Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats / 中国病理生理杂志

[ABSTRACT]AIM:ToevaluatetheeffectsofantisenseTGF-β1oligodeoxynucleotide(ASTGF-β1)ontheex-pression of TGF-β1 , deposition of extracellular matrix ( ECM) and the neointima formation in the arteries after balloon inju-ry.METHODS:The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed.At the same time, sense TGF-β1 oligodeoxynucleotide ( S TGF-β1 ) with the base sequence complement to AS TGF-β1 was synthesized as a control . The oligodeoxynucleotides were introduced into in vivo and in vitro experiments , respectively .RESULTS:The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner , and S TGF-β1 did not have the same effect.Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was ob-served.Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis.Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs . Fibronectin ( FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (0.01~1 μmol/L)-dependent manner .AS TGF-β1 significantly increased the mRNA expression of contractile marker SM 22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla , especially at the concentration of 0.01μmol/L and 0.1 μmol/L.After treatment with AS TGF-β1 (90 μg· kg-1 · d-1 ) for 28 d, the neointima formation was significantly inhibited , and the area ratio of intima/media was markedly decreased by 68% compared with untreated group , but S TGF-β1 had no effect on neointimal formation .CONCLUSION:The AS TGF-β1 specifically inhibits the pro-tein expression of TGF-β1 in the VSMCs derived from injured arteries .Moreover , it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN .Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury .The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the altera-tion of VSMC phenotype after balloon injury .

Effects of Tiam 1 antisense oligodeoxynucleotides transfection on the invasive and migratory potentials of gastric cancer cells in vitro and vivo / 中国癌症杂志

Background and purpose:As a regulatory factor of cytoskeletal structure,a close correlation existed between Tiam 1(T lymphoma invasion and metastasis inducing factor 1) and the invasion and metastasis of gastroenteric tumor.We aimed to observe the effects of Tiam 1 antisense oligodeoxynucleotides(Tiam 1 ASODN) transfection on the invasive and migratory potentials of gastric cancer cells in vitro and vivo.Methods:The higher invasive and migratory subgroup(MH) were separated from human gastric cancer cell line MKN-45(M0) by laminin adhesion method in vitro.Tiam 1 ASODN was transfected into MH cells with liposome,and the expression of Tiam 1 mRNA and protein was determined by RT-PCR and ELISA separately.Changes in the invasive and migratory potentials of MH cells in vitro and vivo after transfected with ASODN were observed by Boyden chamber test and inoculation into nude mice respectively.Results:The expression of Tiam 1 mRNA and protein in MH cells after transfected with 0.43 ?mol/L ASODN(0.162?0.018,0.982?0.119) was significantly lower than that of either Liposome-transfected cells(0.789?0.054,1.237?0.108),sense oligodeoxynucleotides(SODN)-Liposome-transfected cells(0.754?0.039,1.234?0.103) or No-transfected cells(0.801?0.065,1.290?0.182)(P

Bcl-2 Antisense Oligodeoxynucleotides Increase Apoptosis of Lung Cancer Cells Induced by Radiation / 医学研究杂志

Objective To study the effects of Bcl-2 antisense oligodeoxynucleotides(ASODN)on the apoptosis of lung cancer cells induced by radiation in vitro.Methods NCI-H446 lung cancer cell strains were divided into 5 groups:control simple radiation,lipofectin plus radiation,nonsense sqnence radiation and ASODN plus radiation.The cells cultured in five groups were collected at 6h,12h,24h,48h and 72h,with Wright-Giemsa stain,morphology analysis for which was done;the mRNA expression for p53、bcl-2 and PTEN gene was examined by RT-PCR half quantivity and DNA-ploid of the cells in five groups was detected by flow cyfometric method.Results Cell proliferation is obviously restrained and conformation is changed too with the shape crimpled and adherence function decreased obviously after irradiated for 10 Gy dose by the linac;p53 and PTEN expression clearly increased for the combination of Bcl-2 ASODN and bcl-2 mRNA expression clearly decreased.The apoptosis rate after 72 hours among control,pure radiation,lipofectin+radiation,nonsense+radiation and ASODN +radiation grouop is 0.14?0.09,13.17?2.47,11.84?1.76,13.72?1.4,21.26?2.97 respectively,the difference between ASODN combined with radiation grouop and other 4 groups are significant(P

