Barriers Associated with Cancer Research Commercialization.

<p>Respondents were asked to score whether a potential barrier (variable) is important to inhibiting their cancer research commercialization at UK. A) The percentage of respondents agreeing that a particular variable is a barrier to the commercialization of their cancer research. The raw data for panel A is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072268#pone.0072268.s007" target="_blank">Table S7</a>. B) The comparison (by percentage) of respondents indicating that they have attempted to commercialize their research and either agree versus not agree that a particular variable is a barrier to commercializing their research. The raw data for panel B is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072268#pone.0072268.s008" target="_blank">Table S8</a>. * p<0.05.</p

Sp1 mediated NFATc1 regulation of TRAIL expression.

<p>HT29 cells were transiently transfected with control siRNA or siRNA targeting NFATc1. Nuclear protein was extracted and EMSAs performed. Nuclear extracts were incubated with ³²P-labeled probe 4 (<b>A</b>) or probe 5 (<b>B</b>) alone or in the presence of unlabeled wild type (<i>cold</i>) probe 4 or probe 5, the Sp1 oligonucleotide, the NFAT oligonucleotide, or the ETS oligonucleotide, respectively. (<b>C</b>) HT29 cells were subjected to ChIP assay; soluble chromatin was prepared from HT29 cells transfected with control siRNA or siRNA targeting NFATc1 and immunoprecipitated with Sp1 antibody or IgG. Total (Input) and immunoprecipitated DNAs were then PCR-amplified using primer pairs covering the Sp1 binding sites within the human TRAIL promoter. (<b>D</b>) HT29 cells were treated with the Sp1 inhibitor mithramycin for 24 h and total protein extracted and Western blotting performed using anti-TRAIL antibody. (<b>E</b>) HT29 cells were transiently transfected with control siRNA or siRNA targeting Sp1. Forty-eight h after transfection, total protein was extracted and Western blotting performed to assess TRAIL expression (left panel) or to confirm knockdown of Sp1 (right panel). Membranes were stripped and re-probed with anti-β-actin antibody to confirm equal loading.</p

Knockdown of NFAT blocked PMA/Io-induced TRAIL expression in HT29 cells.

<p>(<b>A</b>) HT29 cells were treated with PMA/Io for 60 min and NFAT DNA binding activity was assessed by EMSA using nuclear extracts. In addition, nuclear extracts were incubated with ³²P-labeled NFAT specific DNA probe alone or in the presence of unlabeled wild type (<i>cold</i>) NFAT oligonucleotide or mutant NFAT oligonucleotide or specific antibodies to either NFATc1, NFATc2, NFATc3, or NFATc4. (<b>B</b>) HT29 cells were transfected with NFATc1, c2, c3, c4 or control siRNA. After a 48 h incubation, transfected cells were treated with PMA/Io for 2 h. Total protein was extracted and TRAIL expression levels were determined by Western blotting using anti-TRAIL antibody. The membrane was stripped and reprobed using anti-β-actin antibody to confirm equal loading. TRAIL signals from three separate experiments were quantitated densitometrically and expressed as fold-change with respect to β-actin. (Data shown as mean ± standard error of the mean; *, <i>P</i><0.05, PMA/Io plus control siRNA vs. control siRNA; ⁺, <i>P</i><0.05, PMA/Io plus NFATC1 siRNA or NFATC4 siRNA vs. PMA/Io plus control siRNA). (<b>C</b>) To confirm NFAT suppression, total RNA was extracted from cells and NFAT expression was assessed by RT-PCR.</p

Identification of NFATc1 responsive sequences in the TRAIL promoter.

<p>(<b>A</b>) Double-stranded oligonucleotides corresponding to all of the potential transcriptional binding sites in human TRAIL promoter −169 to −33 were synthesized and used for DNA binding analysis. The putative Sp1 binding sequences are boxed, which are also overlapped with other transcription factor binding sequences. (<b>B</b>) HT29 cells were treated with or without PMA/Io for 60 min and nuclear protein extracted. Double-stranded oligonucleotides (as shown in A) were radiolabeled and tested for DNA binding by EMSA. The arrows indicated the increased binding complexes. (<b>C</b>) Total RNA from HT29 cells stably transfected with control (CON) or four individual shRNAs targeting human NFATc1 (clones 33, 34, 35, 36) was extracted and RT-PCR performed for NFATc1 mRNA expression. (<b>D and E</b>) Nuclear protein (D) or total protein (E) from HT29 cells stably transfected with control (CON) or NFATc1 shRNA (clones 33, 34, 36) was extracted. EMSA was performed (D) using probe 4 and probe 5 (sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019882#pone-0019882-g006" target="_blank">Figure 6</a>). Western blotting was performed for the analysis of TRAIL expression (E); membranes were stripped and re-probed with anti-β-actin to confirm equal loading. TRAIL signals from two separate experiments were quantitated densitometrically and expressed as fold-change with respect to β-actin (Data shown as mean ± standard error of the mean).</p

Overexpression of NFATc1 increased TRAIL expression in HT29 and Caco-2 cells.

