|Year : 2017 | Volume
| Issue : 3 | Page : 147-152
Lithium treatment prior to radiation exposure of human neuroblastoma cells modifies outcome of cellular damage
J Kerawala1, P Shastry2, R Mukopadhyaya3, Medha S Rajadhyaksha1
1 Department of Life Science, Sophia College, Mumbai, Maharashtra, India
2 National Centre for Cell Sciences, Pune, Maharashtra, India
3 Division of Molecular Biology, Bhabha Atomic Research Centre and Homi Bhabha National Institute, Mumbai, Maharashtra, India
|Date of Web Publication||17-Oct-2017|
Medha S Rajadhyaksha
Department of Life Science, Sophia College, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: Radiation exposure to brain is known to cause cognitive deficits. Aim of the current study was to investigate whether lithium protects against radiation. It was further investigated whether this protective effect is associated with neuritogenesis and altered Neural Cell Adhesion Molecule (NCAM) expression. Materials and Methods: Human neuroblastoma cell line, SK-N-MC was exposed to gamma radiation alone and after 24 h pretreatment with lithium. Cells were scored for viability by MTT assay and checked for apoptotic changes by flow cytometry (FCM). Geimsa stained cells were scored for number of cells bearing neuritis and length of neuritis. Real time PCR was used to measure levels of NCAM transcript in the cells. Results: Radiation exposure of 5 Gray resulted in significant decrease in cell viability. Pretreatment with lithium rescued cells from radiation- induced toxicity as indicated by MTT assay and by FCM analysis. Morphological observations suggested radiation caused increase in neurite lengths and decrease in number of cells bearing neurites. This was reversed by lithium treatment. Several fold decrease in NCAM expression was observed post radiation exposure which was reversed by lithium pretreated cells. Conclusion: Lithium protects neuronal cells against radiation damage. The protective effect of lithium is associated with change in differentiation status of cells as indicated by neuritogenesis and with alteration in expression of NCAM, a molecule known to be associated with neurite formation.
Keywords: Altered gene expression, lithium, radiation damage modification, SK-N-MC cells
|How to cite this article:|
Kerawala J, Shastry P, Mukopadhyaya R, Rajadhyaksha MS. Lithium treatment prior to radiation exposure of human neuroblastoma cells modifies outcome of cellular damage. J Radiat Cancer Res 2017;8:147-52
|How to cite this URL:|
Kerawala J, Shastry P, Mukopadhyaya R, Rajadhyaksha MS. Lithium treatment prior to radiation exposure of human neuroblastoma cells modifies outcome of cellular damage. J Radiat Cancer Res [serial online] 2017 [cited 2018 Mar 21];8:147-52. Available from: http://www.journalrcr.org/text.asp?2017/8/3/147/216874
| Introduction|| |
Different cell types exhibit unique responses to radiation stress., Different types of radiation are used for therapy of brain cancer, which may be modified by many factors including the presence of metals such as lithium. In neurons, kinases such as SAPK/JNK, p38, and mitogen-activated protein kinases (MAPKs) are activated on exposure to radiation stress. Few like ERK have been shown to have cytoprotective effect inducing differentiation and proliferation of these cells., On the other hand, MAPKs have been shown to have dual roles and can induce apoptosis in neurons. Development of neurocognitive defects after cranial irradiation for the treatment of primary and metastatic tumors, could be due to apoptosis of proliferating neuronal progenitor cells in the hippocampus which is the important center for learning and memory.
