Combined effect of 2-5A-linked antisense against telomerase RNA and conventional therapies on human malignant glioma cells in vitro and in vivo

We recently showed that therapy with 2'-5'-oligoadenylate (2-5A)-linked antisense against human telomerase RNA component (2-5A-anti-hTR) is a novel telomerase-targeting strategy against malignant gliomas. In this study, we investigated conventional chemotherapeutic agents and gamma-irradiation (IR) to determine whether they could augment the efficacy of 2-5A-anti-hTR against these tumors in vitro and in vivo. Treatment with 2-5A-anti-hTR inhibited the viability of U373-MG and U87-MG malignant glioma cells in a dose-dependent manner; the antitumor effect resulted from induction of apoptosis. Also, telomerase-positive astrocytes with oncogenic Ras were more sensitive to 2-5A-anti-hTR than were those without oncogenic Ras. In addition, we sought to determine the combined effect of 2-5A-anti-hTR with N, N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), cisplatin (CDDP), paclitaxel (PTX), temozolomide (TMZ), or IR. When we administered the combination treatments on the same day, PTX and IR showed a greater combined effect with 2-5A-anti-hTR on both tumor cell lines than did BCNU, CDDP and TMZ. However, all of the combination regimens were synergistic when we first treated tumor cells with 2-5A-anti-hTR for 24 h and then exposed them to the conventional treatments. Apoptosis-inducing agents (CDDP and PTX) but not autophagy-inducing therapies (TMZ and IR) enhanced the incidence of apoptosis caused by 2-5A-anti-hTR. Lastly, we observed a combinatorial effect of 2-5A-anti-hTR and TMZ in vivo in subcutaneous U87-MG tumors in nude mice. Interestingly, treatment with TMZ increased the incidence of apoptosis in subcutaneous tumor cells treated with 2-5A-anti-hTR. These results suggest that 2-5A-anti-hTR is preferable in combination with established cancer therapies.     

Combination therapy of 2-5A antisense against telomerase RNA and cisplatin for malignant gliomas

2',5'-Oligoadenylate (2-5A) linked to an antisense oligonucleotide against human telomerase RNA (2-5A-anti-hTR) is a novel therapeutic modality we have recently developed. We designed the oligonucleotide to target telomerase which maintains chromosome integrity in cancer cells. We have already demonstrated the efficacy of this new therapy in malignant glioma cells with telomerase activity. In the present study, we investigated the effect of 2-5A-anti-hTR in combination with cisplatin, an anti-cancer drug commonly used for malignant glioma patients. Six human malignant glioma cell lines with telomerase activity were treated with 0.5 microM 2-5A-anti-hTR and/or cisplatin (0, 0.1, 1, 5, 10, or 20 microg/ml) for three days, and cell viability was measured using the MTT colorimetric assay. The combination therapy showed synergistic effect at 1 microg/ml cisplatin and additive effect at 5 microg/ml cisplatin. TUNEL staining of the treated cells showed significantly increased apoptotic cells after the combination therapy. Furthermore, tumor growth of subcutaneous xenografts of human malignant glioma cells in nude mice was effectively reduced after the combination therapy for seven days. Our study shows that the tumor-killing effects of the combined treatments are, at least in part, due to induction of apoptosis. 2-5A-anti-hTR is a promising therapy not only when used alone, but also in combination with cisplatin.     

2-5A antisense therapy directed against human telomerase RNA inhibits telomerase activity and induces apoptosis without telomere impairment in cervical cancer cells

Human telomerase RNA (hTR), an important component of telomerase, is a possible target of telomerase-based cancer gene therapy. The present study was undertaken to assess the efficacy of antisense hTR therapy using newly developed 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked oligonucleotides against cervical cancer cells. ME180 and SiHa cells were treated with 2-5A-linked antisense hTR designed to complement the region of hTR between residues 76 and 94. The hTR expression, telomerase activity, cell viability, and apoptosis were then examined. The 2-5A anti-hTR effectively degraded hTR and inhibited telomerase activity. The 2-5A mutant anti-hTR and the anti-hTR without 2-5A were not capable of inhibiting telomerase activity. Inhibition of telomerase by 2-5A anti-hTR rapidly decreased cell viability only in telomerase-positive cells within 3-6 days after the treatment, when telomere length has not yet been shortened. This inhibition was associated with apoptosis, possibly through activation of caspase family members. These findings suggest that 2-5A-linked antisense-hTR therapy has a potent telomerase-inhibitory effect associated with a cytocidal effect from caspase-induced apoptosis, and may therefore be a potential tool in telomerase-based gene therapy against cervical cancers.     

