The polysaccharides from pumpkin fruit (PP) were obtained and purified by hot-water extraction, anion-exchange chromatography, and gel column chromatography. The physicochemical properties of PP were determined by gel filtration chromatography, gas chromatography, fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Results indicated that the molecular weight of PP was about 23 kDa and PP was composed of D-Arabinose, D-Mannose, D-Glucose, and D-Galactose with a molar ratio of 1 : 7.79 : 70.32 : 7.05. FTIR and NMR spectra indicated that PP was the polysaccharide containing pyranose ring. Additionally, PP protected islets cells from streptozotocin (STZ) injury in vitro via increasing the levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) and reducing the production of NO. The experiment of reverse transcriptase-polymerase chain reaction further proved that PP inhibited apoptosis via modulating the expression of Bax/Bcl-2 in STZ-damaged islet cells. In conclusion, PP could be explored as a novel agent for the treatment of diabetes mellitus.
Animals ; Apoptosis ; drug effects ; Chromatography, Gas ; Chromatography, Gel ; Cucurbita ; chemistry ; Diabetes Mellitus, Experimental ; drug therapy ; Islets of Langerhans ; drug effects ; injuries ; Magnetic Resonance Spectroscopy ; Malondialdehyde ; analysis ; Molecular Weight ; Monosaccharides ; analysis ; Nitric Oxide ; biosynthesis ; Polysaccharides ; chemistry ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spectroscopy, Fourier Transform Infrared ; Superoxide Dismutase ; drug effects ; bcl-2-Associated X Protein ; drug effects

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics

To study the function and potential application of nkx2.5, a critical gene for heart development, we constructed a recombinant adenovirus overexpressing nkx2.5 gene (Ad-Nkx2.5) with the AdEasy system. To evaluate the effect and mechanism of Ad-Nkx2.5 against oxidative injury, the H9c2 myocardial cells were infected with the recombinant adenoviruses Ad-Nkx2.5 or Ad-EGFP, and subsequently exposed to H2O2 to induce apoptosis. The anti-apoptotic potential of Ad-Nkx2.5 was validated by MTT assay for cell viability, Hoechst33342 staining for cellular morphology, and immunoblotting for caspase-3 activity. Ad-Nkx2.5 infection led to an increased survival rate of H9c2 cells and decreased the amount of caspase-3 in an active form. Additionally, overexpression of Nkx2.5 inhibited the release of cytochrome C from the mitochondria into the cytosol. Mechanismic studies showed that Nkx2.5 upregulated bcl-2 gene expression and significantly repressed H2O2-induced expression of bax detected by Real-time PCR. Additionally, H2O2 treatment did not affect the nuclear localization of Nkx2.5. These findings indicate that adenovirus-mediated nkx2.5 gene transfer exerted a protective effect on H9c2 cells against H2O2-induced apoptosis via mitochondrial pathway, and the Nkx2.5-mediated expression modulation of apoptosis-associated genes could be involved in this event.
Adenoviridae ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Genetic Vectors ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; biosynthesis ; genetics ; Hydrogen Peroxide ; pharmacology ; Myocytes, Cardiac ; cytology ; Oxidative Stress ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

Bcel-2 family proteins (Bcl-x(L), Bcl-2, Mel-1 etc.) are key regulators of some life processes, including apoptosis and autophagy. They are currently considered as promising targets for developing new anti-tumor therapies. In our study, the human Bcl-2/Bcl-x(L) chimeric gene and the human/mouse Mel-1 chimeric gene were designed and cloned, and the prokaryotic expression vectors for expressing glutathione S-transferase (GST) fusion proteins and histidine tag fusion proteins were constructed respectively. These two proteins as well as the GST-Bcl-x(L) fusion protein were all successfully expressed in E. coli and subsequently purified. In addition, we measured the binding of these Bcl-2 family proteins to the Bid BH3 peptide by fluorescence polarization-based assay. The dissociation constants (Kd) obtained by us were in general agreement with the data reported in literature. The Kd values of all three proteins with or without the GST tag were almost identical. All these results validate the biological functions of these Bcl-2 family proteins obtained by us. These proteins can be used in the experimental screening of small-molecule regulators of Bcl-2 family proteins in vitro.
Escherichia coli ; genetics ; metabolism ; Fluorescence Polarization ; methods ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; bcl-X Protein ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo study the effect of pGCsi-H1-APRIL on the growth of human colorectal cancer cells in transplated tumor in nude mice and to improve the effect of APRIL on proliferation and apoptosis of colorectal cancer (CRC).

METHODSHuman CRC model was established in nude mice, and the nude mice were treated with APRIL siRNA twice per week for 2 weeks. APRIL mRNA expression was surveyed by PCR and APRIL protein expression was detected by immunohistochemistry. The expression of PCNA protein was detected by ELISA. The expression of bcl-2 and bcl-xl was assessed by immunohistochemical staining, and TUNEL staining was used to detect apoptosis.

