HO-1 up-regulation: a key point in high-risk neuroblastoma resistance to bortezomib.

AbstractHigh-risk neuroblastoma (NB) is characterized by the development of chemoresistance, and bortezomib (BTZ), a selective inhibitor of proteasome, has been proposed in order to overcome drug resistance. Considering the involvement of the nuclear factor-erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the antioxidant and detoxifying ability of cancer cells, in this study we have investigated their role in differently aggressive NB cell lines treated with BTZ, focusing on the modulation of HO-1 to improve sensitivity to therapy. We have shown that MYCN amplified HTLA-230 cells were slightly sensitive to BTZ treatment, due to the activation of Nrf2 that led to an impressive up-regulation of HO-1. BTZ-treated HTLA-230 cells down-regulated p53 and up-regulated p21, favoring cell survival. The inhibition of HO-1 activity obtained by Zinc (II) protoprophyrin IX (ZnPPIX) was able to significantly increase the pro-apoptotic effect of BTZ in a p53- and p21-independent way. However, MYCN non-amplified SH-SY5Y cells showed a greater sensitivity to BTZ in relation to their inability to up-regulate HO-1. Therefore, we have shown that HO-1 inhibition improves the sensitivity of aggressive NB to proteasome inhibition-based therapy, suggesting that HO-1 up-regulation can be used as a marker of chemoresistance in NB. These results open up a new scenario in developing a combined therapy to overcome chemoresistance in high-risk neuroblastoma

Glutathione-mediated antioxidant response and aerobic metabolism: two crucial factors involved in determining the multi-drug resistance of high-risk neuroblastoma

Neuroblastoma, a paediatric malignant tumor, is initially sensitive to etoposide, a drug to which many patients develop chemoresistance. In order to investigate the molecular mechanisms responsible for etoposide chemoresistance, HTLA-230, a human MYCN-amplified neuroblastoma cell line, was chronically treated with etoposide at a concentration that in vitro mimics the clinically-used dose. The selected cells (HTLA-Chr) acquire multi-drug resistance (MDR), becoming less sensitive than parental cells to high doses of etoposide or doxorubicin. MDR is due to several mechanisms that together contribute to maintaining non-toxic levels of H2O2. In fact, HTLA-Chr cells, while having an efficient aerobic metabolism, are also characterized by an up-regulation of catalase activity and higher levels of reduced glutathione (GSH), a thiol antioxidant compound. The combination of such mechanisms contributes to prevent membrane lipoperoxidation and cell death. Treatment of HTLA-Chr cells with L-Buthionine-sulfoximine, an inhibitor of GSH biosynthesis, markedly reduces their tumorigenic potential that is instead enhanced by the exposure to N-Acetylcysteine, able to promote GSH synthesis.Collectively, these results demonstrate that GSH and GSH-related responses play a crucial role in the acquisition of MDR and suggest that GSH level monitoring is an efficient strategy to early identify the onset of drug resistance and to control the patient's response to therapy

PKCδ Sensitizes Neuroblastoma Cells to L-Buthionine-Sulfoximine and Etoposide Inducing Reactive Oxygen Species Overproduction and DNA Damage

Neuroblastoma is a type of pediatric cancer. The sensitivity of neuroblastoma (NB) cancer cells to chemotherapy and radiation is inhibited by the presence of antioxidants, such as glutathione (GSH), which is crucial in counteracting the endogenous production of reactive oxygen species (ROS). We have previously demonstrated that cells depleted of GSH undergo apoptosis via oxidative stress and Protein kinase C (PKC) δ activation. In the present study, we transfected PKCδ in NB cells resistant to oxidative death induced by L-buthionine-S,R-sulfoximine (BSO), a GSH-depleting agent. Cell responses, in terms of ROS production, apoptosis and DNA damage were evaluated. Moreover, PKCδ activation was monitored by analyzing the phosphorylation status of threonine 505 residue, carrying out PKC activity assay and investigating the subcellular localization of the kinase. The cell responses obtained in BSO-resistant cells were also compared with those obtained in BSO-sensitive cells subjected to the same experimental protocol. Our results demonstrate, for the first time, that PKCδ induces DNA oxidation and ROS overproduction leading to apoptosis of BSO-resistant NB cells and potentiates the cytotoxic effects induced by BSO in sensitive cells. Moreover, PKCδ overexpression enhances the sensitivity of NB cells to etoposide, a well-characterised drug, commonly used in neuroblastoma therapy. Altogether our data provide evidence of a pro-oxidant role of PKCδ that might be exploited to design new therapeutic strategies aimed at selective killing of cancer cells and overcoming drug resistance. However, it becomes evident that a more detailed understanding of ROS-mediated signaling in cancer cells is necessary for the development of redox-modulated therapeutic approaches

