12 research outputs found

    Table1_Long non-coding RNA H19 enhances the pro-apoptotic activity of ITF2357 (a histone deacetylase inhibitor) in colorectal cancer cells.DOCX

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    Introduction: Long non-coding RNA H19 (lncH19) is highly expressed in colorectal cancer (CRC) and plays critical roles in tumor development, proliferation, metastasis, and drug resistance. Indeed, the expression of lncH19 usually affects the outcomes of chemo-, endocrine, and targeted therapies. ITF2357 (givinostat) is a histone deacetylase inhibitor (HDACi) that revealed a significant anti-tumor action by inducing apoptosis in different tumor models, including leukemia, melanoma, and glioblastoma. However, no data are present in the literature regarding the use of this compound for CRC treatment. Here, we investigate the role of lncH19 in ITF2357-induced apoptosis in CRC cells.Methods: The HCT-116 CRC cell line was stably silenced for H19 to investigate the role of this lncRNA in ITF2357-induced cell death. Cell viability assays and flow cytometric analyses were performed to assess the anti-proliferative and pro-apoptotic effects of ITF2357 in CRC cell lines that are silenced or not for lncH19. RT-PCR and Western blot were used to study the effects of ITF2357 on autophagy and apoptosis markers. Finally, bioinformatics analyses were used to identify miRNAs targeting pro-apoptotic factors that can be sponged by lncH19.Results: ITF2357 increased the expression levels of H19 and reduced HCT-116 cell viability, inducing apoptosis, as demonstrated by the increase in annexin-V positivity, caspase 3 cleavage, and poly (ADP-ribose) polymerase (PARP-1) degradation. Interestingly, the apoptotic effect of ITF2357 was much less evident in lncH19-silenced cells. We showed that lncH19 plays a functional role in the pro-apoptotic activity of the drug by stabilizing TP53 and its transcriptional targets, NOXA and PUMA. ITF2357 also induced autophagy in CRC cells, which was interpreted as a pro-survival response not correlated with lncH19 expression. Furthermore, ITF2357 induced apoptosis in 5-fluorouracil-resistant HCT-116 cells that express high levels of lncH19.Conclusion: This study shows that lncH19 expression contributes to ITF2357-induced apoptosis by stabilizing TP53. Overall, we suggest that lncH19 expression may be exploited to favor HDACi-induced cell death and overcome 5-fluorouracil chemoresistance.</p

    Image1_Long non-coding RNA H19 enhances the pro-apoptotic activity of ITF2357 (a histone deacetylase inhibitor) in colorectal cancer cells.TIF

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    Introduction: Long non-coding RNA H19 (lncH19) is highly expressed in colorectal cancer (CRC) and plays critical roles in tumor development, proliferation, metastasis, and drug resistance. Indeed, the expression of lncH19 usually affects the outcomes of chemo-, endocrine, and targeted therapies. ITF2357 (givinostat) is a histone deacetylase inhibitor (HDACi) that revealed a significant anti-tumor action by inducing apoptosis in different tumor models, including leukemia, melanoma, and glioblastoma. However, no data are present in the literature regarding the use of this compound for CRC treatment. Here, we investigate the role of lncH19 in ITF2357-induced apoptosis in CRC cells.Methods: The HCT-116 CRC cell line was stably silenced for H19 to investigate the role of this lncRNA in ITF2357-induced cell death. Cell viability assays and flow cytometric analyses were performed to assess the anti-proliferative and pro-apoptotic effects of ITF2357 in CRC cell lines that are silenced or not for lncH19. RT-PCR and Western blot were used to study the effects of ITF2357 on autophagy and apoptosis markers. Finally, bioinformatics analyses were used to identify miRNAs targeting pro-apoptotic factors that can be sponged by lncH19.Results: ITF2357 increased the expression levels of H19 and reduced HCT-116 cell viability, inducing apoptosis, as demonstrated by the increase in annexin-V positivity, caspase 3 cleavage, and poly (ADP-ribose) polymerase (PARP-1) degradation. Interestingly, the apoptotic effect of ITF2357 was much less evident in lncH19-silenced cells. We showed that lncH19 plays a functional role in the pro-apoptotic activity of the drug by stabilizing TP53 and its transcriptional targets, NOXA and PUMA. ITF2357 also induced autophagy in CRC cells, which was interpreted as a pro-survival response not correlated with lncH19 expression. Furthermore, ITF2357 induced apoptosis in 5-fluorouracil-resistant HCT-116 cells that express high levels of lncH19.Conclusion: This study shows that lncH19 expression contributes to ITF2357-induced apoptosis by stabilizing TP53. Overall, we suggest that lncH19 expression may be exploited to favor HDACi-induced cell death and overcome 5-fluorouracil chemoresistance.</p

    Effects of CTO on cell adhesion molecules and cytokines mRNA expression.

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    <p>(I) CTO reverts the effects of CML exosome treatment on VCAM1, ICAM1 and IL8 mRNA expression in HUVEC cells. VCAM1 (a), ICAM1(b) and IL8 (c) mRNA expression increased in a dose dependent manner after adding exosomes (Exo) to endothelial cell monolayer. CTO (1-5-10 µM) reverts these effects in a time- and dose dependent manner. (II) VCAM1, ICAM1 and IL8 mRNA expression in HUVEC treated for 6 h either with low serum medium (CN), or with 50 µg/ml exosomes (Exo), or with 10 ng/ml of recombinant IL8 (Rec IL8) with or without CTO 10 µM, or with 50 µg/ml exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8). Values are representative for three independent experiments. *p≤0.05; **p≤0.01.</p

    CTO inhibits the effects of LAMA84R exosomes on HUVEC migration.

