BACKGROUND: Mesenchymal stem cells (MSCs) have strong self-renewal ability and multiple differentiation potential. Some studies confirmed that spreading shape and area of single MSCs influence cell differentiation, but few studies focused on the effect of the circularity of cell shape on the osteogenic differentiation of MSCs with a confined area during osteogenic process.METHODS: In the present study, MSCs were seeded on a micropatterned island with a spreading area lower than that of a freely spreading area. The patterns had circularities of 1.0 or 0.4, respectively, and areas of 314, 628, or 1256 µm² . After the cells were grown on a micropatterned surface for 1 or 3 days, cell apoptosis and F-actin were stained and analyzed. In addition, the expression of β-catenin and three osteogenic differentiation markers were immunofluorescently stained and analyzed, respectively.RESULTS: Of these MSCs, the ones with star-like shapes and large areas promoted the expression of osteogenic differentiation markers and the survival of cells. The expression of F-actin and its cytosolic distribution or orientation also correlated with the spreading shape and area. When actin polymerization was inhibited by cytochalasin D, the shape-regulated differentiation and apoptosis of MSCs with the confined spreading area were abolished.CONCLUSION: This study demonstrated that a spreading shape of low circularity and a larger spreading area are beneficial to the survival and osteogenic differentiation of individual MSCs, which may be regulated through the cytosolic expression and distribution of F-actin.
Actins ; Antigens, Differentiation ; Apoptosis ; Cell Differentiation ; Cell Shape ; Cytochalasin D ; Cytosol ; Mesenchymal Stromal Cells ; Osteogenesis ; Polymerization ; Polymers

OBJECTIVE: To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms. METHODS: The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively. RESULTS: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells. CONCLUSIONS COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.
Biomarkers, Tumor ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Celastrus ; chemistry ; Cell Hypoxia ; drug effects ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cobalt ; Down-Regulation ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins ; metabolism ; Plant Extracts ; pharmacology ; therapeutic use ; Signal Transduction ; drug effects

Dysfunctional sperm maturation is the primary reason for the poor sperm motility and morphology in infertile men. Spermatozoa from infertile men were fractioned on three-layer density gradient (80%, 60%, and 40%). Fraction 1 (F1) refers to the least mature stage having the lowest density, whereas the fraction 4 (F4) includes the most dense and morphologically mature motile spermatozoa. Fraction 2 (F2) and fraction 3 (F3) represent the intermediate stages. Proteins were extracted and separated by 1-dimensional gel. Bands were digested with trypsin and analyzed on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. A total of 1585 proteins were detected in the four fractions of spermatozoa. A dysregulated protein turnover and protein folding may lead to accumulation of defective proteins or proteins that otherwise would have been eliminated during the process of maturation, resulting in the impairment of sperm function. Aberrant chaperone expression may be a major contributing factor to the defective sperm function. Androgen receptor was predicted as a transcription regulator in one of the networks and the affected pathways were chaperone-mediated stress response, proteosomal pathway, and sperm function. The downregulation of key pathways and proteins which compromises the fertilizing potential of spermatozoa may provide insight into the mechanisms that lead to male infertility.
Adult ; Cell Shape/physiology* ; Humans ; Infertility, Male/metabolism* ; Male ; Proteome/metabolism* ; Proteomics ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Tandem Mass Spectrometry

OBJECTIVETo investigate the effects of Compound Zhebei Granule (, CZG) combined with doxorubicin hydrochloride (adriamycin, ADM) on specific surface antigens in mice with KG-1a transplanted cells.

METHODSA subcutaneous tumor xenograft model was established by injection of the acute myeloid leukemia cell line KG-1a into the axillary flfl anks of BALB/c-nude mice. Twenty-four tumor bearing mice were divided into 4 groups according to a random number table, including normal saline control group, ADM group, high-dose CZG group, and mid-dose CZG group, with 6 mice in each group. Drug administration occurred on the 14th day after cell inoculation, and normal saline control group mice were gavaged with normal saline at 0.2 mL/10 g every other day. ADM group mice were intraperitoneally injected with ADM 1 mg/kg [conversion of adults, 40 mg/(m•d)] every other day. High- and mid-dose CZG groups mice were gavaged with CZG at the dose of 8 and 4 g/kg respectively every other day and intraperitoneally injected with ADM (1 mg/kg) every other day. The administration period lasted for 10 days. The tumor xenografts were made into mononuclear cell suspensions after dissection, and the expressions of specific surface antigens, including CD34CD38, CD34CD38CD123, CD34CD38CD96 and CD34CD38CD33, in KG-1a cell line tumor xenografts were detected by flfl ow cytometry.