Effects of Livin antisense oligodeoxynucleotides on the proliferation and apoptosis of HL60 cells / 基础医学与临床

Objective To investigate effects of Livin antisense oligodeoxynucleotides(ASODN) on the proliferation and apoptosis of human leukemia(HL60) cells.Methods Livin protein on HL60 cells was examined by immunohistochemistry.Specific phosphorothioate antisense oligodeoxynucleotides and missense oligodeoxynucleotides target Livin mRNA were synthesized and transfected into HL60 cells following cationic liposome.The proliferation inhibition of HL60 cells was assessed by MTT.The expression of Livin mRNA was detected by RT-PCR.Transmission electron microscope and TUNEL technology were used to detect the apoptosis and morphologic change.ResultsASODN of 600 nmol/L inhibited the HL60 cell proliferation and the expressions of Livin mRNA.The percentage of apoptosis detected by TUNEL was 38.48%?4.37%.cellar ultrastructure was markedly destroyed by Livin ASODN.A significant difference was found when compared with the control group(P

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.

Association between apoptosis induced in hepatoblastoma HepG2 cells by Cdk7 silencing and decreased pRb and Cdk2 phosphorylation / 中国药理学通报

Aim: To evaluate the effect of Cdk7 silencing on the cell cycle control, the phosphorylation level changes of Cdk2 and pRb in human hepatoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel target for anticancer therapeutics. Method: Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA contents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flow cytometry and ultrastructural changes of cells were observed with transmission electron microscopy. Result The phosphorylation levels of pRb and Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner when the concentration was above 100 nmol·L-1. Indice of cells arrested in G0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P < 0.01); Characteristic apoptosis in Cdk7 ASODN treated groups were obvious under the transmission electron microscopy. Conclusion: Bioactivities and phosphorylation levels of pRb and Cdk2 decreased after Cdk7 silencing and thus induced obvious G0/G1 phases arrest and apoptosis in HepG2 cell culture in vitro, it is feasible to consider Cdk7 as a novel target for anticancer therapeutics.

Preparation of antisense oligodeoxynucleotides-loaded butylcyanoacrylate nanoparticles by interfacial polymerization and their stability / 第三军医大学学报

Objective To optimize the preparation of nanoparticles encapsulating antisense oligodeoxynucleotides in a-butyleyanoacrylate carrier (ASODN in NP) and investigate their stability. Methods ASODN in NP were prepared by interfacial polymerization of butyleyanoacrylate (BCA). The formulation and technology of the prepared NP was optimized by using orthogonal design based on the single-factor experiment. The morphology of NP was examined by transmission electron microscope; The size and size distribution of NP were determined by Malvern laser granularity equipment;The encapsulation efficiency and drug loading were determined by HPLC; The ability of protecting oligodeoxynucleotides from serum was investigated on a 20% polyacrylamide-7 Murea sequencing gel (PAGE). Results The nanoparticles in the optimal conditions were of regular spherical surface and discrete. The average size was 97.1 nm,the average encapsulation efficiency and drug loading of ASODN in NP were 96.7% and 10.1% respectively; The oligonucleotides were more efficiently protected from degradation by nucleases than by oligonucleotides adsorbed into nanospheres.Conclusion ASODN in NP has good stability,encapsulation efficiency,drug loading and great potential for ASODN delivery.

Antisense Deoxyoligonucleotides Inhibit Activities of Matrix Metalloproteinase-2 in Human Fibrosarcoma HT1080 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: MMP-2, 72 kDa-type IV collagenase, plays a major role in the migration and growth of tumor cells, a process that requires the disintegration of basement membrane. Activation of MMP-2 is correlated with the invasiveness of various tumors. The aim of this study was to determine the sequence-specific phosphorothioated oligodeoxynucleotides (ODNs) inhibiting the translation of MMP-2 mRNA and the subsequent invasiveness of tumor cells. MATERIALS AND METHODS: Eight types of antisense ODNs were designed and each (8micro gram/ml) were transfected into HT1080 cells. The effects of these antisense ODNs on MMP expression were examined by gelatin zymography, Western blot, Northern blot and matrigel assay. RESULTS: Antisense-5 (+904~923), antisense-6 (+1274~+1293) and antisense-7 (+1646~+1665) reduced the MMP-2 activity of the culture supernatant in HT1080 fibrosarcoma cells. Treatment with antisense-6 showed inhibition of MMP-2 mRNA and protein, and in vitro invasion in a dose-dependent manner. CONCLUSION: Antisense-6 might be one of the therapeutic candidates for tumor invasion and metastasis.

