Bcl-2 Antisense Oligodeoxynucleotides Research Articles

Slow Release and Delivery of Antisense Oligonucleotide Drug by Self-Assembled Peptide Amphiphile Nanofibers

Antisense oligonucleotides provide a promising therapeutic approach for several disorders including cancer. Chemical stability, controlled release, and intracellular delivery are crucial factors determining their efficacy. Gels composed of nanofibrous peptide network have been previously suggested as carriers for controlled delivery of drugs to improve stability and to provide controlled release, but have not been used for oligonucleotide delivery. In this work, a self-assembled peptide nanofibrous system is formed by mixing a cationic peptide amphiphile (PA) with Bcl-2 antisense oligodeoxynucleotide (ODN), G3139, through electrostatic interactions. The self-assembly of PA-ODN gel was characterized by circular dichroism, rheology, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM images revealed establishment of the nanofibrous PA-ODN network. Due to the electrostatic interactions between PA and ODN, ODN release can be controlled by changing PA and ODN concentrations in the PA-ODN gel. Cellular delivery of the ODN by PA-ODN nanofiber complex was observed by using fluorescently labeled ODN molecule. Cells incubated with PA-ODN complex had enhanced cellular uptake compared to cells incubated with naked ODN. Furthermore, Bcl-2 mRNA amounts were lower in MCF-7 human breast cancer cells in the presence of PA-ODN complex compared to naked ODN and mismatch ODN evidenced by quantitative RT-PCR studies. These results suggest that PA molecules can control ODN release, enhance cellular uptake and present a novel efficient approach for gene therapy studies and oligonucleotide based drug delivery.

Enhanced sensitivity of BIU87 cells to cisplatin by bcl-2 antisense oligodeoxynucleotides

Objective To observe the influence of bcl-2 antisense oligodeoxynucleotides (ASON)on reversal of cisplatin resistance in BIU87 cell line of bladder carcinoma. Methods Cisplatin resistance bladder carcinoma cells served as models. The bcl-2 antisense oligodeoxynucleotides were transfected to the cells by lipofectamine and electroporation. The sense-and random-oligodeoxynucleotides were used as controls. The expression level of bcl-2 mRNA was detected by using semi-quantitative reverse transcription polymerase chain reaction ( RT-PCR) . The apoptosis of bladder cancer cell lines was examined by DNA ladder assay. The protein level and 50% inhibitory concentration (IC50) were measured by immunofluorescence technique and methyl thiazol tetrazolium (MTT) assay, respectively. Results Transfecting BIU87/DDP cells with antisense oligonucleotides statistically reduced IC50, values of BID/DDP cells from (66. 000 ±4. 637) to (20. 400 ±2. 590) mg/L. Twelve h after transfection by lipofectamine, 30% cells had apoptotic or necrotic changes under the inverted phase contrast microscopy, and DNA Ladder was observed with agrose gel electrophoresis in the antisense-transfected cells. Forty-eight h after transfection by electroporation followed by treatment of 10 mg/L cisplatin, RT-PCR showed the expression of bcl-2 mRNA in the transfected cells was significantly lower (0. 72 ± 0. 07 ) than before transfection ( 2. 94 ± 0. 09 ) , and that of bcl-2 protein was decreased, as well as the IC50. Conclusion bcl-2 antisense oligodeoxynucleotides can reverse the resistance to cisplatin in BIU87/DDP cells by inhibiting the expression of bcl-2 protein and increasing the apoptosis. Key words: bcl-2; Antisense oligodeoxynucleotides; Bladder carcinoma

Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment

We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.