Lithium which is a drug used in bipolar disorder had been recognized as a potential neuroprotective agent and was recently implicated in reducing damage to neurons in the brain. The possibility that lithium might find wider use as neuroprotective agent against radiation damage emphasizes the relevance of this study. A large body of preclinical literature supporting therapeutic potential of lithium in cases of disorders of the central nervous system (CNS) such as fragile X Syndrome, Parkinson's Disease, retinal degeneration, ischemic stroke, Huntington's Disease, Alzheimer's Disease, spinal cord injury, alcohol-induced degeneration, and HIV-induced neurotoxicity became available in the past decade. While studies on lithium as neuroprotective agent were amply demonstrated, possibility of it being a radiomodifying agent for CNS received little attention, especially in human cancer cells. Systematic study demonstrating "radiomodifying" properties of lithium have been limited.,
Lithium arrests cells in G2+M phase of cell cycle and enhances neurite outgrowth in mouse neuroblastoma cells was shown earlier. Our previous observation confirmed the same in human neuroblastoma cells SK-N-MC in addition to identifying transcripts of ANP32c, neural cell adhesion molecule (NCAM), occludin, and PKD2 genes in response to lithium. So far in this list, only NCAM was reportedly involved with neurite outgrowth besides its role in proliferation, differentiation, and synaptic plasticity of cell., The present study, for the first time, demonstrated that lithium modifies the radiation-induced damage in neuroblastoma cells by blocking cell cycle arrest, and this effect is associated with differentiation status and levels of NCAM. Thus, NCAM could be important for radiomodification by lithium.
| Materials And Methods|| |
Cell culture and reagents
Human neuroblastoma cell line SK-N-MC (HTB-10) was obtained from the American Type Culture Collection, USA. SK-N-MC cells were maintained in Dulbecco's modified eagle's medium
(DMEM) (GIBCO) supplemented with 10% fetal bovine serum, penicillin (100 u/ml), and streptomycin (0.75 μg/ml). Working concentrations of lithium chloride (2.5 mM and 5 mM, Sigma, USA) were prepared in FBS supplemented DMEM. Cells were treated with lithium chloride for 24 h for all experiments. Culture conditions during the study and the treatment parameters were kept constant. Cells were cultured in 96-well plates (5000 cell/well/100 μl) or seeded (7500 cells/100 μl) on glass coverslips for irradiation experiments.
Radiation and cell viability
Gamma radiation source Steritome 780E60 Co unit (Jaslok Hospital, Mumbai, Maharashtra, India) was used for the study. Cells cultured on coverslips were exposed to doses of 3, 5, and 10 Gy gamma radiation. Irradiated cells were washed once in fresh medium before removal for assays. Viability study was done using 3-(4,5 dimethyithiazol-2yl)-2,5 diphenyltetrazolium bromide (MTT) reduction assay, a sensitive colorimetric assay that checks for viability and metabolic state of cells. Briefly, 40 μl MTT (Sigma) (5 mg/ml) was added per well to cells postradiation exposure. After 4 h, the supernatant was discarded and 100 μl of dimethyl sulfoxide (Hi-media) was added and readings were taken at 540 nm using ELISA reader (Biotek, VT, USA). The percentage viability was defined as mean abs 540 nm of treated/Abs 540 nm of untreated cells ×100.
For this cells were cultured on the coverslips. One set of cells were treated with lithium (2.5 mM) for 24 h, and other set was kept without lithium under same growth conditions. After irradiation cells were fixed with methanol: acetic acid (4:1) and stained with Giemsa. The number of cells bearing more than 3 neurites was noted. Images of SK-N-MC cells (≥30) from randomly selected fields were obtained and observed for changes in cell size, adherence, spreading, and neurite formation. Cytoplasmic extension of ≥0.1 mm from the soma was considered as a neurite. Length of the neurite was taken as the distance from the soma to the tip of the neurite (Italia J et al. 2011), and this was quantified using Image J software at 24 h. Neurite formation was assessed as percent neurite bearing cells.
Flow cytometry analysis
Control and irradiated cells (5 Gy) pretreated with 2.5 mM lithium (24 h) and without any lithium treatment were harvested after 24 h and fixed in 70% ethanol. Cells were washed with Phosphate buffered saline (PBS) and treated with RNAse (5 mg/ml, Sigma). Cells were stained with PI overnight (Sigma, MO, USA) and analyzed on a flow cytometer (PACS Vantage, Becton Dickinson, USA) under 488 nm argon-ion laser using Cell Quest and Modfit Softwares.
