OBJECTIVETo investigate the expression of immune-related genes during osteogenesis stimulated by simvastatin.

METHODSAfter treated with simvastatin, the expression of immune-related genes of mouse osteoblast was examined with gene chip (BiostarM-140s).

RESULTSThere were 16 differently expressed genes related to immune function, with nine down-regulated genes and seven up-regulated genes.

CONCLUSIONSAfter treated with simvastatin, expression of inflammation related genes is down-regulated and inflammation inhibitor genes is up-regulated in mouse osteoblasts.

Animals ; Down-Regulation ; Gene Expression Profiling ; Gene Expression Regulation ; Mice ; Osteoblasts ; drug effects ; immunology ; Osteogenesis ; drug effects ; Simvastatin ; pharmacology

OBJECTIVETo investigate the effect of hydroxycamptothecin (HCPT) on the proliferation, cell cycle and apoptosis of human lung carcinoma cell line A549.

METHODSThe growth of A549 cells exposed to HCPT was observed by staining with acridine orange/ethidium bromide dye. Agarose gel electrophoresis was performed to detect DNA fragmentation of the apoptotic cells. The cell cycle distribution of the exposed cells was analyzed using flow cytometry, and cell apoptosis was examined with annexin V-FITC/PI staining. RT-PCR was used to investigate Bcl-2 gene expression changes in the exposed cells.

RESULTSAgarose gel electrophoresis of the DNA from HCPT-treated cells showed a DNA ladder, and typical apoptotic appearance of the exposed cells was observed under fluorescence microscope. Treatment of A549 cells with 1 µmol/L HCPT for 24 h resulted in a cell apoptosis rate of 18.11%, significantly higher than the rate in control cells (0.09%, P<0.05). The treatment also caused a significant reduction of Bcl-2 mRNA expression by 70% (P<0.05).

CONCLUSIONHCPT can significantly inhibit the proliferation, induce apoptosis, and down-regulate Bcl-2 gene expression in human lung carcinoma cell line A549, suggesting the involvement of Bcl-2 gene in the inhibitory effect of HCPT on A549 cells.

Camptothecin ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, bcl-2 ; Humans ; Transfection

This study was purposed to investigate the effect of rapamycin on proliferation, apoptosis, cell cycle progression and the regulation of chemokine receptor CXCR4 on RPMI8226 cells. Different concentrations of rapamycin were used to treat the multiple myeloma cell line RPMI8226 for different times. The proliferation of the cells was detected by MTT assay; the apoptosis rate and cell cycle were determined by flow cytometry (FCM); apoptosis of cells was observed by inverted microscopy; the cylin D1, CXCR4 and mTOR mRNA expressions were detected by RT-PCR or FQ-PCR after treating RPMI8226 cells with different concentrations of rapamycin. The results indicated that the rapamycin could inhibit the proliferation of RPMI8226 cells and induce their apoptosis. The cell cycle was arrested at the G(0)/G(1) phase. PCR results showed the down-regulation of mTOR, cyclin D1 and mTOR mRNA expressions after treating RPMI8226 cells with different concentrations of rapamycin for 24 hours. It is concluded that the rapamycin significantly inhibits the growth of RPMI8226 cells in a dose-and time-dependent mannes and induce cell apoptosis. Cell cycle arrests at the G(0)/G(1) phase, may be due to the down-regulation of the mTOR and cyclin D1 expressions. In additions, the down-regulation of CXCR4 mRNA expression is correlated with the reduction of adhesion between myeloma cells and stromal cells.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Receptors, CXCR4 ; metabolism ; Sirolimus ; pharmacology

