A new approach is described for delivering small interfering RNA (siRNA) into cancer cells by noncovalently complexing unmodifi ed siRNA with pristine single-walled carbon nanotubes (SWCNTs). The complexes were prepared by simple sonication of pristine SWCNTs in a solution of siRNA, which then served both as the cargo and as the suspending agent for the SWCNTs. When complexes containing siRNA targeted to hypoxiainducible factor 1 alpha (HIF-1 ) were added to cells growing in serum containing culture media, there was strong specific inhibition of cellular HIF-1 activity. The ability to obtain a biological response to SWCNT / siRNA complexes was seen in a wide variety of cancer cell types. Moreover, intratumoral administration of SWCNT-HIF-1 siRNA complexes in mice bearing MiaPaCa-2 / HRE tumors signifi cantly inhibited the activity of tumor HIF-1 . As elevated levels of HIF-1 are found in many human cancers and are associated with resistance to therapy and decreased patient survival, these results imply that SWCNT / siRNA complexes may have value as therapeutic agents.
BackgroundHomologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3′-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands.Methodology/Principle FindingsThis paper re-examines heteroduplex-patch-strand polarity using phage λ and the λdv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for λ recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5′ or 3′ DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation.Conclusions/SignificanceThese results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.
Oncogenic mutation of the KRas enzyme occurs frequently in human tumors and results in the constitutive activation of multiple signaling pathways. KRas mutation occurs in three specific codons, 12, 13, or 61, and involves the substitution of multiple amino acids. We observed in cancer cell lines containing activating mutations in KRas but not wild type KRas, that the Hypoxia Inducible Factor 1 α (HIF-1α) is expressed and functional under normoxic conditions. HIF-1α itself has been implicated in multiple aspects of tumorigenesis, including survival and invasion. We have found that exogenous expression of wild type KRas or the introduction of oncogenic KRas into transformed cells, causes the expression of HIF-1α in normoxia, but has little or no effect on HIF-1α induction by hypoxia. Additionally, in cancer cells, the deletion of the oncogenic KRas allele, while preserving the wild type allele, silenced the normoxic expression of HIF-1α. To determine the mechanisms by which KRas elicits the expression of HIF-1α, we quantitated the expression of 176 phospho or total proteins utilized a Reverse Phase Protein Array (RPPA) of 74 highly characterized non small cell lung cancer lines (NSCLC's) including 22 NSCLS with oncogenic KRas under three different culture conditions (with serum, without serum and starved then stimulated with serum for 30 minutes). From this RPPA, we found that NSCLC lines containing oncogenic KRas expressed active serine 473 Akt at differing levels, dependent upon which amino acid substitution was present in KRas. Additionally, we found that the KRas activation of Akt signaling, along with other KRas dependent signals, was responsible for HIF-1 stabilization in a hypoxia independent manner. As previously suggested we find that through KRas and Akt driven signaling, KRas is able to overcome the oxygen driven mechanisms responsible for HIF-1α degradation by increased HIF-1α protein translation However, we additionally found that aerobic glycolysis driven by KRas and Akt, specifically the accumulation of the products of glycolysis, also contribute to HIF-1α expression in normoxia. Finally, genome wide mRNA profiling of a KRas transformed line with a stable shRNA mediated knockdown of HIF-1α compared it a vector control showed that under normoxic conditions, knockdown of HIF-1α down regulates several genes important to transformation, including growth factors previously associated with KRas induced transformation. This data suggests inhibitors of HIF-1α may be effective in tumors harboring the KRas oncogenes. Additionally, Akt and HIF-1α inhibitors may be more active when combined with other agents targeted to other signaling pathways activated by KRas in order to bypass resistance mediated by redundant KRas driven signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 306.
The ability to survive hypoxic stress is an essential component of tumor development. Many rapidly proliferating solid tumors outgrow their vasculature while the abnormal tumor vasculature leads to small, transient pockets of hypoxic cells. In addition to being deprived of oxygen, these cells are also deprived of nutrients and unable to export harmful byproducts of cellular metabolism out of their immediate environment. One mechanism cancer cells employ to survive hypoxic stress is the stabilization and activation of the transcription factor hypoxia inducible factor 1α (HIF-1α), which translocates to the nucleus and induces pro-survival genes such as VEGF, Glut-1, and glycolytic enzymes. MiaPaCa-2 pancreatic carcinoma cells stably transfected with a 5X hypoxia response element (HRE) promoter regulating the firefly luciferase gene were generated, and shown to express luciferase only when HIF-1α was stabilized in hypoxia(1% O2). Using these cells, a genome wide siRNA screen was performed using the Dharmacon 22,000 gene siRNA library under the conditions of normoxia, 16hr and 72 hr hypoxia, to discover novel effectors of the HIF-1α pathway. Luciferase expression was corrected for cell number using a WST-1 cell proliferation assay prior to luciferase detection. Several hits were identified including genes which, when silenced, both increased and decreased HIF-1α transcriptional activity. Genes whose inhibition increased HIF-1α transcriptional activity in normoxia, in some cases up to 19 fold, include members of the E3 ubiquitin ligase complex which bind to and degrade HIF-1α under normal oxygen tensions, and also a number of other genes with uncharacterized function. There were also genes whose inhibition decreased hypoxia induced HIF-1α activity. Many were not well characterized, but there were a number of genes belonging to the MAP kinase pathway; the DNA repair pathway; the NF-κB pathway; and glycolysis genes, that were identified as necessary for HIF-1α transcriptional activity. Hits in these pathways were validated by secondary siRNAs and Western blots. It was discovered that silencing key genes involved in glycolysis inhibited HIF-1α transcriptional activity due to a decrease in HIF-1α protein levels. They include hexokinase-1, pyruvate dependent kinases-1,2 and 3, and 6-phosphofructo-1-kinase(M). These results suggest that increased glycolysis found in many tumors under aerobic conditions (the Warburg effect), which has been suggested to be caused by increased Myc expression, mutant Ras, mutant p53 and PI-3-kinase signaling, has an important role in increasing HIF-1α expression and activity. This stabilization and increase in activity then further induces the expression of target glycolytic genes and glycolytic activity in a feed-forward loop. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5505.
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