C-myc Antisense Oligodeoxynucleotides Research Articles

Magnetic-silk/polyethyleneimine core-shell nanoparticles for targeted gene delivery into human breast cancer cells

The lack of efficient and cost-effective methods for gene delivery has significantly hindered the applications of gene therapy. In this paper, a simple one step and cost effective salting-out method has been explored to fabricate silk-PEI nanoparticles (SPPs) and magnetic-silk/PEI core-shell nanoparticles (MSPPs) for targeted delivery of c-myc antisense oligodeoxynucleotides (ODNs) into MDA-MB-231 breast cancer cells. The size and zeta potential of the particles were controlled by adjusting the amount of silk fibroin in particle synthesis. Lower surface charges and reduced cytotoxicity were achieved for MSPPs compared with PEI coated magnetic nanoparticles (MPPs). Both SPPs and MSPPs were capable of delivering the ODNs into MDA-MB-231 cells and significantly inhibited the cell growth. Through magnetofection, high ODN uptake efficiencies (over 70%) were achieved within 20 min using MSPPs as carriers, exhibiting a significantly enhanced uptake effect compared to the same carriers via non-magnetofection. Both SPPs and MSPPs exhibited a significantly higher inhibition effect against MDA-MB-231 breast cancer cells compared to human dermal fibroblast (HDF) cells. Targeted ODN delivery was achieved using MSPPs with the help of a magnet, making them promising candidates for targeted gene therapy applications.

Galactosylated poly-L-lysine targeted microbubbles for ultrasound mediated antisense c-myc gene transfection in hepatocellular carcinoma cells.

IntroductionThe aim of the study was to investigate the efficiency of delivery and targeted binding of c-myc antisense oligodeoxynucleotide (ASODN) and find a novel therapy for hepatic carcinoma.Material and methodsA targeted ultrasound microbubble compound was synthesized to deliver the c-myc ASODN by ultrasound-targeted microbubble destruction (UTMD) and applied in hepatocellular carcinoma cells (HCC) and cancer bearing mice. Lipid microbubbles were conjugated with biotinylated galactosylated poly-L-lysine (G-PLL) and SonoVue to target the hepatocellular carcinoma SMMC7721 cells with asialoglycoprotein receptors. There were four groups in both in vitro and in vivo studies: control group (group A); c-myc ASODN + G-PLL (CG group, group B); c-myc ASODN + SonoVue (CUS group, group C); c-myc ASODN + G-PLL + SonoVue (CGUS group, group D). The expression of c-myc mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and proliferation investigations of the SMMC7721 cells were also performed. In addition, the tumor volume was calculated and compared among different groups.ResultsThe level of c-myc mRNA in the three experimental groups was significantly lower than that in the control group in vitro (p < 0.05). Furthermore, c-myc gene expression was suppressed more strongly in the CGUS group compared with other groups in both in vitro and in vivo studies (p < 0.05). In addition, ultrasound mediation of targeted microbubbles yielded the highest inhibition of tumor growth and cell proliferation among the four groups.ConclusionsThe use of a G-PLL targeted microbubble contrast agent combined with ultrasound exposure could be a potential method for increasing gene delivery efficiency. This technique is a promising nonviral approach that can be used in liver cancer.

Liposome-mediated induction of apoptosis of human hepatoma cells by c-myc antisense phosphorothioate oligodeoxynucleotide and 5-fluorouracil.

