Polyamine analogues inhibit neuroblastoma cell growth

Abstract Neuroblastoma is a highly malignant neoplasm found in children. Although children with high-risk neuroblastoma respond to chemotherapy, relapses are common and an estimated long-time survival prognosis is approximately 15%. Thus, there is a tremendous need for new chemotherapeutic drugs for the treatment for neuroblastoma. Polyamines are needed for cell proliferation and cell survival. Thus, polyamine pool depletion is considered as a cancer intervention strategy. One means to achieve polyamine pool depletion is by treating with polyamine analogues. Polyamine analogues have shown to have antineoplastic activities in a variety of experimental tumour systems and are therefore tested in clinical trials. Polyamine analogues could be potential new chemotherapeutic agents for treatment of neuroblastoma. The general aim of my thesis was to investigate how polyamine analogue treatment would affect neuroblastoma cell proliferation and survival. Three neuroblastoma cell lines, chosen because of their different genetic backgrounds, were treated with the three polyamine analogues PG-11047, PG-11093, and N1,N11-diethylnorspermine. When the cells were treated with polyamine analogue for one treatment cycle, SH-SY5Y and IMR-32 cells, containing wild type p53, were more sensitive than LA-N-1 cells, containing mutated p53. However, when the cells were treated with PG-11047 for repeated cycles, in a manner more resembling the treatment schedule used for patients, the IMR-32 and LA-N-1 cells, containing MYCN amplification, were more sensitive than SH-SY5Y cells. In fact SH-SY5Y cells appeared to develop resistance to PG-11047 while the MYCN-amplified cell lines died. This work shows the importance of treating cell lines with repeated treatment cycles to obtain a better picture of the fate of the cells. Most importantly, the results show that polyamine analogue treatment may be very effective in aggressive MYCN-amplified neuroblastoma. The polyamine analogue PG-11047 is used in clinical trial and shows low general toxicity and may be used for treatment of children with MYCN-amplified neuroblastoma

Apoptosis induced by the potential chemotherapeutic drug N1, N11-Diethylnorspermine in a neuroblastoma cell line.

Neuroblastoma is a highly malignant neoplasm found in young children. Although children with high-risk neuroblastoma respond to chemotherapy, relapses are common. On account of poor treatment outcome, new treatment strategies are constantly sought for neuroblastoma. Polyamine analogues are potentially novel substances for treatment of neuroblastoma. In this study, we have treated two neuroblastoma cell lines, SH-SY5Y and LA-N-1, with the spermine analogue N, N-Diethylnorspermine (DENSPM). SH-SY5Y was the most sensitive cell line, in which DENSPM treatment resulted in an inhibition of cell proliferation and an induction of cell death. The cell death induced by DENSPM treatment was apoptotic, as evidenced by cleavage of procaspase 3 and induction of caspase-3 activity. In contrast, DENSPM treatment only resulted in a slight inhibition of cell proliferation in LA-N-1 cells. There were several possible causes for the lower sensitivity to DENSPM treatment in the latter cell line when compared with SH-SY5Y cells. DENSPM-induced polyamine depletion was more extensive in SH-SY5Y cells than in LA-N-1 cells. This was partly because of a higher induction of the polyamine catabolic enzyme spermidine/spermine N-acetyltransferase in the cell line SH-SY5Y. The DENSPM-induced polyamine depletion was also caused by the inhibition of ornithine decarboxylase. LA-N-1 cells contained a higher level of the prosurvival protein survivin, which was further increased after DENSPM treatment. In contrast, DENSPM treatment resulted in a decreased survivin level in SH-SY5Y cells

Gold- and silver nanoparticles affect the growth characteristics of human embryonic neural precursor cells.

Rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of nanoparticles (NPs) on human health and environment. NPs can be directly taken up by organs exposed, but also translocated to secondary organs, such as the central nervous system (CNS) after systemic- or subcutaneous administration, or via the olfactory system. The CNS is particularly vulnerable during development and recent reports describe transport of NPs across the placenta and even into brain tissue using in vitro and in vivo experimental systems. Here, we investigated whether well-characterized commercial 20 and 80 nm Au- and AgNPs have an effect on human embryonic neural precursor cell (HNPC) growth. After two weeks of NP exposure, uptake of NPs, morphological features and the amount of viable and dead cells, proliferative cells (Ki67 immunostaining) and apoptotic cells (TUNEL assay), respectively, were studied. We demonstrate uptake of both 20 and 80 nm Au- and AgNPs respectively, by HNPCs during proliferation. A significant effect on the sphere size- and morphology was found for all cultures exposed to Au- and AgNPs. AgNPs of both sizes caused a significant increase in numbers of proliferating and apoptotic HNPCs. In contrast, only the highest dose of 20 nm AuNPs significantly affected proliferation, whereas no effect was seen on apoptotic cell death. Our data demonstrates that both Au- and AgNPs interfere with the growth profile of HNPCs, indicating the need of further detailed studies on the adverse effects of NPs on the developing CNS

