Cinnamomum verum Component 2-Methoxycinnamaldehyde: A Novel Anticancer Agent with Both Anti-Topoisomerase I and II Activities in Human Lung Adenocarcinoma A549 Cells In Vitro and In Vivo.

Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human lung adenocarcinoma A549 cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an upregulation of pro-apoptotic Bax and Bak genes and downregulation of anti-apoptotic Bcl-2 and Bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase-3 and -9, and morphological characteristics of apoptosis, including plasma membrane blebbing and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartment (VAC) and suppressions of nuclear transcription factors nuclear factor-κB (NF-κB) and both topoisomerase I and II activities. Further study reveals that the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against A549 cells is accompanied by downregulations of NF-κB binding activity and proliferative control involving apoptosis and both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

Total synthesis of macrodiolide ionophores aplasmomycin A and boromycin via double ring contraction.

The half structure of the symmetrical macrodiolide aplasmomycin A was synthesized by alkylation of a C3-C10 α-sulfonyl ketone subunit, prepared from (R)-pulegone and protected as a C3 ortholactone with (2R,3R)-butanediol, by a protected 15,16-dihydroxy (12E)-allylic chloride representing C11-C17. The latter was obtained from (2S,3R)-1,2-epoxy-3-butanol and propargyl alcohol. Regio- and stereoselective 5-exo-trig cyclization of the ene diol moiety in this segment, mediated by N-bromosuccinimide, led to the (2R,3S,5R)-tetrahydrofuran substructure of aplasmomycin A. Attachment of an α-acetic ester at the C3 carboxylic acid and esterification of the 3'-hydroxyl group of the tetrahydrofuran as its α-bromoacetate enabled coupling of two aplasmomycin half structures as an α-acyloxy acetate. Mukaiyama macrolactonization of this hydroxy acid afforded a symmetrical 36-membered diolide. Base-mediated double Chan rearrangement of this bis α-acyloxy dilactone caused ring contraction to the 34-membered macrocycle of desboroaplasmomycin A while generating the transannular 2-hydroxy-3-hemiketal motif of the natural product in the correct configuration. Final incorporation of boron into the tetraol core produced aplasmomycin A, isolated as its sodium borate. Extension of this route to the unsymmetrical macrodiolide boromycin was accomplished by modifications that included reversal of C12-C13 olefin geometry to (Z) for the southern half structure along with stereoselective hydride reductions of the C9 ketone that produced (9R) and (9S) alcohols for northern and southern half structures, respectively. Coupling of these half structures was made using an α-acyloxy ester linkage as for aplasmomycin A, but ring closure in this case was orchestrated via a blocked C16 alcohol that left open the C15 hydroxyl group of the southern half for Mukaiyama macrolactonization. A double Chan rearrangement of the resulting 35-membered macrocycle produced the 33-membered diolide of desborodesvalinylboromycin which had been obtained previously by degradation of natural boromycin. Insertion of boron into the tetraol core followed by esterification of the C16 alcohol with a masked d-valine and final deprotection furnished boromycin as its zwitterionic (Böeseken) complex.

Expression of Key Enzymes in Glucose Metabolism in Chronic Mountain Sickness and Its Correlation with Phenotype / 中国实验血液学杂志

OBJECTIVE@#To explore the pathogenesis of erythrocytosis by detecting the key enzymes of glucose metabolism and glucose transporter in bone marrow erythrocytes of chronic mountain sickness (CMS), and analyzing its correlation with hemoglobin.@*METHODS@#Twenty CMS patients hospitalized in Qinghai Provincial People's Hospital from January 2019 to December 2020 were selected as CMS group. Twenty males with leukocyte count > 3.5×109/L who had accepted bone marrow aspiration and had normal result were taken as control group. The mRNA and protein expression of key enzymes and glucose transporter in glucose metabolism in bone marrow CD71+ erythrocytes were detected by real time qPCR and Western blot, respectively. Glucose, lactic acid and 2,3-diphosphoglycerate in the bone marrow supernatant and serum were tested by ELISA. The mRNA and protein expression of key enzymes and glucose transporter, glucose, lactic acid and 2,3-diphosphoglycerate of the two groups were compared. Pearson correlation was used to analyze the correlation between key enzymes, glucose transporter in glucose metabolism in bone marrow CD71+ erythrocytes and hemoglobin.@*RESULTS@#The expression of HK2, GLUT1 and GLUT2 mRNA in the CMS group were higher than those in the control group (P<0.001), while the expression of HK1, OGDH and COX5B mRNA were not different. The expression of HK2, GLUT1 and GLUT2 protein in the CMS group were higher than those in the control group (P<0.05). The levels of glucose and lactic acid in the bone marrow supernatant and serum in the CMS group were not different from those in the control group, while the level of 2,3-diphosphoglycerate was higher (P<0.001). Both HK2 and GLUT2 proteins were positively correlated with hemoglobin (r=0.511, 0.717).@*CONCLUSION@#CMS patients may increase glycolysis by increasing the expression of HK2, and promote the utilization of glucose through high expression of GLUT1 and GLUT2 to meet the need of energy supply.

