Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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On Sep 2018




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On Sep 2018




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"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



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Saraswati Dental College
Lucknow
On Sep 2018




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Best regards,
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Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
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Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


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Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
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Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2023 | Month : January | Volume : 17 | Issue : 1 | Page : FC01 - FC05 Full Version

Antiproliferative and Proapoptotic Effects of Vitamin D3 in Human Neuroblastoma Cell Lines SH-SY5Y: An In-vitro Study


Published: January 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/60147.17298
Kruti N Parikh, Kavitha Ramasamy, Kranthi Karunai Kadal, Punnagai Kumaravelu

1. Postgraduate Student, Department of Pharmacology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India. 2. Professor, Department of Pharmacology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India. 3. Assistant Professor, Department of Pharmacology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India. 4. Professor and Head, Department of Pharmacology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India.

Correspondence Address :
Dr. Kavitha Ramasamy,
No.1 Ramachandra Nagar, Porur, Chennai, Tamil Nadu, India.
E-mail: r.kavitha@sriramachandra.edu.in

Abstract

Introduction: Neuroblastoma is the most common and earliest childhood tumour with complicated biological and clinical characteristics. The treatment includes chemotherapy, radiation therapy, surgical resection, stem cell therapy and many other modes, making the management difficult to tolerate and unacceptable. Thereby increasing the need to develop novel therapies or repurpose already existing ones with anticancer potential. Many studies have shown that vitamin D3 has anticancer properties. Vitamin D3 receptors have been found in neuroblastoma cell lines, according to research. Anticancer property of vitamin D3 hasn’t been studied much in neuroblastoma cell lines.

Aim: To evaluate the antiproliferative and proapoptotic effects of vitamin D3 on human neuroblastoma cell lines-SH-SY5Y.

Materials and Methods: The present study was an in-vitro study in which human neuroblastoma cell lines SY5Y (a total of 2 cell lines) were obtained from National Centre for Cell Science (NCCS), Pune, Maharashtra, India, and control cells are cells from the cell lines that were left untreated. The antiproliferative effect of vitamin D3 in human neuroblastoma cell lines evaluated using the MTT (3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide) assay. After 48 hours of incubation and treatment with six different (0.01, 0.1, 1, 10, 100, and 1000 ng/mL) concentrations of vitamin D3, the percentage of viable cells was determined using spectrophotometry and compared with control cells (untreated cells from cell lines). Different vitamin D3 (250 ng/mL, 500 ng/mL, 1000 ng/mL) doses were applied to cells and they were then incubated for 24 hours, Cell death and malformations were then observed using a phase contrast microscope, and Deoxyribonucleic Acid (DNA) fragmentation was investigated using gel chromatography. The obtained results were expressed as percentage of inhibition and tabulated in Microsoft Excel Sheet Version 16.16.27 and scatter plot graph was used to calculate IC50 (Half maximal inhibitory concentration).

Results: The vitamin D3 showed antiproliferative property in SH-SY5Y cells at an IC50 of 164 ng/mL when tested against human neuroblastoma cells using the MTT assay. Phase contrast microscope demonstrated that vitamin D3 treated cells showed condensation of nuclei, shrinkage of the cytoplasm, convolution of outline and cell peeling demonstrating apoptosis. DNA fragmentation also showed typical DNA ladder formation confirming apoptosis in vitamin D3 treated cells, which showed that the treated cells’ DNA was more damaged than the control cells’ DNA.

Conclusion: Vitamin D3 exhibited both proapoptotic and antiproliferative properties, as demonstrated by the MTT assay, Phase Contrast, and DNA fragmentation.

Keywords

Anticancer activity, Cholecalciferol, Half maximum inhibitory concentration

Neuroblastoma is one of the most prevalent extracranial solid tumours in children with a prevalence rate of 10-15% of all the childhood tumours (1). It is a complicated tumour with a variety of clinical and biological characteristics that arises from primordial neural crest cells (2). It is hypothesised that morphological and bio-genetic characteristics may be beneficial in classifying children with neuroblastoma for proper care (3). The tumour differs from other solid tumours due to its biological diversity, which ranges from spontaneous regression to aggressive metastatic illness (4). The tumour is supposed to develop within the peripheral sympathetic nervous system’s neuronal ganglia (5). It is known that 60% of abdominal paraspinal ganglion tumours and 30% of neuroblastoma originate from the adrenal medulla that has led to the disease’s clinical presentation being variable (6).

