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Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells.

Fiocchetti M, Nuzzo MT, Totta P, Acconcia F, Ascenzi P, Marino M - Cell Death Dis (2014)

Bottom Line: E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells.Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines.E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Science, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy.

ABSTRACT
Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17β-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.

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Effect of E2 on the localization of NGB in HepG2 and MCF-7 cells. Western blot of NGB level in nuclear (Nuclei), cytosolic (Cyt), and mitochondrial (Mit) fractions of (a) HepG2 and (b) MCF-7 cells and related densitometric analyses (c). The purity of fractions was assessed with PARP-1, TRAP1, and PP2A with respect to nucleus, mitochondria, and cytosol, respectively. The amount of proteins was normalized to each fraction marker protein. The figure represents a typical western blot of three independent experiments. Data reported in panel c represent the mean±S.D. of three different experiments. Significant differences (P<0.001) were determined by ANOVA followed by the Turkey–Kramer post-test with respect to unstimulated samples (*). (d) Confocal microscopy analysis of NGB and COX-4 co-immuno-localization. Cells were fixed, permeabilized, and stained with anti-NGB antibody (red) and co-stained with anti-COX4 antibody (green) (original magnification × 63). Merged images by confocal microscopy show NGB distribution in HepG2 cells treated with either vehicle or E2 (10 nM) for 1, 4, and 24 h. White arrows point to examples of NGB and COX-4 co-staining in a single Z-stack plane. Representative images from three different experiments are shown
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fig5: Effect of E2 on the localization of NGB in HepG2 and MCF-7 cells. Western blot of NGB level in nuclear (Nuclei), cytosolic (Cyt), and mitochondrial (Mit) fractions of (a) HepG2 and (b) MCF-7 cells and related densitometric analyses (c). The purity of fractions was assessed with PARP-1, TRAP1, and PP2A with respect to nucleus, mitochondria, and cytosol, respectively. The amount of proteins was normalized to each fraction marker protein. The figure represents a typical western blot of three independent experiments. Data reported in panel c represent the mean±S.D. of three different experiments. Significant differences (P<0.001) were determined by ANOVA followed by the Turkey–Kramer post-test with respect to unstimulated samples (*). (d) Confocal microscopy analysis of NGB and COX-4 co-immuno-localization. Cells were fixed, permeabilized, and stained with anti-NGB antibody (red) and co-stained with anti-COX4 antibody (green) (original magnification × 63). Merged images by confocal microscopy show NGB distribution in HepG2 cells treated with either vehicle or E2 (10 nM) for 1, 4, and 24 h. White arrows point to examples of NGB and COX-4 co-staining in a single Z-stack plane. Representative images from three different experiments are shown

Mentions: Recently, we demonstrated that in SK-N-BE cells E2 stimulation induces a localization of NGB at mitochondrial level, where it exerts an antiapoptotic role.13 Thus, the analysis of the subcellular localization of NGB was performed also in E2-related non-nervous cancer cells to obtain an inkling of its function in these cells. HepG2 and MCF-7 cells were fractionated into three main compartments (i.e., nuclei, mitochondria, and cytosol) using as fraction purity markers nuclear poly (ADP-ribose) polymerase 1 (PARP-1), mitochondrial TNF receptor-associated protein 1 (TRAP1), and cytosolic protein phosphatase 2A (PP2A) (Figures 5a, b, and c). Upon E2 stimulation (i.e., 10 nM; 24 h), a significant reduction of NGB in the nucleus with the parallel increase of NGB in the mitochondrial fraction in both HepG2 (Figure 5a) and MCF-7 (Figure 5b) cells was observed. However, the E2 treatment led to the increase of the NGB level in the cytosol of HepG2 cells, whereas in MCF-7 cells the amount of cytosolic NGB after E2 stimulation was similar to that observed in the control (Figures 5a and b). A further confirmation that E2 induces NGB re-allocation in mitochondria of HepG2 derives from the confocal microscopy analysis, which indicates that E2 increased the colocalization of NGB with cytochrome c oxidase 4 (COX-4) at mitochondrial level beginning 1 h after the stimulation and reaching a maximum 24 h after the hormone treatment (Figure 5d).


Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells.