Both tetrandrine (Tet) and 5-bromotetrandrine (BrTet) can effectively reverse P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). The structure of multidrug resistance associated protein 7 (MRP7) has its own specificity and difference compared with other members of the MRP family. This study was aimed to investigate whether Tet and BrTet can inhibit the expression level of MRP7 so as to further look into the mechanisms of the reversal effects of Tet and BrTet on MDR. The inhibitory effects of daunorubicin (DNR) used alone on the proliferation of K562 and K562/A02 cells were evaluated by MTT assay, the IC(50) of DNR and drug resistant folds were calculated. The mRNA level of MRP7 was tested by real-time PCR, and the protein levels of MRP7 and P-gp were tested by Western blot. The DNR accumulation was analyzed by flow cytometry (FCM). The results showed that the resistance of K562/A02 cells to DNR was 23.65-folds of that of K562 cells. After administration of 1 µmol/L Tet or 2 µmol/L BrTet, the mRNA level of MRP7 in the K562/A02 cells decreased to 2% and 12% respectively, and the protein level of MRP7 decreased by 53.2% and 83.7% respectively. The protein level of P-gp decreased by 58.47% and 52.20% in the 1 µmol/L Tet and 2 µmol/L BrTet groups. FCM detection showed that 1 µmol/L Tet and 2 µmol/L BrTet significantly increased the accumulation of DNR in K562/A02 cells by 94.32% and 271% respectively. It is concluded that Tet and BrTet both can reverse MDR in vitro. The mechanisms may be related to the inhibition of MRP7 overexpression and the increase of anticancer drug concentration in cells. At the same molar concentration, the effects of Tet and BrTet in inhibiting the protein level of MRP7 expression do not show significant difference.
Benzylisoquinolines ; pharmacology ; Down-Regulation ; drug effects ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; K562 Cells ; Multidrug Resistance-Associated Proteins ; metabolism

To explore the effect of chronic high dose of insulin on lipolysis in porcine adipocytes and the underlying molecular regulation mechanisms, we cultured primary porcine adipocytes and incubated them with different concentrations of insulin (0, 200, 400, 800, 1600 nmol/L) for 24-96 h in the absence or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the glycerol release into the culture media as an indicator of the lipolysis, and observed the lipid accumulation morphology by phase-contrast microscopy. Further, we analyzed the gene expressions of perilipin A and peroxisome proliferator-activated receptor-gamma 2 (PPAR gamma 2) with semi-quantitative RT-PCR and Western blotting, respectively. The results showed that chronic high dose of insulin stimulated lipolysis in differentiated porcine adipocytes in a dose- and time-dependent manner, and significantly attenuated the lipolytic response to isoprenaline. Meanwhile, the protein and mRNA expressions of PPAR gamma 2 and perilipin A were significantly reduced. In addition, both PKA and ERK inhibitors significantly suppressed insulin-stimulated lipolysis, however, only ERK inhibitor reversed the insulin-induced down-regulation of perilipin A. These findings imply that chronic high dose of insulin stimulates lipolysis in porcine adipocytes by repressing perilipin A, which is involved in ERK pathway.
Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Carrier Proteins ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Insulin ; pharmacology ; Lipolysis ; drug effects ; Perilipin-1 ; Phosphoproteins ; metabolism ; Swine

Lead exposure is a known potential risk factor for neurodegenerative diseases such as Alzheimer's disease (AD). Exposure to lead during the critical phase of brain development has been linked with mental retardation and hypophrenia in later life. This study was aimed to investigate the effects of lead exposure of pregnant mice on the expressions of insulin-degrading enzyme (IDE) and nerve growth factor (NGF) in the hippocampus of their offspring. Blood samples were collected from the tail vein, and after anesthetizing the pups, the brain was excised on postnatal day 21. Lead concentrations were determined by graphite furnace atomic absorption spectrophotometry, and the expressions of IDE and NGF were determined by immunohistochemistry and Western blotting. Results showed that the reduction in IDE and NGF expression in the hippocampus of pups might be associated with impairment of learning and memory and dementia induced by maternal lead exposure during pregnancy and lactation.
Animals ; Down-Regulation ; Female ; Gene Expression Regulation, Developmental ; drug effects ; Gene Expression Regulation, Enzymologic ; drug effects ; Hippocampus ; drug effects ; growth & development ; metabolism ; Insulysin ; genetics ; metabolism ; Lead ; toxicity ; Mice ; Pregnancy ; Prenatal Exposure Delayed Effects ; chemically induced

OBJECTIVETo investigate the role of protein kinase C (PKC) in the down-regulation of fibroblast proliferation in normal skin (NFb) and hyperplastic scar (SFb) by adrenaline.

METHODSHuman NFb and SFb cells were cultured in vitro. Phentolamine (in final concentrations of 0 and 3 x 10(-6) micromol/L) was added to the culture medium. One hour later, adrenaline in different final concentrations (0.00, 0.05, 0.10, 0.20 micromol/L) was added to the culture medium and incubated for 24 hours. The cellular proliferation activity and cell viability rate were determined with MTT. The cell culture supernatant was harvested for the determination of LDH activity to assess the toxicity of phentolamine and adrenaline. The phosph-PKC activity was determined with Western-blotting and was semiquantitatively analyzed.