Analysis of epidemiological characteristics and pathogenic factors of sepsis-related acute liver injury / 中华急诊医学杂志

Objective:To analyze the epidemiological characteristics, high risk factors and pathogenic factors of sepsis-related liver injury patients by collecting epidemiological data and the sequencing results.Methods:A total of 288 sepsis patients been admited to the Emergency Department of the First Affiliated Hospital of Air Force Military Medical University from January 1, 2018 to December 31,2019 were selected and divided into sepsis liver injury group ( n = 44) and sepsis without liver injury group ( n = 244) according to whether acute liver injury occurred or not. The differences ofthe general data, hematological parameters, severity of illness and other indicators at admission between the two groups were compared and analyzed. Logistic regression was used to analyze the risk factors of sepsis-related liver injury. Total of 8 septic patients with liver injury and 4 septic patients without liver injury were selected for RNA-sequencing. Ribonucleic acid (RNA) was extracted from peripheral blood mononuclear cell of patients, detected using RNA-seq, and differential genes were screened and analyzed. Results:Compared with the sepsis without liver injury group, patients in the liver injury group suffered less hypertension (11.4% vs. 30.3%) and relatively more chronic renal insufficiency (40.9% vs. 12.1%); more patients were admitted to the emergency department due to renal disease (43.2% vs. 24.6%), higher sequential organ failure score (SOFA) and acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score (SOFA (points) 9.86 ± 3.59 vs. 5.41 ± 3.13, APACHE Ⅱ (points) (16.07 ± 4.41) vs. (14.46 ± 3.77), with prolonged hospital days (d): 8 (4.75, 13.75) vs. 6 (2, 9)]; in the liver injury group, the incidence of infection in respiratory and digestive systems (70.5% vs. 18.0%) andthe chance of infection with Staphylococcus aureus were higher (9.1% vs. 2.0%), and laboratory parameters (procalcitonin (PCT), lactate dehydrogenase (LDH), partial thromboplastin time (APTT), direct bilirubin (DBIL), aspartate aminotransferase (ALT), alanine aminotransferase (AST)) were significantly increased [PCT (μg/L) (23.90 ± 33.22) vs. (10.95 ± 20.18), LDH (U/L) 540.00 (370.50, 1177.00) vs. 168.00 (98.65, 875.18), APTT (s) (41.50 ± 3.13) vs. (36.23 ± 5.27), DBIL (μmol/L) 18.50 (10.10, 58.85) vs. 10.30 (7.60, 16.85), ALT (U/L) 67.00 (41.25, 164.00) vs. 29.00 (18.00, 51.25), AST (U/L), 101.00 (51.25, 174.75) vs. 35.00 (25.00, 65.50)], while platelet (PLT) and albumin (Alb) were significantly lower than those in the sepsis without liver injury group [PLT (× 10 9/L) 62.50 (38.50, 164.25) vs. 90.5 (66.25, 165.5), Alb (g/L) (30.17 ± 7.16) vs. (34.20 ± 6.50)] (all P < 0.05).Logistic regression analysis revealed that Staphylococcus aureus infection, thrombocytopenia, elevated procalcitonin, elevated lactate dehydrogenase, elevated total bilirubin, and elevated glutamyltransferase were associated with sepsis with acute liver injury (odds ratio, OR) with 95% confidence interval (95% CI) of 0.1167 (0.0380~0.7300), 0.9836 (1.0060~1.0290), 0.9986 (1.0000~1.0001), 0.9745 (1.0040~1.0170), 1.0020 (0.9940~1.0000), and 0.9931 (1.0000~1.0001), respectively. A total of 311 significantly differential expressed genes (DEGs) were selected, with 151 up-regulated genes and 160 down-regulated genes compared with the septic non-liver injury group. Further bioinformatics analysis reveled that the top 10 GO sequences are:①platelet α granules,② platelet α granule cavity,③wound healing,④cell migration,⑤multicellular organism process,⑥anatomical structure development,⑦cartilage ossification,⑧tissue development,⑨ keratinization,⑨Multicellular biological development. And KEGG pathway enrichment analysis revealed that human disease-related pathways were dominant, mainly including purine metabolism, AGE-RAGE signaling pathway, p53 signaling pathway, porphyrin and chlorophyll metabolism, nitrogen metabolism, mineral nutrient absorption, protein processing in the endoplasmic reticulum, and FoXo signaling pathway. Conclusions:Staphylococcus aureus infection, thrombocytopenia, elevated procalcitonin, elevated lactate dehydrogenase, elevated total bilirubin, and elevated glutamyltransferase were independent risk factors for sepsis liver injury. Coagulation dysfunction, apoptosis, and metabolic level changes may be important mechanisms of sepsis-associated liver injury, which are related to purine metabolism, porphyrin and chlorophyll metabolism and the expression of genes related to FoXo signaling pathway, Hippo signaling pathway, and p53 signaling pathway.

Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism / 中华烧伤杂志

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on pulmonary vascular endothelial cells (PMVECs) injury in septic mice and its mechanism. Methods: The experimental research method was adopted. The primary ADSCs were isolated and cultured from the discarded fresh adipose tissue of 3 patients (female, 10-25 years old), who were admitted to the First Affiliated Hospital of Air Force Medical University undergoing abdominal surgery, and the cell morphology was observed by inverted phase contrast microscope on the 5th day. The expressions of CD29, CD34, CD44, CD45, CD73, and CD90 of ADSCs in the third passage were detected by flow cytometry. The third to the fifth passage of ADSCs were collected, and their exosomes from the cell supernatant were obtained by differential ultracentrifugation, and the shape, particle size, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin of exosomes were detected, respectively, by transmission electron microscopy, nano-particle tracking analysis and Western blotting. Twenty-four adult male BALB/c mice were adopted and were divided into normal control group, caecal ligation perforation (CLP) alone group, and CLP+ADSC-exosome group with each group of 8 according to random number table (the same grouping method below) and were treated accordingly. At 24 h after operation, tumor necrosis factor (TNF-α) and interleukin 1β (IL-1β) levels of mice serum were detected by enzyme-linked immunosorbent assay, and lung tissue morphology of mice was detected by hematoxylin-eosin and myeloperoxidase staining, and the expression of 8-hydroxy-deoxyguanosine (8-OHdG) of mouse lung cells was detected by immunofluorescence method. Primary PMVECs were obtained from 1-month-old C57 mice regardless gender by tissue block method. The expression of CD31 of PMVECs was detected by immunofluorescence and flow cytometry. The third passage of PMVECs was co-cultured with ADSCs derived exosomes for 12 h, and the phagocytosis of exosomes by PMVECs was detected by PKH26 kit. The third passage of PMVECs were adopted and were divided into blank control group, macrophage supernatant alone group, and macrophage supernatant+ADSC-exosome group, with 3 wells in each group, which were treated accordingly. After 24 h, the content of reactive oxygen species in cells was detected by flow cytometry, the expression of 8-OHdG in cells was detected by immunofluorescence, and Transwell assay was used to determine the permeability of cell monolayer. The number of samples in above were all 3. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results: The primary ADSCs were isolated and cultured to day 5, growing densely in a spindle shape with a typical swirl-like. The percentages of CD29, CD44, CD73 and CD90 positive cells of ADSCs in the third passage were all >90%, and the percentages of CD34 and CD45 positive cells were <5%. Exosomes derived from ADSCs of the third to fifth passages showed a typical double-cavity disc-like structure with an average particle size of 103 nm, and the protein expressions of CD9, CD63 and TSG101 of exosomes were positive, while the protein expression of β-actin of exosomes was negative. At 24 h after operation, compared with those in normal control group, both the levels of TNF-α and IL-1β of mice serum in CLP alone group were significantly increased (with t values of 28.76 and 29.69, respectively, P<0.01); compared with those in CLP alone group, both the content of TNF-α and IL-1β of mice serum in CLP+ADSC-exosome group was significantly decreased (with t values of 9.90 and 4.76, respectively, P<0.05 or P<0.01). At 24 h after surgery, the pulmonary tissue structure of mice in normal control group was clear and complete without inflammatory cell infiltration; compared with those in normal control group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP alone group were more obvious; compared with those in CLP alone group, the pulmonary tissue edema and inflammatory cell infiltration of mice in CLP+ADSC-exosome group were significantly reduced. At 24 h after operation, endothelial cells in lung tissues of mice in 3 groups showed positive expression of CD31; compared with that in normal control group, the fluorescence intensity of 8-OHdG positive cells of the lung tissues of mice in CLP alone group was significantly increased, and compared with that in CLP alone group, the fluorescence intensity of 8-OHdG positive cells in the lung tissues of mice in CLP+ADSC-exosome group was significantly decreased. The PMVECs in the 3rd passage showed CD31 positive expression by immunofluorescence, and the result of flow cytometry showed that CD31 positive cells accounted for 99.5%. At 12 h after co-culture, ADSC-derived exosomes were successfully phagocytose by PMVECs and entered its cytoplasm. At 12 h after culture of the third passage of PMVECs, compared with that in blank control group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant alone group was significantly increased (t=15.73, P<0.01); compared with that in macrophage supernatant alone group, the fluorescence intensity of reactive oxygen species of PMVECs in macrophage supernatant+ADSC-exosome group was significantly decreased (t=4.72, P<0.01). At 12 h after culture of the third passage of PMVECs, and the 8-OHdG positive fluorescence intensity of PMVECs in macrophage supernatant alone group was significantly increased; and compared with that in blank control group, the 8-OHdG positive fluorescence intensity of PMVECs in macrophage+ADSC-exosome supernatant group was between blank control group and macrophage supernatant alone group. At 12 h after culture of the third passage PMVECs, compared with that in blank control group, the permeability of PMVECs monolayer in macrophage supernatant alone group was significantly increased (t=6.34, P<0.01); compared with that in macrophage supernatant alone group, the permeability of PMVECs monolayer cells in macrophage supernatant+ADSC-exosome group was significantly decreased (t=2.93, P<0.05). Conclusions: Exosomes derived from ADSCs can ameliorate oxidative damage in mouse lung tissue, decrease the level of reactive oxygen species, 8-OHdG expression, and permeability of PMVECs induced by macrophage supernatant.