RESULTSCompared with the control and ADM groups, expression of CD34CD38 in the two CZG groups was significantly lower (P<0.05). Compared with the control group, expression of CD34CD38CD123 in the two CZG groups decreased (P<0.05). The high-dose CZG group showed more obvious outcomes compared with the ADM group (P<0.05). Compared with the control and ADM groups, the expression of CD34CD38CD96 and CD34CD38CD33 in the two CZG groups decreased (P<0.05).

CONCLUSIONSCZG combined with doxorubicin could reduce the expression of CD34CD38, CD34CD38CD123, CD34CD38CD96 and CD34CD38CD33 in KG-1a cell line tumor xenografts, which shows that CZG could target leukemia stem cells and play a role in chemosensitization.

Animals ; Antigens, Surface ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Doxorubicin ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Humans ; Mice, Inbred BALB C ; Subcutaneous Tissue ; drug effects ; pathology ; Xenograft Model Antitumor Assays

PURPOSE: Bone marrow has long been a source of primary cells. This study was performed to evaluate the effects of age and sex on the cellular viability and expression of stem cell markers of mRNA and on the protein expression of bone marrow stem cells (BMSCs) derived from healthy donors. MATERIALS AND METHODS: Stem cells were isolated from human bone marrow and plated in culture plates. The shape of the BMSCs was observed under inverted microscope. Quantitative cellular viability was evaluated using a Cell-Counting Kit-8 assay. The expression of stem cell surface markers was tested and a series of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot was performed to evaluate the expression in each group. RESULT: The shapes of the cells at 20s, 30s, and 50s were similar to each other. No significant changes in cellular viability were noted among different age groups or sex groups. The BMSCs expressed CD44, CD73, and CD90 surface markers but did not express CD14 and CD34. There were no noticeable differences in CD surface markers among the different age groups. The expressions of CD surface markers were similar between men and women. No significant differences in the secretion of vascular endothelial growth factors (VEGFs) were noted at Day 3 between different age groups. qRT-PCR regarding the expression showed differences between the age groups. However, Western blot analysis showed a decrease in expression but did not reach statistical significance (P>0.05). CONCLUSION: This study clearly showed no significant differences in shape, cell viability, expression of stem cell surface markers, or secretion of human VEGF among different age groups. However, western blot analysis showed a tendency of age-related decrease which did not reach statistical significance. Collectively, autologous or allogeneic BMSCs should be meticulously applied to obtain optimal results regarding age and sex.
Age Factors ; Blotting, Western ; Bone Marrow ; Cell Shape ; Cell Survival ; Female ; Humans ; Male ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Stem Cells ; Tissue Donors ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.

METHODSThe HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.

RESULTSThe bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.

CONCLUSIONSBufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

Apoptosis ; drug effects ; genetics ; Bufanolides ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; genetics ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia, Erythroblastic, Acute ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects ; genetics ; WT1 Proteins ; genetics ; metabolism

OBJECTIVETo investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells.

METHODSEca109 cells were treated with TLD and its separated formulae, including the clearing-heat and detoxification formula (Q), activating-blood and promoting-qi formula (H) and nourishing-yin and blood formula (Z). Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, cell morphology was observed using a microscope, the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B (NF-κB) signal pathway was detected by Western blot.

RESULTSThe half maximal inhibitory concentrations of TLD, Q and H were 386, 771 and 729 mg/L, respectively. TLD, Q and H significantly inhibited cell proliferation, with 69.43%, 60.84% and 61.90% of treated cells in the G phase of the cell cycle. The percentage of cells in S phase increased significantly after treatment with TLD, Q, and H compared with the control group (P<0.05), and TLD showed the strongest effect. Z had no influence on the cell cycle compared with the control group (P>0.05). Western blot detection indicated slight differences in the inhibition of the NF-κB pathway by the different formulae. TLD formula strongly inhibited IKKβ, NF-κB, interleukin-6 and tumor necrosis factor-α expression compared with the control group.

CONCLUSIONSTLD inhibited Eca109 cell proliferation by arresting cells in S phase. The possible mechanism might be related to inhibiting the NF-κB transduction cascade. The combination of the herbs found in the three separate formulae, H, Q and Z, work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.

Blotting, Western ; Cell Count ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Esophageal Neoplasms ; metabolism ; pathology ; Flow Cytometry ; Humans ; Inhibitory Concentration 50 ; NF-kappa B ; metabolism ; S Phase ; drug effects ; Signal Transduction ; drug effects

OBJECTIVETo investigate the neuroprotective effects of icariin on formaldehyde (FA)-treated human neuroblastoma SH-SY5Y cells and the possible mechanisms involved.