RESULTSThe expression of APRIL mRNA in the APRIL siRNA group was (0.13 ± 0.05) × 10(-3), significantly lower than that in the vector group (0.95 ± 0.04) × 10(-3) and the PBS group (0.96 ± 0.05) × 10(-3). The expression of APRIL protein in the APRIL siRNA group was (87.5 ± 5.0)% lower than that in the vector and PBS groups (P < 0.05). APRIL siRNA significantly suppressed the growth of SW480 tumor: the IR (inhibitory rate) of APRIL siRNA group was (60.7 ± 1.5)% (P < 0.05). The expression of PCNA in APRIL siRNA group was (176.8 ± 18.1) ng/ml, was (56.5 ± 2.0)% lower than that of PBS group (328.4 ± 22.8) ng/ml. Furthermore, the expressions of anti-apoptosis proteins bcl-2 and bcl-xl of APRIL siRNA group were (82.6 ± 4.5)% and (79.2 ± 3.5)% lower than those of the PBS group. The apoptotic rate of the APRIL siRNA group was 40.1% ± 2.5%, significantly higher than that in the vector group (2.5 ± 0.1)% and PBS group (2.5 ± 0.2)% (P < 0.05).

CONCLUSIONAPRIL siRNA may significantly suppress the growth and promote apoptosis in transplanted tumor of human colorectal cancer in nude mice. APRIL may become a candidate gene of gene therapy of human colorectal cancer.

Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Female ; Humans ; Ligands ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Proliferating Cell Nuclear Antigen ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Random Allocation ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; biosynthesis ; genetics ; bcl-X Protein ; metabolism

To study the inhibitory effect of a recombinant adenoviral vector carrying human IL-24 gene on SGC-7901 human gastric cancer cell. We infected the SGC-7901 gastric cancer cells with Ad blank adenovirus at various multiplicity of infection (MOIs) to find the optimal infective dose. The SGC-7901 tumor cells were infected with Ad-IL-24 at the optimal MOI in the following experiments. Adenovirus-mediated IL-24 transcription expression in SGC-7901 cells was examined by RT-PCR. The growth-suppressing effect of Ad-IL-24 on SGC-7901 tumor cells was assessed by MTT assay. Apoptosis and cell cycle of SGC-7901 tumor cells infected with Ad-IL-24 was evaluated by flow cytometer (FCM), respectively. The karyomorphology of apoptotic SGC-7901 tumor cells was examined using Hoechst33258 staining under fluorescence microscopy. The expression of apoptosis-related genes was future determined by semi-quantification RT-PCR; We demonstrated that the MOI of 100 was the optimal infective dose in the study on adenovirus-mediated IL-24 gene transfer into SGC-7901 gastric cancer cell; IL-24 gene mediated by adenovirus could successfully transcribe in SGC-7901 tumor cells; Ad-IL-24 could significantly inhibit SGC-7901 tumor cell growth and induce apoptosis, it also can up-regulate the express of bax, caspase-3 and p53 whilst down-regulate the bcl-2 expression. Thus, adenovirus-mediated IL-24 expression had marked anti-tumor effect in suppressing SGC-7901 human gastric cancer cell growth and inducing apoptosis, which may be closely associated with its up-regulation of bax/bcl-2, caspase-3 and p53.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; genetics ; Caspase 3 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Interleukins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; pathology ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Up-Regulation ; bcl-2-Associated X Protein ; genetics ; metabolism

To study the inhibitory effect and anti-cancer mechanisms of interleukin 24 (IL-24) on human osteosarcoma cell MG-63, we delivered IL-24 into MG-63 cells in vitro and in vivo by adenovirus. The expression level of IL-24 was detected by RT-PCR and fluorescence microscope; the growth inhibition, apoptosis rate and apoptosis body were measured by MTT, Flow cytometry and Hoechst staining respectively. Furthermore, we analyzed the expression of bcl-2, bax, caspase3 genes by RT-PCR after overexpression of IL-24. For in vivo study, we first established the MG-63 tumor model by grafting MG-63 cells in athymic nude mice; and then injected Ad-IL-24 into the tumors. Two weeks after injection, we sacrificed the mice, removed the tumors, weighed and calculated the ratios of tumor-suppression. We also detected the expressions of Bcl-2, Bax, Caspase-3 and CD34 with immumohistochemistry. Our in vitro results indicated that Ad-IL-24 was transcribed and translated in MG-63 osteosarcoma cells. More interestingly, IL-24 inhibited the growth of MG-63 cells and induced apoptosis by up-regulation of bax, caspase-3 and down-regulation of bcl-2. The in vivo data showed that IL-24 suppressed the tumor growth conspicuously through down-regulating the expression of bcl-2, and up-regulating the expression of bax, caspase-3. This study would provide evidence for the gene therapy of IL-24 on osteosarcoma.
Adenoviridae ; genetics ; metabolism ; Animals ; Apoptosis ; genetics ; Bone Neoplasms ; pathology ; therapy ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Interleukins ; biosynthesis ; genetics ; Mice ; Mice, Nude ; Osteosarcoma ; pathology ; therapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the protective effect of resvertrol on the intestinal mucosal cells in rats with severe acute pancreatitis and explore the possible mechanism.