Fiocchetti M, Nuzzo MT, Totta P, Acconcia F, Ascenzi P, Marino M - Cell Death Dis (2014)

Effect of E2 on the localization of NGB in HepG2 and MCF-7 cells. Western blot of NGB level in nuclear (Nuclei), cytosolic (Cyt), and mitochondrial (Mit) fractions of (a) HepG2 and (b) MCF-7 cells and related densitometric analyses (c). The purity of fractions was assessed with PARP-1, TRAP1, and PP2A with respect to nucleus, mitochondria, and cytosol, respectively. The amount of proteins was normalized to each fraction marker protein. The figure represents a typical western blot of three independent experiments. Data reported in panel c represent the mean±S.D. of three different experiments. Significant differences (P<0.001) were determined by ANOVA followed by the Turkey–Kramer post-test with respect to unstimulated samples (*). (d) Confocal microscopy analysis of NGB and COX-4 co-immuno-localization. Cells were fixed, permeabilized, and stained with anti-NGB antibody (red) and co-stained with anti-COX4 antibody (green) (original magnification × 63). Merged images by confocal microscopy show NGB distribution in HepG2 cells treated with either vehicle or E2 (10 nM) for 1, 4, and 24 h. White arrows point to examples of NGB and COX-4 co-staining in a single Z-stack plane. Representative images from three different experiments are shown
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237245&req=5

fig5: Effect of E2 on the localization of NGB in HepG2 and MCF-7 cells. Western blot of NGB level in nuclear (Nuclei), cytosolic (Cyt), and mitochondrial (Mit) fractions of (a) HepG2 and (b) MCF-7 cells and related densitometric analyses (c). The purity of fractions was assessed with PARP-1, TRAP1, and PP2A with respect to nucleus, mitochondria, and cytosol, respectively. The amount of proteins was normalized to each fraction marker protein. The figure represents a typical western blot of three independent experiments. Data reported in panel c represent the mean±S.D. of three different experiments. Significant differences (P<0.001) were determined by ANOVA followed by the Turkey–Kramer post-test with respect to unstimulated samples (*). (d) Confocal microscopy analysis of NGB and COX-4 co-immuno-localization. Cells were fixed, permeabilized, and stained with anti-NGB antibody (red) and co-stained with anti-COX4 antibody (green) (original magnification × 63). Merged images by confocal microscopy show NGB distribution in HepG2 cells treated with either vehicle or E2 (10 nM) for 1, 4, and 24 h. White arrows point to examples of NGB and COX-4 co-staining in a single Z-stack plane. Representative images from three different experiments are shown
Mentions: Recently, we demonstrated that in SK-N-BE cells E2 stimulation induces a localization of NGB at mitochondrial level, where it exerts an antiapoptotic role.13 Thus, the analysis of the subcellular localization of NGB was performed also in E2-related non-nervous cancer cells to obtain an inkling of its function in these cells. HepG2 and MCF-7 cells were fractionated into three main compartments (i.e., nuclei, mitochondria, and cytosol) using as fraction purity markers nuclear poly (ADP-ribose) polymerase 1 (PARP-1), mitochondrial TNF receptor-associated protein 1 (TRAP1), and cytosolic protein phosphatase 2A (PP2A) (Figures 5a, b, and c). Upon E2 stimulation (i.e., 10 nM; 24 h), a significant reduction of NGB in the nucleus with the parallel increase of NGB in the mitochondrial fraction in both HepG2 (Figure 5a) and MCF-7 (Figure 5b) cells was observed. However, the E2 treatment led to the increase of the NGB level in the cytosol of HepG2 cells, whereas in MCF-7 cells the amount of cytosolic NGB after E2 stimulation was similar to that observed in the control (Figures 5a and b). A further confirmation that E2 induces NGB re-allocation in mitochondria of HepG2 derives from the confocal microscopy analysis, which indicates that E2 increased the colocalization of NGB with cytochrome c oxidase 4 (COX-4) at mitochondrial level beginning 1 h after the stimulation and reaching a maximum 24 h after the hormone treatment (Figure 5d).

Bottom Line: E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells.Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines.E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Science, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy.

ABSTRACT
Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17β-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.

Show MeSH
Related in: MedlinePlus