OBJECTIVE: To investigate the changes of mitochondrion by transferring antisense oligodeoxynucleotide Stat3 into laryngeal carcinoma Hep-2 cell, for elucidating the mechanism of laryngeal carcinoma Hep-2 cell apoptosis, for developing more effective treatment for laryngeal cancer. METHOD: The designed Stat3 ASODN was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Mitochondrion membrane potential, VDAC and Cyt-c were detected for determining the changes of mitochondrion. RESULT: MMP was fell, Cyt-c and VDAC were increased with the heighten concentration of ASODN. CONCLUSION Mitochondria approach play an important role in the apoptosis mechanism of human Hep-2 cell by Stat3. This research elucidated the regulating mechanism of Hep-2 cell proliferation by Stat3, provided a new research focus for clinical therapy.
Apoptosis ; Cell Proliferation ; Cytochromes c ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; STAT3 Transcription Factor ; genetics ; Transfection ; Voltage-Dependent Anion Channels ; metabolism

OBJECTIVETo explore the effects of antisense oligodeoxynucleotides (ASODN) on proliferation and apoptosis in gastric cancer cell line BGC-823 cells and the molecular mechanisms induced by ASODN.

METHODSsurvivin ASODN-1, survivin ASODN-2 and survivin ASODN-3 were transfected into BGC-823 cells by Lipofectamine(TM) 2000 transfection reagent. The growth activity of BGC-823 cells was detected by MTT assay. Apoptosis index (AI), proliferation index (PI), cell cycle and expressions of survivin, VEGF and Smac/DIABLO proteins were detected by flow cytometry (FCM). The changes of survivin mRNA, VEGF mRNA and Smac/DIABLO mRNA were detected by RT-PCR.

RESULTSThe expression of survivin was down-regulated by the three ASODN sequences, especially the ASODN-2 was best. At 48 hours after transfection with 600 nmol/L survivin ASODN-2, the cells in G(1)/G(0) phase were significantly increased [(72.25 ± 2.95)%], apoptotic index increased [(11.31 ± 0.38)%], proliferation index decreased [(27.77 ± 2.97)%], compared with those in the control group [(56.25 ± 0.75)%, (1.62 ± 0.36)%, (43.80 ± 0.80)%, all P < 0.05]. The survivin mRNA and protein levels (0.523 ± 0.091, 0.733 ± 0.009) were down-regulated compared with those in the control group (0.861 ± 0.047, 0.997 ± 0.233), VEGF (0.519 ± 0.076, 0.75 ± 0.006) were down-regulated compared with those in the control group (0.779 ± 0.059, 1.000 ± 0.01), while those of Smac/DIABLO(0.899 ± 0.113, 1.637 ± 0.023)were up-regulated compared with those in the control group (0.558 ± 0.041, 1.000 ± 0.049, all P < 0.05).

CONCLUSIONSSurvivin ASODN can induce apoptosis and inhibit the proliferation of gastric cancer cell line BGC-823 cells. Those effects are induced through up-regulation of Smac/DIABLO and down-regulation of survivin and VEGF expression simultaneously.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Female ; Ferric Compounds ; chemistry ; Genes, erbB-2 ; genetics ; Humans ; Magnetics ; Microscopy, Atomic Force ; methods ; Molecular Probe Techniques ; Nucleic Acid Probes ; chemistry ; genetics ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; ultrastructure ; RNA, Messenger ; genetics ; metabolism

