Intratracheal versus intravenous liposomal delivery of siRNA, antisense oligonucleotides and anticancer drug.

PURPOSE: To compare systemic intravenous and local intratracheal delivery of doxorubicin (DOX), antisense oligonucleotides (ASO) and small interfering RNA (siRNA). METHODS: "Neutral" and cationic liposomes were used to deliver DOX, ASO, and siRNA. Liposomes were characterized by dynamic light scattering, zeta-potential, and atomic force microscopy. Cellular internalization of DOX, ASO and siRNA was studied by confocal microscopy on human lung carcinoma cells. In vivo experiments were carried out on nude mice with an orthotopic model of human lung cancer. RESULTS: Liposomes provided for an efficient intracellular delivery of DOX, ASO, and siRNA in vitro. Intratracheal delivery of both types of liposomes in vivo led to higher peak concentrations and much longer retention of liposomes, DOX, ASO and siRNA in the lungs when compared with systemic administration. It was found that local intratracheal treatment of lung cancer with liposomal DOX was more efficient when compared with free and liposomal DOX delivered intravenously. CONCLUSIONS: The present study outlined the clear advantages of local intratracheal delivery of liposomal drugs for the treatment of lung cancer when compared with systemic administration of the same drug.

Nonviral nanoscale-based delivery of antisense oligonucleotides targeted to hypoxia-inducible factor 1 alpha enhances the efficacy of chemotherapy in drug-resistant tumor.

PURPOSE: To enhance the efficacy of cancer treatment, we propose a complex approach: simultaneous delivery to the tumor of a chemotherapeutic agent and a suppressor of hypoxia-inducible factor 1 alpha (HIF1A). EXPERIMENTAL DESIGN: The novel complex liposomal drug delivery system was developed and evaluated in vitro and in vivo on nude mice bearing xenografts of multidrug-resistant human ovarian carcinoma. The proposed novel complex drug delivery system consists of liposomes as a nanocarrier, a traditional anticancer drug (doxorubicin) as a cell death inducer, and antisense oligonucleotides targeted to HIF1A mRNA as a suppressor of cellular resistance and angiogenesis. RESULTS: The system effectively delivers active ingredients into tumor cells, multiplies the cell death signal initiated by doxorubicin, and inhibits cellular defensive mechanisms and angiogenesis by down-regulating BCL2, HSP90, and vascular endothelial growth factor proteins. This, in turn, activates caspases, promotes apoptosis, necrosis, and tumor shrinkage. The proposed novel complex multipronged approach enhances the efficiency of chemotherapy. CONCLUSIONS: The proposed combination therapy prevents the development of resistance in cancer cells, and thus, increases the efficacy of chemotherapy to an extent that cannot be achieved by individual components applied separately. It could form the foundation for a novel type of cancer therapy based on simultaneous delivery of an anticancer drug and a suppressor of HIF1A.

Targeted proapoptotic anticancer drug delivery system.

A novel targeted proapoptotic anticancer drug delivery system (DDS) was developed and evaluated both in vitro and in vivo. The system contains poly(ethylene glycol) polymer (PEG) as a carrier, camptothecin (CPT) as an anticancer drug/cell death inducer, a synthetic analogue of luteinizing hormone-releasing hormone (LHRH) peptide as a targeting moiety/penetration enhancer, and a synthetic analogue of BCL2 homology 3 domain (BH3) peptide as a suppressor of cellular antiapoptotic defense. The design of the multicomponent DDS allowed for a conjugation of one or two copies of each active ingredient (CPT, LHRH, and BH3) to one molecule of PEG carrier. The complex structure of the PEG conjugates was visualized at nanometer resolution using atomic force microscopy. We found that the ligand-targeted DDS for cancer cells preferentially accumulated in the tumor and allowed the delivery of active ingredients into the cellular cytoplasm and nuclei of cancer cells. Simultaneous apoptosis induction through the caspase-dependent signaling pathway and inhibition of cellular antiapoptotic defense by the suppression of BCL2 protein enhanced cytotoxicity and antitumor activity of the entire DDS to a level which could not be achieved by individual components applied separately. The DDS containing two copies of each active component (CPT, LHRH, and BH3) per molecule of PEG polymer had the highest anticancer efficiency in vitro and in vivo.

