The chemopreventive retinoid 4HPR impairs prostate cancer cell migration and invasion by interfering with FAK/AKT/GSK3β pathway and β-catenin stability

<p>Abstract</p> <p>Background</p> <p>Prostate cancer shows an extremely slow progression, appearing in its metastatic, hormone refractory phenotype mostly in elderly men. The chemopreventive targeting of this tumor could accordingly delay its malignancy over life expectancy. The cancer chemopreventive retinoid <it>N</it>-(4 hydroxyphenyl)retinamide (4HPR) has already been shown to restrain prostate cancer growth in vitro and in vivo, though its mechanisms of action are only partially explained.</p> <p>Results</p> <p>We found that 4HPR impairs DU145 and PC3 prostate cancer cells migration and invasion by down-regulating FAK and AKT activation and by enhancing β-catenin degradation, causing the downregulation of target genes like cyclin D1, survivin and VEGF. This non-migratory phenotype was similarly produced in both cell lines by stable silencing of β-catenin. 4HPR was able to decrease AKT phosphorylation also when powerfully upregulated by IGF-1 and, consequently, to impair IGF-1-stimulated cell motility. Conversely, the expression of constitutively active AKT (myr-AKT) overcame the effects of 4HPR and β-catenin-silencing on cell migration. In addition, we found that BMP-2, a 4HPR target with antiangiogenic activity, decreased prostate cancer cell proliferation, migration and invasion by down-regulating the pathway described involving AKT phosphorylation, β-catenin stability and cyclin D1 expression.</p> <p>Conclusion</p> <p>These data point to 4HPR as a negative regulator of AKT phosphorylation, effectively targeting the β-catenin pathway and inducing a relatively benign phenotype in prostate cancer cells, limiting neoangiogenesis and cell invasion.</p

The immunological basis of tumor therapy by targeted delivery of TNFa to tumor vessels

ISSN:1742-469

NAC, Tiron and Trolox Impair Survival of Cell Cultures Containing Glioblastoma Tumorigenic Initiating Cells by Inhibition of Cell Cycle Progression

<div><p>Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.</p></div

NAC, tiron and trolox treatments of the PT4 cultures containing GBM TICs inhibit the phosphorylation of the retinoblastoma (RB) protein.

<p>Western blot analyses were performed with lysates of the PT4 cell culture containing GBM TICs treated for 48 h and 6 days with the indicated antioxidant drug. The membrane was challenged first with an anti phosphorylated RB (Ser 807/811) antibody, then with an anti total RB antibody and lastly with an anti tubulin antibody. Stripping and control of effective stripping were performed before re-challenging of the membrane.</p

Validation of gene expression regulation by Real-time RT-PCR and Immunoblot analysis.

<p>(A) Real-Time RT-PCR analysis performed with RNA extracted from the PT4 cell culture containing GBM TICs to validate the microarray data. This was accomplished on randomly selected genes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090085#pone.0090085.s012" target="_blank">Table S3</a> and showed, in arbitrary units. Expression levels are relative to the expression of the housekeeping gene transcript coding for the ribosomal protein L19 (RPL19). Standard deviations are indicated as vertical bars (n = 3 independent assays). Gene name symbols are those approved by the Human Genome Organization Gene Nomenclature Committee (<a href="http://www.genenames.org/" target="_blank">http://www.genenames.org/</a>). Standard deviations are indicated as vertical bars (n = 3 independent assays). #The unpaired t-test was significant at P<0.05. §The unpaired t-test was significant at P<0.01. *The unpaired t-test was significant at P<0.001. (B) Western blot analyses were performed with lysates of the PT4 cell culture containing GBM TICs treated for 48 h with the indicated antioxidant drug and challenged with anti MKi67, Pdz-binding kinase (PBK), transferrin receptor (TFRC), carbonic anhydrase 9 (CA9). (C) Western blot analyses were performed with lysates of the PT4 cell culture containing GBM TICs treated for 6 days with the indicated antioxidant drug and challenged with anti nestin (NES), oligodendrocyte transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2) and tumor protein 53 (TP53) antibodies. (D) Western blot analyses were performed with lysates of the PT4 cell culture containing GBM TICs treated for 48 h with the indicated antioxidant drug and challenged with anti tumor protein 53 (TP53) mAb. (B–D) Immunoblotted membranes were subjected to multiple antibody challenging, stripping, control of effective stripping, and re-challenging with a different antibody. The last antibody used was an anti histone deacetylase 1 (HDAC1) (B) or an anti tubulin alpha (D) to show equal loading.</p

Cell survival of the PT4, MM1 and FO-1 cell cultures containing TICs.

<p>Survival of PT4 cells (A, E); MM1 cells (B) and FO-1 cells (C, D) after 6 days of treatment with the indicated substances and solvent controls. The medium renewal schedule was identical to that used for the cultures containing GBM TICs (see Introduction). Cell survival is expressed in arbitrary units as evaluated by MTT analysis. Standard deviations are indicated as vertical bars (n = 3 independent assays). DMSO concentration in (D) was 0.1% vol/vol. Drug concentrations in (D) were: NAC 20 mM, PLX4032 10 µM. Gefitinib final concentration in (E) was 3.9 µM. #The unpaired t-test was significant at P<0.05. §The unpaired t-test was significant at P = 0.01 or less. *The unpaired t-test was significant at P<0.001. **The unpaired t-test was significant at P<0.0001.</p

Top three gene ontology biological processes resulting from the analysis of differential regulated genes between antioxidant drugs and the respective solvent controls.

<p>This analysis was performed using the DAVID program (see MM). Count indicates the number of genes belonging to the indicated gene ontology biological processes that we found regulated by a factor 2; % indicates the percentage of regulated genes belonging to the indicated biological process compared to the total number of regulated genes and included in the DAVID database; P-value indicates the probability that the indicated biological process would result by random selection.</p

NAC and tiron modify the distribution of the cell cycle phases in the PT4 cell culture containing GBM TICs.

<p>Representative experiment of high resolution FCM analysis of DAPI-stained nuclei of the H₂O control PT4 culture containing GBM TICs after 48 h of exposure (A). Analysis of the percentage of PT4 cells in the cell cycle phases as determined by the ModFit LT™ software after 48 h of exposure with the indicated substances at the IC50 concentration and solvent controls (B). Percentage of PT4 cells in S phase actively synthesizing DNA after 48 h of exposure to tiron at the IC50 concentration with respect to control condition (H₂O) performed by BrdU labeling and FCM analysis (C). Standard deviations are indicated as vertical bars (n = 4 independent assays, B ; n = 3 independent assays, C). #The unpaired t-test was significant at P<0.05. *The unpaired t-test was significant at P<0.001. **The unpaired t-test was significant at P<0.0001. ***The unpaired t-test was significant at P<0.00001.</p