METHODSTwenty-four SD rats were randomly divided into the sham-operation (SO) group, severe acute pancreatitis (SAP) group and resveratrol-treated (RES) group. In the SO group, the pancreases were slightly flipped only. In the SAP and RES groups, SAP model was established by retrograde injection of 40 g/L sodium chrolate (1 ml/kg) through the pancreatic duct, and in the latter group, resveratrol (10 mg/kg) was given intravenously. Specimens were obtained 6 h after SAP model establishment and the endotoxin levels in the portal vein was determined with turbidimetry to evaluate the effect of resversatrol on the intestinal endotoxin translocation in SAP rats. Apoptosis of the mucosal cells was detected by TUNEL methods, and the expression of bax and bcl-2 mRNA were determined by RT-PCR. The mitochondrial membrane potential of the intestinal mucosal cells was measured by confocal microscopy.

RESULTSThe endotoxin levels in the portal vein were significantly lower in RES group than in SAP group (P<0.01). TUNEL assay demonstrated significantly higher apoptotic index of the mucosal cells in SAP group than that in RES group (P<0.01). The expression of Bax mRNA in the intestinal mucosal cell was significantly higher in SAP group than in RES group (P<0.01), whereas the expression of bcl-2 mRNA was significantly lower in SAP group (P<0.01). The mitochondrial membrane potential of the intestinal mucosal cell was significantly lower in SAP group than in RES group (P<0.01).

CONCLUSIONResvertrol can inhibit the apoptosis of the intestinal mucosa cells and maintain the integrity of the intestinal barrier to prevent the bacterial and endotoxin translocation in SAP.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; In Situ Nick-End Labeling ; Intestinal Mucosa ; drug effects ; metabolism ; pathology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Microscopy, Confocal ; Pancreatitis, Acute Necrotizing ; chemically induced ; drug therapy ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sodium Chloride ; Stilbenes ; pharmacology ; therapeutic use ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo study the protective effect of Grape procyanidins (GPC) on radiation injury in radiation-contacted persons.

METHODSSixty radiation-contacted persons were randomly divided into the experimental group and the control group and 15 radiation-uncontacted persons were selected as the normal group. The experimental group was given GPC (100 mg/day), while the control group was given the capsule of starch every day for 60 days. Vein blood samples were taken before and after the study and the total antioxidative capacity (T-AOC), Malondialdehyde (MDA), cell proliferation, expression levels of proliferative cell nuclear antigen (PCNA), Bcl-2 and Bax protein, WBC were measured.

RESULTSThe WBC, T-AOC and cell proliferation rate of the experimental group were (5.62 +/- 0.40) 10(9)/L, (17.07 +/- 1.91) U/ml and 0.87 +/- 0.09 respectively, which were significantly higher than those of the control group. The MDA and Bax expression levels were (4.12 +/- 0.37) nmol/L and 28.06% +/- 5.79% respectively that were significantly lower than those of the control group.

CONCLUSIONGPC should have protective effects on radiation injury of the radiation-contacted persons.

Cell Proliferation ; Glutathione Peroxidase ; blood ; Humans ; Leukocyte Count ; Malondialdehyde ; metabolism ; Occupational Exposure ; Placebos ; Proanthocyanidins ; pharmacology ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Radiation-Protective Agents ; pharmacology ; Superoxide Dismutase ; blood ; bcl-2-Associated X Protein ; biosynthesis

OBJECTIVETo investigate the mechanism of K(ATP)-mediated protection of the hippocampal neurons exposed to hypoxia.

METHODSThe neurons were exposed to hypoxia (0% O(2), 5% CO(2) and 95% N(2)) or treated with tolbutamide or diazoxide for 12 or 24 h 1 week after seeding. MTT assay was used to measure the cell viability. RT-PCR was performed to detect Bax mRNA expression.

RESULTSMTT assay showed a much lower survival rate (75.7-/+2.8%) of the neurons exposed to severe hypoxia (PO(2)=0 mmHg) than that of the neurons in normoxia (P<0.01, n=7). Tolbutamide (100 micromol/L) treatment significantly reduced the survival rate of the neurons to (55.7-/+2.5)%, while diazoxide (100 micromol/L) increased the survival rate to 81.1-/+2.4)% (P<0.01, n=6). In normoxia, neither diazoxide nor tolbutamide significantly affected the cell viability (P>0.05, n=6). A significant increase in Bax (P<0.01) and Fas (P<0.01) mRNA expression occurred in the neurons exposed to severe hypoxia (PO(2)=0 mmHg) as compared with the expressions in cells in normoxia (PO(2)=144 mmHg). In the hypoxic neurons, tolbutamide significantly increased Bax mRNA expression(P<0.05), while diazoxide reduced the expression to a level comparable with that observed in normoxic condition. CONCLUSION Bax is involved in KATP-mediated protection of hippocampal neurons exposed to hypoxia.

Adenosine Triphosphate ; metabolism ; Animals ; Animals, Newborn ; Cell Hypoxia ; Cells, Cultured ; Hippocampus ; cytology ; metabolism ; Immunohistochemistry ; Neurons ; cytology ; metabolism ; Potassium Channels ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; bcl-2-Associated X Protein ; biosynthesis ; genetics