In this study, the effects of apollon antisense oligodeoxynucleotide (ASODN) on the proliferation and apoptosis of human Lovo cells in vitro were investigated. Apollon ASODN was incubated with human colorectal Lovo cells for 48 h, the proliferation inhibition and the clone forming rates were detected by WST method and clone formation assay, respectively. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The percentage of apoptotic cells and cell cycle distribution were determined by flow cytometry. The morphology of apoptotic cells was examined by fluorescence microscope. Lovo cells incubated with apollon ASODN combined with 5-fluorouracil (5-FU), cisplatin (DDP) or epirubicin (EPI) of different concentrations, cell proliferation inhibition rates were detected with WST method and IC50 was calculated. It was found that ASODN targeting apollon gene could all suppress the growth of Lovo cells and induce apoptosis of these cells significantly (P < 0.05). After Lovo cells treated with apollon ASODN for 48 hours, the expression of the apollon mRNA level was suppressed significantly. And a marked concentration-dependent decline of cell proliferation and clone forming, increasing of cell apoptosis levels were observed. The percentage of G0/G1 phage cells was abated and that of S phage cells was increased and the Lovo cells arrested at S phage of the cell cycle detected with flow cytometry. Many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation. Cell proliferation inhibition was detected and their chemo-therapeutic effects of 5-FU, DDP and EPI on Lovo cells combined with apollon ASODN (0.08 micromol x L(-1)) were enhanced independently compared with single 5-FU, DDP and EPI groups, and the sensitivity enhanced about 2.58, 4.47, and 5.33 times respectively. It can be concluded that ASODN targeting apollon can suppress the expression of apollon mRNA, and inhibit the proliferation, induce apoptosis, arrest cell cycle at S phase of colorectal cancer Lovo cells in vitro and enhance the chemo-sensitivity to 5-FU, DDP and EPI.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Colonic Neoplasms ; metabolism ; pathology ; Epirubicin ; pharmacology ; Fluorouracil ; pharmacology ; Gene Knockdown Techniques ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Inhibitory Concentration 50 ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; metabolism ; Sensitivity and Specificity ; Transfection

As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.
ADP-Ribosylation Factors/metabolism/physiology ; Animals ; Basic Helix-Loop-Helix Transcription Factors/metabolism/physiology ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Glucose/*pharmacology ; Insulin/*secretion ; Insulin-Secreting Cells/*drug effects/enzymology/metabolism/secretion ; Intracellular Signaling Peptides and Proteins/metabolism/physiology ; Mice ; Models, Biological ; Oligodeoxyribonucleotides, Antisense/pharmacology ; Phospholipase D/antagonists & inhibitors/genetics/metabolism/*physiology ; Protein-Serine-Threonine Kinases/metabolism/physiology ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism/physiology ; Signal Transduction/drug effects/genetics

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 effect of survivin antisense oligodeoxynucleotide (ASODN) on proliferation and apoptosis of human malignant melanoma cells.

METHODShMMC A375 colonies in log growth phase were collected and divided into control group (C, without transfection), sense chain group [SC, transfected with 600 nmol/L survivin sense oligodeoxynucleotide (ODN)], mismatch chain group (MC, transfected with 600 nmol/L survivin mismatch sense ODN), liposome group (L, treated with liposome), antisense chain group (AC, transfected with survivin ASODN, and subdivided into AC 200, 400, 600 nmol/L subgroups) according to the random number table. Transfection result was observed under inverted fluorescence microscope. Inhibition rate of cell proliferation was calculated after determination of cell viability with MTT method. Cell cycle and apoptosis rate were detected with bi-variable flow cytometry. Expression of survivin protein was determined with Western blot. Activity of caspase-3 was assessed with kinase method. Data were processed with analysis of variance.

RESULTS(1) Cell transfection rates in SC, MC, AC 600 nmol/L groups were all above 80%. (2) Compared with those in SC group [(5.23 +/- 0.25)%], MC group [(5.09 +/- 0.13)%] and L group [(4.70 +/- 0.45)%], inhibition rates of cell proliferation in AC 200, 400, 600 nmol/L groups 24 hours after transfection [(10.30 +/- 0.56)%, (16.69 +/- 0.58)%, (24.67 +/- 0.67)%] were significantly increased (F = 746.91, and P values all below 0.05). As time after transfection went on, proliferation inhibition rate was increased obviously. (3) Apoptosis rate in AC 200, 400, 600 nmol/L groups 24 hours after transfection was respectively (13.5 +/- 1.9)%, (20.1 +/- 1.5)%, (32.1 +/- 2.9)%, which were significantly higher than those in C, SC, MC, and L groups [(6.5 +/- 0.6)%, (5.6 +/- 0.7)%, (6.4 +/- 1.0)%, (6.5 +/- 1.3)%, F = 139.9, P values all below 0.05]. Cells in AC group were blocked in G2/M stage. (4) Compared with those in C group, expression amount of survivin protein decreased, and caspase-3 activity obviously increased (F = 63.1, P values all below 0.05) in AC group. No significant difference in caspase-3 activity between SC, MC, L groups and C group was observed (F = 0.512, P values all above 0.05).