Phase I study of apoptosis gene modulation with oblimersen within preoperative chemotherapy in patients with primary breast cancer

Expression of the Bcl-2 protein confers resistance to chemotherapy-mediated apoptotic signals in patients with breast cancer. We investigated effects of Bcl-2 down-regulation by the Bcl-2 antisense oligodeoxynucleotide oblimersen in breast tumor biopsies. Oblimersen targets Bcl-2 messenger RNA (mRNA), down-regulates Bcl-2 protein translation and enhances antitumor effects of subtherapeutic chemotherapy doses. Within a phase I trial, we administered escalating doses of oblimersen (3, 5 or 7 mg/kg/day) as continuous infusion on days 1–7 in combination with standard-dose docetaxel (Taxotere), Adriamycin and cyclophosphamide (TAC) on day 5 as preoperative chemotherapy in 28 patients with T2–4 tumors. Effects of oblimersen were evaluated in tumor biopsies and peripheral blood mononuclear cells (PBMCs) 4 days after start of oblimersen and before TAC treatment by quantitative microfluidic real-time PCR. Read-outs consisted in measurement of Bcl-2 mRNA modulations and of 18 putative predictive markers. Two of 13 patients showed a diminution of Bcl-2 transcripts after 4 days of treatment with oblimersen 5 mg/kg/day. PBMCs could not be evaluated as a surrogate tissue because no qualified RNA could be isolated. Nevertheless, we demonstrated feasibility to process clinical samples and to obtain good quality RNA from tumor biopsies and indicated the potential of oblimersen to lower Bcl-2 mRNA in breast cancer.

Dual targeting of Bcl-2 and VEGF: A potential strategy to improve therapy for prostate cancer

We previously demonstrated that Bcl-2 overexpression stimulates angiogenesis in PC-3 human prostate cancer cells, thus giving these tumors a growth advantage. To further elucidate the relationship between Bcl-2 and vascular endothelial growth factor (VEGF) in PC-3- Bcl-2 cells, tumorigenicity and angiogenesis were evaluated in our in vitro and in vivo model treated with antisense Bcl-2 oligodeoxynucleotide (ASO) and bevacizumab. In vitro and in vivo angiogenesis assays, as well as a xenograft tumor model of the human prostate cancer cell line PC-3- Bcl-2, were subjected to ASO alone, bevacizumab alone, or the combination of ASO and bevacizumab. Protein-based assays (e.g., immunohistochemical staining and enzyme-linked immunosorbent assay [ELISA]) were utilized to detect molecular changes. Interestingly, targeting Bcl-2 with ASO resulted in the inhibition of in vitro tube formation and inhibition of angiogenesis in Matrigel plugs similar to treatment with bevacizumab. In our PC-3- Bcl-2 xenograft model, ASO alone resulted in 41% reduction in tumor size, bevacizumab alone resulted in a 50% reduction in tumor size, whereas the combination of ASO with bevacizumab was associated with >95% reduction in tumor volume. Reduction in tumor size in all groups was associated with reduction in Bcl-2 and VEGF expression, induction of apoptosis, and inhibition of angiogenesis and its associated chemokine production. These findings confirm that Bcl-2 is a pivotal target for cancer therapy and thus, further study of this novel combination of Bcl-2 reduction and angiogenic targeting in human tumors is warranted.

Phase I study of apoptosis gene modulation with oblimersen within preoperative chemotherapy in patients with primary breast cancer

e11576 Background: The expression of the Bcl-2 protein confers resistance to chemotherapy mediated apoptotic signals in patients with breast cancer. The development of chemo sensitizing drugs may result in new treatment options. We investigated the effects of Bcl-2 down-regulation by the Bcl-2 antisense oligodeoxynucleotide oblimersen in breast tumor biopsies. Oblimersen targets Bcl-2 mRNA, thereby down regulates Bcl-2 protein translation, and enhances the antitumor effects of sub therapeutic doses of docetaxel. Methods: Within a phase I trial we administered escalating doses of oblimersen (3/5/7mg/kg/day) as continuous infusion on day 1–7 in combination with standard dosed docetaxel, doxorubicin, cyclophosphamide (TAC) on day 5 as preoperative chemotherapy in 28 patients with T2–4 breast cancers. Effects of oblimersen were evaluated in tumor biopsies and peripheral blood mononuclear cells (PBMCs) 4 days after start of oblimersen continuous infusion and before TAC treatment by quantitative microfluidic real-time polymerase chain reaction (RT-PCR). Read-outs consisted in measurement of Bcl-2 mRNA modulations and of 18 other putative predictive markers. Results: Two out of 13 patients showed a diminution of Bcl-2 transcripts after 4 days of treatment with oblimersen 5 mg/kg/day. PBMCs could not be evaluated as a surrogate tissue, because no qualified RNA could be isolated. Conclusions: Nevertheless, we demonstrated the feasibility to process clinical samples and to obtain good quality RNA from tumor biopsies, and indicated the potential of oblimersen to lower Bcl-2 mRNA in breast cancer. No significant financial relationships to disclose.