Correction: Gold- and Silver Nanoparticles Affect the Growth Characteristics of Human Embryonic Neural Precursor Cells.

Rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of nanoparticles (NPs) on human health and environment. NPs can be directly taken up by organs exposed, but also translocated to secondary organs, such as the central nervous system (CNS) after systemic- or subcutaneous administration, or via the olfactory system. The CNS is particularly vulnerable during development and recent reports describe transport of NPs across the placenta and even into brain tissue using in vitro and in vivo experimental systems. Here, we investigated whether well-characterized commercial 20 and 80 nm Au- and AgNPs have an effect on human embryonic neural precursor cell (HNPC) growth. After two weeks of NP exposure, uptake of NPs, morphological features and the amount of viable and dead cells, proliferative cells (Ki67 immunostaining) and apoptotic cells (TUNEL assay), respectively, were studied. We demonstrate uptake of both 20 and 80 nm Au- and AgNPs respectively, by HNPCs during proliferation. A significant effect on the sphere size- and morphology was found for all cultures exposed to Au- and AgNPs. AgNPs of both sizes caused a significant increase in numbers of proliferating and apoptotic HNPCs. In contrast, only the highest dose of 20 nm AuNPs significantly affected proliferation, whereas no effect was seen on apoptotic cell death. Our data demonstrates that both Au- and AgNPs interfere with the growth profile of HNPCs, indicating the need of further detailed studies on the adverse effects of NPs on the developing CNS

Inhibition of cell proliferation and induction of apoptosis by N(1),N(11)-diethylnorspermine-induced polyamine pool reduction

Reduction of cellular polyamine pools results in inhibition of cell proliferation and sometimes in induction of cell death. Reduction of cellular polyamine pools can be achieved by several strategies involving all the mechanisms of polyamine homoeostasis, i.e. biosynthesis, catabolism and transport across the cell membrane. In the present paper, we concentrate on results achieved using the polyamine analogue DENSPM (N(1),N(11)-diethylnorspermine) on different cell lines. We discuss polyamine levels in DENSPM-treated cells in relation to effects on cell cycle kinetics and induction of apoptosis. To really understand the role of polyamines in cell cycle regulation and apoptosis, we believe it is now time to go through the vast polyamine literature in a meta-analysis-based manner. This short review does not claim to be such a study, but it is our hope to stimulate such studies in the polyamine field. Such work is especially important from the viewpoint of introducing drugs that affect polyamine homoeostasis in the treatment of various diseases such as cancer

Silver and gold nanoparticles exposure to in vitro cultured retina--studies on nanoparticle internalization, apoptosis, oxidative stress, glial- and microglial activity.

The complex network of neuronal cells in the retina makes it a potential target of neuronal toxicity--a risk factor for visual loss. With growing use of nanoparticles (NPs) in commercial and medical applications, including ophthalmology, there is a need for reliable models for early prediction of NP toxicity in the eye and retina. Metal NPs, such as gold and silver, gain much of attention in the ophthalmology community due to their potential to cross the barriers of the eye. Here, NP uptake and signs of toxicity were investigated after exposure to 20 and 80 nm Ag- and AuNPs, using an in vitro tissue culture model of the mouse retina. The model offers long-term preservation of retinal cell types, numbers and morphology and is a controlled system for delivery of NPs, using serum-free defined culture medium. AgNO3-treatment was used as control for toxicity caused by silver ions. These end-points were studied; gross morphological organization, glial activity, microglial activity, level of apoptosis and oxidative stress, which are all well described as signs of insult to neural tissue. TEM analysis demonstrated cellular- and nuclear uptake of all NP types in all neuronal layers of the retina. Htx-eosin staining showed morphological disruption of the normal complex layered retinal structure, vacuole formation and pyknotic cells after exposure to all Ag- and AuNPs. Significantly higher numbers of apoptotic cells as well as an increased number of oxidative stressed cells demonstrated NP-related neuronal toxicity. NPs also caused increased glial staining and microglial cell activation, typical hallmarks of neural tissue insult. This study demonstrates that low concentrations of 20 and 80 nm sized Ag- and AuNPs have adverse effects on the retina, using an organotypic retina culture model. Our results motivate careful assessment of candidate NP, metallic or-non-metallic, to be used in neural systems for therapeutic approaches

TEM evaluation of the uptake of Au- and AgNPs of HNPCs. A.