Combined transcriptional and translational targeting of EWS/FLI-1 in Ewing's sarcoma.

PURPOSE: To show the efficacy of targeting EWS/FLI-1 expression with a combination of specific antisense oligonucleotides and rapamycin for the control of Ewing's sarcoma (EWS) cell proliferation in vitro and the treatment of mouse tumor xenografts in vivo. EXPERIMENTAL DESIGN: EWS cells were simultaneously exposed to EWS/FLI-1-specific antisense oligonucleotides and rapamycin for various time periods. After treatment, the following end points were monitored and evaluated: expression levels of the EWS/FLI-1 protein, cell proliferation, cell cycle distribution, apoptotic cell death, caspase activation, and tumor growth in EWS xenografts implanted in nude mice. RESULTS: Simultaneous exposure of EWS cells in culture to an EWS/FLI-1-targeted suppression therapy using specific antisense oligonucleotides and rapamycin resulted in the activation of a caspase-dependent apoptotic process that involved the restoration of the transforming growth factor-beta-induced proapoptotic pathway. In vivo, individual administration of either antisense oligonucleotides or rapamycin significantly delayed tumor development, and the combined treatment with antisense oligonucleotides and rapamycin caused a considerably stronger inhibition of tumor growth. CONCLUSIONS: Concurrent administration of EWS/FLI-1 antisense oligonucleotides and rapamycin efficiently induced the apoptotic death of EWS cells in culture through a process involving transforming growth factor-beta. In vivo experiments conclusively showed that the combined treatment with antisense oligonucleotides and rapamycin caused a significant inhibition of tumor growth in mice. These results provide proof of principle for further exploration of the potential of this combined therapeutic modality as a novel strategy for the treatment of tumors of the Ewing's sarcoma family.

End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes.

Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine-homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA-DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.

Specific binding of poly(ADP-ribose) polymerase-1 to cruciform hairpins.

Poly(ADP-ribose) polymerase-1 (PARP-1) participates in DNA cleavage and rejoining-dependent reactions, such as DNA replication, recombination and repair. PARP-1 is also important in transcriptional regulation, although the determinants for its binding to undamaged genomic DNA have not been defined. Previously, we have shown by low-resolution mapping that PARP-1 may bind to the cruciform-forming regions of its own promoter. Here, using DNase I and nuclease P(1) footprinting and atomic force microscopy, we show that PARP-1 binds to stem/loop boundaries of cruciform hairpins. Cleavage of the cruciform by the junction resolvase T4 endonuclease VII is independent of PARP-1, which indicates that PARP-1 does not bind to the four-arm junctions of the cruciform. Thus, PARP-1 differs from other cruciform-binding proteins by binding to hairpin tips rather than to junctions. Furthermore, our data indicate that PARP-1 can interact with the gene regulatory sequences by binding to the promoter-localized cruciforms.

Regulation of poly(ADP-ribose) polymerase-1 by DNA structure-specific binding.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an intracellular sensor of DNA strand breaks and plays a critical role in cellular responses to DNA damage. In normally functioning cells, PARP-1 enzymatic activity has been linked to the alterations in chromatin structure associated with gene expression. However, the molecular determinants for PARP-1 recruitment to specific sites in chromatin in the absence of DNA strand breaks remain obscure. Using gel shift and enzymatic footprinting assays and atomic force microscopy, we show that PARP-1 recognizes distortions in the DNA helical backbone and that it binds to three- and four-way junctions as well as to stably unpaired regions in double-stranded DNA. PARP-1 interactions with non-B DNA structures are functional and lead to its catalytic activation. DNA hairpins, cruciforms, and stably unpaired regions are all effective co-activators of PARP-1 auto-modification and poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. Enzyme kinetic analyses revealed that the structural features of non-B form DNA co-factors are important for PARP-1 catalysis activated by undamaged DNA. K0.5 constants for DNA co-factors, which are structurally different in the degree of base pairing and spatial DNA organization, follow the order: cruciform

Progressive loss of Syk and abnormal proliferation in breast cancer cells.