Cationic Liposomes Loaded with Proapoptotic Peptide d-(KLAKLAK)2 and Bcl-2 Antisense Oligodeoxynucleotide G3139 for Enhanced Anticancer Therapy

The treatment of cancer using macromolecular therapeutics such as oligonucleotides or peptides requires efficient delivery systems capable of intracellular penetration and may also benefit from use of a combination of therapeutics with different mechanisms of action. With this possibility in mind, we constructed cationic liposome loaded with the proapoptotic peptide, d-(KLAKLAK)(2) and the Bcl-2 antisense oligodeoxynucleotide, G3139, and determined whether the combination of the proapoptotic macromolecules in a single cationic liposome can enhance antitumor efficacy. Advantage was taken of alternating charge interaction to entrap macromolecules of opposite charge. The polycationic peptide d-(KLAKLAK)(2) was first condensed with the polyanionic oligodeoxynucleotide G3139 to obtain overall negatively charged peptide/oligodeoxynucleotide complexes. The complexes were then entrapped into DOTAP/DOPE cationic liposomes (CL). This sequential charge interaction ensured efficient entrapment of d-(KLAKLAK)(2) and G3139 with a high loading efficiency (50%) and capacity (7.5 wt %). In vitro treatment of mouse melanoma B16(F10) with CL loaded with d-(KLAKLAK)(2)/G3139 led to significantly enhanced antitumor efficacy, mediated by stimulated induction of apoptotic (caspase 3/7) activity, when compared to CL loaded with G3139 alone. Intratumoral injection of CL loaded with d-(KLAKLAK)(2)/G3139 in B16(F10) mice xenograft also led to suppressed tumor growth associated with enhanced apoptotic activity. Thus, the combination of proapoptotic peptide d-(KLAKLAK)(2) and antisense oligonucleotide G3139 in a cationic liposome led to enhanced apoptotic/antitumor efficacy and may provide a promising tool for cancer treatment.

Optimizing chemotherapy for transitional cell carcinoma by application of bcl-2 and bcl-xL antisense oligodeoxynucleotides

Objective Therapy failure after intravesical and systemic chemotherapy for transitional cell carcinoma (TCC) is still high. Antiapoptotic proteins such as Bcl-2 and Bcl-xL have been reported to promote chemoresistance in TCC. Targeting bcl-2 and bcl-xL messenger ribonucleic acid with antisense oligodeoxynucleotides (AS-ODNs) may enhance the cytotoxic effects of chemotherapeutic agents. Therefore, we investigated the effects of bcl-2 and bcl-xL AS-ODNs in combined treatment with conventional and new chemotherapeutic agents to evaluate the cytotoxic effects in comparison to monotreatment. Methods and materials Western blot analysis or immunohistochemistry verified Bcl-2 and Bcl-xL expression in a panel of human TCC cell lines that had been monotreated with cisplatin, gemcitabine, mitomycin C, and paclitaxel. In addition, bcl-2 or bcl-xL AS-ODNs were applied in combination with each chemotherapeutic agent. Cell viability was determined using a standard MTT assay and Neubauer hemocytometry. Results All cell lines responded to chemotherapeutic monotreatment in a dose-dependent manner. Maximum cell death rates after monotreatment were 47.4% (cisplatin), 39.0% (gemcitabine), 83.4% (mitomycin C), and 54.8% (paclitaxel). After combined treatment with chemotherapy and bcl-2 or bcl-xL AS-ODNs, cell death rates were significantly higher (e.g., 30.3% vs. 87.2% in HT 1197 cells for monotreatment vs. the combination of paclitaxel and bcl-xL AS-ODNs). Three-way analysis of variance revealed that combined treatment had a significant effect on all cell lines. Conclusions Our study confirms that the addition of bcl-2 and bcl-xL AS-ODNs enhances the cytotoxic potential of chemotherapeutic agents in TCC cell lines as a result of combined effects. Further trials in ex vivo and in vivo models have to be performed to promote clinical application in patients.