2-5A antisense telomerase RNA therapy for intracranial malignant gliomas

Malignant gliomas are the most common intracranial tumors and are considered incurable. Therefore, exploration of novel therapeutic modalities is essential. Telomerase is a ribonucleoprotein enzyme that is detected in the vast majority of malignant gliomas but not in normal brain tissues. We, therefore, hypothesized that telomerase inhibition could be a very promising approach for the targeted therapy of malignant gliomas. Thus, 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked antisense against human telomerase RNA component (2-5A-anti-hTER) was investigated for its antitumor effect on an intracranial malignant glioma model. 2-5A is a mediator of one pathway of IFN actions by activating RNase L, resulting in RNA degradation. By linking 2-5A to antisense, RNase L degrades the targeted RNA specifically and effectively. Prior to the experiments using intracranial tumor models in nude mice, we modified the in vitro and in vivo treatment modality of 2-5A-anti-hTER using a cationic liposome to enhance the effect of 2-5A-anti-hTER. Here we demonstrate that 2-5A-anti-hTER complexed with a cationic liposome reduced the viability of five malignant glioma cell lines to 20-43% within 4 days but did not influence the viability of cultured astrocytes lacking telomerase. Furthermore, treatment of intracranial malignant gliomas in nude mice with 2-5A-anti-hTER was therapeutically effective compared with the control (P < 0.01). These findings clearly suggest the therapeutic potentiality of 2-5A-anti-hTER as a novel approach for the treatment of intracranial malignant gliomas.     

Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR

Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.     

Delivering antisense telomerase RNA by a hybrid adenovirus/ adeno-associated virus significantly suppresses the malignant phenotype and enhances cell apoptosis of human breast cancer cells

Activated telomerase is frequently detected in cancer cells and is able to maintain and stabilize the integrity of telomeres; it also contributes to unlimited divisions in cancer cells. Recently, a new generation of selective anticancer strategies is under development targeting the blockage of telomerase activity either at the protein level or telomerase RNA. Here, we report suppression of the malignant phenotype by the expression of the full-length antisense human telomerase RNA (hTR) delivered by a novel hybrid vector recombining adenovirus and adeno-associated virus (vAd-AAV). The hybrid vector vAd-AAV retained the unique traits from two parental viruses, such as high efficiency of gene transfer in mammalian cells and the ability to integrate into the genomic DNA of host cells. The stable expression of antisense hTR in MCF-7 cells significantly suppressed telomerase activity and progressively shortened telomere length for 30 population doublings (PD30). Expression of antisense hTR leads to a telomere-based growth arrest and the induction of spontaneous apoptosis. Antisense hTR decreased soft agar colony formation and reduced the cell proliferation, leading to exit from the cell cycle at G1 at PD15. The expression of antisense hTR also sensitized MCF-7 cells to apoptosis induced by sodium butyrate or serum starvation. Our study demonstrates that delivering antisense hTR by the hybrid Ad/AAV vector is an effective antineoplastic gene therapeutic strategy, which significantly suppresses the malignant phenotype and enhances apoptosis of human breast cancer cells.     

Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.     

Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA (siRNA) has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA (hTR) can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells (HRCCs). METHODS The siRNA duplexes for hTR were synthesized and 786-0 HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined. RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally, significant inhibition of proliferation and induction of apoptosis were observed. CONCLUSION siRNA against the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.     

