To investigate the effect of jianpi-jiedu (JPJD) prescription-contained serum on colorectal cancer SW48 cell proliferation and the underlying mechanisms.
 Methods: Crude extract from JPJD was made by water extract method and the main components of crude extract from JPJD were analyzed by ultra-performance liquid phase high resolution time of flight mass spectrometry (UPLC-Q-TOF/MS). The low, medium, and high-concentration of JPJD-contained serum were prepared by the serum pharmacological method. The effect of serum containing JPJD on SW48 cell proliferation was determined by MTT assay. The cell cycle was detected by flow cytometric method. The protein levels of mammalian target of rapamycin (mTOR), phospho-mTOR, P-P53, and -P21, and the mRNA level of mTOR were examined by Western blot and RT-PCR, respectively.
 Results: Seven compounds including calycosin-7-glucoside, astragaloside, ginsenoside-Re, ginsenoside-Rb1, glycyrrhizinic acid, apigenin, atractylenolide-II were identified. MTT assays demonstrated that the SW48 cell proliferation was inhibited by medium and high concentration of JPJD-contained serum and the percentages of cells at G1 phase in SW48 cell cultured in the medium and high concentration of JPJD serum group were significantly higher than those in the control group (P<0.05). Meanwhile, the levels of mTOR mRNA and phospho-mTOR protein in the medium and high concentration of JPJD serum groups were substantially lower than those in the control group (P<0.05). Conversely, the expressions of phospho-P53 and P21 protein were significantly increased in the medium and high concentration of JPJD serum group compared with those in the control group.
 Conclusion: JPJD prescription-contained serum can inhibit SW48 cell proliferation, which may be related to mTOR-P53-P21 signaling pathways.
Animals ; Apigenin ; Blotting, Western ; Cell Cycle ; Cell Division ; Cell Proliferation ; drug effects ; genetics ; Colorectal Neoplasms ; Cyclin-Dependent Kinase Inhibitor p21 ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Ginsenosides ; Glycyrrhizic Acid ; Humans ; Lactones ; Phosphorylation ; genetics ; RNA, Messenger ; Saponins ; Sesquiterpenes ; Signal Transduction ; TOR Serine-Threonine Kinases ; drug effects ; Triterpenes ; Tumor Suppressor Protein p53 ; drug effects

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.
Adenine ; administration & dosage ; analogs & derivatives ; Apoptosis ; drug effects ; Autophagy ; drug effects ; genetics ; CDC2 Protein Kinase ; Cell Cycle ; drug effects ; genetics ; Cell Division ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin B ; biosynthesis ; Cyclin-Dependent Kinases ; Gene Expression Regulation ; drug effects ; Humans ; Membrane Proteins ; biosynthesis ; Microtubule-Associated Proteins ; biosynthesis ; Mitochondrial Dynamics ; drug effects ; genetics ; Mitochondrial Proteins ; biosynthesis ; Neuroblastoma ; Signal Transduction ; drug effects

Aminolevulinic acid (ALA) is biosynthesized by the enzyme ALA synthase (ALAS). The ALA production has been studied using the overproducing ALAS from several bacteria in Escherchia coil, respectively. However, ALAS from eucaryote expressed in E. coli for producing ALA in the culture is not known. The extracellular ALA production and cell growth were investageted respectively using the recombinant E. coli overproducing Saccharomyces cerevisiae ALAS in shake-flask fermentations. The ALAS activity from the cell extract was assayed. The extracellular ALA was purified by the national-made large-dimension resins and confirmed by the capillary electrophoresis measurements. At 12h after induction at 37 degrees C, the extracellular ALA production was up to 162mg per liter LB culture at initial pH 6.5 with exogenous levulinate, succinate and and glycine at the concentration of 20 mmol/L respectively. The purity of ALA after purification is up to 90%.
5-Aminolevulinate Synthetase ; genetics ; metabolism ; Aminolevulinic Acid ; isolation & purification ; metabolism ; Cell Division ; drug effects ; Dose-Response Relationship, Drug ; Electrophoresis, Capillary ; Escherichia coli ; genetics ; growth & development ; metabolism ; Extracellular Space ; drug effects ; metabolism ; Glycine ; pharmacology ; Hydrogen-Ion Concentration ; Levulinic Acids ; pharmacology ; Recombinant Proteins ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Succinic Acid ; pharmacology

OBJECTIVETo investigate the effects of mutant exogenous P27(kip1) gene on chemosensitivity of human cholangiocarcinoma cell line.