Understanding the intricate pathological diversity of the tumour by looking at its pathophysiology in connection to brain cells might assist uncover chemicals and, in turn, routes for targeted intervention (7). The neural crest, also known as the fourth germ layer, is an embryological tissue that develops from neuroectoderm (8). Through maturity, the first neural crest precursor develops multipotent differentiation potential and, as a result, acquires the ability to self-renew (9). Many signaling pathways (like Bone Morphogenetic Protein (BMP), Wingless related integration site (Wnt), Notch, and others) are responsible for the cell differentiation (10). Inhibiting these maturation pathways would likely make early multipotent neural crest precursors more likely to develop malignant transformation. In addition to this, numerous gene mutations have also been linked to the emergence of neuroblastoma (10). Anaplastic Lymphoma Kinase (ALK), Protein Tyrosine Phosphate Non-Receptor-11 (PTPN-11), Alpha-Thalassaemia/mental retardation, X-linked (ATRX), MYCN protooncogene (MYCN), and Neuroblastoma Rat Sarcoma (NRAS) gene alterations are the ones that are shown to be related with it most frequently (11).

Chemotherapy with drugs including vincristine, carboplatin, etoposide, cyclophosphamide, and cisplatin is the mainstay of treatment for neuroblastoma (12). Further therapeutic options include surgical resection, radiotherapy, immunotherapy and other modes (12). Based on the severity of the tumour, this multimodal therapy is divided into four phases: induction, local control, consolidation, and maintenance (13). The total treatment time can take upto 18 months (14). There is a need to develop innovative medicines with higher tolerability and acceptance because the existing therapy method is linked to adverse effects and low patient compliance (14).

Many malignant and non malignant cells have demonstrated significant physiological effects of vitamin D3 on proliferation and differentiation (15). It is a strong seco-steroid with a significant impact on neuroprotection (15). According to some studies, vitamin D3 may help to prevent neuronal damage brought on by various types of injury (15). Several researches have shown that vitamin D3 has anticancer properties in prostate, colon, and breast cancer cell lines (16). In the liver and kidney, vitamin D precursors are methodically converted into active hormone in two steps (17). Vitamin D3 (cholecalciferol) and vitamin D2 are the two main physiologically inactive precursors of vitamin D (ergocalciferol) (17). When they enter the liver, vitamin D precursors from the diet and sunlight exposure are both transformed to 25-hydroxyvitamin D [25(OH)D] (calcidiol) by the enzyme 25-hydroxylase (18). The main form of vitamin D that circulates and is used to assess vitamin D levels is 25(OH)D. 1,25-dihydroxyvitamin D [1,25(OH)2D] requires further hydroxylation in the kidneys to become physiologically active (calcitriol) by the enzyme 1α-Hydroxylase (17),(18). The active hormone calcitrol interacts to the Vitamin D Receptor (VDR), which then affects the expression of several genes for skeletal and non skeletal homeostasis through a sequence of activation and co-activation events (19). Several investigations have shown the existence of VDR in human neuroblastoma cell line (20). Studies have also shown that VDR is necessary for vitamin D3’s anticancer impact in cancer cells (21). The amount of VDR present in the transfected cell lines correlated with the degree of vitamin D3-induced antiproliferative activity (21). Vitamin D’s antiproliferative effect results from its capacity to halt cell cycle progression (21). P21 and other gene associated to the cell cycle have been shown to be directly impacted by vitamin D3 (22). It has been demonstrated to suppress the Endothelial Growth Factor Receptor (EGFR) signaling cascade (23). and inhibit the Hedgehog pathway (24). By inhibiting the expression of antiapoptotic proteins including Bcl-2 and Bcl-xl and activating proapoptotic proteins BAX, BAK, and BAD, vitamin D3 has also been demonstrated to promote apoptosis in several cancer cells (25). Vitamin D3 deficiency has been linked with many diseases including cancer, vitamin D3 has shown to be effective in treating many cancers including breast, prostate, squamous cell carcinoma and melanoma in-vitro (26). The same principle has been applied in this study to determine anticancer effects of vitamin D3 in neuroblastoma cell lines, which has not been explored much and in this study both antiproliferative and antiapoptotic effects of vitamin D3 has been explored.

The aim of the current study was to determine the antiproliferative effects of vitamin D3 against the human neuroblastoma cell line SH-SY5Y by examining its effects on cell viability percentage inhibition, cell migration inhibition, DNA damage inhibition, and apoptosis induction.

