Antineoplastic Agents ; therapeutic use ; Apoptosis ; drug effects ; Caspase Inhibitors ; Drug Resistance, Neoplasm ; Gene Expression Regulation, Neoplastic ; Inhibitor of Apoptosis Proteins ; metabolism ; Neoplasms ; drug therapy ; metabolism ; pathology

OBJECTIVETo observe the effect of survivin antisense oligodeoxynucleotide (ASODN) on the apoptosis of human carcinoma of larynx cell line Hep2 and the inhibitory rate in nude mice model so as to discuss the selective blocking activity of antisense technique on gene expression seeking a new way for gene therapy of carcinoma of larynx.

METHODSAntisense oligodeoxynucleotides survivin were transformed into human carcinoma of larynx cell line Hep2 by liposome Lipofectamine 2000. Within 72 h after transfection, 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cellular proliferation. Forty eight hours after transfection, reverse transcription-polymerase chain reaction (RT-PCR) assay was used to observe the expression of survivin gene, Western Blot assay for the protein, and terminal deoxynucleotide mediated nick end labeling (TUNEL) and flow cytometer for cellular apoptosis.

RESULTSCellular inhibition rate of 72 h went up to 52. 5% and 71.4% at 1.0 micromol/L and 2.0 micromol/L value in Lipo-ASODN groups which differed statistically remarkably (P = 0.046), higher than that in controls in MTT assay (P =0. 003 and 0. 0004). Forty eight hours after transfection survivin gene expression in Lipo-ASODN groups were less than that in control group in RT-PCR assay. Survivin protein expression decreased in Western blot. In TUNEL assay, nuclear positive staining was observed and the apoptosis peak was observed in flow cytometer test, which were absent in controls. In nude mice of carcinoma of larynx model, the inhibitory rate in Lipo-ASODN groups got up to 48.1% and 61.3% higher than that of controls (P < 0.004 and 0. 0006), which differed remarkably (P = 0.032) in a dose-dependently way.

CONCLUSIONSThe findings showed that the expression of survivin gene and protein induced cellular apoptosis in Hep2 cells after transfection of Lipo-ASODN and that the carcinoma of larynx in the nude mice model were inhibited by Lipo-ASODN which suggested that antisense technique can be an effective means in the gene therapy of carcinoma of larynx.

Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Laryngeal Neoplasms ; therapy ; Male ; Mice ; Mice, Nude ; Microtubule-Associated Proteins ; genetics ; pharmacology ; therapeutic use ; Oligonucleotides, Antisense ; genetics ; pharmacology ; therapeutic use ; Transfection

OBJECTIVE: To investigate the effect of adenovirus-mediated ING4 with RGD on proliferation, apoptosis and cell cycle of human nasopharyngeal carcinoma cell CNE and explore its probable mechanism. METHOD: CNE cells were infected with Ad. RGD-ING4 and adenovirus vector, ING4 gene expression level was detected by RT-PCR and the target protein expression was tested by Western blot. MTT assay was adopted to evaluate the efect of ING4 on cell growth of CNE, Annexin -V-PE/7-AAD Double staining was used to measure the efect of ING4 on apoptosis, and PI staining was used to measure the efect of ING4 on the cell cycle. Differential expression of P21, Bcl-2 and Bax gene was detected by RT-PCR,and Differential expression of Survivin and Caspase 3 protein was detected by Western blot. RESULT: CNE cells were cultured with Ad. RGD-ING4 for 72 h ,the results showed that ING4 was overexpressed in CNE cells ,the growth of CNE cells was obviously inhibited , apoptosis rate was significantly increased and G2/M phase was arrested apparently. The results of RT-PCR showed that Ad. RGD-ING4 significantly down-regulated the Bcl-2 and up-regulates the Bax and P21 expression in CNE cells, and the difference was statistically significant(P < 0.01). Western blot showed that the expression of Survivin was decreased and Cleaved-Caspase 3 was increased. CONCLUSION Ad. RGD-ING4 can play the role of tumor suppressor synergies on nasopharyngeal carcinoma cell CNE by down-regulating Bcl-2, Survivin expression and up-regulating P21, Bax and Cleaved-Caspase 3 expression.
Adenoviridae ; Apoptosis ; Carcinoma ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Cycle Proteins ; genetics ; therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression ; Genetic Vectors ; Homeodomain Proteins ; genetics ; therapeutic use ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; therapy ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Survivin ; Tumor Suppressor Proteins ; genetics ; therapeutic use

OBJECTIVESTo investigate the apoptosis induced by antisense oligodeoxynucleotide (ASODN) against survivin and the mechanisms after the hepatocellular carcinoma SMMC-7721 cells transfected with the ASODN.