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice / 中华烧伤杂志

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice. Methods: The experimental research methods were adopted. The discarded adipose tissue was collected from 3 female patients (aged 10-25 years) who underwent abdominal surgery in the First Affiliated Hospital of Air Force Medical University. ADSCs were extracted from the adipose tissue by collagenase Ⅰ digestion and identified with flow cytometry. Exosomes were extracted from the human ADSCs by differential ultracentrifugation, the morphology of the exosomes was observed by transmission electron microscopy, the particle diameter of the exosomes was detected by nanoparticle tracking analyzer, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin were detected by Western blotting. The human ADSCs exosomes (ADSCs-Exos) and RAW264.7 cells were co-cultured for 12 h, and the uptake of RAW264.7 cells for human ADSCs-Exos was observed. The RAW264.7 cells were divided into phosphate buffer solution (PBS) group stimulated with PBS for suitable time, endotoxin/lipopolysaccharide (LPS) stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group stimulated with LPS for corresponding time, with 3 wells in each group, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-10 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) method. The RAW264.7 cells were divided into PBS group, LPS alone group, and LPS+ADSCs-Exos group, with 3 wells in each group, which were dealt correspondingly for the time screened out in the previous experiment, the mRNA expressions of IL-1β, TNF-α, IL-6, IL-10, trasforming growth factor β (TGF-β,) and vascular endothelial growth factor (VEGF) were detected by real time fluorescence quantitative RT-PCR method, and the protein expressions of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) were detected by Western blotting. Twenty-four 8-week-old male BALB/c mice were divided into PBS group and ADSCs-Exos group according to the random number table, with 12 mice in each group, and a full-thickness skin defect wound with area of 1 cm×1 cm was inflicted on the back of each mouse. Immediately after injury, the wounds of mice in the two groups were dealt correspondingly. On post injury day (PID) 1, the concentration of IL-1β and TNF-α in serum were detected by enzyme-linked immunosorbent assay, and the mRNA expressions of IL-1β, TNF-α, and IL-6 were detected by real time fluorescence quantitative RT-PCR method. On PID 3, 6, 9, 12, and 15, the wound healing was observed and the wound non-healing rate was calculated. On PID 15, the defect length of skin accessory and collagen volume fraction (CVF) were detected by hematoxylin eosin staining and Masson staining, respectively, the CD31 expression and neovascularization were detected by immunohistochemistry, and the ratio of Ki67 positive cells, the ratio of iNOS and Arg1 double positive cells, and the ratio of iNOS positive cells to Arg1 positive cells and their fluorescence intensities were detected by immunofluorescence method. The number of samples in animal experiments was 6. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: At 12 h of culture, the cells exhibited a typical spindle shape, which were verified as ADSCs with flow cytometry. The exosomes with a vesicular structure and particle diameters of 29-178 nm, were positively expressed CD9, CD63, and TSG101 and negatively expressed β-actin. After 12 h of co-culture, the human ADSCs-Exos were endocytosed into the cytoplasm by RAW264.7 cells. The mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group were significantly higher than those in PBS group (with t) values of 39.10, 14.55, 28.80, 4.74, 48.80, 22.97, 13.25, 36.34, 23.12, 18.71, 29.19, 41.08, 11.68, 18.06, 8.54, 43.45, 62.31, 22.52, 21.51, and 37.13, respectively, P<0.01). The stimulation 12 h with significant expressions of all the inflammatory factors was selected as the time point in the following experiment. After stimulation of 12 h, the mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS alone group were significantly higher than those in PBS group (with t values of 44.20, 51.26, 14.71, and 8.54, respectively, P<0.01); the mRNA expressions of IL-1β, TNF-α, and IL-6 of RAW264.7 cells in LPS+ADSCs-Exos group were significantly lower than those in LPS alone group (with t values of 22.89, 25.51, and 8.03, respectively, P<0.01), while the mRNA expressions of IL-10, TGF-β, and VEGF were significantly higher than those in LPS alone group (with t values of 9.89, 13.12, and 7.14, respectively, P<0.01). After stimulation of 12 h, the protein expression of iNOS of RAW264.7 cells in LPS alone group was significantly higher than that in PBS group and LPS+ADSCs-Exos group, respectively (with t values of 11.20 and 5.06, respectively, P<0.05 or P<0.01), and the protein expression of Arg1 was significantly lower than that in LPS+ADSCs-Exos group (t=15.01, P<0.01). On PID 1, the serum concentrations of IL-1β and TNF-α and the mRNA expressions of IL-1β, TNF-α, and IL-6 in wound tissue of mice in ADSCs-Exos group were significantly those in lower than PBS group (with t values of 15.44, 12.24, 9.24, 7.12, and 10.62, respectively, P<0.01). On PID 3, 6, 9, 12, and 15 d, the wound non-healing rates of mice in ADSCs-Exos group were (73.2±4.1)%, (53.8±3.8)%, (42.1±5.1)%, (24.1±2.8)%, and 0, which were significantly lower than (82.5±3.8)%, (71.2±4.6)%, (52.9±4.1)%, (41.5±3.6)%, and (14.8±2.5)% in PBS group, respectively (with t values of 4.77, 8.93, 5.54, 7.63, and 7.59, respectively, P<0.01). On PID 15, the defect length of skin accessory in wounds of mice in PBS group was significantly longer than that in ADSCs-Exos group (t=9.50, P<0.01), and the CVF was significantly lower than that in ADSCs-Exos group (t=9.15, P<0.01). On PID 15, the CD31 expression and the number of new blood vessels (t=12.99, P<0.01), in wound tissue of mice in ADSCs-Exos group were significantly more than those in PBS group, and the ratio of Ki67 positive cells was significantly higher than that in PBS group (t=7.52, P<0.01). On PID 15, the ratio of iNOS and Arg1 double positive cells in wound tissue of mice in PBS group was (12.33±1.97)%, which was significantly higher than (1.78±0.29)% in ADSCs-Exos group (t=13.04, P<0.01), the ratio of iNOS positive cells and the fluorescence intensity of iNOS were obviously higher than those of ADSCs-Exos group, and the ratio of Arg1 positive cells and the fluorescence intensity of Arg1 were obviously lower than those of ADSCs-Exos group. Conclusions: The human ADSCs-Exos can alleviate inflammatory response of mouse RAW264.7 cells, decrease macrophage infiltration and secretion of the pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines to promote neovascularization and cell proliferation in full-thickness skin defect wounds of mice, hence accelerating wound healing.