Inhibitory effects of phosphorothioate-modified antisense VEGF oligodeoxynucleotides formulated in cationic liposome on angiogenesis and metastasis of lung cancer / 中国免疫学杂志

Objective:The study aims to investigate whether phosphorothioate-modified antisense vascular endothelial growth factor oligodeoxynucleotides(VEGF ASODN)micro-encapsulated in cationic liposome could inhibit angiogenesis and metastasis of lung cancer.Methods:The phosphorothioate-modify VEGF ASODN, VEGF SODN(sense oligodeoxynucleotides) and VEGF MODN(mismatch oligonucleotides)micro-encapsulated in cationic liposome were added into in vitro culture of Lewis lung carcinoma(LLC ) cells,respectively. Expression of VEGF protein was detected by immunohistochemistry staining. And the inhibitory effects on bovine aortic endothelial cell proliferation by conditioned media obtained from LLC cells treated with ODN were observed. 40 mice of Lewis lung cancer models were established and subsequently were randomized into 4 groups: control group, ASODN group, SODN group,and MSODN group. Liposome, ASODN, SODN or MODN were given by twice a week for 4 weeks,respectively.The weights of subcutaneous tumors were measured. The rates of lung metastasis were detected, while the microvessel density(MVD) in tumor mass was detected by imuunohistochemistry staining. Peak systolic flow velocity(PS) and resistance index(RI) were measured with a sonographic scanner.Results:ASODN downregulated the expression of VEGF in LLC cells at the level of protein in vitro. The conditioned media obtained from LLC cells treated with ASODN significantly inhibited the proliferation of bovine aortic endothelial cells. ASODN significantly suppressed the growth of subcutaneous tumors and lung metastasis. MVD, PS and RI of VEGF ASODN group were remarkably different from those of other 3 groups(P

Effect of vascular endothelial cell growth factor (VEGF) antisense oligodeoxynucleotides on expression of VEGF in rats with oxygen-induced retinopathy / 中华眼底病杂志

0.05), which were both lower than those in the normal control group (P

EFFECT ON C-myc AND N-ras EXPRESSION OF ANTISENCE OLIGODEOXYNUCLEOTIDES / 肿瘤研究与临床

0.05).The C myc and N ras protein expression of 2.2.15 were reduced after ASONs were used 3 days.There was a significant difference between ASON group and control group ( P

Inhibition of Overexpression of c-myb Protooncogene in K562 Cells Using c-myb Antisense Oligodeoxynucleotides / 대한소아혈액종양학회지

PURPOSE: The c-myb protooncogene encodes MYB protein that is critical for normal and leukemic hematopoietic cell proliferation and development. It is known that c-myb plays an important role in leukemogenesis as well. Aberrant expression of c-myb is seen in acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), and chronic myeloid leukemia (CML). We reasoned that down regulation of c-myb expression using synthetic antisense oligomers targeted to c-myb mRNA might prove useful antileukemic agents if leukemia cells were more dependent on c-myb function for proliferation than their normal counterparts. To investigate the applying possibility of c-myb antisense oligodeoxynucleotides as the useful antileukemic agents, we examined the cell viability, cloning efficiency and the expression of c-myb mRNA and MYB protein after the exposure of c-myb oligomers on normal bone marrow cells and leukemia cells. MATERIALS AND METHODS: We maintained in short-term suspension culture for 4 days to analyze the effect of c-myb oligomers on normal bone marrow cells and chronic myelocytic leukemia cell line K562. During suspension culture, cell counts and viability were periodically determined, and immediately seeded into duplicate methylcellulose cultures containing recombinanat human interleukin 3 and GM-CSF. Cells placed in semisolid cultures were allowed to grow for an additional 10~12 days. We counted the colonies, and then RNA and protein was extracted from cells cloned in liquified methyl- cellulose cultures. We detected the c-myb mRNA expression by RT-PCR and Southern hybridization analysis and MYB expression by Western hybridization analysis. RESULTS: c-myb sense oligomers had negligible effects on K562 cells growth in short- term suspension culture, while exposure to c-myb antisense oligomers resulted in a daily decline in cell number. In contrast, normal bone marow cell viability and numbers were unaffected by c-myb sense or antisense oligomers exposure. c-myb antisense oligodeoxy nucleotides strongly inhibited or completely abolished growth and colony formation of K562 cells. In contrast, c-myb sense oligomers did not affect. At antisense dose that inhibited leukemic cell growth, normal bone marrow cells survived. Thus, normal and leukemic cells showed the differential sensitivity to the toxic effect of c-myb antisense oligomers. RT-PCR, Southern hybridization analysis and Western hybridization analysis of c-myb antisense-treated K562 cells revealed a complete absence of c-myb mRNA expression and MYB expression. CONCLUSION: Results obtained from these studies suggest that inhibition of c-myb function with antisense oligodeoxynucleotides might eventually form the basis for a molecular biologic approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agent.