CONCLUSIONSSurvivin ASODN can inhibit the proliferation of hMMC A375 in a concentration-time dependent manner, and it induces G2/M stage block and promotes its apoptosis.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Inhibitor of Apoptosis Proteins ; Melanoma ; metabolism ; pathology ; Microtubule-Associated Proteins ; genetics ; pharmacology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Transfection

OBJECTIVETo better assess the role of p38 MAPK, this project was designed to investigate whether intraventricular injection of antisense oligodeoxynucleotide (As-ODN) directed against the p38 MAPK of pyramidal neurons in hippocampus could affect the brain ischemic tolerance induced by limb ischemic preconditioning (LIP).

METHODSThe rat 4-vessel occlusion global cerebral ischemic model was used. Forty-eight male Wistar rats with permanently occlusion of the bilateral vertebral arteries were divided into 8 groups (n=6): sham, LIP, brain ischemic insult, LIP + brain ischemic insult, distilled water + LIP + brain ischemic insult, p38 MAPK As-ODN and p38 MAPK As-ODN + LIP + brain ischemic insult (two doses of 5 nmol/5 microl and 10 nmol/5 microl were used) groups. Thionin staining was used for observing histological changes of the hippocampus.

RESULTSNo significant delayed neuronal death (DND) was detected in the CA1 hippocampus of the rats that underwent sham and LIP operation. Brain ischemic insult for 8 min induced obvious DND as represented with the increase in histological grade (HG) and decrease in neuronal density (ND) significantly compared with sham and LIP groups. LIP protected the CA1 hippocampal pyramidal neurons against DND induced by global brain ischemic insult, suggesting the occurrence of brain ischemic tolerance. However, pretreatment with p38 MAPK As-ODN effectively blocked the ischemic tolerance induced by LIP in a dose dependent manner.

CONCLUSIONIt could be concluded that p38 MAPK plays an important role in the brain ischemic tolerance induced by LIP.

Animals ; Brain Ischemia ; physiopathology ; Cell Death ; Extremities ; blood supply ; Hippocampus ; pathology ; Ischemic Preconditioning ; methods ; Male ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Rats ; Rats, Wistar ; Reperfusion Injury ; prevention & control ; p38 Mitogen-Activated Protein Kinases ; metabolism ; physiology

OBJECTIVETo investigate the effect of hTERT antisense oligodeoxynucleotide on telomerase activity in bladder cancer cells.

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by telomerase PCR ELISA kit. hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR), and hTERT protein by immunohistochemistry and flow cytometry.

RESULTSTelomerase activity was decreased in T24 cells 48 h after treatment with AS PS-ODN, and was significantly inhibited at 72 h.

CONCLUSIONAS PS-ODN can significantly inhibit telomerase activity by down-regulating hTERT mRNA and protein expression in bladder cancer cells.

Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Urinary Bladder Neoplasms ; enzymology ; pathology

OBJECTIVE: To study the mechanism of apoptosis in laryngeal carcinoma cell induced by Stat3 antisense oligodeoxynucleotide (ASODN). METHOD: The designed Stat3 ASODN was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Expression of Bcl-2, Bax and C-Myc were detected by Western blot and PCR. RESULT: Western blot and PCR results demonstrated that Stat3 ASODN could significantly increased the expression of Bax and decreased the expression of Bcl-2 and C-Myc when the concentration of antisense oligodeoxynucleotide were heightened. CONCLUSION Stat3 ASODN participate in apoptosis by enhancing the expression of Bax and reducing the expression of Bcl-2 and C-Myc.
Apoptosis ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; STAT3 Transcription Factor ; genetics ; bcl-2-Associated X Protein ; metabolism