Targeting LncRNA LLNLR-299G3.1 with antisense oligonucleotide inhibits malignancy of esophageal squamous cell carcinoma cells in vitro and in vivo

Accumulating evidence has indicated that long non-coding RNAs(lncRNAs)play critical roles in the development and progression of cancers,including esophageal squamous cell carcinoma(ESCC).However,the mechanisms of lncRNAs in ESCC are still incompletely understood and therapeutic attempts for in vivo targeting cancer-associated lncRNA remain a challenge.By RNA-sequencing analysis,we identified that LLNLR-299G3.1 was a novel ESCC-associated lncRNA.LLNLR-299G3.1 was up-regulated in ESCC tissues and cells and promoted ESCC cell proliferation and invasion.Silencing of LLNLR-299G3.1 with ASO(antisense oligonucleotide)resulted in opposite effects.Mechanistically,LLNLR-299G3.1 bound to cancerassociated RNA binding proteins and regulated the expression of cancer-related genes,including OSM,TNFRSF4,HRH3,and SSTR3.ChIRP-seq(chromatin isolation by RNA purification and sequencing)revealed that these genes contained enriched chromatin binding sites for LLNLR-299G3.1.Rescue experiments confirmed that the effects of LLNLR-299G3.1 on ESCC cell proliferation were dependent on interaction with HRH3 and TNFRSF4.Therapeutically,intravenous delivery of placental chondroitin sulfate A binding peptide-coated nanoparticles containing antisense oligonucleotide(pICSA-BP-ANPs)strongly inhibited ESCC tumor growth and significantly improved animal survival in vivo.Overall,our results suggest that LLNLR-299G3.1 promotes ESCC malignancy through regulating gene-chromatin interactions and targeting ESCC by pICSA-BP-ANPs may be an effective strategy for the treatment of lncRNA-associated ESCC.

Antisense therapy:a potential breakthrough in the treatment of neurodegenerative diseases

Neurodegenerative diseases are a group of disorders characterized by the progressive degeneration of neurons in the central or peripheral nervous system.Currently,there is no cure for neurodegenerative diseases and this means a heavy burden for patients and the health system worldwide.Therefore,it is necessary to find new therapeutic approaches,and antisense therapies offer this possibility,having the great advantage of not modifying cellular genome and potentially being safer.Many preclinical and clinical studies aim to test the safety and effectiveness of antisense therapies in the treatment of neurodegenerative diseases.The objective of this review is to summarize the recent advances in the development of these new technologies to treat the most common neurodegenerative diseases,with a focus on those antisense therapies that have already received the approval of the U.S.Food and Drug Administration.

Sequencing of PCR amplified HBV DNA pre-c and c regions in 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence

AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and thecombination of MK-AS and ADM has a much better in vitro and in vivo synergism.

Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.

Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin- eosin (HE) staining. RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

Multi?targeted Antisense Oligonucleotide Delivery by a Framework Nucleic Acid for Inhibiting Biofilm Formation and Virulence

Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge.Bacteria and the extracellular polysaccharides(EPS)cause biofilms to become adherent,toxic,resistant to antibiotics,and ultimately difficult to remove.Inhibition of EPS synthesis can prevent the formation of bacterial biofilms,reduce their robustness,and promote removal.Here,we have developed a framework nucleic acid delivery system with a tetrahedral configuration.It can easily access bacterial cells and functions by delivering antisense oligonucleotides that target specific genes.We designed antisense oligonucleotide sequences with multiple targets based on conserved regions of the VicK protein-binding site.Once delivered to bacterial cells,they significantly decreased EPS synthesis and biofilm thickness.Compared to existing approaches,this system is highly efficacious because it simultaneously reduces the expression of all targeted genes(gtfBCD,gbpB,ftf).We demonstrate a novel nucleic acid-based nanomaterial with multi-targeted inhibition that has great potential for the treatment of chronic infections caused by biofilms.

Effects of HSP70 Antisense Oligonucleotide on the Proliferation and Apoptosis of Human Hepatocellular Carcinoma Cells

The study investigated the effects of heat shock protein 70(HSP70) antisense oligonucleotide(ASODN) on the proliferation and apoptosis of a human hepatocellular carcinoma cell line(SMMC-7721 cells) in vitro.HSP70 oligonucleotide was transfected into SMMC-7721 cells by the mediation of SofastTM transfection reagent.Inhibition rate of SMMC-7721 cells was determined by using MTT method.Apoptosis rate and cell cycle distribution were measured by flow cytometry.Immunocytochemistry staining was used to observe the expression of HSP70,Bcl-2 and Bax.The results showed that HSP70 ASODN at various concentrations could significantly inhibit the growth of SMMC-7721 cells,and the inhibition effect peaked 48 h after transfection with 400-nmol/L HSP70 ASODN.Cytometric analysis showed the apoptotic rate was increased in a dose-and time-dependent manner in the HSP70 ASODN-treated cells.The percentage of cells in the G2/M and S phases was significantly decreased and that in the G0/G1 phase increased as the HSP70 ASODN concentration was elevated and the exposure time prolonged.Immunocytochemistry showed that treatment of SMMC-7721 cells with HSP70 ASODN resulted in decreased expressions of HSP70 and Bcl-2 proteins,and an increased expression of Bax protein.It was concluded that the HSP70 ASODN can inhibit the growth of the SMMC-7721 cells and increase cell apoptosis by down-regulating the expression of HSP70.HSP70 ASODN holds promise for the treatment of hepatocellular carcinoma.

Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD44-specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

Summary： The proliferating cell nuclear antigen （PCNA） gene expression was blocked and retinal pigment epithelium （RPE） proliferation was inhibited by using antisense oligonucleotides （AS-ODN） mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy （PVR）. RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN （0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides （S-ODN with the same concentrations as AS-ODN） mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by ^3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression ofPCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density （AOD） was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN （0.28 and 1.12 μmol/L） markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant （P〈0.05 and P〈0.01, repectively） as compared with blank control group. AOD was well correlated with CPM （r=0.975）. It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Antisense Oligonucleotide of Hypoxia-inducible Factor-1alpha Suppresses Growth and Tumorigenicity of Lung Cancer Cells A549

Summary： To study the role and mechanisms of hypoxia-inducible factor-lalpha （HIF-1α on the growth and tumorigenicity of lung cancer cells A549, the antisense oligonucleotide of HIF-1α was transfected to A549 cells. The effect of the antisense oligonucleotide on tumor growth in vitro and in vivo was evaluated by the growth rate suppression of A549 cells and subcutaneous implanted tumor in nude mice, and the effect on tumorigenicity was evaluated by the expression inhibition of angiogenic factors, the microvessel density （MVD）and vascular endothelial growth factor （VEGF） protein expression which were detected by immohistochemistry and western blot respectively. This study revealed that in vitro the growth rate of antisense oligonucleotide group was significantly decreased as compared with that of control group, sense oligonucleotide group and false-sense oligonucleotide group; in vivo the weight of implanted tumors in nude mice of antisense oligonucleotide group was 1.51±0.40 g, which was significantly lower than that of control group （2.79±0.33 g）, sense oligonucleotide group （2.81±0.45g） and false-sense oligonucleotide group （2.89±0.39 g） and the inhibitory rate was 47 %. Both MVD and VEGF protein expression were significantly inhibited in antisense oligonucleotide group compared with those in other groups. These results indicated that antisense oligonucleotide of HIF-1α could inhibit lung cancer cells A549 growth in vitro and in vivo, and the mechanism may be due to the inhibition of vascular growth and VEGF protein expression.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

Obstructive Effects of Ultrasonic Microbubble Intensifier on CHG-5 Cell with Survivin Antisense Oligonucleotides Transfection

Objective： To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides （ASODN） transfection. Methods： Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. Four regimen groups were designed, group A： survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation, group B： survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation, group C： survivin antisense oligonucelotides with lipofectamine transfection, group D： blank control. The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting, and MTT assay was used to measure the changes of proliferation. Results： Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups（P〈0.05）, and the proliferating rate of CHG-5 in group A was also significantly inhibited（P〈0.05）. Conclusion： Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.

Effect of Antisense Oligonucleotide to AnnexinⅡon the t-PA-mediated Plasmi nogen Activation in vitro

In order to study the role of annexin Ⅱ, a recombinant expression vector, pZeoSV2(+)ANN Ⅱ, containing the annexin Ⅱ cDNA , was developed. The 1.1 kb length annexin Ⅱ cDNA was inserted into a express ion vector, PZeoSV(+) and transfected into HL 60 cells which had low baseline e xpression of Ann Ⅱ. pZeoSV(+) ANNⅡ was analyzed by restriction mapping and th e Ann Ⅱ sequence identified. The ability of the transfected cells, non transf ected and mock transfected cells to stimulate t PA depend plasminogen activat ion was compared. The results showed that HL 60 with pZeoSV(+)ANNⅡ transfectio n could significantly increase the plasminogen activation (8.9±1.2 U) in vitr o with the difference being significant as compared with non transfected (1.5±0. 4 U) and mock transfected cells (4.2±0.9 U), respectively. AntiannexinⅡoligon ucleotides significantly inhibited the binding ability of t PA and plasminogen to annexinⅡ, and obviously reduced the plasminogen activation in vitro . The above findings showed human umbilical vein endothelial cells (HUVECs) treated w ith sense or missense oligonucleotides indicated no significant change in bindin g of t PA and PLG. Treatment of HUVECs with antiannexin Ⅱ oligonucleotides cou ld significantly reduce the plasminogen activation by 2.4±0.3 U as compared wit h sense oligonucleotide group in binding of t PA and PLG. These results, theref ore, suggest that Ann Ⅱ can bind plasminogen and participate in the stimulatio n of t PA dependent activation of plasminogen, and that interference with Ann Ⅱ mRNA by antisense oligonucleotide may be a new strategy for the therapy of bleeding in patients with hyperfibrinolysis.