The tumor suppressor gene Syk tyrosine kinase is absent or reduced in invasive breast cancer tissues and cell lines; its loss in breast tissues is linked to poor prognosis and metastasis. Also, evidence shows that in vitro Syk is involved in regulating proliferation. Here, we show by in situ hybridization on breast tissue sections that the loss of Syk expression is progressive during tumor development. Strikingly, Syk is already partially lost in normal epithelial tissue adjacent to the cancer lesion. In vivo, cell proliferation (as measured by the proliferative index Ki67) increased from normal to ductal carcinoma in situ to invasive, whereas Syk in situ staining in the same tissues decreased. In vitro, the presence of Syk was associated with reduced cell proliferation in an epidermal growth factor receptor-overexpressing breast cancer cell line, BT549, whereas changes in apoptosis were undetected. Concomitantly, the kinase activity of the proto-oncogene Src was reduced by approximately 30%. A 5-fold increase in abnormal mitoses was observed in the Syk-transfected cells compared with vector control. We propose that Syk is involved in the regulation of cell proliferation, possibly by controlling mechanisms of mitosis and cytokinesis via Src signal transduction pathway(s). Because of its progressive and early loss during tumor onset and development, monitoring of Syk loss in breast epithelial cells by noninvasive techniques such as ductal lavage may be a powerful tool for screening purposes.

Targeting Ku protein for sensitizing of breast cancer cells to DNA-damage.

Targeting molecular components that are critically involved in the maintenance of genome stability is a promising approach for overcoming intrinsic tumor cell resistance to DNA-damaging treatments. In mammalian cells, the Ku-dependent non-homologous end-joining repair pathway is the predominant process for the repair of double-strand breaks (DSBs) in DNA. Previously, RNA aptamers were selected to efficiently block DNA-binding activity of the Ku protein in vitro. In the present study, we have tested the efficacy of RNA aptamers against the Ku protein as molecular sensitizer of MCF-7 breast carcinoma cells to DNA-damage. Toward this end, we established MCF-7 cell sublines stably expressing SC4 aptamer RNAs under the control of the human 7SL small nuclear RNA gene promoter. Vector-transfected (MCF/7SL) cells and cells stably expressing SC4 aptamers (MCF/SC4) were exposed to the anticancer drug etoposide and cellular responses to DNA-damage were evaluated. We found that the presence of RNA aptamers against Ku protein enhanced etoposide-induced growth inhibition of MCF-7 breast cancer. The SC4 aptamer-mediated sensitization of MCF-7 cells to the anticancer drug is attributable to an increased susceptibility of these cells to apoptosis. The observed effects cannot be accounted for by the differential expression levels of Ku protein in control and SC4 aptamer-expressing cells, but are rather due to augmented DNA binding-capacity of Ku protein, as demonstrated in in vitro studies. Thus, RNA aptamers against Ku protein show potential to sensitize MCF-7 breast carcinoma cells to DNA-damaging agents.

Gene therapy for prostate cancer by targeting poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) has strong affinity for DNA strand breaks and cycles on and off the DNA ends to allow DNA repair. A DNA-binding domain of PARP (PARP-DBD) acts as a dominant-negative mutant by binding to DNA strand breaks irreversibly and sensitizing mammalian cells to DNA-damaging agents. Therefore, expression of PARP-DBD in prostate carcinoma cells offers a strategy to achieve sensitization to genotoxic treatments. Toward this end, we developed recombinant plasmids expressing the PARP-DBD under the control of the 5'-flanking sequences of the human prostate-specific antigen (PSA) gene. Tissue specificity of PARP-DBD expression in human tumor cells was confirmed using the PSA-producing (LNCaP) and PSA-negative (PC-3) prostate cancer cells, as well as cells of nonprostate origin, Ewing's sarcoma (A4573 cells). LNCaP cells stably transfected with the PSA-regulated cDNA for PARP-DBD exhibit an androgen-dependent induction of PARP-DBD expression as determined by Western blotting, reverse transcription-PCR, and in situ immunofluorescence. Furthermore, we found that PARP-DBD sensitized LNCaP cells to DNA-damaging agents, such as ionizing radiation and etoposide. Androgen (R1881) -dependent stimulation of PARP-DBD expression resulted in a 2-fold growth inhibition in LNCaP cells as compared with controls, and an augmented apoptotic cell death in response to ionizing radiation or etoposide. Taken together, the plasmid vector developed in this study permits the expression of the human PARP-DBD in an androgen-inducible and PSA-dependent fashion, and sensitizes prostatic adenocarcinoma cells to DNA-damaging treatments. These results provide proof-of-principle for a novel therapeutic strategy for the treatment of prostate cancer.