Downregulation of BCL-2 Induces Downregulation of Carbonic Anhydrase IX, Vascular Endothelial Growth Factor, and pAkt and Induces Radiation Sensitization

Our group previously demonstrated that expression of the oncogene, Bcl-2, was associated with radiation resistance. The aim of the present study was to determine whether Bcl-2 expression in radiation-resistant tumors was associated with expression of carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), and pAkt and whether downregulation of Bcl-2 could modulate the expression of CAIX, VEGF, and pAkt. Two prostate cancer cell lines, PC-3-Bcl-2 and PC-3-Neo, were injected into the subcutaneous flanks of 192 athymic male nude mice; 96 received PC-3 Bcl-2 and 96 PC-3-Neo. The mice were then treated with antisense Bcl-2 oligodeoxynucleotide (ASODN), reverse control ODN, or vehicle only (mock treatment) with or without irradiation (4 Gy). The tumors were monitored for growth (ie, volume) over time, resected at various points, and processed and sectioned for protein analyses. Bcl-2 ASODN treatment was associated with downregulation of Bcl-2, VEGF, pAkt, and CAIX. PC-3-Bcl-2 and PC-3-Neo prostate tumor xenografts in mice treated with the combination of Bcl-2 ASODN and irradiation were significantly (ie, one third) smaller than those in mice treated with reverse control ODN alone, Bcl-2 ASODN alone, irradiation alone, or reverse control ODN plus irradiation (P = 0.0001). An increased tumor response to the combined therapy was associated with decreased expression of Bcl-2, VEGF, and pAkt proteins. These findings have demonstrated an intimate relationship among CAIX, VEGF, pAkt, and Bcl-2 in prostate tumors. Thus, CAIX might prove effective as a potential marker of tumor radiation resistance. Downregulation of CAIX might lead to radiation sensitization. Consequently, the combination of CAIX reduction and radiotherapy warrants further consideration as a new strategy for therapy.

Therapeutic integration of c‐myc and bcl‐2 antisense molecules with docetaxel in a preclinical model of hormone‐refractory prostate cancer

The response of hormone-refractory prostate cancer (HRPC) to chemotherapy remains modest, necessitating the search for new forms of treatment to improve the prognosis. Since an increased expression of oncogenes, including c-myc and bcl-2, accompanies the transition to HRPC, we evaluated whether the concomitant downregulation of these oncogenes by antisense strategy sensitized HRPC to chemotherapy. PC-3 prostate cancer cells were exposed in vitro to c-myc (INX-6295) and bcl-2 (G3139) antisense oligodeoxynucleotides (ODNs) and docetaxel given alone or in combination. Therapeutic efficacy of the different treatments was also evaluated in xenografts. We show that the triple combination of drugs given in the sequence G3139/docetaxel/INX-6295 was the most active in reducing the survival of PC-3. Likewise, the combination triggered apoptosis in more than 80% of cells. A marked tumor weight inhibition was observed in PC-3 xenografts after G3139/docetaxel/INX-6295 treatment, with a complete tumor regression being noted in half the mice. A 111% overall increase in life survival and a complete cure in two out of eight mice was also reported. This treatment remained effective even when started at a very late stage of tumor growth producing about 80% tumor weight inhibition (TWI), with tumor regression being maintained for 1 month. Finally, the antitumor effect resulted in a significant increase (70%) in mice survival. These data indicate that the combined targeting of genes involved in uncontrolled proliferation and evasion of apoptosis renders HRPC responsive to chemotherapy making this treatment a promising antineoplastic strategy.