Material and Methods

The present study was an in-vitro study, which was carried out on the Human Neuroblastoma cell lines SH-SY5Y at Sri Ramachandra Medical College and Research Institute, Chennai, from December 2020-January 2021. The study has been carried out in SH-SY5Y cell lines which is a triple cloned cell line from a well-established SK-N-SH neuroblastoma cell lines (well established cell lines are usually cloned and cultured from already existing cell lines and not obtained directly from humans / animals) and have been obtained from NCCS, Pune , Maharashtra, India which produces and generates cultured and cloned cell lines. Since the cells haven’t been harvested directly from humans/animals, Ethics approval was exempted for this study as per the standard guidelines (27).

Chemicals and reagents

Crystalline vitamin D3 was obtained from FERMENTA biotech, Thane, Maharashtra. DMSO (Dimethyl sulfoxide) obtained from SRL (Sisco research laboratories) chemicals, Chennai. MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) from Sigma Aldrich, India.

The different media used are Foetal Bovine Serum (FBS), Ham’s F12 medium and Minimum Essential Medium (MEM) which were obtained from Genetix Biotech, India.

Cell line

Human Neuroblastoma cell line, SH-SY5Y obtained from NCCS Pune, Maharashtra, India. The cell lines were cultured in an equal mixture of MEM and Ham’s F12 medium which was supplemented with 10% FBS and antibiotics. For the control, the cells were left untreated.

Cytotoxicity assay (MTT ) (28): By using the MTT assay, the cytotoxicity of vitamin D3 was evaluated against the SHSY-5Y cell line (29). The cells cultured with equal parts of MEM and F12 medium (that were treated with FBS, penicillin and streptomycin) at 37°C and allowed to grow to a convergence of 70-80%, post this they were plated in 96-well microplates (1 X 106 cells/wall), cultured for 24 hours at 37°C in 5% CO2. After that, different Vitamin D3 concentrations (0.01, 0.1, 1, 10, 100, and 1000 ng/mL) were prepared from the stock (Vitamin D3 was prepared as 10 mg/mL stock by adding 100% ethanol), as serial dilution of 1:10 works well for initial experiment (30) and added to the medium and cells, and everything was incubated for 48 hours at 37°C in 5% CO2. Following a Phosphate Buffer Saline (PBS, pH-7.3) wash, each well received 50 μL of MTT solution (5 mg/mL in PBS) and incubated for 3 hours. The plates were then let to sit for 30 minutes at 37° Celsius in the dark. The absorbance of the Formazan crystals that gathered in well plates was measured after they were dissolved in 100 μL of DMSO spectrophotometrically at 570 nm. The following formula was used to compute the proportion of viable cells:

Cell Viability (%)=(Absorbance of sample/Absorbance of control)×100 (31).

Phase Contrast Microscopy (32): To study the morphological effects of vitamin D3 on the human neuroblastoma cell lines SH-SY5Y, phase contrast microscopy was used. Carefully sown in eight wells of six well plates, the neural cancer cell line (SHSY5Y) was allowed to adhere and proliferate for 24 hours. Vitamin D3 sample was supplied to the cells at dosage concentrations of 250 ng/mL, 500 ng/mL, and 1000 ng/mL (the reason for selecting 250 mg as dose was based on the IC50 value, based on the first dose the dose was doubled until response was obtained); each dose was given to the cells in two wells (duplicates) in maintenance media with 2% FBS. For 24 hours, the treated cell line was incubated. A phase contrast microscope was used to check the cells for cell death, deformities and cell shrinkage under 10X magnification, no special staining was done/used.

DNA Fragmentation using Agarose Gel Electrophoresis (33): It was carried out to research how vitamin D3 affected the molecular behaviour of SH-SY5Y cell lines. Four T25 flasks were seeded with the cells, and they were given 24 hours to adhere and proliferate. Vitamin D3 was administered to the cells in maintenance media containing 2% FBS at dosage concentrations of 250 ng/mL, 500 ng/mL, and 1000 ng/mL. For 24 hours, the treated cell line was incubated. The cells were then removed for DNA isolation after being proteolysed. The manual Phenol-Chloroform DNA isolation process was used to isolate the DNA, and the pellet was then dissolved in nuclease-free water. A 0.8% agarose gel containing the DNA samples was loaded, and it was run at 50 volts. The gel was examined with a UV light source to look for DNA bands, which indicate DNA fragmentation.

Statistical Analysis

Obtained results were expressed as percentage of inhibition and Standard Deviation (SD) and it has been tabulated in Microsoft Excel Sheet Version 16.16.27. IC50 values are calculated using scattered plot graph.