Tailoring the activity of restriction endonuclease PleI by PNA-induced DNA looping.

DNA looping is one of the key factors allowing proteins bound to different DNA sites to signal one another via direct contacts. We demonstrate that DNA looping can be generated in an arbitrary chosen site by sequence-directed targeting of double-stranded DNA with pseudocomplementary peptide-nucleic acids (pcPNAs). We designed pcPNAs to mask the DNA from cleavage by type IIs restriction enzyme PleI while not preventing the enzyme from binding to its primary DNA recognition site. Direct interaction between two protein molecules (one bound to the original recognition site and the other to a sequence-degenerated site) results in a totally new activity of PleI: it produces a nick near the degenerate site. The PNA-induced nicking efficiency varies with the distance between the two protein-binding sites in a phase with the DNA helical periodicity. Our findings imply a general approach for the fine-tuning of proteins bound to DNA sites well separated along the DNA chain.

Poly(ADP-ribose) polymerase turnover alterations do not contribute to PARP overexpression in Ewing's sarcoma cells.

Ewing's sarcoma (EWS) cells contain significantly higher levels of poly(ADP-ribose) polymerase (PARP) mRNA, protein and enzymatic activities than any other eukaryotic cells. Evidence from our laboratory showed that increased transcription, rather than mRNA stability, contributes to the elevated PARP levels. It has been proposed that alterations in the normal turnover rate of PARP may also contribute to the total cellular PARP content as well as to the apoptotic response of Ewing's sarcoma cells to ionizing radiation. To address this possibility, we compared the turnover of PARP in EWS cells (A4573), which contain high PARP and are relatively radiosensitive, with that in laryngeal squamous cell carcinoma cells (SQ-20B), which have low PARP and are radioresistant. Results showed that PARP turnover parameters are nearly identical in both cells types. These data conclusively demonstrate that PARP turnover is not a determinant of either the elevated PARP content or the radiation response of EWS cells.

Differential regulation of the response to DNA damage in Ewing's sarcoma cells by ETS1 and EWS/FLI-1.

Ewing's sarcoma (EWS) cells contain levels of poly(ADP-ribose) polymerase (PARP) significantly higher than other eukaryotic cells. Previously, we cloned the PARP gene promoter region from EWS cells, showed that it contained multiple ETS-binding sites and demonstrated a positive regulation of PARP by ETS1. We now report that, contrary to ETS1, EWS/FLI-1, an aberrant ETS transcription factor present in most EWS cells, is a negative effector of PARP transcription. Because PARP levels have been associated with cellular resistance or sensitivity to genotoxic agents, we studied the effect of modifying PARP levels in EWS cells on their response to DNA damage by modulating the expression of ETS1 or EWS/FLI-1 using antisense methodology. Results show that stable down-regulation of ETS1 increases the resistance of EWS cells to various genotoxic agents, whereas down-regulation of EWS/FLI-1 has pro-apoptotic effects. Because down-regulation EWS/FLI-1 does not dramatically change PARP levels, these results suggest a direct effect for EWS/FLI-1 in the DNA damage response of EWS cells. Since expression of the aberrant fusion proteins by EWS cells is essential for maintaining their neoplastic phenotype, our results suggest that the use of antisense oligonucleotides in combination with chemotherapeutic agents or radiation may be doubly effective by causing both an increase in sensitivity to therapeutic agents and a simultaneous down-regulation, or reversion, of the neoplastic phenotype of EWS cells.

Transcriptional repression by binding of poly(ADP-ribose) polymerase to promoter sequences.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD+ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4-5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP(-/-) cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.