Neuroglobin in Breast Cancer Cells: Effect of Hypoxia and Oxidative Stress on Protein Level, Localization, and Anti-Apoptotic Function

Abstract

The over-expression of human neuroglobin (NGB), a heme-protein preferentially expressed in the brain, displays anti-apoptotic effects against hypoxic/ischemic and oxidative stresses enhancing neuron survival. As hypoxic and oxidative stress injury frequently occurs in fast proliferating neoplastic tissues, here, the effect of these stressors on the level, localization, and anti-apoptotic function of NGB in wild type and NGB-stable-silenced MCF-7 breast cancer cells has been assessed. The well-known endogenous NGB inducer 17β-estradiol (E2) has been used as positive control. The median pO2 present in tumor microenvironment of breast cancer patients (i.e., 2% O2) does not affect the NGB level in breast cancer cells, whereas hydrogen peroxide and lead(IV) acetate, which increase intracellular reactive oxygen species (ROS) level, enhance the NGB levels outside the mitochondria and still activate apoptosis. However, E2-induced NGB up-regulation in mitochondria completely reverse lead(IV) acetate-induced PARP cleavage. These results indicate that the NGB level could represent a marker of oxidative-stress in MCF-7 breast cancer cells; however, the NGB ability to respond to injuring stimuli by preventing apoptosis requires its re-allocation into the mitochondria. As a whole, present data might lead to a new direction in understanding NGB function in cancer opening new avenues for the therapeutic intervention.

Dissecting the 17β‐estradiol pathways necessary for neuroglobin anti‐apoptotic activity in breast cancer

“…Intriguingly, in MCF-7 cells, the ROS-inducing PbIV triggers this same pathway to enhance NGB level (Fiocchetti, Cipolletti, Leone, Naldini, et al, 2016) suggesting that E2 and PbIV could share a common up-stream pathway to enhance NGB levels and a divergent downstream pathway to re-allocate NGB into the cells.…”

“…Our result indicate the crucial role of transcription factor CREB (one of the AKT downstream transcriptional factors) in the E2-induced modulation of NGB levels in both MCF-7 and T47D cells. Therefore, the rapid and persistent E2/ERα-induced AKT assure the rapid NGB accumulation into the cells, the long-term indirect transcription of NGB gene, and the translocation of NGB into mitochondria were NGB exerts its role in promoting cell survival and avoiding the triggers of apoptotic cascade (Fiocchetti et al, 2014(Fiocchetti et al, , 2015Fiocchetti, Cipolletti, Leone, Naldini, et al, 2016).…”

“…Total proteins were quantified using the Bradford Protein Assay. Solubilized proteins (20 μg) were resolved by 7% or 15% SDS-PAGE at 100 V for 1 hr at 24.0°C and then transferred to nitrocellulose with the Trans-Blot (Fiocchetti et al,2014;Fiocchetti, Cipolletti, Leone, Ascenzi, et al, 2016;Fiocchetti, Cipolletti, Leone, Naldini, et al, 2016)…”

Neuroglobin and friends

“…These data open new possibilities about the role of Ngb in sensing the intracellular redox state and in translating a stress condition to the signal transduction pathway . Furthermore, Ngb levels represent a marker of oxidative stress also in breast cancer cells MCF‐7 in a similar way …”

“…33,41 Furthermore, Ngb levels represent a marker of oxidative stress also in breast cancer cells MCF-7 in a similar way. 104 The involvement of Ngb in intracellular signaling processes is limited not only to the interaction with heterotrimeric G proteins but also in the regulation of small GTP-binding proteins of the Rho family, 105 consisting of at least 14 distinct members including RhoA, Rac1, and Cdc42. 106 represents the first event required to convert Rho GTPase to the active GTP-bound form and to translocate it to the membrane.…”