Cinnamomum verum component 2-methoxycinnamaldehyde: a novel antiproliferative drug inducing cell death through targeting both topoisomerase I and II in human colorectal adenocarcinoma COLO 205 cells.

BACKGROUND: Cinnamomum verum is used to manufacture the spice cinnamon. In addition, the plant has been used as a Chinese herbal medication. METHODS: We investigated the antiproliferative effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the cortex of the plant, and the molecular biomarkers associated with tumorigenesis in human colorectal adenocarcinoma COLO 205 cells. Specifically, cell viability was evaluated by colorimetric assay; apoptosis was determined by flow cytometry and morphological analysis with bright field, acridine orange, and neutral red stainings, as well as comet assay; topoisomerase I activity was determined by assay based upon DNA relaxation and topoisomerase II by DNA relaxation plus decatentation of kinetoplast DNA; lysosomal vacuolation and volume of acidic compartments (VACs) were determined by neutral red staining. RESULTS: The results demonstrate that 2-MCA inhibited proliferation and induced apoptosis as implicated by mitochondrial membrane potential (ΔΨm) loss, activation of both caspase-3 and -9, increase of annexin V(+)PI(+) cells, as well as morphological characteristics of apoptosis. Furthermore, 2-MCA also induced lysosomal vacuolation with elevated VAC, cytotoxicity, and inhibitions of topoisomerase I as well as II activities. Additional study demonstrated the antiproliferative effect of 2-MCA found in a nude mice model. CONCLUSIONS: Our data implicate that the antiproliferative activity of 2-MCA in vitro involved downregulation of cell growth markers, both topoisomerase I and II, and upregulation of pro-apoptotic molecules, associated with increased lysosomal vacuolation. In vivo 2-MCA reduced the tumor burden that could have significant clinical impact. Indeed, similar effects were found in other tested cell lines, including human hepatocellular carcinoma SK-Hep-1 and Hep 3B, lung adenocarcinoma A549 and squamous cell carcinoma NCI-H520, and T-lymphoblastic MOLT-3 (results not shown). Our data implicate that 2-MCA could be a potential agent for anticancer therapy.

Cuminaldehyde from Cinnamomum verum Induces Cell Death through Targeting Topoisomerase 1 and 2 in Human Colorectal Adenocarcinoma COLO 205 Cells.

Cinnamomum verum, also called true cinnamon tree, is employed to make the seasoning cinnamon. Furthermore, the plant has been used as a traditional Chinese herbal medication. We explored the anticancer effect of cuminaldehyde, an ingredient of the cortex of the plant, as well as the molecular biomarkers associated with carcinogenesis in human colorectal adenocarcinoma COLO 205 cells. The results show that cuminaldehyde suppressed growth and induced apoptosis, as proved by depletion of the mitochondrial membrane potential, activation of both caspase-3 and -9, and morphological features of apoptosis. Moreover, cuminaldehyde also led to lysosomal vacuolation with an upregulated volume of acidic compartment and cytotoxicity, together with inhibitions of both topoisomerase I and II activities. Additional study shows that the anticancer activity of cuminaldehyde was observed in the model of nude mice. Our results suggest that the anticancer activity of cuminaldehyde in vitro involved the suppression of cell proliferative markers, topoisomerase I as well as II, together with increase of pro-apoptotic molecules, associated with upregulated lysosomal vacuolation. On the other hand, in vivo, cuminaldehyde diminished the tumor burden that would have a significant clinical impact. Furthermore, similar effects were observed in other tested cell lines. In short, our data suggest that cuminaldehyde could be a drug for chemopreventive or anticancer therapy.