<p>Gold and silver NPs (20 and 80 nm) imaged by TEM. Scale bars equal 0.2 µm. <b>B.</b> NPs were added to the medium 48 h after seeding and spheres were prepared for transmission electron microscopy two weeks later. All four different types of NPs were taken up by HNPCs. Arrowheads show the NPs that have been taken up by HNPCs. Scale bars equal 0.5 µm.</p

AgNP exposure increased numbers of apoptotic TUNEL+ HNPC. A.

<p>AgNP-exposure for two weeks increased the cell death in HNPCs, seen as increased number of TUNEL-+ cells. Both Au- and AgNPs induced cell death of HNPCs indicated by the increased number of TUNEL-+ cells and -aggregates. The presented data are representative images of 6 spheres per section from two independent experiments. Blue staining, DAPI; red staining, TUNEL; Au 20∶800, gold 20 nm and 800 particles/cell; Au 80∶800, gold 80 nm and 800 particles/cell; Ag 20∶800, silver 20 nm and 800 particles/cell; Ag 80∶800, silver 80 nm and 800 particles/cell; AgNO₃ 1.0, silver nitrate 1.0 mg/ml. Scale bar equal 214 µm. <b>B.</b> Only AgNPs significantly induced cell death in HNPCs. Evaluation of the data presented under A, showing the number of TUNEL-+ cells/mm₂ of the HNPC neurospheres. Au 20∶50, gold 20 nm and 50 particles/cell; Au 20∶800, gold 20 nm and 800 particles/cell; Au 80∶50, gold 80 nm and 50 particles/cell; Au 80∶800, gold 80 nm and 800 particles/cell; Ag 20∶50, silver 20 nm and 50 particles/cell; Ag 20∶800, silver 20 nm and 800 particles/cell; Ag 80∶50, silver 80 nm and 50 particles/cell; Ag 80∶800, silver 80 nm and 800 particles/cell; AgNO₃ 0.5, silver nitrate 0.5 mg/ml; AgNO₃ 1.0, silver nitrate 1.0 mg/ml. *<i>p</i><0.05 compared to control, **<i>p</i><0.01 compared to control, ***<i>p</i><0.001 compared to control.</p

NP exposure increases numbers of HNPC proliferating Ki67-+ cells. A.

<p>AgNP-exposure for two weeks increased the cell proliferation in HNPCs, seen as increased numbers of Ki67+ cells (green). Representative images of 6 spheres per section from two independent experiments. Blue staining, DAPI. Au 20∶800, gold 20 nm and 800 particles/cell; Au 80∶800, gold 80 nm and 800 particles/cell; Ag 20∶800, silver 20 nm and 800 particles/cell; Ag 80∶800, silver 80 nm and 800 particles/cell; AgNO₃ 1.0, silver nitrate 1.0 mg/ml. Scale bars equal 214 µm. <b>B.</b> Only AgNPs significantly induced cell proliferation in HNPCs. Evaluation of the data presented under A, showing the number of Ki67-+ cells/mm² of the HNPC neurospheres. Au 20∶50, gold 20 nm and 50 particles/cell; Au 20∶800, gold 20 nm and 800 particles/cell; Au 80∶50, gold 80 nm and 50 particles/cell; Au 80∶800, gold 80 nm and 800 particles/cell; Ag 20∶50, silver 20 nm and 50 particles/cell; Ag 20∶800, silver 20 nm and 800 particles/cell; Ag 80∶50, silver 80 nm and 50 particles/cell; Ag 80∶800, silver 80 nm and 800 particles/cell; AgNO₃ 0.5, silver nitrate 0.5 mg/ml; AgNO₃ 1.0, silver nitrate 1.0 mg/ml. *<i>p<</i>0.05 compared to control, **<i>p<</i>0.01 compared to control.</p