METHODSThe recombinant vector was constructed and the mutant P27(kip1) gene was transfected into human cholangiocarcinoma cell line (QBC(939)). RT-PCR and Western blot were used to determine the expression of target genes. The effects of 5-fluorouracil (5-FU), mitomycin C (MMC) and cyclophosphamide (CTX) on the transfected cells were detected by assaying the apoptotic rate and growth inhibition by methabenzthiazuron (MTT) assay and flow cytometry (FCM).

RESULTSThe mutant exogenous P27(kip1) gene was expressed effectively in the cells, and the expression enhanced the apoptosis and growth inhibition of QBC(939) inducted by 5-FU, MMC and CTX. The ratio of growth inhibiting increased significantly from 41.89% (5-FU), 45.59% (MMC), 38.91% (CTX) to 56.15% (5-FU), 55.65% (MMC), 51.69% (CTX), and apoptosis index from 13.76% +/- 3.03% (5-FU), 11.76% +/- 3.99% (MMC), 10.46% +/- 2.10% (CTX) to 41.39% +/- 4.32% (5-FU), 35.94% +/- 2.71% (MMC), 34.46% +/- 2.32% (CTX) (P < 0.05).

CONCLUSIONSThe exogenous P27(kip1) gene transfer can remarkably increase the drug sensibility of the cholangiocarcinoma cells. The strategy targeted to control the cell cycle may be more effective in cancer treatment by combination of P27(kip1) gene therapy.

Adenoviridae ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bile Duct Neoplasms ; pathology ; Bile Ducts, Intrahepatic ; Cell Division ; drug effects ; Cell Line, Tumor ; Cholangiocarcinoma ; pathology ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; pharmacology ; Drug Synergism ; Genetic Vectors ; Humans ; Transfection

OBJECTIVETo observe the effect of Chinese herbs for supplementing Shen and strengthening bone (HB) on myelogenic osteoclasts formation, and gene expression of interleukin-6 (IL-6), IL-6 receptor (IL-6R) and gp130 in bone marrow.

METHODSSeventy-two healthy female SD rats of 3 months, were randomly divided into three groups, 24 in the sham-operated group (A), 24 in the ovariectomized group (B) and 24 in the after ovariectomy HB treated group (C). Bone marrow cells of 6 rats from each group were respectively collected and cultured at four time points (2nd, 4th, 6th and 12th weeks after operation). After 6 days of culture, the bone marrow cells were differentiated by Wright-Giemsa stain and TRAP stain, and total RNA in them was extracted by TRIZOL.

RESULTSBeginning from the 2nd week, the osteoclasts formation in Group B was higher than that in Group A (P < 0.05), and IL-6, IL-6R gene expression significantly increased in Group B (P < 0.05 or P < 0.01). These changes reached the peak in the 4th to 6th week, with the level maintained to the 12th week. As for comparison of Group B and C, the above-mentioned changes were significantly weakened in the latter (P < 0.05 or P < 0.01). No significant change of gp130 gene expression revealed in the whole course in either group.

CONCLUSIONHB could inhibit the myelogenic osteoclasts formation in ovariectomized rats, this effect may be correlated with, partially at least, its inhibitory effect on the over-expressed IL-6 and IL-6R gene expression in myelocytes after ovariectomy.