Discovery of a novel anticancer agent with both anti-topoisomerase I and II activities in hepatocellular carcinoma SK-Hep-1 cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine for various applications. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human hepatocellular carcinoma SK-Hep-1 cell line. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase-3 and caspase-9, increase in the DNA content in sub-G1, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments, suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2, prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against SK-Hep-1 cells is accompanied by downregulations of NF-κB-binding activity, inflammatory responses involving cyclooxygenase-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and volume of acidic compartments. Similar effects (including all of the above-mentioned effects) were found in other tested cell lines, including human hepatocellular carcinoma Hep 3B, lung adenocarcinoma A549, squamous cell carcinoma NCI-H520, colorectal adenocarcinoma COLO 205, and T-lymphoblastic MOLT-3 (results not shown). Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

Discovery of a Novel Anti-Cancer Agent Targeting Both Topoisomerase I & II as Well as Telomerase Activities in Human Lung Adenocarcinoma A549 Cells In Vitro and In Vivo: Cinnamomum verum Component Cuminaldehyde.

Cinnamomum verum is used to make the spice cinnamon and has been used for more than 5000 years by both of the two most ancient forms of medicine in the words: Ayurveda and traditional Chinese herbal medicines for various applications such as adenopathy, rheumatism, dermatosis, dyspepsia, stroke, tumors, elephantiasis, trichomonas, yeast, and virus infections. We evaluated the anticancer effect of cuminaldehyde (CuA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human lung adenocarcinoma A549 cells. The results show that cuminaldehyde suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase 3 and 9, increase in annexin V+PI+ cells, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and comet with elevated tail intensity and moment. In addition, cuminaldehyde also induced lysosomal vacuolation with increased volume of acidic compartments (VAC), suppressions of both topoisomerase I & II as well as telomerase activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of cuminaldehyde was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of cuminaldehyde against A549 cells is accompanied by downregulations of proliferative control involving apoptosis, both topoisomerase I & II as well as telomerase activities, together with an upregulation of lysosomal vacuolation and VAC. Similar effects (including all of the above-mentioned effects) were found in other cell lines, including human lung squamous cell carcinoma NCI-H520 and colorectal adenocarcinoma COLO 205 (results not shown). Our data suggest that cuminaldehyde could be a potential agent for anticancer therapy.

Effect of a potent cyclooxygenase inhibitor, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), on human platelets.

Because the metabolites of arachidonic acid participate in many physiopathological responses, including inflammation and platelet aggregation, cyclooxygenase inhibitors are important in the treatment of associated diseases. A biologically active compound, 5-ethyl-4-methoxy-2-phenylquinoline (KTC-5), selectively and concentration dependently inhibited aggregation of platelets from man and ATP release caused by arachidonic acid (200 microM) and collagen (10 microg mL(-1)) without affecting the aggregation caused by thrombin (0.1 U mL(-1)) and U46619 (2 microM). The IC50 value (drug concentration inhibiting maximum response by 50%) of KTC-5 for aggregation induced by arachidonic acid and collagen was 0.11+/-0.04 microM and 0.20+/-0.03 microM, respectively. This inhibitory effect of KTC-5 was reversible and time dependent. KTC-5 specifically inhibited intracellular calcium mobilization initiated by arachidonic acid or collagen without affecting that caused by thrombin or U46619 in human platelets. Furthermore, KTC-5 inhibited thromboxane B2 and prostaglandin D2 formation provoked by arachidonic acid. The IC50 value of KTC-5 for arachidonic-acid-induced thromboxane B2 formation was 0.07+/-0.02 microM. Based on these observations, the data indicated that KTC-5 potently inhibited human platelet aggregation and ATP release mainly via the inhibition of the cyclooxygenase-1 activity. Moreover, KTC-5 inhibited lipopolysaccharide-induced prostaglandin E2 formation in RAW264.7 cells in the presence of external arachidonic acid with an IC50 value of 0.17+/-0.06 microM. Immunoblot analysis showed that KTC-5 did not affect the cyclooxygenase-2 expression in the presence of lipopolysaccharide on RAW264.7 cells. This result indicated that KTC-5 affects the activity of cyclooxygenase-2. According to these data, we concluded that KTC-5 is a cyclooxygenase inhibitor for both subtypes.

A New CoII Complex as a Bulge-Specific Probe for DNA.

Bulge cleavage of two or three bases occurs when a DNA substrate is specifically cleaved oxidatively by [CoII (tfa)2 (happ)] (see picture). Hydrogen peroxide is necessary for the activation of this octahedral complex, which suggests that hydroxyl radicals are the reactive species. The complex has no significant reactivity towards the corresponding sequence in a single-stranded DNA region, and it exhibits only a low affinity towards double-stranded DNA. happ=macrocyclic ligand based on 1,10-phenanthroline, tfa=trifluoroacetate.

Acoustic characteristics of different target vowels during the laryngeal telescopy.

