GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo

We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC

Ferritin Heavy Subunit Silencing Blocks the Erythroid Commitment of K562 Cells via miR-150 up-Regulation and GATA-1 Repression

Erythroid differentiation is a complex and multistep process during which an adequate supply of iron for hemoglobinization is required. The role of ferritin heavy subunit, in this process, has been mainly attributed to its capacity to maintain iron in a non-toxic form. We propose a new role for ferritin heavy subunit (FHC) in controlling the erythroid commitment of K562 erythro-myeloid cells. FHC knockdown induces a change in the balance of GATA transcription factors and significantly reduces the expression of a repertoire of erythroid-specific genes, including α- and γ-globins, as well as CD71 and CD235a surface markers, in the absence of differentiation stimuli. These molecular changes are also reflected at the morphological level. Moreover, the ability of FHC-silenced K562 cells to respond to the erythroid-specific inducer hemin is almost completely abolished. Interestingly, we found that this new role for FHC is largely mediated via regulation of miR-150, one of the main microRNA implicated in the cell-fate choice of common erythroid/megakaryocytic progenitors. These findings shed further insight into the biological properties of FHCand delineate a role in erythroid differentiation where this protein does not act as a mere iron metabolism-related factor but also as a critical regulator of the expression of genes of central relevance for erythropoiesis

<<A>> cross- talk between estrogens and IGF1R pathways controls leydig and adrenocortical tumor cell proliferation

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXIV Ciclo, a.a. 2010-2011Università della Calabri

Chemoresistance in H-Ferritin Silenced Cells: The Role of NF-κB

Nuclear Factor-&kappa;B (NF-&kappa;B) is frequently activated in tumor cells contributing to aggressive tumor growth and resistance to chemotherapy. Here we demonstrate that Ferritin Heavy Chain (FHC) protein expression inversely correlates with NF-&kappa;B activation in cancer cell lines. In fact, FHC silencing in K562 and SKOV3 cancer cell lines induced p65 nuclear accumulation, whereas FHC overexpression correlated with p65 nuclear depletion in the same cell lines. In FHC-silenced cells, the p65 nuclear accumulation was reverted by treatment with the reactive oxygen species (ROS) scavenger, indicating that NF-&kappa;B activation was an indirect effect of FHC on redox metabolism. Finally, FHC knock-down in K562 and SKOV3 cancer cell lines resulted in an improved cell viability following doxorubicin or cisplatin treatment, being counteracted by the transient expression of inhibitory of NF-&kappa;B, I&kappa;B&alpha;. Our results provide an additional layer of information on the complex interplay of FHC with cellular metabolism, and highlight a novel scenario of NF-&kappa;B-mediated chemoresistance triggered by the downregulation of FHC with potential therapeutic implications

Ferritin Heavy Subunit Silencing Blocks the Erythroid Commitment of K562 Cells via miR-150 up-Regulation and GATA-1 Repression

Erythroid differentiation is a complex and multistep process during which an adequate supply of iron for hemoglobinization is required. The role of ferritin heavy subunit, in this process, has been mainly attributed to its capacity to maintain iron in a non-toxic form. We propose a new role for ferritin heavy subunit (FHC) in controlling the erythroid commitment of K562 erythro-myeloid cells. FHC knockdown induces a change in the balance of GATA transcription factors and significantly reduces the expression of a repertoire of erythroid-specific genes, including α- and γ-globins, as well as CD71 and CD235a surface markers, in the absence of differentiation stimuli. These molecular changes are also reflected at the morphological level. Moreover, the ability of FHC-silenced K562 cells to respond to the erythroid-specific inducer hemin is almost completely abolished. Interestingly, we found that this new role for FHC is largely mediated via regulation of miR-150, one of the main microRNA implicated in the cell-fate choice of common erythroid/megakaryocytic progenitors. These findings shed further insight into the biological properties of FHCand delineate a role in erythroid differentiation where this protein does not act as a mere iron metabolism-related factor but also as a critical regulator of the expression of genes of central relevance for erythropoiesis

FTH1P3, a Novel H-Ferritin Pseudogene Transcriptionally Active, Is Ubiquitously Expressed and Regulated during Cell Differentiation.