Real-time polymerase chain reaction
Real-time polymerase chain reaction (PCR) amplification reactions were carried out in Roche real-time sequence detection system and quantitation done by comparative Ct method. CT-value was the cycle number at which the fluorescence generated in a reaction crossed the threshold within the linear phase of the amplification profile. Expression of endogenous genes β-actin, and 18S rRNA were measured as reference for CT-values. Briefly, the ΔCT-value was obtained by subtracting the average β-actin/18s sRNA CT-values from the average CT-value of gene of interest (NCAM). The fold change was calculated according to the formula 2 − (ΔΔCT), where ΔΔCT was the difference between ΔCT of sample and the ΔCT calibrator (RNA of untreated sample). Reaction mixture of 10 μl consisted of 5 μl 1× PCR master mix with SYBR-Green, 2 pm of each primer, and cDNA templates. All reactions were carried out in triplicates and the PCR conditions were 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, and 60°C for 30 s and 72°C for 30 s.
| Results|| |
Lithium modifies effect of radiation by enhancing cell viability
Percent viability of cells as indicated by MTT assay suggested that exposure of cells to 3 Gy gamma radiation did not affect viability [Figure 1]a. Exposure to 5 Gy dose reduced cell viability by 60% compared to that of unirradiated cells. Henceforth, all further experiments were carried out with 5 Gy dose of gamma rays. Lithium by itself did not affect cell viability and were comparable to that of control cells grown under same conditions for 24 h (data not shown). Pretreatment of cells with lithium (2.5 mM) for 24 h before radiation exposure not only maintained cell integrity but also showed slightly increased viability compared to the control cells. However, a significant increase was observed in lithium-treated cells compared to cells exposed to radiation without lithium treatment [Figure 1]b.
|Figure 1: (a) Effect of various doses radiation (3 and 5 Gy) on viability of cells using 3-(4,5 dimethyithiazol-2yl)-2,5 diphenyltetrazolium bromide assay (b) Viability of SK-N-MC cells by 3-(4,5 dimethyithiazol-2yl)2,5 diphenyltetrazolium bromide assay 24h after irradiation (5Gy) and 24 h lithium (2.5mM/5mM) pretreatment|
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Flow cytometric analysis
A typical flow cytometric profile of SK-N-MC cells stained with DNA specific dye PI is depicted in [Figure 2]a. The profile is essentially bimodal with majority of cells in G1/G0 phase and a smaller fraction of cells in S+G2 + M phase of the cell cycle [Figure 2]a. The sub-G1/G0 fraction of cells is marked as M1 and is a measure of cell death in the population. This M1 fraction of cells was dramatically and consistently heightened in cells irradiated with 5 Gy. Pretreatment of cells with lithium substantially reduced the sub-G1 cell population (dead cells). Protective effect was observed in cell pretreated with lithium (24 h) at both the doses (2.5 and 5 mM) [Figure 2]b.
|Figure 2: Flow cytometry profile of SK-N-MC cells – percent cell death 24 h after irradiation. (b) Fraction of dead cells was substantially reduced in cells exposed to gamma rays (5Gy) after lithium (2.5 mM/5 mM) treatment|
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Cell morphology studies
Comparative morphology of cells [Figure 3] observed up to 48 h showed that cells treated with lithium alone were enlarged with enhanced neurite extensions. Cells pretreated with lithium before radiation exposure also appeared enlarged and had developed neurites by this period in culture. Contrary to this, cells directly subjected to radiation appeared vacuolated had significantly reduced percent of cells bearing two and more neurites. This suggested increase in differentiation state in the former 2 groups and pronounced effect of radiation-induced oxidative damage phenotype in the latter. Radiation affected neurite outgrowth in these cells. To reduce any bias during scoring for neurite outgrowth per cell this was done blindfolded.
|Figure 3: Photomicrographs of SK-N-MC cells stained with Giemsa (×40) at 48 hr. after treatment. (a) Control, untreated cells (b) cells treated with lithium alone (c) Irradiated cells (d) Irradiated cells pretreated with lithium|
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Lithium restores differentiation block induced by radiation
Cytoplasmic extensions (neurites), a characteristic of neuronal cells and are indicative of cell differentiation. Enhanced neuritogenesis have been quantitated by measuring two parameters, mean length of neurites, and number of cells bearing 3 or more neurites [Table 1]. No significant difference in mean length of neurite was observed but a significantly larger number of cells had 3 or more neurites in the lithium-treated cells suggesting that lithium-induced neurite formation. Irradiated cells (without pretreatment of lithium) had longer neurites compared to the control but number of cells bearing two or more neurites was significantly reduced compared to the control cells. Lithium-treated cells, on the other hand, had reduced neurite lengths but increasing number of cells with two neurites as like in control phenotype indicating modification (P < 0.01) and subsequent evasion from radiation induced block in differentiation.