OBJECTIVE: The aim of this study was to investigate the acoustic characteristics of target vowels phonated in normal voice persons while performing laryngeal telescopy. The acoustic characteristics are compared to show the extent of possible difference to speculate their impact on phonation function. METHODS: Thirty-four male subjects aged 20-39 years with normal voice were included in this study. The target vowels were /i/ and /ɛ/. Recording of voice samples was done under natural phonation and during laryngeal telescopy. The acoustic analysis included the parameters of fundamental frequency, jitter, shimmer and noise-to-harmonic ratio. RESULTS: The sound of a target vowel /ɛ/ was perceived identical in more than 90% of the subjects by the examiner and speech language pathologist during the telescopy. Both /i/ and /ɛ/ sounds showed significant difference when compared with the results under natural phonation. There was no significant difference between /i/ and /ɛ/ during the telescopy. CONCLUSION: The present study showed that change in target vowels during laryngeal telescopy makes no significant difference in the acoustic characteristics. The results may lead to the speculation that the phonation mechanism was not affected significantly by different vowels during the telescopy. This study may suggest that in the principle of comfortable phonation, introduction of the target vowels /i/ and /ɛ/ is practical.

Significance of Bone Marrow Microvessel Density and Vascular- Related Factors in Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of bone marrow microvessel density(MVD) and angiogenesis related factors in multipic myeloma(MM).@*METHODS@#Twenty cases of MM and 20 cases of simple fracture were selected and enrolled in MM group and control group respectively. The clinical data and results of laboratorial tests were collected; the bone marrow MVD of patients was detected by using the modified plastic-embedded pathologic sections of bone marrow tissue and histochemistry staining, the expression levels of amgiogenesis-related factors including VEGF, TNF-α, HGF, TGF-α, TGF-β1, bFGF, Ang-Ⅰ, Ang-Ⅱ in bone marrow supernatant were detected by ELISA; the mRNA expression levels of above-mentioned cytokines in bone marrow mononuclear cells were detected by real time-PCR; the pearson correlation analysis was used to analyze the correlation of MVD with VEGF, HGF and bFGF levels.@*RESULTS@#The MVD in MM group was significantly higher than that in control group (P＜0.001); the mRNA expression of VEGF, TGF-α, TGF-β1 and HGF in bone marrow mononuclear cells of MM group was higher than that of control group(P＜0.001); the levels of VEGF, HGF, bFGF and THF-α in bone marrow supernatant of MM group were higher than those in control group(P＜0.05), moreover, the MVD positively correlated with levels of VEGF, HGF and bFGF in bone marrow(r=0.488, 0.472 and 0.457).@*CONCLUSION@#The MVD and levels vessel-related factors in bone marrow supernatant of MM patients increase, among which the levels of VEGF and HGF in bone marrow supernatant are consistant with those mRNA expression level in bone marrow mononuclear cells, moreover, the MVD possitively cerrelates with levels of VEGF, HGF and bFGF in bone marrow supernatant, suggesting that the changes of bone marrow microenvironment vassel-related factors play an important role in angiogenesis and pathogenesis of multiple myeloma.

Effect of HIF-1α on Angiogenesis-Related Factors in K562 Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the mechanisms of angiogenesis in chronic myeloid leukemia (CML) through detecting the levels of angiogenesis-related factors secreted from K562 cells after overexpression and interference of HIF-1α gene in K562 cells.@*METHODS@#The K562 cells were transfected by lentiviruses carried and interfered HIF-1α gene, then the transtected K562 cells with carried and interfered with HIF-1α gene were enrolled in overexpression and interference groups respectively, at the same time the K562 cells transfected by the empty virus were enrolled in control group. The cells were harvested after culture for 72 hours under normoxid condition. The transfection efficient in 3 groups was detected by fluorescence microscopy; the mRNA expression of HIF-1α gene and angiogenesis-related factors was detected by RT-PCR; the concentration of angiogenesis-related factors in the caltured supernatant was detected by ELISA.@*RESULTS@#The optimal MOI of K562 cells transfected with lentivirus was 10 and the transfection efficiency was about 50%. The positive rate of transfection after screening by puromycin was more than 90%. The mRNA expression of ANG-I, ANG-II, TGF-α and VEGF in the interference group was lower than that in the over-expression group, and the TGF-β1 mRNA expression in the interference group was higher than in the over-expression group. The mRNA expression of ANG-I and VEGF in the interference group was lower than that in the control group. TGF-αdid not could be detected, and the culture supernatant concentration of ANG-I and TNF-α in the interference group was lower than in the over-expression group, while the VEGF concentration in the interference group was higher than that in the over-expression group. All of the above-mentioned differences were statistically significant (P＜0.05).@*CONCLUSION@#The positive K562 cells transfected with leutivirus have been harvested by screening with puromycin. The HIF-1α mRNA positively regulates the mRNA expression of ANG-1, ANG-2, TGF-α, VEGF in K562 cells, promotes the antocrine ability of ANG-1 and TNF-α, moreover not stimulates the autocrine of TGF-α, the up-regulation of HIF-1α expression can inhibit the expression TGF-β1 in K562 cells and the autocrine of TGF-β1.