Animals ; Antigens, CD ; biosynthesis ; genetics ; Bone Marrow ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Cytokine Receptor gp130 ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Granulocyte Precursor Cells ; metabolism ; Interleukin-6 ; biosynthesis ; genetics ; Isoflavones ; pharmacology ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; pathology ; Osteoporosis ; metabolism ; pathology ; Ovariectomy ; RNA ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin-6 ; biosynthesis ; genetics

AIMTo investigate the effect of antisense oligonucleotides (ASON) of ryanodine receptor on proliferation and [Ca2+]i concentration of airway smooth muscle cells (ASMCs).

METHODSASMCs were cultivated with collagen enzyme digestion method. Different concentrations of ASON were added to the cultures with Lipofectamine 2000 to observe the ASMCs proliferation using MTS/PES method. The changes of ASMCs [Ca2+]i were also observed by flow cytometry. The expression of mRNA of subtypes of RyR was assayed by RT-PCR.

RESULTSRyR ASON restrained the proliferation of ASMCs, decreased the expression of RyR and reduced the concentration of [Ca2+]i.

CONCLUSIONRyR ASON could inhibit the proliferation of ASMCs by influencing the concentration of [Ca2+]i after excited.

Animals ; Calcium ; metabolism ; Calcium Channels ; Cell Division ; Cell Proliferation ; drug effects ; Cells, Cultured ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; Rats ; Respiratory System ; Ryanodine Receptor Calcium Release Channel ; genetics ; pharmacology

OBJECTIVETo study the relationship between the expression of dipeptidyl peptidase IV (DPP IV) gene and malignant behavior of cells of ovarian carcinoma.

METHODSThe differences of the malignant behavior of A2780, SKOV-3, HO-8910 and HO-8910PM cell lines were examined by drawing cell proliferative curves, adhesive test, assay of incursion and chemotaxis. The expression of DPP IV among the cell lines and its relationship with the malignant behavior of ovarian carcinoma cell were detected by techniques of DPP IV activity assay, cytometry and reverse transcription polymerase chain reaction.

RESULTSAmong all cell lines, the ability of proliferation, adhesion, incursion and chemotaxis of HO-8910PM were the highest, while those of A2780 were the lowest. The transcription of mRNA in A2780, SKOV-3, HO-8910 and HO-8910PM cell lines were 0.7512 +/- 0.0012, 0.5596 +/- 0.0015, 0.3369 +/- 0.0009, and 0.2777 +/- 0.0006, respectively. The activity of DPP IV were 0.79 +/- 0.02, 0.64 +/- 0.03, 0.21 +/- 0.02, and 0.18 +/- 0.01, respectively; and the protein expression of DPP IV gene were 657.83 +/- 1.14, 538.53 +/- 5.29, 130.50 +/- 1.46, and 33.14 +/- 0.47, respectively, as assayed by cytometry. The correlation coefficients of the transcription of DPP IV gene and the adhesive, incursive and migratory ability of ovarian carcinoma cells were -0.987, -0.983, and -0.991, respectively; the correlation coefficients of the expression of DPP IV and those ability of cells were -0.959, -0.988, and -0.968; the correlation coefficients of the activity of DPP IV and those ability of cells were -0.952, -0.868, and -0.983.

CONCLUSIONThere is a negative correlation between the expression of DPP IV gene and the adhesive and incursive capability of cells of ovarian carcinoma.

Cell Adhesion ; Cell Division ; drug effects ; Cell Line, Tumor ; Cystadenocarcinoma ; genetics ; pathology ; Dipeptidyl Peptidase 4 ; biosynthesis ; genetics ; Female ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Ovarian Neoplasms ; genetics ; pathology ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo study the effect of tumor suppressor gene PTEN on proliferation and cell cycle of hepatocellular carcinoma cell line HHCC.

METHODSFirstly, eukaryotic expression vectors of wild type and mutated type of PTEN gene were constructed, named as pEGFP-WT-PTEN and pEGFP-PTEN; G129R, respectively. Lipofectamine 2000 was used to transfect the constructed expression vectors into hepatocellular carcinoma cell line HHCC which was PTEN protein negative. G418 was used to select the cell clones constantly expressing PTEN protein. Flow cytometry was used to assay the cell cycle of HHCC transfected by above mentioned eukaryotic expression vectors and non-transfected cell line HHCC. Intrinsic 473-phosphorylated AKT representing the level of active AKT was assayed by Western blot. The non-transfected HHCC served as control.