端粒酶反义RNA 对人喉癌细胞系Hep-2 生长的影晌

 目的　探讨抗端粒酶在喉癌细胞治疗中的意义。方法　用脂质体介导法将 pBBS2 12 hTR(反义端粒酶RNA真核表达载体 )转染入人喉癌Hep 2细胞系中 ,采用TRAP法测定端粒酶活性 ,细胞动力学检测 ,电镜下观察等方法研究其对Hep 2细胞的端粒酶活性、细胞增殖及细胞凋亡影响。 结果　端粒酶反义RNA能显著抑制细胞的端粒酶活性 ,抑制细胞增殖并促进其凋亡。结论　以端粒酶反义RNA抑制端粒酶活性是治疗喉癌的潜在途径。     

Telomerase: A novel target of antitumor agents

Anticancer drug screenings include cytotoxicitybased and mechanism-oriented strategies. Several reviewshave covered the topic.['--'] Although cytotoxicity-basedsystemic chemotherapy has had some success inselectively killing rapidly growing tumor cells, it has themajor disadvantage of killing rapidly proliferatingnonmalignant cells, resulting in serious side effects. Theresearch of mechanism-oriented antitumor agents notnecessmp producing significantly better effectivenessthan those that are a…     

Acquisition of chemoresistant phenotype by overexpression of the antiapoptotic gene testosterone-repressed prostate message-2 in prostate cancer xenograft models

Testosterone-repressed prostate message-2 (TRPM-2) expression is highly up-regulated in normal and malignant prostate cells after androgen withdrawal. Although recent studies have suggested a protective role of TRPM-2 expression against apoptosis in several experimental models, the functional role of TRPM-2 in chemotherapy-induced apoptosis remains undefined. Here, we demonstrated that overexpression of TRPM-2 in human androgen-dependent LNCaP prostate cancer cells by stable transfection rendered them highly resistant to paclitaxel treatment than control LNCaP cells, with a 20-fold higher IC50 through the inhibition of apoptotic cell death. In mice bearing TRPM-2-overexpressing LNCaP tumors, tumor volume and serum prostate-specific antigen increased two to three times faster after castration and paclitaxel treatment compared with mice bearing control tumors. We then tested the efficacy of combined treatment with antisense TRPM-2 oligodeoxynucleotide (ODN) and paclitaxel in the mouse androgen-dependent Shionogi tumor model. Antisense TRPM-2 ODN treatment significantly enhanced paclitaxel chemosensitivity of Shionogi tumor cells in a dose-dependent manner, reducing the IC50 by 75%. Combined treatment of Shionogi cells with 500 nM antisense TRPM-2 ODN and 10 nM paclitaxel-induced apoptosis, either agent alone did not. Adjuvant administration of antisense TRPM-2 ODN and polymeric micellar paclitaxel after castration resulted in reduced TRPM-2 levels in vivo and a significant delay of emergence of androgen-independent recurrent Shionogi tumors compared with administration of either agent alone. Furthermore, combined treatment of mice bearing androgen-independent recurrent Shionogi tumors with antisense TRPM-2 ODN and micellar paclitaxel inhibited tumor growth compared with treatment with either agent alone. Collectively, these findings demonstrate that TRPM-2 overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis, and that antisense TRPM-2 ODN may be useful in enhancing the effects of cytotoxic chemotherapy in hormone-refractory prostate cancer.     

Inhibition of cyclooxygenase (COX)-2 expression by Tet-inducible COX-2 antisense cDNA in hormone-refractory prostate cancer significantly slows tumor growth and improves efficacy of chemotherapeutic drugs.

PURPOSE: Overexpression of the proinflammatory enzyme cyclooxygenase (COX)-2 is associated with the progression of various malignancies; the role of COX-2 in prostate cancer is less clear. The significance of COX-2 in prostate cancer growth and response to chemotherapy was investigated in an androgen-refractory prostate cancer cell line using a Tet-inducible antisense COX-2 expression system. EXPERIMENTAL DESIGN: An antisense COX-2 cDNA construct under the control of a doxycycline-inducible promoter was transfected into a prostate cancer cell line, PC-3ML. Modulations of cell growth, apoptosis, and chemosensitivity in the presence or absence of doxycycline were analyzed. Tumor incidence, growth rate, and response to two cytotoxic drugs, COL-3 [chemically modified tetracycline-3-(6-demethyl-6-deoxy-4-dedimethylamino-tetracycline)] and Taxotere (docetaxel), were investigated in tumor xenografts. Apoptotic incidences and tumor microvessel density in tumors were determined by immunohistochemistry. RESULTS: Conditional suppression of COX-2 in PC-3ML caused reduced cell proliferation, decreased levels of phosphorylated AKT, G(0)-G(1) arrest, and increased apoptosis and caspase-3 activity. Suppression of COX-2 increased Bax protein and decreased Bcl-x(L) protein in vitro. COX-2 antisense-expressing PC-3ML tumors showed a 57% growth delay compared with nontransfected or vector controls. Oral administration of COL-3 (40 mg/kg, oral gavage) or Taxotere (2.3 mg/kg, intraperitoneally; 3x per week) in tumor-bearing mice further slowed tumor growth (65% and approximately 94%, respectively). Compared with the control group, the occurrence of apoptosis in antisense COX-2 tumors was eight times higher, and the tumor microvessel density was three times lower. CONCLUSIONS: These results provide direct evidence that constitutive expression of COX-2 in prostate cancer has both angiogenic and cytoprotective functions. Suppression of tumor cell COX-2 is sufficient to enhance chemotherapy response in prostate cancer.     