Vitamin E and neurodegenerative diseases.

Vitamin E is essential for neurological function. This fact, together with a growing body of evidence indicating that neurodegenerative processes are associated with oxidative stress, lead to the convincing idea that several neurological disorders may be prevented and/or cured by the antioxidant properties of vitamin E. In this review, some aspects related to the role of vitamin E against Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and ataxia with vitamin E deficiency will be presented

Hormesis and Oxidative Distress: Pathophysiology of Reactive Oxygen Species and the Open Question of Antioxidant Modulation and Supplementation

Alterations of redox homeostasis leads to a condition of resilience known as hormesis that is due to the activation of redox-sensitive pathways stimulating cell proliferation, growth, differentiation, and angiogenesis. Instead, supraphysiological production of reactive oxygen species (ROS) exceeds antioxidant defence and leads to oxidative distress. This condition induces damage to biomolecules and is responsible or co-responsible for the onset of several chronic pathologies. Thus, a dietary antioxidant supplementation has been proposed in order to prevent aging, cardiovascular and degenerative diseases as well as carcinogenesis. However, this approach has failed to demonstrate efficacy, often leading to harmful side effects, in particular in patients affected by cancer. In this latter case, an approach based on endogenous antioxidant depletion, leading to ROS overproduction, has shown an interesting potential for enhancing susceptibility of patients to anticancer therapies. Therefore, a deep investigation of molecular pathways involved in redox balance is crucial in order to identify new molecular targets useful for the development of more effective therapeutic approaches. The review herein provides an overview of the pathophysiological role of ROS and focuses the attention on positive and negative aspects of antioxidant modulation with the intent to find new insights for a successful clinical application

Physicochemical Characterization of two Cationic Copolymers Effective on Etoposide-Sensitive and Resistant Neuroblastoma Cells

New therapeutic agents as antibiotics and chemotherapeutics capable of inducing cytotoxic effects and employed in the treatment of lethal human diseases as cancer and infections need also to circumvent the increasing chemoresistance, which limits their action. Natural and synthetic cationic peptides and polymers have given appealing results both in microbiology, and in the oncological field, where they resulted effective against several tumors, including human neuroblastoma (NB) [1,2]. To this end, we recently synthetized two polystyrene-based cationic copolymers (P5 and P7), which proved a potent ROS-related cytotoxicity against both etoposide-sensitive (HTLA-230) and -resistant (HTLA-ER) NB cells [3]. Interestingly, the cytotoxic effects of P5 and P7 were even higher on HTLA-ER cells, thus proving that they could be promising template macromolecules for the development of new chemotherapeutic agents able to fight NB chemoresistance.Water-solubility, surface charge, protonation profile in the physiological pH range, Z-potential, polydispersity index, buffer capacity, and particles size are pivotal features for the feasibility of biomedical application of new bioactive macromolecules. In our poster, in addition to show the spectroscopic characterization of P5 and P7, we have reported their complete physicochemical characterization. [1] L.T. Eliassen, et al. Int. J. Cancer. 2006, 119, 493\u2013500. [2] J. Tan, et al. Biomat. 2020, 252, 120078. [3] S. Alfei, et al. Nanomaterials 2021, 11, 977