Irradiation of Human Prostate Cancer Cells Increases Uptake of Antisense Oligodeoxynucleotide

To investigate whether irradiation before antisense Bcl-2 oligodeoxynucleotide (ODN) administration enhances tissue uptake, and whether periodic dosing enhances cellular uptake of fluorescently labeled ODN relative to constant dosing. PC-3-Bcl-2 cells (prostate cancer cell line engineered to overexpress Bcl-2) were subjected to increasing doses of irradiation (0-10 Gy) with or without increasing concentrations of fluorescently labeled antisense Bcl-2 ODN (G4243). The fluorescent signal intensity was quantified as the total grain area with commercial software. In addition, PC-3-Bcl-2 subcutaneous xenograft tumors were treated with or without irradiation in combination with various dosing schemas of G4243. The uptake of fluorescent G4243 in tumors was quantitated. The uptake of G4243 was increased in prostate cancer cells exposed to low doses of irradiation both in vitro and in vivo. Irradiation before G4243 treatment resulted in increased fluorescent signal intensity in xenograft tumors compared with those irradiated after G4243 treatment. A single weekly dose of G4243 produced higher G4243 uptake in xenograft tumors than daily dosing, even when the total dose administered per week was held constant. These findings suggest that ionizing radiation increases the uptake of therapeutic ODN in target tissues and, thus, has potential to increase the efficacy of ODN in clinical applications.

Knock-down of Bcl-2 by antisense oligodeoxynucleotides induces radiosensitization and inhibition of angiogenesis in human PC-3 prostate tumor xenografts

Expression of the proto-oncogene Bcl-2 is associated with tumor progression. Bcl-2's broad expression in tumors, coupled with its role in resistance to chemotherapy and radiation therapy-induced apoptosis, makes it a rational target for anticancer therapy. Antisense Bcl-2 oligodeoxynucleotide (ODN) reagents have been shown to be effective in reducing Bcl-2 expression in a number of systems. We investigated whether treating human prostate cancer cells with antisense Bcl-2 ODN (G3139, oblimersen sodium, Genasense) before irradiation would render them more susceptible to radiation effects. Two prostate cancer cell lines expressing Bcl-2 at different levels (PC-3-Bcl-2 and PC-3-Neo) were subjected to antisense Bcl-2 ODN, reverse control (CTL), or mock treatment. Antisense Bcl-2 ODN alone produced no cytotoxic effects and was associated with G(1) cell cycle arrest. The combination of antisense Bcl-2 ODN with irradiation sensitized both cell lines to the killing effects of radiation. Both PC-3-Bcl-2 and PC-3-Neo xenografts in mice treated with the combination of antisense Bcl-2 ODN and irradiation were more than three times smaller by volume compared with xenografts in mice treated with reverse CTL alone, antisense Bcl-2 ODN alone, irradiation alone, or reverse CTL plus radiotherapy (P = 0.0001). Specifically, PC-3-Bcl-2 xenograft tumors treated with antisense Bcl-2 ODN and irradiation had increased rates of apoptosis and decreased rates of angiogenesis and proliferation. PC-3-Neo xenograft tumors had decreased proliferation only. This is the first study which shows that therapy directed at Bcl-2 affects tumor vasculature. Together, these findings warrant further study of this novel combination of Bcl-2 reduction and radiation therapy, as well as Bcl-2 reduction and angiogenic therapy.

G3139 induces cell death by caspase-dependent and -independent apoptosis on human melanoma cell lines

G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors.

Systemic Bcl‐2 antisense oligodeoxynucleotide in combination with cisplatin cures EBV+ nasopharyngeal carcinoma xenografts in SCID mice

Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV), and the EBV oncoprotein, latent membrane protein 1 (LMP-1), is expressed in the majority of NPCs. LMP-1 upregulates antiapoptotic genes, including bcl-2, and Bcl-2 protein is overexpressed in NPC. Given the antiapoptotic and chemoprotective effects of Bcl-2, it represents a rational therapeutic target in NPC. We have investigated the antitumor and chemosensitizing effects of the Bcl-2 antisense oligodeoxynucleotide G3139 (oblimersen, Genasense) in NPC. For these studies, we used the C666-1 line, a stably infected NPC-derived line that co-expresses LMP-1 and Bcl-2. We have shown that G3139 treatment of C666-1 in vitro caused sequence-dependent suppression of Bcl-2 protein, inhibition of cell growth and enhanced sensitivity to cisplatin (CDDP), as measured by increased antiproliferative and apoptotic effects. In vivo, G3139 treatment (25 mg/kg every 3 days x 5 doses) delayed engraftment and significantly inhibited growth of established C666-1 xenografts in SCID mice compared to control oligo-treated animals. However, G3139 alone did not prevent engraftment or cure established tumors in any animals. In contrast, G3139 treatment (25 mg/kg every 3 days x 5 starting on day 7) in combination with CDDP (8 mg/kg on day 14) completely abrogated tumor engraftment in 80% of animals compared to CDDP (0%) or CDDP + control oligo (0%). When treatment was delayed until tumor was established, G3139 in combination with CDDP significantly inhibited tumor growth compared to CDDP or CDDP + control oligo, and cured 69% animals with established tumors. No animals treated with G3139, CDDP or CDDP + control oligo were cured. Tumor burden and response to treatment correlated with EBV DNA load in serum, measured by real-time PCR. Western blots of tumor extracts obtained during oligo treatment showed that Bcl-2 levels were significantly decreased in G3139-treated animals. Our studies have demonstrated that the Bcl-2 antisense oligodeoxynucleotide, G3139, has proapoptotic effects in C666-1, and in combination with CDDP, is curative in C666-1 NPC xenograft tumors in vivo. The sequence-dependency of these effects is consistent with an antisense mechanism. These studies suggest that Bcl-2 may represent a biologically relevant target for the development of novel combinatorial therapies for NPC.

Bcl-2 and Mn-SOD Antisense Oligodeoxynucleotides and a Glutamine-enriched Diet Facilitate Elimination of Highly Resistant B16 Melanoma Cells by Tumor Necrosis Factor-α and Chemotherapy

Mitochondrial glutathione (mtGSH) depletion increases sensitivity of Bcl-2-overexpressing B16 melanoma (B16M)-F10 cells (high metastatic potential) to tumor necrosis factor-alpha (TNF-alpha)-induced oxidative stress and death in vitro. In vivo, mtGSH depletion in B16M-F10 cells was achieved by feeding mice (where the B16M-F10 grew as a solid tumor in the footpad) with an L-glutamine (L-Gln)-enriched diet, which promoted in the tumor cells an increase in glutaminase activity, accumulation of cytosolic L-glutamate, and competitive inhibition of GSH transport into mitochondria. L-Gln-adapted B16M-F10 cells, isolated using anti-Met-72 monoclonal antibodies and flow cytometry-coupled cell sorting, were injected into the portal vein to produce hepatic metastases. In l-Gln-adapted invasive (iB16M-Gln+) cells, isolated from the liver by the same methodology and treated with TNF-alpha and an antisense Bcl-2 oligodeoxynucleotide, viability decreased to approximately 12%. iB16M-Gln+ cell death associated with increased generation of O2*- and H2O2, opening of the mitochondrial permeability transition pore complex, and release of proapoptotic molecular signals. Activation of cell death mechanisms was prevented by GSH ester-induced mtGSH replenishment. The oxidative stress-resistant survivors showed an adaptive response that includes overexpression of manganese-containing superoxide dismutase (Mn-SOD) and catalase activities. By treating iB16M-Gln+ cells with a double anti- antisense therapy (Bcl-2 and SOD2 antisense oligodeoxynucleotides) and TNF-alpha, metastatic cell survival decreased to approximately 1%. Chemotherapy (taxol plus daunorubicin) easily removed this minimum percentage of survivors. This contribution identifies critical molecules that can be sequentially targeted to facilitate elimination of highly resistant metastatic cells.