端粒酶反义RNA对人肾癌细胞系GRC—1生长的影响

目的 探讨抗端粒酶在肾细胞癌治疗中的意义。方法 采用脂质体介质导法将端粒酶反义RNA真核表达载体（pBBS212-hTR)导入人肾癌GRC－1细胞系中,采用TRAR法测定端粒酶活性,采用MTT法检测细胞生长动力,电镜下观察细胞增殖情况及端粒酶反义RNA对细胞凋亡的影响。结果 端粒酶反应RNA能显著抑制细胞的端粒酶活性,抑制细胞增殖并促进其凋亡。结论 应用端粒酶反义RNA抑制肾癌端粒酶活性是治疗肾癌的潜在途径。     

AKT-1, -2, and -3 are expressed in both normal and tumor tissues of the lung, breast, prostate, and colon.

PURPOSE: The AKT/PKB kinase controls many of the intracellular processes that are dysregulated in human cancer, including the suppression of apoptosis and anoikis and the induction of cell cycle progression. Three isoforms of AKT have been identified: AKT-1, -2, and -3. Selective up-regulation of AKT-3 RNA expression has been reported in hormone-independent breast and prostate cancer cell lines suggesting that AKT-3 expression may be increased with breast or prostate tumor progression. To determine whether AKT-3 RNA expression is selectively up-regulated in human cancers and whether the patterns of AKT RNA expression may change with tumor development, we examined AKT isoform expression by RT-PCR in human cancer cell lines, primary human cancers, and normal human tissues. EXPERIMENTAL DESIGN: AKT-1, -2, and -3 RNA expression was examined by RT-PCR. Because up-regulated AKT-3 expression has been implicated in human breast and prostate cancer progression, we also examined AKT-3 expression levels by semiquantitative RT-PCR using matched normal/tumor first-strand cDNA pairs from colon, breast, prostate, and lung cancers. RESULTS: Our data reveal that the overwhelming majority of both normal and tumor tissues express all three of the AKT isoforms. Moreover, semiquantitative RT-PCR of matched normal/tumor pairs confirmed similar AKT-3 RNA expression levels in both normal and tumor tissue. CONCLUSIONS: Our data show that both normal and tumor tissues express all three of the AKT isoforms and indicate that tumorigenesis does not involve a dramatic shift in the RNA expression patterns of the three AKT isoforms.     

Tunicamycin sensitizes human prostate cells to TRAIL-induced apoptosis by upregulation of TRAIL receptors and downregulation of cIAP2

The addition of tunicamycin to prostate cancer cells enhances cell death mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we investigated whether tunicamycin, an endoplasmic reticulum stress inducer, can potentiate TRAIL-induced apoptosis in human prostate cancer cells. We evaluated the combination of tunicamycin and TRAIL and found synergistic promotion of apoptosis in prostate cancer cells. The combined treatment with tunicamycin and TRAIL significantly induced apoptosis, and stimulated caspase-3, -8 and -9 activity, as well as the cleavage of poly (ADP-ribose) polymerase. We found that tunicamycin promoted TRAIL-induced apoptosis by the upregulation of death receptor (DR)4 and DR5 and the downregulation of cellular inhibitor of apoptosis 2 (cIAP2). In addition, downregulation of cIAP2 expression using small interfering RNA significantly attenuated the apoptosis induced by TRAIL. Taken together, our results demonstrate that the combination of tunicamycin and TRAIL may provide a novel strategy for treating prostate cancer by overcoming critical mechanisms of apoptosis resistance.     