RESULTSThe proliferation of HHCC constantly expressing PTEN protein was obviously inhibited compared with HHCC cells transfected with mutated PTEN gene and empty vectors, and non-transfected HHCC cells. The number of HHCC cells transfected with wild type PTEN gene at G(1) phase, G(2) phase and S phase was 70.8%, 6.8% and 22.4%, respectively. Compared with control group transfected with empty vector, the number of G(1) phase HHCC cells constantly expressing wild type-PTEN protein was significantly higher than that of control. The number of cells in G(2) and S phase was significantly lower than that of control. However, the number of cells in G(1) phase, G(2) phase and S phase of HHCC transfected with mutant PTEN was 63.2%, 10.1% and 26.7%, respectively. There was no significant difference compared with control group. Western blot result showed that the intrinsic level of 473-phosphorylated AKT of HHCC constantly expressing wild type PTEN protein was down-regulated, and that of HHCC transfected with mutated PTEN gene was equal to that of control.

CONCLUSIONWild type PTEN gene can inhibit the proliferation of hepatocellular carcinoma cells at G(1) phase. The mechanism is possibly related with intrinsic activity of AKT, which is down-regulated by wild type PTEN.

Carcinoma, Hepatocellular ; pathology ; Cell Division ; drug effects ; Cell Line, Tumor ; Genes, Tumor Suppressor ; Humans ; Liver Neoplasms ; pathology ; PTEN Phosphohydrolase ; genetics ; pharmacology

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.
Acetates ; pharmacology ; Carbon ; pharmacology ; Cell Division ; drug effects ; Culture Media ; chemistry ; pharmacology ; DNA ; biosynthesis ; genetics ; Escherichia coli ; cytology ; drug effects ; genetics ; Genetic Therapy ; instrumentation ; Genetic Vectors ; biosynthesis ; genetics ; Humans ; Nitrogen ; pharmacology ; Phosphates ; pharmacology ; Plasmids ; biosynthesis ; genetics

AIMTo determine the effect of saposin C (a known trophic domain of prosaposin) on proliferation, migration and invasion, as well as its effect on the expression of urokinase plasmonogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMP)-2 and -9 in normal and malignant prostate cells. In addition, we tested whether saposin C can activate p42/44 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signal transduction pathways of the mitogen-activated protein kinase (MAPK) superfamily.

METHODSWe employed Western blot analysis, phospho-specific antibodies, cell proliferation assay, reverse transcriptase-polymerase chain reaction, in vitro kinase assays and migration and invasion to determine the effect of saposin C on various biological behaviors of prostate stromal and cancer cells.

RESULTSSaposin C, in a cell type-specific manner, upregulates uPA/uPAR and immediate early gene c-Jun expression, stimulates cell proliferation, migration and invasion and activates p42/44 and SAPK/JNK MAPK pathways in prostate stromal and cancer cells. Normal prostate epithelial cells were not responsive to saposin C treatment in the above studies.

CONCLUSIONSaposin C functions as a multipotential modulator of diverse biological activities in prostate cancer and stromal cells. These results strongly suggest that saposin C functions as a potent growth factor for prostatic cells and may contribute to prostate carcinogenesis and/or the development of hormone-refractory prostate cancer.

Cell Division ; drug effects ; Enzyme Activation ; Humans ; Male ; Mitogen-Activated Protein Kinases ; metabolism ; Neoplasm Invasiveness ; Prostatic Neoplasms ; enzymology ; metabolism ; pathology ; Receptors, Cell Surface ; genetics ; Receptors, Urokinase Plasminogen Activator ; Reverse Transcriptase Polymerase Chain Reaction ; Saposins ; pharmacology ; Signal Transduction ; Stromal Cells ; enzymology ; metabolism ; pathology ; Up-Regulation ; Urokinase-Type Plasminogen Activator ; genetics