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Induction of Apoptosis on C6 & U87mg Cells by Bark Extract of Spathodea Campanulata

Researchers: Shrawan Kumar and Shila Elizabeth Besra

INTRODUCTION

Cancer has become the most dominant disease in our society that continues to demand action. The burden of cancer continues to grow globally and has become the 2nd leading cause of death. It is estimated at 9.6 million deaths globally; about 1 in 6 deaths is due to cancer (WHO 2018).

There are various types of cancer responsible for global death in which Gliomas are one of them. Gliomas are the most common type of tumors of the central nervous system (CNS) that originate from glial cells. It is classified into four grades (I-IV) according to their molecular and histological features, with glioblastoma multiforme (GBM, grade IV) being the most aggressive, invasive, and difficult-to-treat. Glioblastomas exhibit a high rate of growth, high vascularization, and are considered to be highly infiltrative with the lowest survival rate. There is no efficient drug or therapy present for the treatment of GBM. However, a combination of surgery and radiotherapy and additionally alkylation chemotherapy with temozolomide (TMZ) has only modestly increased the survival of glioma patients to a median of 14.6 months. In previous researches, it is stated that Doxorubicin has an anti-glioma effect. The blood-brain barrier (BBB) prevents the entry of doxorubicin. To overcome this, liposome encapsulation is adapted which has many side effects, and reappearance of symptoms after discontinuation. Therefore, major attention is being given to look for better and safer plants derived products, which might be a promising therapeutic modality for gliomas.

India has a rich history of traditional systems of medicine which is strongly plant based. Spathodea campanulata is an African plant that is used in tropical and subtropical areas for ornamental purposes as well as for the treatment of various types of diseases. In this study, we investigated the effects of bark extract of Spathodea campanulata (BESC) in glioma cells because of its high fatality rate for brain tumors. Therefore, we evaluated the anti-glioma effect in C6 & U87MG cell line.

METHODOLOGY AND RESULTS

Chemicals

The following chemicals were used: DMEM medium, Foetal bovine serum (FBS), trypsin (Gibco, USA), HEPES, L- glutamine, Penicillin- Streptomycin (Bio-west, Germany), Gentamycin (Nicholas, India), MTT, Ethidium bromide and Acridine orange, Annexin V- FITC apoptosis detection kit, RNase, Propidium iodide, were purchased from Sigma (St. Louis, MO, USA). Proteinase k (SRL), DMSO, Chloroform, isoamyl alcohol, Methanol (Merk), and all other chemicals and reagents were of analytical grade and procured locally.

Cell culture

Glioma cell lines C6 & U87MG were purchased from the National Facility for Animal Tissue and Cell Culture, Pune, India. And the cells were maintained in DMEM medium. Cultures were maintained at 37˚C in a humidified atmosphere containing 5% CO2 in air. In all the experiments untreated leukemic cells were termed as control group.

Plant Material

The bark of Spathodea campanulata (family: Bignoniaceae) was collected from the park of Kasba, Gariahat, Kolkata, India in the month of May, 2015. The bark of this plant was authenticated by Dr.V. P. Prasad, Scientist – D, Central National Herbarium, Botanical Survey of India, Ministry of Environment & Forests, and Government of India at Howrah, West Bengal. A voucher specimen No is CNH/Tech. II/2015/30/309 was deposited in the Cancer Biology & Inflammatory Disorder Division, CSIR, Indian Institute of Chemical Biology, Kolkata.

Detection of Cytotoxicity by MTT assay

C6 & U87MG cells (1×105) were separately seeded in 96-well sterile plates for 24, 48 and 72 hrs. All the treated cells were grown in humidified atmosphere containing 5% CO2 in an incubator at 37℃ and the untreated cells were considered as control. After desired incubation 20μl of MTT was added to each well and incubated again for 3 to 4 hrs at 37℃. The intensity of the colour was measured at 570nm by micro-plate manager. The IC50 values were determined for the all the carcinoma cells.

