Effects of oridonin nanosuspension on cell proliferation and apoptosis of human prostatic carcinoma PC-3 cell line

This study aims to investigate the inhibitory effects of oridonin nanosuspension on human prostatic carcinoma PC-3 cell line in vitro. The PC-3 cells were incubated with increasing concentrations of oridonin solution and nanosuspensions for 12 hours, 24 hours, and 36 hours. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to measure cellular viability and investigate the effect of oridonin on cell growth of PC-3. Annexin V-FITC/PI staining method was used to determine the effect of oridonin by fluorescence microscope and flow cytometry, respectively. Nanosuspension on early apoptosis of PC-3 cells was also evaluated. Oridonin significantly inhibited the growth of PC-3 cells after 12 hours, 24 hours, and 36 hours of treatment in a dose-dependent manner (P < 0.05). Compared with the same concentration of oridonin solution, oridonin nanosuspension enhanced the inhibition ratio of proliferation. The observation of propidium iodide fluorescence staining confirmed the MTT assay results. The cell proportion of PC-3 at the G2/M phase in the nanosuspension treatment group was upregulated compared with that of the control and oridonin solution groups. Both oridonin solution and nanosuspension promoted the early apoptosis of PC-3 cells. Furthermore, while improving the ratio of early apoptosis, oridonin nanosuspensions also enhanced growth suppression, and induced apoptosis of PC-3 cells. This shows great potential in the treatment of androgen-independent carcinoma of prostate by oridonin nanosuspensions

Comparative Analyses by Sequencing of Transcriptomes during Skeletal Muscle Development between Pig Breeds Differing in Muscle Growth Rate and Fatness

Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illumina's Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement

Knockdown of glutamate cysteine ligase catalytic subunit by siRNA.

<p>(A) GCLC mRNA levels in A549 cells following 24 hours transfected with negative control siRNA, GCLC siRNA-1, GCLC siRNA-2 and GCLC siRNA-3. GCLC siRNA significantly decreased the GCLC mRNA levels in A549 cells. (B) Representative Western blot gel documents for GCLC and summarized data showing that efficiency of gene silencing of GCLC by siRNA. Cytosolic proteins were isolated from transfected cells. GCLC protein levels in cell extracts were measured by Western blot analysis and were normalised to β-actin expression levels. *** P<0.001, compared to negative control.</p

Effect of GNPs on intracellular ROS levels in GCLC siRNA pretreated cells.

<p>Exponentially growing cells were transfected with negative control and GCLC siRNA-1 for 24 hours, following treated with GNPs (20μM), GNPs (20μM) and GSH (1mM), GNPs (20μM) and NAC (1mM). ROS levels were measured. Graphs indicate ROS (as determined by DCF) levels (%) compared with negative control cells. Each bar represents the mean (±SD n = 4) of triplicate determinations. **p<0.01</p

Knockdown of Glutamate Cysteine Ligase Catalytic Subunit by siRNA Causes the Gold Nanoparticles-Induced Cytotoxicity in Lung Cancer Cells

<div><p>Gold nanoparticles (GNPs) have shown promising medical applications in cancer treatment involved in the regulation of intracellular redox balance. Previously, we have reported that GNPs can trigger apoptosis and necrosis in human lung cancer cells (A549) when L-buthionine-sulfoximine (BSO) was used to decrease the expression of intracellular glutathione (GSH). Herein, we investigated the cytotoxicity of GNPs toward lung cancer cells under the glutamate cysteine ligase catalytic subunit (GCLC) was silenced by siRNA. Our results showed that GNPs cause apoptosis and necrosis in cells transfected with GCLC siRNA by elevating intracellular reactive oxygen species (ROS). These findings demonstrated that the regulation of glutathione synthesis by GCLC siRNA in A549 cells can initiate the gold nanoparticles-induced cytotoxicity.</p></div

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis

145-151Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo — the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, β-1,6-GlcNAc-branching, ⍺-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(β-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis

Flow cytometry analysis of mitochondrial membrane potential in GNPs-treated cells.

<p>After transfected with negative control siRNA or GCLC siRNA-1, cells were treated with GNPs (20μM) for additional 72 hours then collected and stained with JC-1 in darkness at 37°C, rinsed by PBS. The fluorescence shift (red to green) of samples was measured by flow cytometry. Each bar represents the mean (±SD n = 4) of triplicate determinations. *<i>p</i><0.05, compared with negative control group.</p

GNPs induce caspase activation in cells transfected with GCLC siRNA.

<p>Activation of caspase-3 was measured using specific antibodies by flow cytometry. Intracellular GSH was depleted by GCLC siRNA-1, after approximately 72 hours of GNPs treatment, the cells were collected, treated with 0.1% Triton X-100 and blocked with 1% BSA, then incubated with cleaved caspase-3 (Asp175) antibody (Alexa fluor 488 conjugate) for 30 minutes. The fluorescence intensity was measured by flow cytometry. Each bar represents the mean (±SD n = 4) of triplicate determinations. *<i>p</i><0.05, compared with negative control group.</p

Biological study of the angiogenesis inhibitor n-(8-(3-ethynylphenoxy)octyl-1-deoxynojirimycin

The alpha-glucosidase inhibitors N-methyl-1-deoxynojirimycin (MDNJ) and castanospermine have been shown to inhibit angiogenesis. A hybrid of 1-deoxynojirimycin (DNJ) and an aryl-1,2,3-triazole, which inhibits both an alpha-glucosidase and methionine aminopeptidase-2 (MetAP2), displayed properties associated with inhibition of angiogenesis (Bioorg. Med. Chem., 16, 2008, 6333-7). The biological evaluation of a structural analogue N-(8-(3-ethynylphenoxy)octyl-1-deoxynojirimycin is described herein. Although this alkyne derivative did not inhibit MetAP2, it inhibited a bacterial alpha-glucosidase, altered bovine aortic endothelial cell (BAEC) surface oligosaccharide expression and inhibited BAEC proliferation by inducing G1 phase cell cycle arrest. Experiments showed G1 arrest was attributable to the alpha-glucosidase inhibitor inducing an increase in p27Kip1 expression and high phosphorylation of ERK1/2 without a reduction in cyclin D1. The DNJ derivative (0.1 mm) prevented capillary tube formation from bovine aortic endothelial cells, whereas DNJ or other analogues were unable to inhibit tube formation at the same concentration. Stress fiber assembly in bovine aortic endothelial cells was abolished, and BAEC migration was inhibited indicating the inhibition of tube formation by this derivative is partially a result of a reduction in cell motility. The agent also caused a reduction in secretion of MMP-2 from bovine aortic endothelial cells. Therefore, the new alpha-glucosidase inhibitor has a different mechanism by which it inhibits angiogenesis in vitro when compared with deoxynojirimycin, the deoxynojirimycin -triazole hybrid, N-methyl-1-deoxynojirimycin and castanospermine

GNPs induce apoptosis and necrosis in cells transfected with GCLC siRNA.

<p>Cells were transfected with either non-target control siRNA or GCLC-specific siRNA-1. One day later, cells were treated with GNPs (20μM), GNPs (20μM)﹢GSH (1mM) and GNPs (20μM)+NAC(1mM) for additional 72 hours. AnnexinV-FITC and PI cells were measured with flow cytometry. (A) The fluorescence pattern of AnnexinV-FITC and PI-stained A549 cells after treatment. (B) Percentages of Annexin V positive or PI positive cells for different treatments. Each bar represents the mean (±SD n = 3). **p<0.01, ***P<0.001, versus control.</p