METHODSThe ASODN was transfected into SMMC-7721 cells mediated by liposomal reagent. The changes of cell cycle and apoptotic rate were detected by flow cytometry. The changes of cell skeleton was observed through confocal microscope. The activity of p38MAPK and caspase-3 were detected by immuno-precipitation and kinase activity assess methods, respectively.

RESULTSThere were control, sense control, 400, 600, 800, and 1 000 ng/ml ASODN groups (I - VI). The apoptotic rats were 0.70%, 0.76%, 2.43%, 7.82%, 23.11%, and 31.35% in groups I - VI, respectively, which in the ASODN-transfected groups were higher than that in the control group (tor=20.9, P<0.01). The activity of p38MAPK increased significantly, when the ASODN was transfected at the concentration of 600 ng/ml or more, so did the caspase-3 activity (the p38MAPK and caspase-3 activity in groups I - VI were 7.03, 7.07, 13.47, 16.37, 43.97, 47.87 and 0.015+/-0.010, 0.014+/-0.002, 0.026+/-0.003, 0.042+/-0.001, 0.093+/-0.001, 0.100+/-0.001, respectively).

CONCLUSIONSASODN targeting at survivin mRNA can induce G2/M stop, activate p38MAPK and caspase-3. The activated caspase-3 destroys the cell skeleton microfilament system, resulting in apoptosis.

Apoptosis ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Microtubule-Associated Proteins ; antagonists & inhibitors ; genetics ; Neoplasm Proteins ; Oligonucleotides, Antisense ; therapeutic use

PURPOSE: To determine whether renal injury induced by ischemia-reperfusion (I/R) could be further improved by mesenchymal stem cells (MSCs) modified with survivin. MATERIALS AND METHODS: Lentiviral vectors were used to introduce the survivin gene into MSCs and the MSCs modified with survivin were transplanted into established mice models of renal I/R injury. Seven days later, serum creatinine (Scr) and blood urea nitrogen (BUN) were measured and the survival of MSCs was determined. Hematoxylin and eosin staining was used to assess renal pathological change. The expressions of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) in kidney tissue were detected by western blot. RESULTS: Mice transplanted with survivin-modified MSCs demonstrated good renal function recovery with Scr and BUN decline close to normal levels and improvement of renal I/R injury repair. Additionally, the survival of transplanted MSCs modified with survivin was enhanced and the expression of HGF and bFGF in kidney tissue was increased. CONCLUSION: Our results demonstrated that MSCs engineered to over-express survivin could enhance their therapeutic effect on renal I/R injury in mice, probably via the improved survival ability of MSCs and increased production of protective cytokines in ischemic tissue.
Animals ; Bone Marrow Cells/*cytology ; Inhibitor of Apoptosis Proteins/*therapeutic use ; Male ; Mesenchymal Stem Cell Transplantation/*methods ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury/drug therapy/*therapy ; Repressor Proteins/*therapeutic use

OBJECTIVETo investigate the effects of small interfering RNA (siRNA) recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil.

METHODSsiRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis.

RESULTSRestriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treating alone (P<0.05).

CONCLUSIONThe siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil.

Antimetabolites, Antineoplastic ; therapeutic use ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; drug therapy ; genetics ; metabolism ; Fluorouracil ; therapeutic use ; Genetic Vectors ; genetics ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism

OBJECTIVETo investigate the inhibitory effect of survivin antisense oligodeoxynuleotides (ASODN) mediated by polyethylenimine (PEI) on hepatocelluar carcinoma SMMC-7721 cell proliferation and its effect on chemosensitivity to 5-FU in tumor-bearing mice.