The aim of this study was to investigate the effect of a c-myc antisense oligodeoxynucleotide and 5-fluorouracil on the expression of c-myc, invasion and proliferation of HEPG-2 liver cancer cells. HEPG-2 cells were treated with lipiosome-mediated c-myc ADSON and 5-fluorouracil. The proliferation inhibition rate and invasion were measured by MTT and invasion assay, respectively. Cell apoptosis was detected by flow cytometry and expression of c-myc by RT-PCR and immunohistochemistry. The proliferation inhibition rate was significantly higher in the antisense oligodeoxynucleotide added-5-fluorouracil group than single antisense oligodeoxynucleotide or 5-fluorouracil group (p<0.05). G0/G1 cells in the antisense oligodeoxynucleotide group and S cells in the 5-fluorouracil groups were significantly increased than that in the control group, respectively (P<0.01). The amplification strips of PCR products in 5-FU, ASODN and combination groups were significantly weaker than that in the control group (P<0.01). The percentage of c-myc-protein- positive cells were significantly lower in antisense oligodeoxynucleotide, 5-fluorouracil and combination groups than that in the control group (P<0.01). A liposome-mediated c-myc antisense oligodeoxynucleotide and 5-fluorouracil can inhibit the proliferation and invasion of liver cancer cells by reducing the expression of c-myc. A c-myc antisense oligodeoxynucleotide can increase the sensitivity of liver cancer cells to 5-fluorouracil and decrease the dosage of the agent necessary for efficacy, providing an experimental basis for the clinical therapy of liver cancer.

Nucleic Acid Drugs for Preventing Restenosis after Coronary Revascularization

Recent progress in molecular and cellular biology has resulted in the development of numerous effective drugs. However, there are still a number of diseases for which no known effective therapy exists, such as ischemic heart disease, vascular bypass graft failure and heart failure. Despite its limitations, oligodeoxynucleotide (ODN)-based therapy is emerging as a potential strategy for the treatment of patients with cardiovascular disease that is resistant to current therapeutic approaches. Indeed, several nucleic acid drugs and delivery methods for heart disease have been developed and their efficacy has been investigated in animal models. Among them, some agents have undergone clinical trials, such as cmyc antisense ODN, E2F and NFκB decoy ODN. However, none of the large randomized placebo-controlled trials has shown conclusive evidence of clinical benefit. Recent experimental studies suggested that siRNA- and miRNA-based strategies have potential as a potent therapeutic approach for the treatment of restenosis. In addition, simultaneous regulation of multiple intracellular signaling pathways is expected to enhance the therapeutic effects. This review focuses on the potential of recent ODN-based gene therapy for the treatment of heart disease, especially restenosis after revascularization.

Inhibition of apoptosis induced by galactose by c-myc antisense oligodeoxynucleotide in lens epithelial cells

Background Various studies demonstrated that the apoptosis of lens epithelial cells(LECs) is associated with the overexpression of the c-myc gene in LECs induced by galactose.Inhibiting the abnormal expression of the c-myc gene in LECs is an effective approach to mitigate the pathogenesis and development of cataract.Objective The goal of this study is to investigate the inhibitory effects of c-myc antisense oligodeoxynucleotide(c-myc ASODN) on the apoptosis of LECs in the eye with galactose-induced cataract.Methods Galactose-induced cataract models were established by the retrobulbar injection of 0.2 mL of 20% galactose once per day.Lipo-antisense oligodeoxynucleotide(Lipo-ASODN,0.2 mL) was retrobulbarly injected 4 hours after the injection of galactose at one-day intervals.The animals were sacrificed and lenses were obtained to evaluate the apoptosis of LECs and the effect of c-myc ASODN on LECs apoptosis induced by galactose was examined by TUNEL assay after 7,14 and 24 days.The ultrastructural changes of LECs were examined under the transmission electron microscopy(TEM).Results A significant difference in the apoptotic rate of LECs was found among the 7 day,14 day and 24 day groups(F_(7 days)=3 418.495,P＜0.01;F_(14 days)=1137.555,P＜0.01;F_(24 days)=2198.871,P＜0.01).The apoptotic rate of LECs in the galactose group was markedly higher than that in the normal saline solution group 7 days,14 days and 24 days after the experiment(P＜0.01).The apoptotic rate of LECs in the galactose+lipo+ ASODN group significantly declined in comparison to the galactose group after 7 days,14 days and 24 days(P＜0.05).TUNEL assay showed the condensation,breakage and irregularity of the nuclei of apoptotic cells in the galactose group.The destruction of the ultrastructure of the cells and organelles were observed under the transmission electron microscope.Conclusion Galactose induces apoptosis of LECs in cataractogenesis.C-myc ASODN inhibits apoptosis of LECs induced by galactose. Key words: c-myc antisense oligodeoxynucleotide; galactose; cataract; cell apoptosis