Morphological study by Fluorescence & confocal Microscope

C6 & U87MG cells (1×106) were treated with different IC50 doses of BESC for 24 hrs and observed using a fluorescence and confocal microscope for determining morphological changes.

The untreated control cells and BESC treated cells were harvested separately (centrifuged at 1000 rpm for 5 min), the pellets were washed twice with PBS and then stained with 100μg/ml of acridine orange (Sigma, USA) and 100μg/ml of ethidium bromide in a ratio of 1:1. In confocal microscopy both treated and untreated cells were harvested and washed with ice cold PBS. The cells were then stained with 10μg/ml of Propidium iodide (Sigma) for 5 min. The cells were then immediately mounted on slides and observed under a microscope for the morphological and nuclear determination of the cells undergoing apoptosis.

Agarose gel electrophoresis study

C6 & U87MG cells were treated with IC50 dose of BESC for 14 hrs then cells were resuspended in 500μl of lysis buffer, 100μg/ml of proteinase K was added and incubation was done at 50ºC for 1 h and 37ºC overnight respectively. DNA extraction was done by following the general phenol-chloroform extraction procedure and kept at -20ºC overnight. After centrifugation, DNA precipitates were washed with 70% ethanol, dried and evaporated at room temperature and dissolved in TE buffer (pH 8.0) at 4ºC overnight. To detect the DNA fragments, the isolated DNA samples were electrophoresed overnight at 20 V in 1% Agarose gel and stained with ethidium bromide. DNA fragmentation was observed in UV transilluminator

Apoptosis Assay

In order to investigate the type of cell death induced by BESC, flow Cytometric analysis was done by performing dot plot assay. The C6 & U87MG cells (1x 106) were treated with individual IC50 dose (18 hrs) of BESC for 18 hrs. The cells were centrifuged at 2000 rpm for 8 min at 4°C and pelleted down. Then washed with AnnexinV- FITC binding buffer provided in apoptosis kit (Sigma). Again after centrifuging at 2000 rpm at 4°C, the cell pellets were dissolved in AnnexinV- FITC binding buffer containing AnnexinV- FITC and Propidium iodide. After 15 min incubation in dark at room temperature flow Cytometric analysis was done. All data were acquired with a Becton-Dickinson FACS LSR Fortessa 4 laser Cytometry. Flow-Cytometry reading was taken using 488 nm excitation and band pass filters of 530/30 nm (for FITC detection) and 585/42 nm (for PI detection).

Statistical Analysis

Statistical analysis was done by Student’s t-test. P < 0.05 was considered as significant.

The percentage cell inhibition was calculated by the following formula:
%Cell inhibition= 100 × (O.D of control- O.D of treated)/O. D of control O. D= Optical Density.

The percentage cell viability was calculated by the formula: – Viable cells (%) = (Total number of viable cells per ml/Total number of cells per ml) × 100.

RESULTS

(Please note that to view the results of this research you can refer to the published work: https://wjpr.net/dashboard/abstract_id/14833)

DISCUSSION & CONCLUSION

Cancer has become a significant problem worldwide, which attracts the attention of the researcher for the discovery of the potent drug. The production of a new drug is still challenging because of its side effect and drug resistance. The selection of natural method helps us to overcome from such problem. Indeed, natural products provide a helpful resource for efficient drug development. Our plant of interest is Spathodea campanulata which has been used in the treatment of various ailments. We investigated the anti-gliomal effect of bark extract of Spathodea campanulata in C6 and U87MG cell line. The results of the present investigation revealed the cytotoxic effect of bark extract of Spathodea campanulata (BESC) on glioma cell line C6 & U87MG. BESC induced cytotoxicity involved DNA fragmentation, membrane blebbing, phosphatidylserine externalization, etc. These all are the hallmark of apoptosis which induced cell death pathways and significant promise for future cancer therapeutics. However, furthermore study should be done and identify the active compound which are responsible for anti-gliomal activity on cancer cells. This result possibility and hope of developing natural and novel anti-cancer drug.

Reference (Sept-20-A4)

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