METHODSSH-SY5Y cells were divided into FA treatment group, FA treatment group with icariin, and the control group. Cell viability, apoptosis, and morphological changes were determined by cell counting kit-8 (CCK 8), flow cytometry, and confocal microscopy, respectively. The phosphorylation of Tau protein was examined by western blotting.

RESULTSFA showed a half lethal dose (LD50) of 0.3 mmol/L in SH-SY5Y cells under the experimental conditions. Icariin (1-10 µmol/L) prevented FA-induced cell death in SH-SY5Y cells in a dose-dependent manner, with the optimal effect observed at 5 µmol/L. After FA treatment, the absorbance in FA group was 1.31±0.05, while in the group of icariin (5 µmol/L) was 1.63±0.05. Examination of cell morphology by confocal microscopy demonstrated that 5 µmol/L icariin significantly attenuated FA-induced cell injury (P <0.05). Additionally, Icariin inhibited FA-induced cell apoptosis in SH-SY5Y cells. Results from western blotting showed that icariin suppressed FA-induced phosphorylation at Thr 181 and Ser 396 of Tau protein, while having no effect on the expression of the total Tau protein level. Furthermore, FA activated Tau kinase glycogen synthase kinase 3 beta (GSK-3β) by enhancement of Y216 phosphorylation, but icariin reduced Y216 phosphorylation and increased Ser 9 phosphorylation.

CONCLUSIONIcariin protects SH-SY5Y cells from FA-induced injury poßsibly through the inhibition of GSK-3β-mediated Tau phosphorylation.

Blotting, Western ; Cell Death ; drug effects ; Cell Line, Tumor ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; DNA Fragmentation ; drug effects ; Flavonoids ; pharmacology ; Formaldehyde ; Glycogen Synthase Kinase 3 beta ; antagonists & inhibitors ; metabolism ; Humans ; Neuroprotective Agents ; pharmacology ; Phosphorylation ; drug effects ; tau Proteins ; metabolism

OBJECTIVETo determine the effect of medicated serum of Chinese herbal compound Naofucong (, NFC) on the microglia BV-2 cells viability and the transcription and expression of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in microglia BV-2 cells to further explore the mechanisms underlying the protective effect of NFC on inflammatory process induced by high glucose.

METHODSThe microglia BV-2 cells incubated in vitro were divided into different groups: the control group (25 mmol/L glucose), the model group (75 mmol/L glucose), high glucose media containing different dose medicated serum of NFC. After being cultured for 24 h, changes in IL-6 and TNF-α were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The expression of surface marker CD11b of activated microglia was measured by confocal laser scanning microscope and Western blot. Nuclear factor-κB (NF-κB) p-p65 expression was analyzed by Western blot.

RESULTSThe model group obviously increased the expression of microglial surface marker CD11b and NF-κB p-p65 (all P<0.01), induced a signifificant up-regulation of release and the mRNA expression of IL-6 and TNF-α (P<0.01 or P<0.05). The medicated serum of NFC could obviously down-regulate the transcription and expression of surface marker CD11 b and NF-κB p-p65 (all P<0.01), and inhibit the mRNA and protein expression (P<0.01 or P<0.05) of inflflammatory cytokines, such as IL-6 and TNF-α, in microglia BV-2 cells cultured with high glucose for 24 h.

CONCLUSIONSThe inhibition of microglial activation and IL-6 and TNF-α expression induced by high glucose may at least partly explain NFC therapeutic effects on diabetes-associated cognitive decline diseases. Its underlying mechanism could probably be related to the inhibition of NFC on NF-κB phosphorylation.

Animals ; Biomarkers ; metabolism ; Blotting, Western ; CD11b Antigen ; genetics ; metabolism ; Cell Line ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Glucose ; toxicity ; Inflammation ; drug therapy ; pathology ; Interleukin-6 ; genetics ; metabolism ; Male ; Mice ; Microscopy, Confocal ; RNA, Messenger ; genetics ; metabolism ; Rats, Wistar ; Real-Time Polymerase Chain Reaction ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer.

METHODSThe inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis.

RESULTSThe abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed.

CONCLUSIONMajor ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.

A549 Cells ; Apoptosis ; drug effects ; Blotting, Western ; Butanols ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Flow Cytometry ; Ginsenosides ; chemistry ; isolation & purification ; pharmacology ; therapeutic use ; Humans ; Inhibitory Concentration 50 ; Lung Neoplasms ; drug therapy ; pathology ; Panax ; chemistry ; Plant Extracts ; pharmacology ; therapeutic use ; Staining and Labeling