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    <p>(a) Effects on migration of CTO-treated endothelial cells using 50 µg/ml of exosomes as chemoattractant. (b) 50 µg/ml of exosomes (Exo) with or without 10 µg/ml of neutralizing anti-IL8 antibody (N Ab IL8), or 10 ng/ml of recombinant IL8 (Rec IL8) with or without neutralizing anti-IL8 antibody were added as chemoattractants to the bottom wells., Motility of endothelial cells with or without increasing doses of CTO (1–10 µM) was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a>. *p≤0.05; **p≤0.01.</p

    <i>in vitro</i> inhibition of exosome-stimulated angiogenesis by CTO.

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    <p>(a) Phase contrast micrographs showing the effects of LAMA84R exosomes and CTO treatment on endothelial network formation (matrigel assay). Few cables are observed when HUVEC are plated in low serum medium (CN). Addition to HUVEC cells of 10 ng/ml of recombinant IL8 (Rec IL8) or 50 µg/ml of LAMA84R exosomes (Exo) induces the formation of capillary-like structures. No tube formation is observed when HUVEC are plated in the presence of 50 µg/ml of exosomes plus neutralizing anti-IL8 antibody (Exo + N Ab IL8). CTO inhibits the effects of recombinant IL8 (10 µM CTO + Rec IL8) or exosomes (Exo +10 µM CTO) on tube formation by HUVEC on matrigel. (b) Histograms showing the quantitative analysis of the cables length by Image J software.</p

    Antitumor activity of CTO on human CML xenografts.

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    <p>LAMA84R cells were injected subcutaneously in NOD/SCID mice as described. After palpable tumor formation, mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a>. Comparison of the median tumor weight was used as index of the antitumor efficacy of the compounds.</p

    Effects of CTO on cell adhesion molecules and cytokines production.

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    <p>CTO inhibits the exosomes-stimulated increase of VCAM1, ICAM1 expression and IL8 secretion on HUVEC cells. Representative overlay histograms showing an increase of surface expression of VCAM1 (a) and ICAM1 (b) on HUVEC treated with 50 µg/ml of LAMA84R exosomes (blue line) compared to HUVEC treated with 50 µg/ml of LAMA84R exosomes plus 10 µM CTO (red line) or untreated HUVEC, as control (green line). (c) ELISA for IL8 release by HUVEC. Aliquots of conditioned medium (CM) from cells were collected after 6 h of stimulation either with 50 µg/ml of exosomes (CM HUVEC + Exo) or with 50 µg/ml of exosomes plus 10 µM CTO (CM HUVEC + CTO + Exo); low serum medium (CM HUVEC) or low serum medium plus 10 µM CTO (CM HUVEC + CTO) were used as negative controls. The amount of IL8 in 50 µg/ml of exosomes was also quantified.</p

    CTO inhibits cell proliferation of LAMA84R and K562R cell lines.

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    <p>(a) Cell growth was measured by MTT assay after 24, 48, 72, 96 h of treatment with increasing doses of CTO (0.1-1-5-10 µM). The values were plotted as a percentage of the control (cells treated with DMSO). Each point represents the mean ± SD for three independent experiments. *p≤0.001. (b) CTO treatment decreases Bcr–Abl expression, inhibits Bcr–Abl tyrosine phosphorylation and its downstream substrate CrkL on LAMA84R and K562R cell lines. These cell lines were treated with increasing doses of CTO (0.1–5 µM) or DMSO (CN) for 72 h and 96 h; afterwards protein lysates were subjected to western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a> using anti-phospho-Abl, anti-cAbl, anti-phospho-CrkL and anti-CrkL antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.</p

    CTO inhibits the adhesion of LAMA84R cells to exosome-treated HUVEC monolayer.

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    <p>(a) Phase contrast micrographs showing the adhesion of LAMA84R cells (arrows) on HUVEC monolayer treated with 50 µg/ml of exosomes (Exo) and with 50 µg/ml of exosomes after pre-treatment of 24 h with 10 µM CTO. (b) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo) and exosomes plus increasing doses of CTO (1–10 µM); (c) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo), 50 µg/ml of exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8), 10 ng/ml of recombinant IL8 (Rec IL8), 10 ng/ml of recombinant IL8 plus 10 µg/ml of a neutralizing anti-IL8 antibody and 10 ng/ml of recombinant IL8 with increasing doses of CTO (1–10 µM). Values are representative for three independent experiments. *p≤0.05; **p≤0.01.</p

    LAMA84R exosomes characterization.

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    <p>(a) Exosomes released by LAMA84R cells observed with a scanning electron microscope. (b) Detection of Hsc70 and CD63 in 30 µg of cell lysate (lane 1) and 30 µg of exosomes lysate (lane 2). (c) Acetylcholinesterase assay. The activity of acetylcholinesterase, an exosome-specific protein marker, was determined in 10 µg either of total cell lysate (Cells) or of Exosomes (Exo); exosome-deprived conditioned medium (CM-Exo) and exosome-deprived Fbs (Fbs–Exo) were used as negative controls.</p
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