|Table 1: The mean length of neurites and the mean number of cells bearing varying number of neurites as seen in SK-N-MC cells under various treatments|
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Pretreatment with lithium altered expression of neural cell adhesion molecule
Real-time PCR profiles of test and 2 housekeeping genes, namely, β-actin and 18s rRNA showed marked decrease in levels of NCAM expression on radiation exposure. This effect was reversed in case of cells pretreated with lithium [Table 2]. Relative Quantitation (2−ΔΔCT) of NCAM expression could well become indicator for differentiation state of these neuroblastoma cells before intervention of radiation exposure.
|Table 2: Differentially expressed genes in lithium (2.5 mM) alone, irradiated (5 grays) alone, and irradiated cells with lithium pretreatment as compared to control SK-N-MC cells|
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| Discussion|| |
Neuroprotective effects of lithium have been recognized over the past 2 decades, but there are very few studies that demonstrated its modifying effects on gamma radiation-induced damage on neuronal cancer cells. Most of the reports discussed protection of cells by lithium from variety of environmental insults. Lithium inhibits the canonical Wnt signaling pathway by inhibiting glycogen synthase kinase-3β (GSK-3β) and stabilizing β-catenin. Lithium is known to modulate stress and mitogen activated kinases. Its protective role could be executing by shift in levels of stress proteins involved with cell survival. We used SK-N-MC cells of neuronal origin for studying cell death, survival, and differentiation and demonstrated that lithium could rescue cells from radiation induced cellular damage.
A measure of differentiation of neuronal cells is increase in length or number of cytoplasmic extensions, the neurites. Lithium has been known to stimulate neuritogenesis.
An increase in number of cells bearing neurites in lithium protected cells as compared to irradiated cells suggested cell differentiation and increase in adherence. Our data demonstrated that with 5 Gy exposure to cells there was reduced cell viability and the fraction of apoptotic cells, as measured by flow cytometric parameters, were significantly higher. Interestingly, cells that survived radiation exposure as a result of pretreatment with lithium also had enhanced neurite extensions. This suggested that pretreatment with lithium could be modulating cell fate by inducing neurites and consequently improving adherence of neuronal cells 2 physical features that affected the outcome of radiation-induced damage in these neuronal cells. One of the molecules involved in cell adherence is NCAM, and its levels in cells that survived radiation were monitored to check for its role in lithium pretreatment. Molecules of NCAM expressed on cell surfaces are known to play a role in neural cell differentiation, proliferation, neurite outgrowth, and plasticity. NCAM belongs to the immunoglobulin superfamily, and alternate splicing gives rise to several isoforms of this single gene. NCAM is also known to participate in canonical Wnt signaling. In PC12 cell lines, neurite extensions are known to involve NCAM/fibroblast growth factor receptor signaling pathway with β-catenin binding NCAM in complex containing FGFR and GSK 3-β. Posttranslational modifications such as polysialic acid forms polysialic acid/NCAM, are antiadhesive and has been implicated in axon growth and synaptogenesis during development. NCAM expression is enhanced in neuroblastoma cells that are destined to differentiate and form neurites on the action of retinoic acid. Our observations of heightened NCAM expression and outgrowth of neurites were consistent with other studies done on neuronal cells. Thus, increase of NCAM in lithium-treated cells seems to be associated with increase in neurite bearing cells and could well be a molecule to watch for its use in radiation protection in future.