Control of Scarring in Adult Wounds using Antisense Connective Tissue Growth Factor Ollgodeoxynucleotides / 대한면역학회지

Adult wounds heal with scar formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. Recently connective tissue growth factor (CTGF) is known to play an important role in wound healing. We reasoned that a strategy employing antisense oligodeoxynucleotides (ODN) complementary to CTGF mRNA by topical application of the ODN on the skin wound. Phosphorothioation of ODN to retard their degradation. When antisense CTGF ODN were applied on the wound site, there was a marked reduction of scarring compared with a control wound site. This effect of antisense CTGF ODN on scar forrnation was associated with decreased expression of the CTGF gene. However, sense CTGF ODN had no effect on the expression of the CTGF gene. In addition, control wounds healed with excessive fibrosis compared with the antisense-treated wounds. In conclusion, our results indicate that antisense CTGF ODN could be used for ameliorating scar formation during wound healing.

Effect of survivin antisense oligodeoxynucleotide on invasive ability of hilar cholangiocarcinoma cell FRH-0201 / 中国现代普通外科进展

Objective: To study the inhibition effect of survivin antisense oligodeoxynucleotide (ASODN)on apoptosis and invasion of hilar cholangiocarcinoma cell line FRH-0201. Methods: ASODN was transfected into cholangiocarcinoma cell line FRH-0201 by LipofectamineTM2000. The level of MMP-2 and TIMP-2 in the supernatant was detected by enzyme linked immunosorbent assay (ELISA). The effect of ASODN transfection was observed by Transwell cell culture chamber. Results: The cell proliferation was inhibited in the ASODN group. The expression of MMP-2 in ASODN group was lower than those in the SODN and blank group (P

Effects on different concentrations of antisense oligodeoxynucleotides on human pancreatic cancer cell line PaTu8988s / 中华消化杂志

Objective To study the effects of different concentrations of antisense oligodeoxynucleotides (ASODN) on human pancreatic cancer cell line PaTu8988s. Methods Human pancreatic cancer cell line PaTu8988s in exponent growth stage are adopted. We observe the effect of different time point on the PaTu8988s cell at 12、24、48 and 72 hour. Results The inhibitory rate on PaTu8988s cell line is 42.25%、66.29%、69.55%、74.58% and 66.20%、91.43%、98.18%、98.33% for ASODN concentrations of 50 ?g/ml and 100 ?g/ml at 12、24、48 and 72 hour, respectively. Conclusion The inhibitory effect of ASODN began from 12 hour and becomes more obvious at 48～72 hour. The higher the concentration of ASODN, the earlier the peak of inhibited rate.

Influence of phosphorothioate-modified antisense VEGF oligodeoxynucleotides formulated in cationic liposome on blood flow of lung cancer / 中国肿瘤生物治疗杂志

Objective: To investigate whether phosphorothioate-modified antisense vascular endothelial growth factor oligodeoxynucleotides ( VEGF-ASODN) formulated in cationic liposome can inhibit the blood flow of lung cancer. Methods: Lewis lung tumor models were established by subcutaneous injection of Lewis lung carcinoma cells into the right flank of 40 C57BL/6 mice. Twenty-four hours later, mice were randomly assigned into 4 groups; pure liposome, liposome containing ASODN, SODN and MODN. Mice in each group were treated twice a week for 4 weeks. Tumor growth were determined. Peak systolic flow velocity (PS) and resistance index (RI) were measured with a sonographic scanner. Expression of VEGF mRNA were detected by hybridization in situ and RT-PCR. Results: ASODN significantly suppressed the growth of subcutaneous tumors. PS and RI in VEGF-ASODN group were significantly different from those in other 3 groups ( P

