Resistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1

doi:10.1016/j.freeradbiomed.2011.11.007

Free Radical Biology and Medicine

Volume 52, Issue 2, 15 January 2012, Pages 488-496

https://doi.org/10.1016/j.freeradbiomed.2011.11.007 Get rights and content

Abstract

Cancer cell survival is known to be related to the ability to counteract oxidative stress, and glutathione (GSH) depletion has been proposed as a mechanism to sensitize cells to anticancer therapy. However, we observed that GI-ME-N cells, a neuroblastoma cell line without MYCN amplification, are able to survive even if GSH-depleted by l-buthionine-(S,R)-sulfoximine (BSO). Here, we show that in GI-ME-N cells, BSO activates Nrf2 and up-regulates heme oxygenase-1 (HO-1). Silencing of Nrf2 restrained HO-1 induction by BSO. Inhibition of HO-1 and silencing of Nrf2 or HO-1 sensitized GI-ME-N cells to BSO, leading to reactive oxygen/nitrogen species overproduction and decreasing viability. Moreover, targeting the Nrf2/HO-1 axis sensitized GI-ME-N cells to etoposide more than GSH depletion. Therefore, we have provided evidence that in GI-ME-N cells, the Nrf2/HO-1 axis plays a crucial role as a protective factor against cellular stress, and we suggest that the inhibition of Nfr2/HO-1 signaling should be considered as a central target in the clinical battle against neuroblastoma.

Graphical abstract

Highlights

► GI-ME-N neuroblastoma cell survival is independent from GSH depletion by BSO. ► BSO activates Nrf2 and up-regulates heme oxygenase-1 (HO-1) in GI-ME-N cells. ► Inhibition of HO-1 and silencing of Nrf2 or HO-1 sensitizes GI-ME-N cells to BSO. ► Inhibition of Nrf2/HO-1 axis sensitizes GI-ME-N cells to etoposide more than BSO. ► Inhibition Nfr2/HO-1 should be a potential target in neuroblastoma therapy.

Section snippets

Cell culture and treatments

GI-ME-N neuroblastoma cells were maintained in RPMI 1640 medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS; Euroclone), 2 mM glutamine (Sigma–Aldrich, Milan, Italy), 1% penicillin/streptomycin (Sigma–Aldrich), and 1% amphotericin B (Sigma–Aldrich), at 37 °C in a 5% CO₂ humid atmosphere and subcultured every 5 days at 1:5.

Cell treatments included 1 mM BSO (Sigma–Aldrich), 2.5 μM zinc(II) protoporphyrin IX (ZnPP; Sigma–Aldrich), a combined treatment of BSO + ZnPP at the

BSO treatment induces severe GSH depletion in GI-ME-N cells

Cell exposure to 1 mM BSO was able to induce deep depletion of tGSH content. As shown in Fig. 1, after 9 and 12 h of BSO treatment, tGSH levels decreased by about 50 and 75%, respectively, in comparison to the untreated cells. After 24 h, tGSH decreased to very low levels (< 5% vs CTR) and these levels were stable after 48 and 72 h. After BSO treatment (24, 48, 72 h) MTT assay showed only a slight reduction in cell viability in comparison to the untreated samples (data not shown).

BSO treatment reduces cell proliferation rate

Cell counts

Discussion

In this study, we investigated the molecular mechanisms involved in GI-ME-N cell resistance to GSH depletion induced by BSO. Our findings provide evidence that (1) BSO activates Nrf2 and up-regulates HO-1 in GI-ME-N cells; (2) Nrf2 is crucial in HO-1 up-regulation by BSO; (3) HO-1 inhibition, as well as Nrf2 or HO-1 silencing, sensitizes GI-ME-N cells to BSO; and (4) BSO is able to sensitize GI-ME-N cells to etoposide but inhibition of the Nrf2/HO-1 axis is by far more effective in this

Acknowledgments

This work was supported by grants from the Italian Ministry of University (PRIN 2009M8FKBB_002 and 2008N9N9KL_002) and Genova University (Progetto Ricerca Ateneo 2010). We thank Mr. Giuseppe Catalano for technical assistance. We thank Ms. Suzanne Patten for reviewing the use of English in the manuscript.

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Original ContributionResistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1

Abstract

Graphical abstract

Highlights

Section snippets

Cell culture and treatments

BSO treatment induces severe GSH depletion in GI-ME-N cells

BSO treatment reduces cell proliferation rate

Discussion

Acknowledgments

Mol. Aspects Med.

Int. J. Radiat. Oncol. Biol. Phys.

Biochem. Pharmacol.

Int. J. Radiat. Oncol. Biol. Phys.

Free Radic. Biol. Med.

Cancer Lett.

J. Pathol.

Biochem. Biophys. Res. Commun.

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Inorg. Biochem.

Urology

Exp. Cell Res.

Free Radic. Biol. Med.

Methods Enzymol.

Free Radic. Biol. Med.

Cytotherapy

Methods Enzymol.

Free Radic. Biol. Med.

Trends Biochem. Sci.

Free Radic. Biol. Med.

Biochem. Pharmacol.

Free Radic. Biol. Med.

Nitric Oxide

Neuroscience

Neurosci. Lett.

Eur. J. Cancer

The role of glutathione in cancer

Cell Biochem. Funct.

Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous reactive oxygen species

Cell Death Differ.

Oxidative stress induces apoptosis in embryonic cortical neurons

J. Neurochem.

Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogen-induced apoptosis

Breast Cancer Res.

Glutathione depletion-induced apoptosis of Ha-ras-transformed NIH3T3 cells can be prevented by melatonin

Oncogene

Glutathione depletion causes cell growth inhibition and enhanced apoptosis in pancreatic cancer cells

Cancer

Original Contribution
Resistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1