Results

MTT assay

Cell growth inhibition property: SH-SY5Y cells were screened against vitamin D3. A range of vitamin D3 concentrations (0.01, 0.1, 1, 10, 100, and 1000 ng/mL) were examined. Each measurement of vitamin D3 concentration was carried out four times, and the cumulative difference between the data points was kept to under 20%, as shown in (Table/Fig 1). A scatter plot in (Table/Fig 2) shows how at an IC50 of 164 ng/mL, which was obtained using the previously given equations.

Phase Contrast

Vitamin D3 inhibited the proliferation of SH-SY5Y human neuroblastoma cells and caused them to undergo apoptosis, according to analysis utilising phase contrast microscopy. [Table/Fig-3a-d] demonstrates the action of different doses of vitamin D3 (250 ng/mL, 500 ng/mL and 1000 ng/mL) on treated cells as compared to the control (untreated cells). Blebbing of the membrane was seen with vitamin D3 and cell shrinkage was observed with increased dosage. With increasing vitamin D3 concentration, the number of cells with changed shape increased, reaching a maximum at 1000 ng/mL dose of vitamin D3.

DNA Fragmentation

Vitamin D3-treated SH-SY5Y cells had shown morphological changes as well. DNA fragmentation was investigated by observing DNA ladder production. (Table/Fig 4)a,b,c,d illustrates how, after 48 hours of exposure, the DNA ladder became more noticeable with increasing dosages of vitamin D3 (250, 500, and 1000 ng/mL doses), which showed DNA fragmentation. However, the control group showed no signs of DNA fragmentation. (A DNA ladder/band is a piece of DNA that has undergone digestion and characterisation, yielding known base pair sizes. It is more evidence that vitamin D3 caused cell death via inducing apoptosis because DNA fragmentation is a characteristic of this process).

Discussion

Neuroblastoma is a common tumour of paediatric age group with a complex management regimen and with a higher relapse rate in the post consolidation phase (34). The presence of VDR in neuroblastoma cell lines, their quantities, and the active metabolite of vitamin D3 impacting the signalling via VDRs have all been connected to the mechanism of action of vitamin D3 in neuroblastoma cell lines programmed cell death (apoptosis) is a key tactic in the eradication of malignant cells (20). In a review it has been noted that the prostate is a vitamin D3-target organ, which has supported the existence of VDR in prostate epithelial cells (35). This resulted in the first in-vitro studies showing the antiproliferative effect of vitamin D3 on human CaP cells (35). Additionally, vitamin D3 has been shown to have antiproliferative effects in some in-vitro experiments using prostate and breast cancer cell models (36).

The functional condition of the mitochondria is determined by the MTT colorimetric assay, which indicates cell viability. Living cells include an enzyme called mitochondrial dehydrogenase that converts yellow tetrazolium MTT salt to blue MTT formazan, which precipitates in healthy cells (37).The anticancer potential of vitamin D3 in numerous tumour types has been studied using MTT assay (38). Vitamin D3 and its analogues has shown antiproliferative effects in an in-vitro investigation against the human breast cancer cell lines T47D and MCF-7, human epithelial squamous cell cancer cell line SCC-25, human leukaemia cell line HL-60, mouse derived leukaemia cell line WEHI-3, and mouse fibroblast cell line BALB/3T3 (38). The idea that vitamin D3 binds to the VDR in epithelial cells and induces the differentiation of epidermal and leukaemic cells while also having antiproliferative properties against squamous cell cancer, breast cancer, and leukaemic cells in-vitro has been proven by this action (38).

Calcitrol (vitamin D3) showed to cause inhibition of 50% of cells (IC50) in SCC-25 cell lines at a dose of 137 ng/mL, in MCF-7 breast cancer cell lines, IC50 of vitamin D3 was found to be at a dose of 43.6 ng/mL and in T47D cell lines it was found to be 67.7 ng/mL (38). In the present study, the MTT assay indicated that vitamin D3 inhibits cell proliferation, with the IC50 of cell growth in the human neuroblastoma cell line being reached at a dosage of 164 ng/mL. In this study, no positive control was used to compare the antiproliferarive effect of vitamin D3 using MTT assay as the main aim was to first establish the antiproliferative effect before the effect of vitamin D3 can be compared with a standard positive.