Expression and mechanism of plasma miR-372 in acute myeloid leukemia patients / 实用医学杂志

Objective To investigate the expression of miR-372 in the plasma of patients with acute my-eloid leukemia(AML)and the possible mechanism to participate in the development of AML. Methods Real-time quantitative PCR was used to detect the level of miR-372 in plasma. Bioinformatics software predicted the pos-sible target genes of miR-372 and dual luciferase reporter assay was performed to validate the prediction. In HL-60 cells,miR-372 was knocked down,and the effects on cell migration and cloning were detected by scratch test and clone formation. Results The level of miR-372 was significantly up-regulated in the plasma of AML patients. ROC analysis showed that miR-372 could distinguish between AML patients and healthy controls. Dual luciferase report-er assay showed that miR-372 could inhibit the activity of PTEN-3'UTR. Inhibition of miR-372 in HL-60 cells can significantly reduce the cell migration rate and clone formation ability. Conclusion In summary,for the first time,we showed novel data that the level of miR-372 was increased in the plasma of AML patients. By targeting the tumor suppressor gene PTEN,miR-372 may become a potential noninvasive biomarker for the screening and di-agnosis of AML.

Canonical Transient Receptor Potential Channels and Their Link with Cardio/Cerebro-Vascular Diseases

The canonical transient receptor potential channels (TRPCs) constitute a series of nonselective cation channels with variable degrees of Ca2+ selectivity. TRPCs consist of seven mammalian members, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7, which are further divided into four subtypes, TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7. These channels take charge of various essential cell functions such as contraction, relaxation, proliferation, and dysfunction. This review, organized into seven main sections, will provide an overview of current knowledge about the underlying pathogenesis of TRPCs in cardio/cerebrovascular diseases, including hypertension, pulmonary arterial hypertension, cardiac hypertrophy, atherosclerosis, arrhythmia, and cerebrovascular ischemia reperfusion injury. Collectively, TRPCs could become a group of drug targets with important physiological functions for the therapy of human cardio/cerebro-vascular diseases.

Expression of SHIP 1 in the Patients with Acute Myeloid Leukemia and Its Influence on the Apoptosis of Human Leukemia Cells / 现代生物医学进展

Objective:To investigate the expression of SHIP1 in the patients with acute myeloid leukemia and its effect on the apoptosis of human leukemia cells.Methods:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was detected by Westem blot.U937 cells was transfected with SHIP1 expression vector (pEGFP-SHIP1 group) and empty vector control (pEGFP group) respectively,U937 cells without transfection were used as the control group.Flow cytometry was used to detect the apoptosis of the cells,the expression of SHIP1,Bcl-2,Bax,Akt,p-Akt were detected by western blot.Results:The expression of SHIP1 in the bone marrow of patients with acute myeloid leukemia was significantly lower than that of the normal human bone marrow SHIP 1 (P＜0.01).The SHIP1 and Bax expressions as well as the apoptotic rate ofpEGFP-SHIP1 group were significantly higher than those of the control group(P＜0.01),while the Bcl-2 and p-Akt expressions were significantly lower than those in the control group(P＜0.01).Conclusions:SH-P1 expression was down regulated in the bone marrow of patients with acute myeloid leukemia.SHIP1 could promote the apoptosis of human leukemia cells via Akt signaling pathway.

Implication of canonical transient receptor potential in cardiovascular disease / 国际药学研究杂志

The canonical transient receptor potential(TRPC)regulating non-voltage-gated cation entry in plasma-membrane is widely expressed in cardiovascular system. Cytosolic Ca2+takes charge of multifunctional biogenic activities and its stabilization is criti?cal for physiological function. Almost all TRPC channels are non-selectively permeable for Ca2+. Overexpression or transgenation of TRPC leading to the change of Ca2+may contribute to fundamental malfunctions and engage in the physiological and pathophysiological processes of some diseases. Evidence suggests that regulation of TRPC channels plays important roles in the development of hyperten?sion,pulmonary arterial hypertension,cardiac hypertrophy,atherosclerosis,and arrhythmia. This review gives an overview of the functional properties of mammalian TRPC channels,describes their roles in cardiovascular diseases and discusses their potential as drug targets for therapeutic intervention.

A boy with a large bladder stone.

Despite the frequent association of urinary tract infection with vesicoureteral reflux and urinary calculi, since vesicouretal reflux is induced by bladder stones, the coexistence of vesicoureteral reflux and bladder stones is rare. Because of its occurrence in children belonging to poor socioeconomic groups, it is believed to be a deficiency disorder. Most cases of bladder stones occur between the ages of 2 and 5 years. Common clinical presentations of bladder stones include urinary dribbling and enuresis, frequency of micturition, pain during micturition, pelvic pain and hematuria. We report the occurrence of a large bladder stone in a boy, who experienced intermittent Lower abdominal pain and urinary incontinence, both during the day and at night. He had been diagnosed with enuresis and treated in pediatric clinics for 1 year. Delayed diagnosis resulted in bladder stone formation. The stone was larger than 2.5cm and open vesicolithotomy was therefore selected as the best and safest treatment choice. His symptoms disappeared after surgery. Thorough metabolic and environmental evaluations of such cases are required on an individual basis. Bladder stones should be considered as a possible diagnosis in children presenting with urinary incontinence.