, OCT, and angiography were collected at Cardiovascular Research Foundation (CRF) using approved protocol with informed consent obtained. 348 slices with lipid core and fibrous caps were selected for study. Convolutional Neural Network (CNN)-based and expert-based data segmentation were performed using established methods previously published. Cap thickness data were extracted to quantify differences between IVUS and OCT measurements. RESULTS: For the 348 slices analyzed, the mean value difference between OCT and IVUS cap thickness measurements was 1.83% (p = 0.031). However, mean value of point-to-point differences was 35.76%. Comparing minimum cap thickness for each plaque, the mean value of the 20 plaque IVUS-OCT differences was 44.46%, ranging from 2.36% to 91.15%. For cap index values assigned to the 348 slices, the disagreement between OCT and IVUS assignments was 25%. However, for the OCT cap index = 2 and 3 groups, the disagreement rates were 91% and 80%, respectively. Furthermore, the observation of cap index changes from baseline to follow-up indicated that IVUS results differed from OCT by 80%. CONCLUSIONS: These preliminary results demonstrated that there were significant differences between IVUS and OCT plaque cap thickness measurements. Large-scale patient studies are needed to confirm our findings.

Chloropupukeananin and Pestalofone C Regulate Autophagy through AMPK and Glycolytic Pathway.

Chloropupukeananin (RN56-6) and Pestalofone C (RN56-49), isolated from the culture of the plant endophytic fungus Pestalotiopsis fici, have been shown cytotoxic, anti-HIV, and antimicrobial activities. However, the underlying mechanism of their regulatory roles in autophagy remains unknown. In the present study, we revealed that both compounds increased the formation of autophagosome and enhanced autophagic flux. While RN56-6 upregulated the expression of HK2, one of the key rate-limiting enzymes of glycolysis, the inhibition of glycolysis chemically attenuated RN56-6-induced autophagy. On the contrary, RN56-49 downregulated the expression of HK2, while the suppression of glycolysis promoted RN56-49-dependent autophagic flux. Moreover, the knockdown of AMPKß1, a scaffolding subunit of AMPK, decreased autophagy induced by these two compounds. Collectively, these findings revealed that RN56-6 and RN56-49 regulated autophagic process through AMPK and glycolytic pathway.

Integrated Analysis of lncRNA-Associated ceRNA Network Identifies Two lncRNA Signatures as a Prognostic Biomarker in Gastric Cancer.

BACKGROUND: Gastric cancer (GC) is a malignant tumour that originates in the gastric mucosal epithelium and is associated with high mortality rates worldwide. Long noncoding RNAs (lncRNAs) have been identified to play an important role in the development of various tumours, including GC. Yet, lncRNA biomarkers in a competing endogenous RNA network (ceRNA network) that are used to predict survival prognosis remain lacking. The aim of this study was to construct a ceRNA network and identify the lncRNA signature as prognostic factors for survival prediction. METHODS: The lncRNAs with overall survival significance were used to construct the ceRNA network. Function enrichment, protein-protein interaction, and cluster analysis were performed for dysregulated mRNAs. Multivariate Cox proportional hazards regression was performed to screen the potential prognostic lncRNAs. RT-qPCR was used to measure the relative expression levels of lncRNAs in cell lines. CCK8 assay was used to assess the proliferation of GC cells transfected with sh-lncRNAs. RESULTS: Differentially expressed genes were identified including 585 lncRNAs, 144 miRNAs, and 2794 mRNAs. The ceRNA network was constructed using 35 DElncRNAs associated with overall survival of GC patients. Functional analysis revealed that these dysregulated mRNAs were enriched in cancer-related pathways, including TGF-beta, Rap 1, calcium, and the cGMP-PKG signalling pathway. A multivariate Cox regression analysis and cumulative risk score suggested that two of those lncRNAs (LINC01644 and LINC01697) had significant prognostic value. Furthermore, the results indicate that LINC01644 and LINC01697 were upregulated in GC cells. Knockdown of LINC01644 or LINC01697 suppressed the proliferation of GC cells. CONCLUSIONS: The authors identified 2-lncRNA signature in ceRNA regulatory network as prognostic biomarkers for the prediction of GC patient survival and revealed that silencing LINC01644 or LINC01697 inhibited the proliferation of GC cells.