Controlled delivery of anti-sense oligodeoxynucleotide from multilayered biocompatible phosphorylcholine polymer films

Fabrication of polymeric multilayered films based on the electrostatic self-assembly of polycations and polyanions is a promising approach for controlled loading and release in gene delivery. In this study, we have fabricated a series of multilayered films based on alternate deposition between positively-charged cationic phosphorylcholine copolymer (PC copolymer) and negatively-charged c-myc anti-sense oligodeoxynucleotide (AS-ODN). The growth of film thickness and increase of ODN loading capacity were monitored by spectroscopic ellipsometry (SE) and confocal laser scanning microscopy (CLSM). After elution into PBS buffer under physiological conditions, the elution profile was monitored by UV spectrometry and gel electrophoresis. Employing a secondary transgenic vector, the cellular uptake of the eluted AS-ODN into HeLa cells was evaluated by fluorescent microscopy and FACS analysis. The biological effect of eluted AS-ODN was evaluated by cell growth inhibition. The results showed that AS-ODN loading capacity increased almost linearly with the number of PC polymer/ODN bilayers and was also strongly dependent upon the cationic charge density. Through swelling, a non-degradable release mechanism, the AS-ODN release was characterized by two distinguishable release regimes: a fast release regime during the first 6 hour period and a slow release regime from 6 hour to the 8th day, both of which were characterized by zero-order kinetics. Gel electrophoresis showed excellent DNA integrity and strong transfection was observed when the eluted ODN was transfected into HeLa cells. Cell growth was significantly inhibited by eluted AS-ODN, indicating its full bioactivity. These results demonstrate that PC multilayered polymer films are capable of delivering a prescribed amount of anti-sense ODN with a controllable kinetic profile and that the multilayer process is more efficient and reliable than most other existing coating approaches largely based on single-layer fabrication.

Increase of therapeutic effects by treating melanoma with targeted combinations of c-myc antisense and doxorubicin

Patients with advanced or metastatic melanoma have a very poor prognosis, due to the resistance of melanoma cells to conventional chemotherapy. We previously reported that coated cationic liposomes targeted with a monoclonal antibody against the disialoganglioside GD 2 and containing c-myc antisense oligodeoxynucleotides (αGD 2-CCL[c-myc-as]) induced partial tumor growth arrest in melanoma xenografts. Here we addressed the role of c-myc-asODN treatment in the susceptibility to doxorubicin (DXR) in human melanoma cells. Cytotoxicity studies revealed that growth of melanoma cells was inhibited to a greater extent by αGD 2-CCL[c-myc-as] than by the corresponding non-targeted formulations or by free c-myc-as. Targeted c-myc-as sensitized cells to DXR, reducing the IC 50 by approximately 10-fold. Scrambled ODNs had no effect on the IC 50 of DXR. Compared to either treatment alone, combination of targeted c-myc-as and DXR resulted in earlier apoptosis and in cell death after 2 days of treatment. In vivo experiments revealed that liposomal formulations of c-myc-as and DXR, both targeted via GD 2, led to the most pronounced delay in tumor growth when administered in a sequential manner. As a result, their combination translates into a statistically significant suppression of blood vessel density and an enhanced apoptosis, compared to all treatments given separately. Our data indicate the increasing cell sensitivity to DXR by c-myc-asODNs as a promising basis for developing novel anti-tumor strategy against advanced melanoma.

The effect of locally deliveredc-mycantisense oligonucleotides combined with intravascular brachytherapy of188Re liquid-filled balloon therapy on vascular smooth muscle cell proliferation in rabbit iliac arteries after injury with a balloon catheterThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual

Poststenting restenosis is a significant clinical problem that involves vascular smooth muscle cell (VSMC) proliferation. We primarily investigated the effect of c-myc antisense oligodeoxynucleotides (ASODNs) combined with 188Re radiation therapy on VSMC proliferation in rabbit common iliac arteries injured by the porous balloon catheter to explore the therapeutic potential of the combined therapy for the prevention of restenosis. The iliac arteries in rabbits were injured with balloon catheters, and radiation therapy was carried out with a 2.5 mm balloon catheter filled with 188Re (8 or 15 Gy), and ASODNs (300 microg) were applied to the adventitia introduced using a pluronic gel releasing system and a lipofectin delivery system. After 3 weeks, the animals were killed and defined segments of arteries were sectioned. The histological sections were stained using alpha-actin immunohistochemistry staining. The positive alpha-actin ratios were calculated and analyzed statistically among groups. In contrast to the rate of alpha-actin positive cell staining in the control group, the rate of alpha-actin positive cell staining did not decrease (P > 0.05) in the 188Re-irradiated group (8 Gy). However, in the ASODN-treated group, the 188Re-irradiated group (15 Gy), and the combined ASODN - 188Reirradiated groups (8 or 15 Gy), the ratios had markedly decreased (P < 0.01). The effect of the combined group (ASODNs + 188Re (15 Gy)) provided the lowest level of alpha-actin positive cell staining (P < 0.01). The ASODNs (300 microg) effectively decreased VSMC poliferation. The effect of the 188Re radiation on the VSMCs depended on the dosage. The ASODNs (300 microg) and combined 188Re irradiation effectively lowered VSMC proliferation, and the effect was better than that achieved with any other treatment.

Mechanism of c-myc Antisense Oligodeoxynucleotide Mediated by Receptor on the Proliferative Inhibition of Bel-7402 Cells

Objective To observe the mechanism of c-myc antisense oligodeoxynucleotide mediated by galactose receptor on the proliferative inhibition of hepatocarcinoma Bel-7402 cells.Methods c-myc ASODN mixed with Galactose(Gal)- polyethyleneimine(PEI) reagent forming Gal-PEI-ASODN complex,then incubated with Bel-7402 cells for 48h,using Propidium Iodide(PI) dying,AnnexinV-FITC and PI double staining,DNA gel electrophoresis to detected the proliferative inhibition mechanism of Gal-PEI-ASODN on Bel-7402 cells.Results Cell cycle was determined by flow cytometry.In the control group,Gal-PEI group and ASODN group,cell proliferative index was found to be 35.04% , 33.95% , 32.90% ,respectively.However,cell proliferative index for Gal-PEI-ASODN group was found to be 23.65% ,decreased by 11.39% compared to the control group(P 0.01 ).Necrotic cell percentage was determined using AnnexinV-FITC/ PI double staining test.The results showed cell apoptotic rate of 2.77% ,cell necrotic rate of 3.06% and the normal cell percentage was detected as 94.13% in the control group;cell apoptotic rate below 4%,cell necrotic rate below 6.8% were detected in both ASODN group and Gal-PEI group; cell apoptotic rate of 6.5% ,cell necrotic rate of 15.9% and the normal cell percentage of 76% were determined in Gal-PEI-ASODN group.There was no DNA ladder morphology,on the contrary,there was DNA smear morphology using DNA gel electrophoresis in Gal-PEI-ASODN group.Conclusion The mechanism by which Gal-PEI-ASODN inhibits cell proliferation maybe that it can restrain the change from G_ 0 /G_ 1 period to S period in cell cycle and induces cell necrosis.

Controlled Delivery of Antisense Oligodeoxynucleotide from Cationically Modified Phosphorylcholine Polymer Films

Antisense strategy is a promising approach for the prevention of in-stent restenosis if therapeutic agents such as antisense oligodeoxynucleotides (AS-ODNs) can be successfully delivered to the implant site. Optimizing the routes and conditions for controlled loading and release of therapeutic agents from a biocompatible polymer coating is still required. In this study, phosphorylcholine (PC) polymer films bearing different cationic charge densities were deposited onto smooth silicon substrates. The thickness of these films was determined by spectroscopic ellipsometry (SE). Human c-myc AS-ODNs were incorporated into the PC polymer films by immersion in concentrated AS-ODN solution and eluted into PBS under physiological conditions. The elution profile was monitored by UV spectrometry and gel electrophoresis. Cellular uptake of the eluted AS-ODN into vascular smooth muscle cells (VSMCs) was evaluated by fluorescence microscopy. The results showed that ODN loading capacities increased with film thickness and were also strongly dependent on the cationic charge density. AS-ODN release was characterized by a slight initial burst in the first half hour followed by a period of sustained release up to 8 days. Gel electrophoresis demonstrated DNA integrity, and different transfection efficiencies were observed when the eluted ODNs were transfected into VSMCs. These results demonstrated that cationically modified PC polymers are capable of delivery of antisense ODNs in a controlled manner and that they are well suited for specific biomedical devices such as DNA-eluting stents.

Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc.

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27(kip1) levels.

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G(1) phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27(kip1) levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27(kip1) in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G(1) arrest and activates apoptosis by increasing p27(kip1) content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G(1) checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

A mechanistic study of proliferation induced by Angelica sinensis in a normal gastric epithelial cell line

It has been reported that an extract from Angelica sinensis mainly consisting of polysaccharides (95%) prevented ethanol- or indomethacin-induced gastric mucosal damage (Cho CH et al. Planta Med 2000;66:348–51). However, it is not known whether Angelica sinensis has a direct stimulatory effect on the healing of gastric mucosal lesions. To study the hypothesis that Angelica sinensis has a direct mucosal healing effect in rats and in isolated gastric epithelial cells, we assessed the wound repair in both animals and normal cell culture (RGM-1), as well as [ 3H]thymidine incorporation, ornithine decarboxylase (ODC) activity, and ODC protein and c-Myc protein expression after different treatments in RGM-1 cells. We found that Angelica sinensis crude extract (ASCE) dose-dependently enhanced gastric ulcer healing in rats and promoted wound repair in RGM-1 cells. It also significantly stimulated [ 3H]thymidine incorporation and ODC activity in RGM-1 cells in a concentration-dependent manner. ODC and c-Myc protein expression was also increased as a result of this process. dl-α-difluoromethyl-ornithine repressed the [ 3H]thymidine incorporation and ODC activity induced by ASCE. Pretreatment with c-Myc antisense oligodeoxynucleotides blocked the stimulatory action of ASCE on [ 3H]thymidine incorporation and ODC protein expression. These data suggest that ASCE has a direct mucosal healing effect on gastric epithelial cells, while ODC and c-Myc are closely associated with this effect.

Effect of c-myc antisense oligodeoxynucleotides on hypoxia-induced proliferation of pulmonary vascular pericytes.

To study the effect of c-myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin-11-dUTP-labeled cDNA, 3H-thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c-myc antisense ODNs on expression of c-myc gene and proliferating cell nuclear antigen (PCNA), and 3H-thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c-myc and PCNA (P < 0.01), and elevate 3H-thymidine incorporation of PC (P < 0.01), but antisense ODNs could significantly inhibit the expression of c-myc and PCNA (P < 0.05), and 3H-thymidine incorporation of PC (P < 0.01). It was suggested that hypoxia could promote the proliferation of PC by up-regulating the expression of c-myc gene, but c-myc antisense ODNs could inhibit hypoxia-induced proliferation of PC by downregulating the expression of c-myc gene.

Identification of a Good C-MYC Antisense Oligodeoxynucleotide Target Site and the Inactivity at This Site of Novel NCH Triplet-Targeting Ribozymes

A region of c-myc mRNA was identified which permitted very efficient antisense effects to be achieved in living cells using chimeric methylphosphonate-phosphodiester antisense effectors. Novel inosine—containing ribozymes (which cleave after NCH triplets) were directed to an ACA triplet within this region and delivered into living cells. No ribozyme intracellular activity could be identified. Very low ribozyme function was also observed in in vitro assays using a 1700nt substrate RNA.

