Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma
Behrouz Hassannia, Bartosz Wiernicki, Irina Ingold, Feng Qu, Simon Van Herck, Yulia Y. Tyurina, Hülya Bayır, Behnaz A. Abhari, Jose Pedro Friedmann Angeli, Sze Men Choi, Eline Meul, Karen Heyninck, Ken Declerck, Chandra Sekhar Chirumamilla, Maija Lahtela-Kakkonen, Guy Van Camp, Dmitri V. Krysko, Paul G. Ekert, Simone Fulda, Bruno G. De Geest, Marcus Conrad, Valerian E. Kagan, Wim Vanden Berghe, Peter Vandenabeele, Tom Vanden Berghe
Behrouz Hassannia, Bartosz Wiernicki, Irina Ingold, Feng Qu, Simon Van Herck, Yulia Y. Tyurina, Hülya Bayır, Behnaz A. Abhari, Jose Pedro Friedmann Angeli, Sze Men Choi, Eline Meul, Karen Heyninck, Ken Declerck, Chandra Sekhar Chirumamilla, Maija Lahtela-Kakkonen, Guy Van Camp, Dmitri V. Krysko, Paul G. Ekert, Simone Fulda, Bruno G. De Geest, Marcus Conrad, Valerian E. Kagan, Wim Vanden Berghe, Peter Vandenabeele, Tom Vanden Berghe
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Research Article Neuroscience Oncology

Nano-targeted induction of dual ferroptotic mechanisms eradicates high-risk neuroblastoma

Abstract

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor–like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

Authors

Behrouz Hassannia, Bartosz Wiernicki, Irina Ingold, Feng Qu, Simon Van Herck, Yulia Y. Tyurina, Hülya Bayır, Behnaz A. Abhari, Jose Pedro Friedmann Angeli, Sze Men Choi, Eline Meul, Karen Heyninck, Ken Declerck, Chandra Sekhar Chirumamilla, Maija Lahtela-Kakkonen, Guy Van Camp, Dmitri V. Krysko, Paul G. Ekert, Simone Fulda, Bruno G. De Geest, Marcus Conrad, Valerian E. Kagan, Wim Vanden Berghe, Peter Vandenabeele, Tom Vanden Berghe

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Figure 2

WA increases intracellular labile Fe(II) upon excessive activation of HMOX1, which is sufficient to induce ferroptosis.

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WA increases intracellular labile Fe(II) upon excessive activation of HM...
(A) IPA of genes differentially expressed after treatment with WA (1μM) in IMR-32 cells. (B) Gene expression profiles of Ctrl, WA-treated, and WN-treated IMR-32 cells. Color key legend represents Z score values. (C) Relative HMOX1 mRNA expression in IMR-32 cells after WA-treatment. (D) Western blot revealing KEAP1, NRF2, and HMOX1 in IMR-32 cells treated with WA. (E) Western blot revealing KEAP1 after immunoprecipitation of biotin-WA in lysates of IMR-32 cells treated with biotin-WA (1 μM). (F) Percentage of cell death induced by WA in the presence/absence of HMOX1 inhibitor (ZnPP). (G) Western blot revealing HMOX1 in response to WA (1 μM). (H) Percentage of cell death induced by WA upon RNAi-mediated knockdown of HMOX1. The results are representative of 2 independent experiments. (I) Percentage of cell death induced by WA in the presence/absence of hemin. (J) Western blot revealing HMOX1 in response to hemin (10 μM). (K) Heatmap representing the sensitivity of IMR-32 cells in response to hemin in the presence of ferroptosis inhibitors. See also Supplemental Table 5. (L) Cellular levels of labile Fe(II), using RhoNox-1, in function of time in response to WA (1 μM). (M) Cellular levels of labile Fe(II), using RhoNox-1, 8 hours after treatment with WA (1 μM), hemin (10 μM), or the combination. MFI, mean fluorescence intensity. (N) Heatmap representing cell death sensitivity of IMR-32 cells in response to (NH4)2Fe(SO4)2 in the absence/presence of ferroptosis inhibitors. See also Supplemental Table 6. The combined results of 2 or 3 independent experiments are shown for F, I, K, and N. Error bars represent SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001, 2-way ANOVA test (F, I, L,and M). NDGA, nordihydroguaiaretic acid.