Expression of NCAM was increased in glial cell lines on exposure to radiation, whereas here we report drastic decrease in NCAM expression levels in human neural cell line. This could probably be a novel observation suggesting cell specific differential responses to radiation. Variability in the capacity of radioresistance among heterogeneous cell types could be inherent in multicellular organism. Association of NCAM expression, lithium pretreatment, and consequent neuromodification are effects of probable complex molecular interactions with or without involvement of Wnt signaling which deserves further investigations.
| Conclusion|| |
This is the first report demonstrating that lithium modifies the radiation effects in SK-N-MC cells, which seems to be associated with heightened neurite growth and alteration in expression of NCAM. However, this study provides further scope for the detailed mechanistic insight of radiomodifying ability of lithium and its potential implications in cancer radiotherapy.
Financial support and sponsorship
Financial support was provided by Board of Research in Nuclear Sciences, India.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Cortellino S, Turner DP, Bellacosa A. Induction of deoxyribonucleic acid damage by gamma irradiation. Methods Mol Biol 2004;285:127-32.
Kawamura K, Fujikawa-Yamamoto K, Ozaki M, Iwabuchi K, Nakashima H, Domiki C, et al.
Centrosome hyperamplification and chromosomal damage after exposure to radiation. Oncology 2004;67:460-70.
Munshi A, Ramesh R. Mitogen-activated protein kinases and their role in radiation response. Genes Cancer 2013;4:401-8.
Dent P, Yacoub A, Contessa J, Caron R, Amorino G, Valerie K, et al.
Stress and radiation-induced activation of multiple intracellular signaling pathways. Radiat Res 2003;159:283-300.
Miloso M, Scuteri A, Foudah D, Tredici G. MAPKs as mediators of cell fate determination: An approach to neurodegenerative diseases. Curr Med Chem 2008;15:538-48.
Reddick WE, Glass JO, Palmer SL, Wu S, Gajjar A, Langston JW, et al.
Atypical white matter volume development in children following craniospinal irradiation. Neuro Oncol 2005;7:12-9.
Paulino AC, Simon JH, Zhen W, Wen BC. Long-term effects in children treated with radiotherapy for head and neck rhabdomyosarcoma. Int J Radiat Oncol Biol Phys 2000;48:1489-95.
Madsen TM, Kristjansen PE, Bolwig TG, Wörtwein G. Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat. Neuroscience 2003;119:635-42.
Inouye M, Yamamura H, Nakano A. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum. J Radiat Res 1995;36:203-8.
Chiu CT, Wang Z, Hunsberger JG, Chuang DM. Therapeutic potential of mood stabilizers lithium and valproic acid: Beyond bipolar disorder. Pharmacol Rev 2013;65:105-42.
Mines MA, Jope RS. Glycogen synthase kinase-3: A promising therapeutic target for fragilexsyndrome. Front Mol Neurosci 2011;4:35.
Lauterbach EC, Fontenelle LF, Teixeira AL. The neuroprotective disease-modifying potential of psychotropics in Parkinson's disease. Parkinsons Dis 2012;2012:753548.
Huang X, Wu DY, Chen G, Manji H, Chen DF. Support of retinal ganglion cell survival and axon regeneration by lithium through a bcl-2-dependent mechanism. Invest Ophthalmol Vis Sci 2003;44:347-54.
Takahashi T, Steinberg GK, Zhao H. Lithium treatment reduces brain injury induced by focal ischemia with partial reperfusion and the protective mechanisms dispute the importance of akt activity. Aging Dis 2012;3:226-33.
Danivas V, Moily NS, Thimmaiah R, Muralidharan K, Purushotham M, Muthane U, et al.
Off label use of lithium in the treatment of Huntington's disease: A case series. Indian J Psychiatry 2013;55:81-3.
] [Full text]
Forlenza OV, de Paula VJ, Machado-Vieira R, Diniz BS, Gattaz WF. Does lithium prevent Alzheimer's disease? Drugs Aging 2012;29:335-42.
Yang ML, Li JJ, So KF, Chen JY, Cheng WS, Wu J, et al.
Efficacy and safety of lithium carbonate treatment of chronic spinal cord injuries: A double-blind, randomized, placebo-controlled clinical trial. Spinal Cord 2012;50:141-6.
Sadrian B, Subbanna S, Wilson DA, Basavarajappa BS, Saito M. Lithium prevents long-term neural and behavioral pathology induced by early alcohol exposure. Neuroscience 2012;206:122-35.