METHODSThe inhibitory effect of PEI-ASODN on SMMC-7721 cell proliferation was assayed by WST-8 test, Trypan blue exclusion test, and cell clone formation assay. In mouse models of transplanted H22 cell hepatocarcinoma and ascites tumor, the effect of 5-FU combined with PEI-ASODN on the weight and volume of the subcutaneous tumors was examined. The tumor inhibition rate in the tumor-bearing mice was calculated and the average survival time recorded.

RESULTSSMMC-7721 cells incubated with different concentrations of PEI-ASODN for 48 h showed significantly reduced cell proliferation in comparison with the control cells, while PEI or ASODN alone produced no such inhibitory effect. Incubation of SMMC-7721 cells with 0.75 micromol/L PEI-ASODN for 24, 48, 72, and 96 h resulted in significantly suppressed cell proliferation, and a 7-day incubation of the cells with PEI-ASODN at different concentrations (0.25-0.75 micromol/L) significantly inhibited the cell clone formation. In the tumor-bearing mice, the tumor weight and volume were obviously reduced with a tumor inhibition rate of 56.91% and volume inhibition rate of 57.83%, significantly different from those in saline-treated mice (P<0.01). In the mice bearing ascites tumor, the average survival time was 22.0 days in saline group and 42.7 days in 5-FU+PEI-ASODN treatment group, showing a a life-prolonging rate of 94.09% in the latter group. A synergetic effect was noted between 5-FU and PEI-ASODN.

CONCLUSIONPEI-ASODN complex can significantly inhibit the proliferation of hepatocarcinoma SMMC-7721 cells and enhance 5-FU chemosensitivity of the tumor cells in vitro and transplanted H22 tumors in mice.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; therapeutic use ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Synergism ; Female ; Fluorouracil ; pharmacology ; therapeutic use ; Inhibitor of Apoptosis Proteins ; genetics ; pharmacology ; therapeutic use ; Liver Neoplasms, Experimental ; drug therapy ; pathology ; Male ; Mice ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; therapeutic use ; Repressor Proteins ; genetics ; pharmacology ; therapeutic use

OBJECTIVETo investigate the expression of apoptotic protein inhibitors, survivin and XIAP, in patients with myelodysplastic syndromes (MDS) and in the cell line MUTZ-1, as well as to explore the possible mechanisms of homoharringtonine (HHT) in the treatment of MDS.

METHODSBone marrow samples from 47 patients with de novo MDS at diagnosis were examined and bone marrow samples from 15 normal donors were used as control. A MDS-RAEB cell line MUTZ-1 was used as in vitro model. Detection of apoptotic cells and cell cycle analysis were performed with flow cytometry (FACS). The expression of apoptotic protein inhibitor survivin and XIAP in the MDS cells were detected by RT-PCR technique. MUTZ-1 were treated with antisense oligodeoxynucleotide (AS-ODNs) of survivin and or HHT, the effects were evaluated by cell viability and cell apoptosis.

RESULTSSurvivin mRNA positive rate in MDS were significantly higher than that in normal controls (38.3% and 0, respectively, P < 0.01), and the positive rate in high risk group (RAEB, RAEBT and CMML) was significantly higher than that in RA/RAS group (53.6% and 16.7%, respectively, P < 0.05). XIAP was expressed in all untreated MDS and healthy controls. XIAP mRNA expression in high risk group was significantly higher than that in RA/RAS subtypes and healthy controls (1.55 +/- 0.34, 0.74 +/- 0.24, and 1.01 +/- 0.28, respectively, P < 0.01). However, XIAP mRNA expression was significantly lower in RA/RAS subtypes than in healthy control (0.74 +/- 0.24 and 1.01 +/- 0.28, P < 0.054). Apoptosis peak detected by FACS analysis and positive Annexin V FITC staining on cell membrane indicated that HHT could induce MUTZ-1 cell undergoing apoptosis in dose- and time-dependent manners. Treatment of MUTZ-1 cells with HHT revealed that HHT could significantly down-regulate survivinexpression but had no significant effect on XIAP expression in the cells. AS-ODNs of survivin could inhibit MUTZ-1 cells growth, induce them to apoptosis and sensitize them to HHT.