<p>HT29 cells were transfected with either the control plasmid (pDF30) or Flag-tagged NFATc1. At 48 h posttransfection, cells were harvested. (<b>A</b>) Expression of TRAIL and NFATc1 protein was detected by Western blot using anti-TRAIL or anti-Flag antibodies. Membranes were reprobed with anti-β-actin antibody to assess loading. (<b>B and C</b>) Total RNA was extracted, and RT-PCR (<b>B</b>) or real time PCR (<b>C</b>) performed. (Data shown as mean ± standard error of the mean. *, <i>P</i><0.05, vs. control plasmids). (<b>D</b>) Caco-2 cells were transfected with a proximal 1371 bp TRAIL promoter construct together with either control plasmid pDF30, pCDNA3.1 or Flag-tagged NFATc1, NFATc2, NFATc3 or NFATc4. At 48 h posttransfection, cells were harvested; luciferase activity was then assayed. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). (Data shown as mean ± standard error of the mean. *, <i>P</i><0.05, vs. control plasmids). (<b>E</b>) Expression of NFATc1, NFATc2, NFATc3 and NFATc4 in Caco-2 cells was confirmed by Western blot using anti-Flag antibody. (<b>F</b>) Caco-2 cells were transfected with control plasmid, pDF30, or Flag-tagged NFATc1. At 48 h posttransfection, total RNA was extracted, and RT-PCR performed using primers to human TRAIL and β-actin. TRAIL signals from three separate experiments were quantitated densitometrically and expressed as fold-change with respect to β-actin (Data shown as mean ± standard error of the mean).</p

PMA/Io-mediated induction of TRAIL expression was attenuated by cyclosporin A, a potent calcineurin inhibitor in HT29 cells.

<p>(<b>A</b>) HT29 cells were pre-treated with cyclosporin A (CsA) for 30 min followed by the combination of PMA (100 nM) plus Io (2.5 µM) with CsA for 2 h. Total protein was extracted from cells and resolved by SDS-PAGE, transferred to a PVDF membrane and probed with anti-TRAIL and anti-β-actin antibodies. TRAIL signals from three separate experiments were quantitated densitometrically and expressed as fold-change with respect to β-actin. (Data shown as mean ± standard error of the mean; *, <i>P</i><0.05, PMA/Io or PMA/Io plus CSA vs. control; ⁺, <i>P</i><0.05, PMA/Io plus CSA vs. PMA/Io). (<b>B</b>) Cells were treated with PMA/Io for 60 min in the presence or absence of CsA. Nuclear protein was extracted and NFAT DNA binding was analyzed by EMSA. Unlabeled NFAT oligonucleotide was added in molar excess to confirm binding specificity (<i>competitor</i>).</p

Deletion of NFAT binding sites did not eliminate NFATc1 increased TRAIL promoter activity.

<p>(<b>A</b>) Nucleotide sequences of the putative NFAT binding sites in the human TRAIL promotor. The 1371 bp TRAIL promoter contains five putative NFAT binding sites (N1, N2, N3, N4, and N5). The numbers on the left are the nucleotide positions relative to the transcriptional start site. (<b>B</b>) HT29 cells were treated with or without PMA/Io for 60 min and nuclear protein extracted; EMSA was performed with ³²P-labeled probes spanning each of the five NFAT binding sites (N1, N2, N3, N4, and N5) in the human TRAIL promoter. (<b>C</b>) Specific binding of various NFAT isoforms with probe N4 or N5 was determined by addition of antibodies to either NFATc1, NFATc2, NFATc3, or NFATc4. Specific binding of NFAT was also confirmed by cold competition using unlabeled wild type (WT) and mutant probes at 100-fold molar excess. (<b>D</b>) Caco-2 cells were transfected with constructs containing −35 or −165 TRAIL promoter sequences together with either control plasmid pDF30, or Flag-tagged NFATc1, respectively. At 48 h posttransfection, cells were harvested; and luciferase activity was assayed. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). (Data shown as mean ± standard error of the mean; *, <i>P</i><0.05, DF30 or NFATc1 plus −165 bp promoter vs. DF30 or NFATc1 plus −35 promoter, respectively; ⁺, <i>P</i><0.05, DF30 plus −165 bp promoter vs. NFATc1 plus −165 bp promoter).</p

Delivery of RNA Nanoparticles into Colorectal Cancer Metastases Following Systemic Administration

The majority of deaths from all cancers, including colorectal cancer (CRC), is a result of tumor metastasis to distant organs. To date, an effective and safe system capable of exclusively targeting metastatic cancers that have spread to distant organs or lymph nodes does not exist. Here, we constructed multifunctional RNA nanoparticles, derived from the three-way junction (3WJ) of bacteriophage phi29 motor pRNA, to target metastatic cancer cells in a clinically relevant mouse model of CRC metastasis. The RNA nanoparticles demonstrated metastatic tumor homing without accumulation in normal organ tissues surrounding metastatic tumors. The RNA nanoparticles simultaneously targeted CRC cancer cells in major sites of metastasis, such as liver, lymph nodes, and lung. Our results demonstrate the therapeutic potential of these RNA nanoparticles as a delivery system for the treatment of CRC metastasis