Ferritin, the major iron storage protein, performs its essential functions in the cytoplasm, nucleus and mitochondria. The variable assembly of 24 subunits of the Heavy (H) and Light (L) type composes the cytoplasmic molecule. In humans, two distinct genes code these subunits, both belonging to complex multigene families. Until now, one H gene has been identified with the coding sequence interrupted by three introns and more than 20 intronless copies widely dispersed on different chromosomes. Two of the intronless genes are actively transcribed in a tissue-specific manner. Herein, we report that FTH1P3, another intronless pseudogene, is transcribed. FTH1P3 transcript was detected in several cell lines and tissues, suggesting that its transcription is ubiquitary, as it happens for the parental ferritin H gene. Moreover, FTH1P3 expression is positively regulated during the cell differentiation process

FHC silencing increases cell proliferation.

<p>(A) Western blot analysis for FHC was performed on 50μg of total proteins extracted from FHC-silenced H460 (H460^siFHC) or from H460 control cells (H460^siRNA). Blots are representative of three independent experiments. γ-Tubulin was used as a loading control. The graph represents the mean of the optical densities (*<i>p</i> < 0.05 compared with H460^siRNA). (B) Real-time PCR analysis of FHC mRNA amounts performed on total RNA from H460^siFHC and H460^siRNAcells. Results are representative of three different experiments (*<i>p</i><0,05 compared with H460^siRNA). (C) H460^siRNAand H460^siFHCcells were incubated for 15 min with 20 μM of 2’-7’-DCF and washed with HBSS solution. Fluorescence was measured at 485 nm and 535 nm after60 min. (D) Cell proliferation was assessed using the MTT method as indicated in the Materials and Methods section. Final results represent mean ± SD of three independent experiments each performed in octuplicate (*<i>p</i>< 0.05 compared with H460^siRNA). (E) Western blot analysis for CCND1, p53 and pAKT were performed on 50μg of total proteins extracted from H460^siFHC and H460^siRNA. Blots are representative of three independent experiments. γ-Tubulin and AKT were used as loading controls.(F) Western blot analysis for FHC was performed on 50μg of total proteins extracted from FHC-stably silenced H460 (H460^shFHC) or from H460 control cells (H460^shRNA). Blots are representative of three independent experiments. γ-Tubulin was used as a loading control. The graph represents the mean of the optical densities (*<i>p</i> < 0.05 compared with H460^shRNA). (G) Cell proliferation of stably silenced cells was assessed using the MTT method. Final results represent mean ± SD of three independent experiments each performed in octuplicate (*<i>p</i>< 0.05 compared with H460^shRNA).</p

FHC is required for caffeine modulation of H460 proliferation.

<p>(A) Real-time PCR analysis of FHC mRNA amounts was performed on total RNA from H460^siFHC and H460^siRNA cells treated with the indicated doses of caffeine. Results are representative of two different experiments (*<i>p</i> < 0.05 of each caffeine concentration compared with untreated cells; NS not significant). (B) Cell proliferation was assessed using the MTT method on H460^siFHC and H460^siRNAcells treated with caffeine at the indicated doses. Final results represent mean ± SD of three independent experiments each performed in triplicate (*<i>p</i> < 0.05 of each caffeine concentration compared withuntreated cells; NS not significant). (C) Direct cell counting of H460^siFHC and H460^siRNAcells treated with caffeine at the indicated doses. Final results represent mean ± SD of two independent experiments (*<i>p</i> < 0.05 of each caffeine concentration compared with untreated cells; NS not significant). (D) Western blot analysis forCCND1, p53 and pAKT were performed on 50μg of total proteins extracted from H460^siFHC and H460^siRNA treated with 80μM caffeine or untreated. γ-Tubulin and AKT were used as loading controls.</p

Caffeine reduces H460 cell proliferation.

<p>(A) Cell proliferation was assessed using the MTT method as indicated in the Materials and Methods section. Final results represent mean ± SD of three independent experiments each performed in triplicate (*<i>p</i> < 0.05 of each caffeine concentration compared with NT-untreated cells). (B) Direct cell counting of NT-untreated and 80μM caffeine<b>-</b>treated cells. The results are the mean of two independent experiments (*<i>p</i> < 0.05 compared with NT-untreated cells). (C) and (D)Western blot analysis for CCND1, p53 and pAKT were performed on 50μg of total proteins. Blots are representative of three independent experiments. γ-Tubulin and AKT were used as loading controls. (E) DNA was extracted from cells and analyzed on a 2% agarose gel as described in Materials and Methods. The image is a representative experiment.</p