Implication of c-Myc in apoptosis induced by the retinoid CD437 in human lung carcinoma cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been recently identified to be a potent inducer of apoptosis in human non-small cell lung carcinoma (NSCLC) cells through a nuclear retinoic acid receptor independent mechanism. To approach the mechanism by which CD437 induces apoptosis in NSCLC cells, we investigated the involvement of c-Myc in CD437-induced apoptosis. CD437 (1 microM) up-regulated the expression of c-Myc and of its downstream target genes ornithine decarboxylase (ODC) and cdc25A in all three NSCLC cell lines (i.e., H460, SK-MES-1 and H1792) used. These effects were correlated with cellular susceptibilities to induction of apoptosis by CD437. Furthermore, CD437-induced apoptosis could be blocked by the ODC inhibitor difluoromethylornithine, the caspase inhibitors Z-VAD FMK and Z-DEVD FMK, and c-Myc antisense oligodeoxynucleotide, respectively. These data indicate that c-Myc gene plays an important role in mediating CD437-induced apoptosis in human NSCLC cells.

C-myc antisense oligodeoxynucleotides can induce apoptosis and down-regulate Fas expression in rheumatoid synoviocytes.

To investigate the role of c-myc in the pathogenesis of rheumatoid arthritis (RA) and the mechanism of synovial apoptosis. Using cultured human synoviocytes from patients with RA and c-myc antisense oligodeoxynucleotides (AS ODN), we examined the inhibition of cell proliferation by the MTT assay and the induction of apoptosis with TUNEL staining and fluorescence microscopy. In addition, the effect of c-myc on down-regulation of Fas expression was analyzed by flow cytometry, cytotoxicity assay, and reverse transcriptase-polymerase chain reaction. Treatment with c-myc AS ODN induced inhibition of cell proliferation, along with down-regulation of c-Myc protein and c-myc messenger RNA (mRNA) expression. The morphologic changes of synovial cell death were typical of apoptosis. In addition, c-myc AS ODN treatment down-regulated expression of Fas mRNA but not Fas antigen. Analysis of the involvement of the caspase cascade revealed that the cytotoxic activity of c-myc AS ODN was completely blocked by inhibitors of both caspase 1 (YVAD-FMK) and caspase 3 (DEVD-FMK). Our results strongly suggest that c-myc AS ODN might be a useful therapeutic tool in RA and clarify that cell death by c-myc AS ODN is induced through the caspase cascade, similar to Fas-induced apoptosis. In addition, combination therapy with anti-Fas antibody and c-myc AS ODN reduced Fas-dependent cytotoxicity.

Glucose deprivation-induced cytotoxicity in drug resistant human breast carcinoma MCF-7/ADR cells: role of c-myc and bcl-2 in apoptotic cell death.

We investigated the effect of glucose deprivation treatment on clonogenicity in multidrug-resistant human breast carcinoma MCF-7/ADR cells. Survival of MCF-7/ADR cells decreased exponentially up to 8 hours of incubation in the glucose-free medium. The surviving fraction of these cells for 8 hours of glucose-deprivation treatment was 1.5 x 10(-3). Photomicrographs and gel electrophoresis data suggest that glucose deprivation-induced cell death is associated with apoptosis. Data from western and northern blots showed an induction of c-myc gene expression during treatment with glucose-free medium in MCF-7/ADR cells. MCF-7/ADR cells transfected with c-myc antisense oligodeoxynucleotides became resistant to glucose deprivation-induced apoptosis. Overexpression of bcl-2 gene protected MCF-7/ADR cells from this apoptotic cell death. Taken together, these results indicate that c-myc expression is a necessary component of glucose-free medium induced apoptosis and bcl-2 prevents apoptotic death induced by c-myc.

Antisense Oligodeoxynucleotide Combination Therapy of Primary Chronic Myelogenous Leukemia Blast Crisis in SCID Mice

The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC. To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c-myc antisense phosphorothioate oligodeoxynucleotides ([S]ODNs), individually or in combination. Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense [S]ODNs resulted in a synergistic antiproliferative effect. Colony formation of normal bone marrow cells was not affected by either treatment. To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense [S]ODNs or with a combination of both antisense [S]ODNs. Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both [S]ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions. These effects correlated with a markedly increased survival of leukemic mice treated with both antisense [S]ODNs. Leukemic cells harvested from antisense [S]ODN-treated mice were sensitive to the effects of antisense [S]ODNs in vitro, suggesting that the treatment can be successfully repeated. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.