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

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 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

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 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 investigate the inhibitory effect of survivin antisense oligodeoxynucleotides (ASODN) mediated by polyethylenimine(PEI) on the proliferation of hepatocellular carcinoma SMMC-7721 cells, and assess its detect on the chemosensitivity of the cells to 5-FU.

METHODSThe inhibitory effect of PEI-ASODN on SMMC-7721 cell proliferation was assessed using WST-8 test, trypan blue staining, and cell clone formation test. In mice bearing transplanted hepatocarcinoma and ascites tumor derived from H22 cells, 5-FU combined with PEI-ASODN was administered, and the weight and volume of the subcutaneous tumors were measured to calculate the tumor inhibition rate, and the average survival time of the mice was calculated.

RESULTSIncubation of the cells with different concentrations of PEI-ASODN for 48 h significantly inhibited the cell proliferation as compared with the control group, but PEI or ASODN alone produced no significant inhibitory effects. At 24, 48, 72, 96 h of incubation of the SMMC-7721 cells with 0.75 micromol/L PEI-ASODN, the cell proliferation was suppressed significantly, and incubation with PEI-ASODN at 0.25-0.75 micromol/L for 7 days resulted in significantly inhibited cell clone formation. No significant inhibition was detected in ASODN and PEI group. The tumor weight and volume were reduced in all the treated groups. The tumor inhibition rate was 56.91% and volume inhibition rate was 57.83% in 5-FU+PEI-ASODN group, significantly different from those in the normal saline group (P<0.01). In mice bearing ascites tumor, the average survival time was 22.0 days in saline group, and 42.7 days 5-FU+PEI-ASODN group. The life-prolongation rate of 5-FU+PEI-ASODN was 94.09% when compared with the survival time in saline group. A cooperative effect was detected between 5-FU and PEI-ASODN.

CONCLUSIONPEI-ASODN complex can significantly inhibit the proliferation of hepatocarcinoma SMMC-7721 cells and enhance the chemosensitivity of the tumor cells to 5-FU.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Fluorouracil ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; pharmacology ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Repressor Proteins ; genetics ; pharmacology

The aim of the paper is to prepare stable antisense oligodeoxynucleotides-loaded cationic liposomes and evaluate the transfection efficiency of asODN to MCF-7 oophoroma cells and study their distribution to different tissues in mice. Antisense oligodeoxynucleotides (asODN)-loaded cationic liposomes were prepared by a thin film-adsorption-lyophilization method which is simple and can overcome crucial pharmaceutical defects (e.g. instability) of liposomes during storage. The morphology was investigated by transmission electron microscope. The size and surface charge of the liposomes were determined by laser particle analyter. The dissociated ligodeoxynucleotides were separated from the liposomes by sephadex column and the entrapment efficiency was determined by using an ultraviolet photometer. Trehalose, mannitol, and glycine were suitable for lyophilization especially trehalose. The resulting liposomes were global microcapsule in a narrow particle size with a mean diameter of 175 nm and 320 nm before and after lyophilization, and a high zeta potentials of +32 mV. The dissociated asODN were separated from the liposomes by sephadex G-50 column and the entrapment coefficient of asODN was 88.4% pre and 83.2% post-lyophilization separately for trehalose. The growth of MCF-7 oophoroma cells were inhibited in vitro obviously (P < 0.05) and transfection efficiency of asODN was 18%, 26%, 44% after 2 h, 4 h and 8 h, respectively. The formulation and method can be used to prepare stable cationic liposomes which can effectively inhibit the growth of MCF-7 oophoroma cells and obtain a high transfection efficiency. This system can improve distribution amount of asODN to tissues especially tumors in mice.
Animals ; Breast Neoplasms ; metabolism ; pathology ; Cations ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Carriers ; Female ; Freeze Drying ; Humans ; Liposomes ; chemistry ; pharmacokinetics ; pharmacology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Oligodeoxyribonucleotides, Antisense ; chemistry ; genetics ; Particle Size ; Transfection

OBJECTIVETo study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells.

METHODSWe examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells.

RESULTSThe compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly.

CONCLUSIONWith a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.

Angiogenesis Inhibitors ; genetics ; Breast Neoplasms ; blood supply ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dendrimers ; Gene Expression Regulation ; drug effects ; Humans ; Hydrogen-Ion Concentration ; Microscopy, Electron, Transmission ; Nylons ; Oligodeoxyribonucleotides, Antisense ; genetics ; pharmacology ; ultrastructure ; RNA, Messenger ; genetics ; Transgenes ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; ultrastructure

OBJECTIVESTo study the inhibitory effect of HBx antisense oligodeoxynucleotide on the formation of transplanted hepatocellular carcinoma in nude mice.

METHOD50 nude mice were randomly divided into 5 groups: 1 control group and 4 experimental groups. Log-phase Hep3B cells endogenously expressing HBX were injected subcutaneously in nude mice. From the second day, the PAGE purified AS1, AS2, AS3 and AS4 HBx antisense oligodeoxynucleotides were injected intraperitoneally into the 4 experimental groups, respectively, on alternate days for 5 times, and distilled water was injected into the control group. Growth information of subcutaneous transplantation tumor in nude mice was recorded for 30 days. Incidence rate of transplanted tumor in different groups was compared and analyzed by survival analysis. Statistics software SPSS12.0 was used to analyze the data.

RESULTSIncidence rate of transplanted tumor was 100% in AS1, AS2, AS3 and control groups, and 90% in AS4 group (x2 = 3.995, P = 1.0). The median latency period for transplanted tumor formation was 19 days (17.48-20.52), 12 days (9.93-14.07), 11 days (9.45 to 12.55), 21 days (19.48 to 22.52), and 10 days (8.99 to 11.01) in AS1, AS2, AS3, AS4 and control group, respectively. The latency period for tumor formation was prolonged by treatment of mice with AS1 and AS4 antisense oligodeoxynucleotide (P less than 0.01).

CONCLUSIONAntisense oligodeoxynucleotide targeting to the appropriate sites of HBx gene can prolong the latency period of subcutaneously transplanted tumor in nude mice, however, the formation of transplanted tumor can not be completely blocked by limited treatment with these antisense oligos. In addition, our results suggest that peritoneal injection may be an effective way to deliver antisense oligodeoxynucleotide to living organisms.

Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Expression Regulation, Neoplastic ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms, Experimental ; genetics ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Oligodeoxyribonucleotides, Antisense ; administration & dosage ; genetics ; pharmacology ; Random Allocation ; Trans-Activators ; genetics ; metabolism ; Xenograft Model Antitumor Assays