From neurons to neuroblastoma: dietary flavonoids as regulators of life, death & differentiation

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Alshangiti, Amnah M.
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University College Cork
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Neuroblastoma (NB) is an embryonal malignancy that arises from cells of sympathoadrenal lineage during the development of the nervous system. Patients classified as stage 4 with aggressive tumors are considered to belong to a high-risk group. MYCN-amplification is detected as a hallmark of NB in 20% of high-risk cases. Based on the International Neuroblastoma Risk Group (INRG), the relapse survival rate of high-risk patients with MYCN-amplification is only 4%. Despite extensive studies, metastasis and tumor relapse lead to debilitating morbidity and frequently death, in high-risk NB patients. Moreover, NB chemotherapy can result in very debilitating long-term sequelae. Errors in the molecular mechanisms that control the development of cell of the sympathetic lineage have been linked to NB development. One group of signals, known as the bone morphogenetic proteins (BMPs), plays an important role in sympathetic neuritogenesis, by signaling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB, but it is unknown if the expression of BMPR1A or BMPR1B is altered. The use of neurotrophic factors such as nerve growth factor (NGF) and growth differentiation factor (GDF)5 to promote cell differentiation has been reported in postnatal day (P)1 mouse NC cultures and MYCN-amplified NB SK-N-BE(2), respectively. GDF5 induces cell differentiation in NB cells through activation of BMP-Smad signaling. However, recombinant BMPs are expensive to produce and their clinical use is limited, due to several factors. Therefore, there is an urgent need to identify novel therapeutic agents that could induce cell differentiation in post-chemotherapy tissue with the minimum side effects. Recently, several screening studies have identified small molecules that modulate the BMP pathway. These include natural dietary compounds, including flavonoids and in particular the chalcone subfamily. Interestingly, flavonoids have potential anti-tumor properties with selective cytotoxicity to cancer cells. Additionally, chalcones, which are the most potent flavonoid subclass, have also demonstrated anti-tumor activity in some contexts. This thesis first investigated, in chapter 2, the association of distinct type1 BMPRs with different molecular pathways and survival outcomes in NB. Lower expression of BMPR2 and BMPR1B, and higher BMPR1A expression, were observed in stage 4 and in MYCN-amplified NB, and they were associated with poor survival. In contrast, higher expression of BMPR2 and BMPR1B were associated with better survival. Regarding MYCN status, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This study therefore showed that expression of distinct BMPRs is associated with different survival outcomes in NB. In chapter 3, the effects of low doses of the chalcones isoliquiritigenin (ISLQ) and 4-hydroxychalcone (4HC), small molecule activator of BMP-Smad signaling, on the morphological differentiation of NB cells, as well as the molecular mechanism of their action, was investigated. ISLQ and 4HC promoted neurite growth in NB cells by activation of BMP-Smad signaling. Moreover, treatment with these chalcones lead to a significant reduction of cell aggregates (tumor-like aggregations) in SK-N-BE(2) cells. After these findings, in chapter 4, the cytotoxic effect of ISLQ in MYCN-amplified NB cells was investigated in detail. ISLQ induced cell death in MYCN-amplified NB cells SK-N-BE(2) and IMR-32 through an elevation of oxidative stress and necroptosis. Combined treatment of ISLQ with the chemotherapy agent cisplatin led to a greater cell toxicity in MYCN-amplified NB cells. In chapter 5, the cytotoxic effect of 4HC on NB cells, and its mode of action, was examined. 4HC demonstrated selective cytotoxicity on MYCN-amplified NB cells, which were more sensitive to 4HC than non-amplified SH-SY5Y cells. Cell death induced by 4HC was shown to result from glutathione (GSH) depletion, elevation of oxidative stress and mitochondrial impairment. This thesis, therefore, demonstrates the role of various types of BMPRs in different molecular pathways and survival outcomes in NB, It also highlights the potential of dietary chalcones for either the induction of NB cell differentiation at lower concentrations, through the activation of BMP-Smad signaling, or induction of selective NB cell death at higher concentrations, by elevation of oxidative stress.
Neuroblastoma , Tumor , BMP-Smad signaling , Small molecule , Chalcone , MYCN amplification , Oxidative stress
Alshangiti, A. M. 2020. From neurons to neuroblastoma: dietary flavonoids as regulators of life, death & differentiation. PhD Thesis, University College Cork.
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