The Study of Inhibition of Malignant Phenotype of SAC-IIB2 Cell Strains by Antisense Oligodeoxynucleotide of c-fos / 中国肿瘤生物治疗杂志

It was found that protooncogene c-fos was over-expressing in SAC-IIB_2 cell strains. Targeting the code 2-7 sequence of c-fos cDNA of mice, we designed and synthesised eighteen mers antisense. sense and nonsense oligodeoxynucleotides(ODN). The results indicated that antisense c-fos ODN could induce SAC- IIB_2 maturative differentiation, inhibit its growth rate, decrease its ability of forming tumor in nude mice and expression of Fos protein, however, antisense ODN has no remarkable effect on cellular dynamics of SAC-IIB2. But sense and nonsense c-fos ODN have no similar effect on SAC-IIB_2 cell strains. The results demonstrated that antisense c-fos oligodeoxynucleotide has inhibition effects on malignant phenotype of mice SRS lymphoma cell.

Inhibitory effects of c-raf-1 antisense oligodeoxynucleotides transfactionon proliferation of the human ovarian carcinoma SKOV_3 cell lines / 重庆医科大学学报

Objective: To investigate the effects and the mechanisms of c-raf-1 genes antisense oligodeoxynucleotides(ASODN) transfection in inhibiting the human ovarian carcinoma SKOV3 cell lines.Methods: There were 3 groups in our study: normal control group,c-raf-1 sense oligodeoxynucleotides(SODN) experimental group,and c-raf-1 antisense experimental group.at the different time points after liposome-mediated transfection,the cell proliferation,apoptosis,protein expressing level were observed by MTT assay,flow cytometry,fluorescent microscope and cloning test.Results: In the ASODN experimental group and SODN group,the OD-value were 0.272 and 1.307 respectively(P

Anti-cytomegalovirus (CMV) effect of antisense oligodeoxynucleotides phosphorothioates (AS S OND) / 中国药理学通报

AIM To study anti CMV effect of S ODNs complementary to the initial code region of major immediate early (MIE) mRNA, and to the intron1/exon2 boundary of MIE mRNA precursor. METHODS To evaluate the anti CMV effect by determining viral antigen on infected 2BS cells by in situ ELISA. RESULTS Both of 2 AS S ODNs and their sense sequence control showed anti CMV effect. The medium effective concentration (EC 50 ) were 4 53, 10 2, 26 2 and 30 1 ?mol?L -1 . The secretion of CMV antigens were delayed by 3 d～4 d by 8 0 ?mol?L -1 AS 1. It showed increased effect by administrating earlier postinfection, by supplementing the S ODN at intervals, by conbinating with ganciclovir, and by packaging with liposome. A slight cytotoxicity was observed at the concentration of 64 0 ?mol?L -1 . Northern blot analysis indicated that the MIE mRNA decreased after the treatment of AS 1. It suggests that disintegration of the mRNA in the hybridized duplex by activated RNase H played an important role as the mechanism of specific action, and "time and effect" analysis suggested that interference of viral adsorbtion and penetration may be the important mechanism of nonspecificity. CONCLUSION AS S ODNs targeted to MIE gene are effective anti CMV agents which can be developed as a new type of chemotherapy drugs against CMV infection.

Effect of liposome-c-raf-1 antisense oligodeoxynucleotides on human ovarian carcinoma transplanted subcutaneously in nude mice / 重庆医科大学学报

Objective:To explore the effect of c-raf-1 antisense oligodeoxynucleotides (ASODN) treatment in the human ovarian epithelial cancer transplanted subcutaneously in nude mice.Methods:The models of human ovarian epithelial cancer transplanted subcutaneously were established in 15 nude mice,then divided randomly into 3 groups and different treatment were given respectively (control group,senseexperimental group and antisense experimental group).The weight of nude mice and tumor volume were observed,the tumor growth inhibitory rate and the tumor response rate calculated,too.Results:The growth inhibitory rate in sense experimental group and antisense experimental group were 6.8% and 68.1%,respectively,the tumor response rate of antisense experimental group was 16.7%.There was no statistical difference in nude mice weight among the 3 groups.Conclusion:The results suggest that there is a positive value in the human ovarian epithelial cancer transplanted subcutaneously in nude mice treated by c-raf-1 antisense oligodeoxynucleotides,which will be an important gene therapeutic strategy for the ovarian epithelial carcinoma in the future.