抑制端粒酶活性对人肾癌细胞生长的抑制作用

目的 :探讨抗端粒酶在肾细胞癌治疗中的意义。方法 :采用脂质体介导法将pBBS2 12 hTR(反义端粒酶RNA真核表达载体 )导入人肾癌GRC 1细胞系中 ,采用TRAP法测定端粒酶活性 ,通过MTT法检测细胞动力学 ,观察细胞的增殖 ,电镜下观察其对细胞凋亡的影响。结果 :端粒酶反义RNA能显著抑制细胞的端粒酶活性 ,抑制细胞增殖并促进其凋亡。结论 :以端粒酶反义RNA抑制端粒酶活性是治疗肾癌的潜在途径     

A novel telomerase template antagonist (GRN163) as a potential anticancer agent

Telomerase, the enzyme responsible for proliferative immortality, is expressed in essentially all cancer cells, but not in most normal human cells. Thus, specific telomerase inhibition is potentially a universal anticancer therapy with few side effects. We designed N3'-->P5' thio-phosphoramidate (NPS) oligonucleotides as telomerase template antagonists and found that their ability to form stable duplexes with the telomerase RNA subunit was the key factor for antitelomerase activity. In biochemical assays 11-13-mer NPS oligonucleotides demonstrated sequence- and dose-dependent inhibition of telomerase with IC(50) values <1 nM. Optimization of the sequence, length, and bioavailability resulted in the selection of a 13-mer NPS oligonucleotide, GRN163, as a drug development candidate. GRN163 inhibited telomerase in a cell-free assay at 45 +/- 7 pM, and in various tumor cell lines at approximately 1 nM and approximately 0.3-1.0 micro M in the presence and absence of carriers, respectively. GRN163 was competitive with telomeric primer binding, primarily because of hybridization to human telomerase RNA (hTR) component. Tumor cells treated with GRN163 in culture underwent telomere shortening, followed by cellular senescence or apoptosis after a period of time that generally correlated with initial telomere length. In a flank DU145 (prostate cancer) xenograft model, parenterally administered GRN163 caused suppression of tumor growth in the absence of gross toxicity. These data demonstrate that GRN163 has significant potential for additional development as an anticancer agent.     

A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity

Androgen deprivation therapy causes a paradoxical elevation of matrix metalloproteinases (MMPs) including MMP-9 resulting in aggressive tumor phenotype in many patients with prostate cancer. In this study, we have evaluated a novel antisense phosphorodiamidate Morpholino oligomer (PMO) targeted against MMP-9 in models of angiogenesis and in human prostate xenograft in athymic mice. The treatment of androgen-independent DU145 human prostate cells with a 21-mer MMP-9 antisense PMO caused a dose-dependent inhibition of cell proliferation compared to scrambled or MMP-2 antisense PMO at similar concentrations. This was associated with decreases in MMP-9 expression, gelatinolytic activity and increased stability of the insulin-like growth factor-binding protein (IGFBP-3), a proapoptotic factor and MMP-9 substrate. In vitro invasion assays revealed a 40-60% inhibition of DU145 cell invasion in the presence of 25 microM MMP-9 antisense PMO. A significant decrease in endothelial cell migration and vascularization was observed in the Matrigel plug assay in mice when treated intraperitoneally with 300 microg/day MMP-9 antisense for 21 days. In the highly vascular DU145 tumor xenografts, MMP-9 inhibition caused decreased tumor growth with regression in 50% of the animals. Histological analysis revealed increased apoptosis and fibrous tissue deposits in the MMP-9 antisense-treated tumors compared to the scrambled and saline controls. No apparent toxicity or mortality was associated with the MMP-9 PMO treatment. In summary, the MMP-9 antisense PMO inhibited in vitro prostate cancer cell proliferation, invasion and in vivo angiogenesis. These data establish the feasibility of developing a site-directed, nontoxic antisense therapeutic agent for inhibiting local invasion and metastasis.