Everall IP, Bell C, Mallory M, Langford D, Adame A, Rockestein E, et al.
Lithium ameliorates HIV-gp120-mediated neurotoxicity. Mol Cell Neurosci 2002;21:493-501.
Zhou K, Xie C, Wickström M, Dolga AM, Zhang Y, Li T, et al.
Lithium protects hippocampal progenitors, cognitive performance and hypothalamus-pituitary function after irradiation to the juvenile rat brain. Oncotarget 2017;8:34111-27.
Yang ES, Wang H, Jiang G, Nowsheen S, Fu A, Hallahan DE, et al.
Lithium-mediated protection of hippocampal cells involves enhancement of DNA-PK-dependent repair in mice. J Clin Invest 2009;119:1124-35.
Rajadhyaksha MS, Jagtap JC, Kelkar R, Gokahle P. Lithium induces morphological and functional differentiation in neuronal cells. Neurosci Res Commun 2000;26:9-16.
Italia J, Mukhopadhyaya R, Rajadhyaksha MS. Differential display RT-PCR reveals genes associated with lithium-induced neuritogenesis in SK-N-MC cells. Cell Mol Neurobiol 2011;31:1021-6.
Seidenfaden R, Krauter A, Hildebrandt H. The neural cell adhesion molecule NCAM regulates neuritogenesis by multiple mechanisms of interaction. Neurochem Int 2006;49:1-1.
Rønn LC, Berezin V, Bock E. The neural cell adhesion molecule in synaptic plasticity and ageing. Int J Dev Neurosci 2000;18:193-9.
Kindich R, Florl AR, Jung V, Engers R, Müller M, Schulz WA, et al.
Application of a modified real-time PCR technique for relative gene copy number quantification to the determination of the relationship between NKX3.1 loss and MYC gain in prostate cancer. Clin Chem 2005;51:649-52.
Quiroz JA, Machado-Vieira R, Zarate CA Jr., Manji HK. Novel insights into lithium's mechanism of action: Neurotrophic and neuroprotective effects. Neuropsychobiology 2010;62:50-60.
Aminzadeh A, Dehpour AR, Safa M, Mirzamohammadi S, Sharifi AM. Investigating the protective effect of lithium against high glucose-induced neurotoxicity in PC12 cells: Involvements of ROS, JNK and P38 MAPKs, and apoptotic mitochondria pathway. Cell Mol Neurobiol 2014;34:1143-50.
Shah SM, Patel CH, Feng AS, Kollmar R. Lithium alters the morphology of neurites regenerating from cultured adult spiral ganglion neurons. Hear Res 2013;304:137-44.
Weledji EP, Assob JC. The ubiquitous neural cell adhesion molecule (N-CAM). Ann Med Surg (Lond) 2014;3:77-81.
Hinsby AM, Lundfald L, Ditlevsen DK, Korshunova I, Juhl L, Meakin SO, et al.
ShcA regulates neurite outgrowth stimulated by neural cell adhesion molecule but not by fibroblast growth factor 2: Evidence for a distinct fibroblast growth factor receptor response to neural cell adhesion molecule activation. J Neurochem 2004;91:694-703.
Bonfanti L, Theodosis DT. Polysialic acid and activity-dependent synapse remodeling. Cell Adh Migr 2009;3:43-50.
De Laurenzi V, Raschellá G, Barcaroli D, Annicchiarico-Petruzzelli M, Ranalli M, Catani MV, et al.
Induction of neuronal differentiation by p73 in a neuroblastoma cell line. J Biol Chem 2000;275:15226-31.
Kleene R, Mzoughi M, Joshi G, Kalus I, Bormann U, Schulze C, et al.
NCAM-induced neurite outgrowth depends on binding of calmodulin to NCAM and on nuclear import of NCAM and fak fragments. J Neurosci 2010;30:10784-98.
Yamanaka R, Tanaka R, Yoshida S. Effects of irradiation on the expression of the adhesion molecules (NCAM, ICAM-1) by glioma cell lines. Neurol Med Chir (Tokyo) 1993;33:749-52.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]