Using Optical Coherence Tomography and Intravascular Ultrasound Imaging to Quantify Coronary Plaque Cap Stress/Strain and Progression: A Follow-Up Study Using 3D Thin-Layer Models.

Accurate plaque cap thickness quantification and cap stress/strain calculations are of fundamental importance for vulnerable plaque research. To overcome uncertainties due to intravascular ultrasound (IVUS) resolution limitation, IVUS and optical coherence tomography (OCT) coronary plaque image data were combined together to obtain accurate and reliable cap thickness data, stress/strain calculations, and reliable plaque progression predictions. IVUS, OCT, and angiography baseline and follow-up data were collected from nine patients (mean age: 69; m: 5) at Cardiovascular Research Foundation with informed consent obtained. IVUS and OCT slices were coregistered and merged to form IVUS + OCT (IO) slices. A total of 114 matched slices (IVUS and OCT, baseline and follow-up) were obtained, and 3D thin-layer models were constructed to obtain stress and strain values. A generalized linear mixed model (GLMM) and least squares support vector machine (LSSVM) method were used to predict cap thickness change using nine morphological and mechanical risk factors. Prediction accuracies by all combinations (511) of those predictors with both IVUS and IO data were compared to identify optimal predictor(s) with their best accuracies. For the nine patients, the average of minimum cap thickness from IVUS was 0.17 mm, which was 26.08% lower than that from IO data (average = 0.23 mm). Patient variations of the individual errors ranged from ‒58.11 to 20.37%. For maximum cap stress between IO and IVUS, patient variations of the individual errors ranged from ‒30.40 to 46.17%. Patient variations of the individual errors of maximum cap strain values ranged from ‒19.90 to 17.65%. For the GLMM method, the optimal combination predictor using IO data had AUC (area under the ROC curve) = 0.926 and highest accuracy = 90.8%, vs. AUC = 0.783 and accuracy = 74.6% using IVUS data. For the LSSVM method, the best combination predictor using IO data had AUC = 0.838 and accuracy = 75.7%, vs. AUC = 0.780 and accuracy = 69.6% using IVUS data. This preliminary study demonstrated improved plaque cap progression prediction accuracy using accurate cap thickness data from IO slices and the differences in cap thickness, stress/strain values, and prediction results between IVUS and IO data. Large-scale studies are needed to verify our findings.

Bioassay-guided separation of citreorosein and other oestrogenic compounds from Polygonum cuspidatum.

Citreorosein was isolated from P. cuspidatum as a new oestrogenic compound, together with emodin and its glucoside, by silica gel column chromatography and preparative high-performance liquid chromatography sequentially. Oestrogenic activity was determined by a recombinant yeast assay.

Grb2 binds to PTEN and regulates its nuclear translocation to maintain the genomic stability in DNA damage response.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein critical for signal transduction and endocytosis, but its role in DNA damage response (DDR) remains unknown. Here, we report that either knockdown of Grb2 or overexpression of the mutated Grb2 promotes micronuclei formation in response to oxidative stress. Furthermore, Grb2 was demonstrated to interact with phosphatase and tensin homologue (PTEN; a tumor suppressor essential for nuclear stability), and the loss of Grb2 reduced the nuclear-localized PTEN, which was further decreased upon stimulation with hydrogen peroxide (H2O2). Overexpression of the T398A-mutated, nuclear-localized PTEN reduced micronuclei frequency in the cells deficient of functional Grb2 via rescuing the H2O2-dependent expression of Rad51, a protein essential for the homologous recombination (HR) repair process. Moreover, depletion of Grb2 markedly decreased the expression of Rad51 and its interaction with PTEN. Notably, Rad51 showed a preference to immunoprecipation with the T398A-PTEN mutant, and silencing of Rad51 alone accumulated micronuclei concurring with decreased expression of both Grb2 and PTEN. Our findings indicate that Grb2 interacts with PTEN and Rad51 to regulate genomic stability in DDR by mediating the nuclear translocation of PTEN to affect the expression of Rad51.