Effect of Bcl-2 Antisense Oligodeoxynucleotides on Human Lung Carcinoma Transplanted Subcutaneously in Nude Mice

Objective: To investigate the inhibitory effects of the Bcl-2 antisense oligodeoxynucleotides (ASODN) on tumor formation and growth of human lung carcinoma transplanted subcutaneously in nude mice. Methods: Human NCI-H460 cells treated with Bcl-2 ASODN or nonesense oligodeoxynucleotide (NSODN) and untreated NCI-H460 cells were respectively implanted subcutaneously into nude mice.When the diameters of tumor were above 0.5 cm after untreated NCI-H460 cells injection, the mice bearing tumor were randomly divided into three groups: saline control group, Bcl-2 ASODN group, NSODN group. ODN was directly injected into the tumor body for 3 weeks. The weight and volume of subcutaneous tumors were measured, and the morphology of tumor cells was observed. Results: The tumorigenic ability of the treated NCI-H460 cells by Bcl-2 ASODN was reduced. The mean time at which tumor can be detected was prolonged up to 12.6 days (P<0.01). The maximum tumor growth inhibitory rate was 87.5%. In therapeutic efficacy, growth of tumor was significantly inhibited in Bcl-2 ASODN group as compared with that in NSODN group, saline-treated group (P<0.01). The NSODN control was ineffective. In comparison with NSODN-treated, saline-treated mice, those treated with Bcl-2 ASODN showed a significant decrease in median weight of subcutaneous tumors (P<0.01). The growth inhibitory rate was 71.0% in ASODN group. Conclusion: Bcl-2 ASODN could inhibit tumor formation and tumor growth in nude mice.

Targeted therapy against Bcl-2-related proteins in breast cancer cells

IntroductionBcl-2 and Bcl-xL confer resistance to apoptosis, thereby reducing the effectiveness of chemotherapy. We examined the relationship between the expression of Bcl-2 and Bcl-xL and chemosensitivity of breast cancer cells, with the aim of developing specific targeted therapy.MethodsFour human breast cancer cell lines were examined, and the effects of antisense (AS) Bcl-2 and AS Bcl-xL phosphorothioate oligodeoxynucleotides (ODNs) on chemosensitivity were tested in vitro and in vivo. Chemosensitivity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, and the antitumor effect was assessed in vivo by the success of xenograft transplantation into athymic mice.ResultsTreatment with AS Bcl-2 and Bcl-xL ODNs resulted in a sequence-specific decrease in protein expression, compared with controls. Treatment of BT-474, ZR-75-1, and MDA-MB-231 cells with AS Bcl-2 increased chemosensitivity to doxorubicin (DOX), mitomycin C (MMC), paclitaxel (TXL), and docetaxel (TXT). Transfection of the Bcl-2 gene into MDA-MB-453 cells decreased sensitivity to DOX and MMC. Treatment of MDA-MB-231, BT-474, and ZR-75-1 cells with AS Bcl-xL increased chemosensitivity to DOX, MMC and taxanes to a smaller extent than AS Bcl-2. This occurred in the setting of increased Bax and cleaved poly(ADP-ribose) polymerase, as well as decreased Bcl-2 and pAkt. AS Bcl-2 ODNs induced splenomegaly in association with increased serum IL-12, which was attenuated by methylation of the CpG motifs of AS Bcl-2; however, methylated CpG failed to negate the increased antitumor effect of AS Bcl-2. Bcl-2 and Bcl-xL, to a smaller extent, are major determinants of chemosensitivity in breast cancer cells.ConclusionTargeted therapy against Bcl-2 protein with the use of AS ODNs might enhance the effects of chemotherapy in patients with breast cancer.

Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective: To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods: Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results: The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion: The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.

Enhancement of radiation-induced apoptosis in raji cell line by Bcl-2 antisense oligodeoxynucleotide

Objective: To investigate whether the Bcl-2 antisense oligonucleotide (ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of Bcl-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bcl-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bcl-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bcl-2 ASODN combined with radiation could significantly inhibit expression of Bcl-2 protein in Raji cells (P<0.05). Cells treated with Bcl-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bcl-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bcl-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

Acceleration of Glutathione Efflux and Inhibition of γ-Glutamyltranspeptidase Sensitize Metastatic B16 Melanoma Cells to Endothelium-induced Cytotoxicity

Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP-/-1) B16M-F10 transfected with the Bcl-2 gene or in MRP-/-1 B16M-F10 cells incubated with l-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an l-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide- and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of gamma-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to approximately 90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.