CONCLUSIONThe expression levels of survivin; Institute of Hematology, Oncology and Tumor Immunology, Robert Roessle Clinic, Humboldt University, Berlin, Germany (Wolf Dieter Ludwig, Christian Wuchter) and XIAP vary in different subtypes of MDS patients, suggesting that the proteins may play an important role in the pathogenesis of the disease. Down-regulation of survivin in MUTZ-1 cells may be one of the mechanisms that HHT induces apoptosis of MDS cells.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Harringtonines ; therapeutic use ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; physiology ; Myelodysplastic Syndromes ; drug therapy ; pathology ; Neoplasm Proteins ; Oligonucleotides, Antisense ; pharmacology ; Proteins ; genetics ; physiology ; RNA, Messenger ; analysis ; X-Linked Inhibitor of Apoptosis Protein

OBJECTIVETo study the protective effect of bone marrow stromal cells (BMSCs) upon childhood leukemia cells and the influence of VLA-4 antibody in vitro on leukemia cell apoptosis.

METHODSBMSCs from children with acute leukemia-were isolated by human lymphocyte separation medium. BMSCs (adherent) and leukemia cells (suspended) were cultured in vitro. This study included four groups: leukemia cells alone (control), leukemia cells+BMSCs, leukemia cells+BMSCs supernatant and leukemia cells+BMSCs+VLA-4 antibody. The apoptosis rate of leukemia cells in the four groups was determined by Annexin Ⅴ-FITC double-labeled flow cytometry. The expression of survivin and bcl-2 genes in leukemia cells was ascertained by RT-PCR.

RESULTSThe apoptosis rate of leukemia cells in the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups was lower than that in the control group (P<0.05). Compared with the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups, the apoptosis rate of leukemia cells in the VLA-4 antibody group increased significantly (P<0.05). In the VLA-4 antibody group, the apoptosis rate of leukemia cells increased with prolonged culture time. There were significant differences in the apoptosis rate between 12 hrs and 24 hrs after VLA-4 antibody treatment (P<0.01). The expression of survivin and bcl-2 genes in leukemia cells from the VLA-4 antibody groups was reduced compared with that from the leukemia cells+BMSCs and the leukemia cells+BMSCs supernatant groups (P<0.05).

CONCLUSIONSBMSCs play protective roles on leukemia cells. VLA-4 antibody can block the adhesion between BMSCs and leukemia cells and promote leukemia cell apoptosis.

Adolescent ; Antibodies ; therapeutic use ; Apoptosis ; Bone Marrow Cells ; physiology ; Child ; Child, Preschool ; Female ; Genes, bcl-2 ; Humans ; Infant ; Inhibitor of Apoptosis Proteins ; Integrin alpha4beta1 ; immunology ; Leukemia ; pathology ; therapy ; Male ; Microtubule-Associated Proteins ; genetics ; Stromal Cells ; physiology

OBJECTIVE: To detect the correlation between survivin and rheumatoid arthritis (RA) to determine the possible mechanism of RA and multidrug resistance in refractory rheumatoid arthritis (RRA). METHODS: We collected 15 normal controls, 35 early untreated RA patients, 20 effectively treated RA patients and 25 RRA patients according to selection standard. The expression of survivin in the peripheral blood lymphocytes was detected by immunocytochemical method. RESULTS: There was significant difference in the survivin expression in the peripheral blood lymphocytes between the early untreated and normal control group (χ(2)=29.59, P<0.01). The survivin expression in the peripheral blood lymphocytes of effectively treated RA group slightly elevated, but had no significant difference with the normal control group (χ(2)=1.591, P>0.05). The survivin expression in the peripheral blood lymphocytes of the RRA group was significantly stronger than in the effectively treated RA group (χ(2)=24.35, P<0.01), and normal control group (χ(2)=26.53, P<0.01), with no significant difference compared with early untreated group (χ(2)=0.014,P>0.05). CONCLUSION Survivin has an influential role in the occurrence and development of rheumatism arthritis. Survivin might be involved in refractory multidrug resistance of RA and be one of the multidrug resistance mechanism of RRA.
Adult ; Antirheumatic Agents ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; metabolism ; Case-Control Studies ; Drug Resistance, Multiple ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Inhibitor of Apoptosis Proteins ; metabolism ; Lymphocytes ; metabolism ; Male ; Middle Aged ; Survivin ; Young Adult