SQSTM1/p62 loss reverses the inhibitory effect of sunitinib on autophagy independent of AMPK signaling.

Sunitinib (ST), a multitargeted receptor tyrosine kinase inhibitor, has been demonstrated to be effective for the treatment of renal carcinoma. It has been reported that ST is involved in the mediation of autophagy; however, its regulatory role in the autophagic process remains controversial. Furthermore, the mechanism by which activated AMP-activated protein kinase (AMPK) negatively regulates autophagy remains nearly unexplored. In the present study, we revealed that ST inhibited AMPK activity and regulated autophagy in a cell type- and dose-dependent manner. In a number of cell lines, ST was demonstrated to inhibit H2O2-induced autophagy and the phosphorylation of acetyl-CoA carboxylase (ACC), whereas alone it could block the autophagic flux concurrent with increased expression of p62. An immunoprecipitation assay revealed that LC3 directly interacted with p62, whereas ST increased punctate LC3 staining, which was well colocalized with p62. Taken together, we reveal a previously unnoticed pathway for ST to regulate the autophagic process, and p62, although often utilized as a substrate in autophagy, plays a critical role in regulating the inhibition of ST in both basal and induced autophagy.

Intramuscular injection of human umbilical cord-derived mesenchymal stem cells improves cardiac function in dilated cardiomyopathy rats.

BACKGROUND: Stem cells provide a promising candidate for the treatment of the fatal pediatric dilated cardiomyopathy (DCM). This study aimed to investigate the effects of intramuscular injection of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) on the cardiac function of a DCM rat model. METHODS: A DCM model was established by intraperitoneal injections of doxorubicin in Sprague-Dawley rats. hUCMSCs at different concentrations or cultured medium were injected via limb skeletal muscles, with blank medium injected as the control. The rats were monitored for 4 weeks, meanwhile BNP, cTNI, VEGF, HGF, GM-CSF, and LIF in the peripheral blood were examined by ELISA, and cardiac function was monitored by echocardiography (Echo-CG). Finally, the expression of IGF-1, HGF, and VEGF in the myocardium was examined by histoimmunochemistry and real-time PCR, and the ultrastructure of the myocardium was examined by electron microscopy. RESULTS: Injection of hUCMSCs markedly improved cardiac function in the DCM rats by significantly elevating left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS). The BNP and cTNI levels in the peripheral blood were reduced by hUCMSCs, while HGF, LIF, GM-CSF, and VEGF were increased by hUCMSCs. Expression of IGF-1, HGF, and VEGF in the myocardium from the DCM rats was significantly increased by hUCMSC injection. Furthermore, hUCMSCs protected the ultrastructure of cardiomyocytes by attenuating mitochondrial swelling and maintaining sarcolemma integrity. CONCLUSIONS: Intramuscular injection of UCMSCs can improve DCM-induced cardiac function impairment and protect the myocardium. These effects may be mediated by regulation of relevant cytokines in serum and the myocardium.

Analysis of estrogenic compounds in Polygonum cuspidatum by bioassay and high performance liquid chromatography.

The estrogenic activity of traditional Chinese herb-Polygonum cuspidatum Sieb. et Zucc. was investigated by a recombinant yeast screening (YES) assay. Anthraquinones are the main components in the plant, of which emodin is the most abundant one. The ethyl acetate fraction of the ethanol extract of Polygonum cuspidatum was separated on a silica gel TLC plate and seven sub-fractions were collected. The results of bioassay demonstrated that Hzs1 and Hzs6 showed higher estrogenic activities than that of others and the potency of these two compounds were approximately 10(-4) g/L and 10(-3) g/L, respectively. HPLC analysis was performed to determine the activities and the active components. Combining the results of HPLC analysis and estrogenic activity test by YES led to the conclusion that an unknown bioactive compound might exist in the extraction of Polygonum cuspidatum.