Al-Tayar, Badr Abdullah Saeed
(2022)
Cytotoxic and molecular effects of
betel quid and areca nut extracts on
selected oral epithelial cell lines.
PhD thesis, Universiti Sains Malaysia.
Abstract
The habit of betel quid chewing is widely prevalent in many parts of Asia.
Betel quid comprises areca nut, betel leaf, lime, and other potential constituents
such as tobacco and essences. The main ingredient of betel quid is areca nut. Many
epidemiological studies link betel quid and areca nut chewing to oral cancer.
Therefore, this study aimed to investigate the effect of areca nut and betel quid on
mouth-ordinary-epithelium1 (MOE1) and human oral squamous carcinoma (HSC-2).
Phytochemical compounds were identified using GC–MS. MOE1 and HSC-2 cells
were treated with high concentrations (25–100%) and low concentrations (0.0122–
25%) and subjected to MTT assay. The cell and nuclei morphological changes were
observed under inverted phase contrast and fluorescence microscopes. Following
microarray analysis, the gene expression data was subjected to bioinformatic
analysis. Microarray data was validated by analysing the expression of six selected
genes through real-time reverse transcription-quantitative polymerase chain reaction
(RT-qPCR). Arecoline was identified as the major chemical compound in areca nut
alkaloids, while phenol, 2 methoxy4 (1propenyl) was the major chemical compound
in betel quid phenolics. MOE1 treated with areca nut decreased in viability starting
from 0.0244% until 1.56%. However, at concentrations 6.25% and above, the cells
viability increased. With betel quid treatment, MOE1, cell viability started to
decrease at extract concentration of 0.78% (24 hours) and 3.125% (48 and 72
hours). However, at concentration 50% and 100%, the cell viability increased
significantly. Areca nut treatment on HSC-2 decreased the cell viability
tremendously at all concentrations and treatment time. Betel quid concentration from 6.25% to 25% decreased HSC-2 viability significantly at all treatment duration.
Based on the cytotoxicity data, two concentrations were selected, which were
0.0976% for areca nut and 6.25% for betel quid for the subsequent experiments.
Microscopy findings indicated that the extracts caused noticeable morphological
changes such as cell shrinkage and ballooning, nuclei condensation, and
fragmentation. The microarray gene expression analysis revealed that the total
number of DEGs in MOE1 treated with areca nut and betel quid compared to
controls was 3,038 and 1,985, respectively, while in HSC-2, it was 4,413 and 1,110,
respectively. The shared upregulated DEGs of areca nut treatment were CLDN4,
PIM1, and HBEGF. KEGG analysis suggested the genes are associated with few
main pathways, including the ErbB signalling pathway. The shared upregulated
DEGs for betel quid treatment are HMOX-1, GCLM, and EGLN3. Its KEGG analysis
suggested an association with a few pathways, mainly ferroptosis. RT-qPCR results
of selected genes validated the microarray gene expression. In conclusion, areca
and betel quid showed different effects on normal and malignant epithelial cells,
whereby cell survival mechanism might play an important role in cells treated with
betel quid compared to the areca nut treatment.
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