Antisense Oligonucleotide Targeting TGF-β1 Abrogates Tumorigenicity of Rhabdomyosarcoma in vivo

OBJECTIVE Over-expression of transforming growth factor β1 （TGF-β1） has been observed in many advanced cancers. The present study was aimed at developing potential antisense oligonucleotides （ASONs） to repress TGF-β1 expression in rhabdomyosarcoma （RMS） RD cells, and to examine their effect on tumorigenicity of RD cells in vivo. METHODS ASONs targeting the region surrounding the start codon of TGF-β1 were synthesized and transferred into cells in the form of complexes with Lipofectamine 2000. The TGF-β1 protein was determined by immunofluorescence and ELISA. The cell viability and cell cycle were examined by MTT and flow cytometry. The RD cells, with or without TGF-β1ASON, in 50 μl of serum-free EMDM medium were injected subcutaneously into the right flank of nude mice. The tumors were then measured and weighed. RESULTS The ASON sequence targeting the first start site at bases 841-855 of the human TGF-β1 gene had the greatest effect on attenuating the expression of TGF-β1 （P 〈 0.05）. The ASONs induced a decrease in OD values after 6 d （P 〈 0.05）. Analysis of the cell cycle revealed that the ASON induced a significant decrease in cells in the S phase and an increase in cells in the G1 phase （P 〈 0,05）. In the nude mice model, the mean tumor volume, after 2 weeks of treatment with Lipofectamine or ASON, decreased to 88.5% or 55% respectively, compared to the control tumor size, resulting in a significant difference （P 〈 0.01）. CONCLUSION The sequence of the ASON, which targeted the start condon at the bases 841-855 of the human TGF-β1 gene, was demonstrated to be a useful agent for studying the regulation of TGF-β1 over-expression in RD cells, and has important therapeutic potential for suppressing the tumorigenicity of human RMS in vivo.

Suppression of intracranial glioma tumorigenesis with vascular endothelial growth factor antisense oligonucleotide in rats

Objective: To observe the inhibition of intracranial glioma tumorigenesis by vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide (ODN) in rats. Methods: Totally 20 μ1 Hank's liquid containing 1×106 C6 glioma cells was seeded into rat right caudate putaraen in high-flow microinfusion with stereotactic technique. VEGF antisense ODN was simultaneously used with glioma cell. Each rat of the treated group Ⅰ and the treated group Ⅱ was treated with 1 000 μmol/L VEGF antisense ODN. Each rat of the treated group Ⅲ and the treated group Ⅳ was treated with 2 000 μmol/L VEGF antisense ODN. The experimental periods of the treated group Ⅰ , the treated group Ⅲ and the control group Ⅰ were 2 weeks, those of the treated group Ⅱ , the treated group Ⅳ and the control group Ⅱ were 3 weeks. Before sacrifice, MRI was performed on each rat. Tumor magnitude and pathologic examination were detected after samples were dissected. Results: The survival state of all treated rats was better, and that of the control rats was in severe danger. The tumor volumes of the treated group Ⅰ and the treated group Ⅱ were remarkably lessened. Tumor tissue could not be found macroscopically in the brain samples of the treated group Ⅲ and the treated group Ⅳ, but tumor nest could be found with microscopy. Tumors of the treated group I and the treated group Ⅱ had weak expressions of VEGF mRNA and VEGF, while normal brains and the samples of the treated group Ⅲ and the treated group Ⅳ had negative expressions, but tumors of the control groups had strong expressions. Conclusion: VEGF antisense ODN used early in situ can suppress angiogenesis and growth of rat intracranial glioma to retard tumorigenesis.

Effects of antisense oligonucleotides targeting VEGF on radio sensitivity of uterine cervix cancer Hela cells

Objective： To determine the impact of antisense oligonucleotides targeting vascular endothelial growth factor （VEGF） on radiosensitivity of uterine cervix cancer Hela cells. Methods： VEGF antisense oligodeoxynucleotides （ASODN） was transfected into Hela cells by liposome-mediated method. Cells transfected with the oligodeoxynuclecotide and saline were used as control groups. Cells were irradiated by 6 MV X ray at the dose of 0 Gy, 2 Gy, 4 Gy and 6 Gy respectively. The expression of VEGF mRNA was determined by RT-PCR. Apoptosis were evaluated using FCM. Cloning efficiency was determined by colony formation assay. Results： The expression of VEGF mRNAwas inhibited by ASODN （P 〈 0.01） in Hela cells. The inhibited activation which was influenced by radiation resulted in increasing apoptosis （P 〈 0.01） and inhibiting plating efficiency （P 〈 0.01）. Conclusion： The expression of VEGF induced by X irradiation in Hela cells can be blocked by VEGF ASODN. Treatment with VEGF might increase apoptosis in HeLa cells and enhance radiosensitivity.

Following Inhibition of BCL-2 by Antisense Oligonucleotides Compensatory Suppression of Apoptosis Involves the Direct Signal Transduction Pathway of LNCaP Cells

Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)];and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.