Apoptosis functions as a defence mechanism in healthy cells to get rid of damaged cells before they become cancerous (39). Numerous chemotherapy medications work primarily by inducing apoptosis, which kills malignant cell (40). Apoptosis is a process that is characterised by morphological changes such as DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage, and loss of mitochondrial membrane potential (41). Extrinsic and intrinsic pathways have been identified as the two main types of apoptosis in living cells (41). There are many methods to determine apoptosis in-vitro such as Morphology-based methods, Immunohistochemistry-based methods, Biochemistry-based methods, Immunology-based methods and Array-based methods. Phase contrast microscopy is one type of morphology based method in which phase contrast microscope can be used to examine cells that have been cultured in flasks or plates. Dead cells start to float in the culture medium as they separate from the substratum to which they are attached. The phase contrast microscope makes it simple to identify these floating cells. A phase contrast microscope used with care can reveal blebs on apoptotic cells. Vacuoles that form in the cytoplasm indicate that the cells are still dispersed on the substratum which can be easily observed. Normally no special dyes are needed to stain the cells in phase constrast microscopy, in some specail cases fluroscent dyes can be used to stain and observe the cells under phasse contrast microscopy (41). Vitamin D3 has also shown to cause apoptosis in SH416–ER and PC9–ER lung cancer cell lines in a study done (42). In this study, vitamin D3 has shown to cause apoptosis in SH-SY5Y cell lines by causing cell peeling, chromatin condensation, cell blebbing and other features of apoptosis.

Agarose gel electrophoresis is a type of biochemical method used for detection of apoptosis. The genomic DNA is divided into 180 base pairs or its multiples at specific locations (internucleosomal areas) during apoptosis, which causes it to appear like a ladder when electrophoresed. Because of this, this discovery is a distinctive, distinguishing property of apoptosis and is absent in necrosis (When cells are necrotic, they leave a smear pattern on the gel). As a result, it is one of the techniques used to distinguish between necrosis and apoptosis (41),(43). Vitamin D3 has shown to cause apoptosis in human breast cancer cell lines MCF-7, MDA-MB-435 cell lines, human prostate cancer cell lines LnCaP and Human Osteosarcoma cell lines U2OS, and has shown DNA ladder formation in them (44). In this study, vitamin D3 has shown to cause DNA laddering in human Neuroblastoma cell lines-SH-SY5Y under agarose gel electrophoresis.

One study has observed that vitamin D3 has shown to have proliferative effects on normal germline related stemcells (Very Small Embryonic Like Stem cells- VSELS) but at the same time it is inhibitory to tumour cells, which is correlated to the pleotropic effects of vitamin D3 in normal and cancerous cells, thereby making supplementation with vitamin D3 important for both normal and pathological conditions (45).

Despite extensive research examining vitamin D3’s anticancer effectiveness in many tumour forms, very few studies have examined its impact on neuroblastoma. In this study, from the results has been clearly demonstarted that vitamin D3 has proapoptotic effects as evidenced by cell shrinkage, nuclear condensation, membrane degradation, and cell peeling, chromatin clevage and cell death under the phase contrast microscopy and this was confirmed by typical laddering of DNA fragmnents formed using the DNA fragmentation on agarose gel electrophoresis which is considered to be the biochemical halmark of apoptosis.

With these findings, it can be definitively stated that vitamin D3 inhibits cell proliferation while promoting apoptosis in the human neuroblastoma SH-SY5Y cell line. But more assays need to be done to strongly establish the role of vitamin D3 in neuroblastoma. With this study it can be implied that the anticancer property of vitamin D3 can be used in the future to establish it as an adjunctive therapy with the standard chemotherapy of neuroblastoma, provided it shows similar positive effects in future in-vivo and clinical trials.

Limitation(s)

This study’s limitation was the lack of a positive control, which would have allowed vitamin D3’s antiproliferative activity to be amplified.

Conclusion

Within the limits of this study, it has been concluded that vitamin D3 has demonstrated antiproliferative and proapoptotic activity with the help of the MTT assay, phase contrast microscopy, and DNA fragmentation, in human neuroblastoma cell line SH-SY5Y. However, to strengthen the proof of vitamin D3’s anticancer effect in human neuroblastoma, more tests and in-depth research must be conducted both in-vitro and in-vivo.

Acknowledgement

The authors are thankful to the Department of Pharmacology, SRMC&RI, Porur, Chennai, Tamil Nadu, India.

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DOI and Others

DOI: 10.7860/JCDR/2023/60147.17298

Date of Submission: Sep 09, 2022
Date of Peer Review: Oct 17, 2022
Date of Acceptance: Dec 03, 2022
Date of Publishing: Jan 01, 2023

Author declaration:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? No
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. No

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