RESULTS(1) After stimulation with adrenaline alone, or combined 0.20 micromol/L adrenaline with 3 x 10(-6) micromol/L phentolamine, the cell viability of both NFb and SFb decreased significantly (P < 0.05 or 0.01). (2) There was no difference in the LDH activity between the cells either stimulated by adrenaline in all concentrations or by combination of adrenaline and phentolamine (P > 0.05). (3) The phosphorylation of PKC in NFb and SFb cells stimulated by 0.05, 0.10, 0.20 micromol/L adrenaline was obviously higher than that before stimulation (P < 0.01). When phentolamine in the concentration of 3 x 10(-6) micromol/L was used alone for stimulation, the phosphorylation of PKC in NFb cells (123 +/- 5) was also evidently higher than that before stimulation (80 +/- 5, P < 0.01). But there was no such effect on SFb cells (P > 0.05). When adrenaline in the concentration of 0.05, 0.10 or 0.20 micromol/L was separately added together with phentolamine in the dose of 3 x 10(6) micromol/L for the stimulation, the phosphorylation of PKC in NFb and SFb cells was evidently lower than that when 3 different concentrations of adrenaline was used alone for stimulation (P < 0.01).

CONCLUSIONAdrenaline can inhibit the proliferation of NFb and SFb by activating PKC through binding alpha adrenaline receptor.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Down-Regulation ; Epinephrine ; adverse effects ; Fibroblasts ; cytology ; Humans ; Phentolamine ; adverse effects ; Phosphorylation ; Protein Kinase C ; metabolism ; Skin ; drug effects

OBJECTIVETo investigate the effect of down-expression of inhibitor of differentiation-1 (Id-1) on the differentiation of dendritic cell sarcoma (DCS) cells in vitro.

METHODSDown-regulation of the expression of Id-1 in DCS cells was performed by RNAi, and confirmed by protein and mRNA quantitative analyses. Cellular differentiation and biological behavior including malignant phenotypes of the cells were evaluated. All experiments included negative (no treatment group and no-target siRNA) and positive (induction-differentiation drug sodium butyrate) controls.

RESULTSWhen the expression of Id-1 was down regulated, the DCS cells showed more mature morphology including cell enlargement, longer cellular extensions, more branches, and decreased nuclear/plasma ratio. Differentiation marker expression (Id-2 and CD86) was also increased. RNAi treated cells at 24 and 48 hours, showed increase percentage of cells at G0/G1 phase and less cells at S phase (P < 0.01). Importantly, the abilities of cell proliferation, colony formation and invasiveness were significantly decreased (P < 0.01), as evidenced by MTT, colony formation and transwell assays respectively.

CONCLUSIONRNAi inhibition of Id-1 protein can induce differentiation of malignant solid tumor cells along with reversion of their malignant phenotype.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Dendritic Cells ; cytology ; drug effects ; Down-Regulation ; Inhibitor of Differentiation Proteins ; pharmacology ; Mice ; Tumor Cells, Cultured

The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 mumol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G(0)/G(1) phase and decrease in S phase. After treatment with 0, 20, 60, 100 mumol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00+/-1.25)% to (58.84+/-0.32)% in G(0)/G(1) phase, whereas it was decreased from (61.45+/-1.04)% to (35.82+/-1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G(0)/G(1) phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.
Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/*drug effects ; Cell Proliferation/*drug effects ; Cyclin D3/metabolism ; Down-Regulation/drug effects ; Jurkat Cells ; Triterpenes/*pharmacology ; bcl-X Protein/metabolism

OBJECTIVETo observe the in vitro effect of compound 861 (Cpd 861) on tissue inhibitor of metalloprotenase 1 (TIMP1) mRNA levels of HSC-T6 cell.

METHODSHSC-T6 cells were exposed in different concentrations of Cpd 861 (0.25~1.0 mg/ml) for 48 hours. The TIMP1 level was measured by the quantitative reverse-transcription polymerase chain reaction (RT-PCR).

RESULTSThe TIMP1 mRNA levels of HSC-T6 cells at different concentrations of Cpd 861 were lower (2.50 0.71, 0.50 0.01, 0.11 0.03) than those of the normal control (3.78 0.67, P<0.05 or P<0.01).

CONCLUSIONSThe antifibrotic mechanism of Cpd 861 is partly due to its downregulation on TIMP1 mRNA levels of HSC-T6 cells.

Animals ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; RNA, Messenger ; biosynthesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics