Ismail, Aziana
(2019)
Antioxidant and antiproliferative activities of six Malaysian medicinal plants and mechanism of action through microrna in thyroid cell lines.
Masters thesis, Universiti Sains Malaysia.
Abstract
Thyroid cancers, including papillary, follicular, medullary and anaplastic
carcinomas, have different growth rates and can metastasize if left untreated. Even
though most thyroid cancer cases have good prognosis, the risk of recurrence among
the patients is high and some patients die from progressive tumor. Medicinal plants are
commonly used as alternative to cancer treatment in many countries. The antiproliferative
activity of phytochemicals has been demonstrated in various cancers, yet
the effect of medicinal plants on thyroid cancer remain under-investigated. Despite
various reports on the anti-cancer activities of plant extracts on a variety of cancer cell
lines, the cellular targets and the underlying mechanisms remain unclear. The overall
goal of the present study was to investigate the effect of crude extracts from six
medicinal plants (Annona muricata L., Angelica keiskei Ito, Chromolaena odorata (L.)
R.M.King & H. Rob, Clinacanthus nutans (Burm. f.) Lindau, Euphorbia hirta L., and
Leea indica (Burm. f.) Merr.) on thyroid cell lines (Nthy-ori 3-1, FTC-133 and Hth-
74) and the mechanism involved. For antioxidant and anti-proliferative activities of
the plant extracts, methods such as Folin-Ciocalteu’s, aluminum chloride, DPPH free
radical scavenging assays and MTT assay were utilized. Flow cytometric analysis of
treated cells stained with propidium iodide and with Annexin V-FITC/propidium
iodide, and TUNEL assay further confirmed the anti-proliferative activity of the plant
extract. Quantitative real-time RT-PCR of apoptotic related genes, candidate
microRNAs and their putative target genes were also performed. Methanolic extract
of E. hirta had high amount of total phenolic and flavonoid contents at 307.59 ± 3.57
mg GAE/g dw and 76.43 ± 4.34 mg QE/g dw, respectively. The extract had 32% of
DPPH scavenging with IC50 of 0.013 mg/mL. Significant reduction of cell viability
was observed when treated with A. muricata and C. odorata methanolic extracts with
IC50 of 20.8 and 74.9 mg/mL in FTC-133 cells at 72 hours of treatment. FTC-133 cells
treated with A. muricata methanolic extract showed significant increase in percentage
of cells at the S- and G2/M-phase with subsequent significant decrease in G0/G1-phase.
Flow cytometric analysis of cells treated with Annexin V-FITC/propidium iodide and
analysis of TUNEL assay further confirmed that A. muricata methanolic extract
induced apoptosis in FTC-133 cells. Gene expression analysis of apoptosis-related
genes suggest that intrinsic apoptosis pathway was induced by the significant increase
in expression of pro-apoptotic gene, BAX and significant decrease in expression of
anti-apoptotic gene, BCL2. In addition, significant reduction in the expression of
several microRNAs (miR-192, miR-197, miR-328 and miR-346) in FTC-133 cells
treated with A. muricata methanolic extract suggest for potential underlying
mechanism of the anti-proliferative property. In conclusion, large amount of phenolic
and flavonoid compounds in E. hirta contributed to its high antioxidant activity.
Treatment of A. muricata methanolic extract resulted in significant anti-proliferative
activity in FTC-133 cells through cell cycle arrest and induction of apoptosis. The antiproliferative
activity may also be due to modulation of several microRNAs aberrantly
expressed in FTC-133 cells. The potential of A. muricata methanolic extract in the
prevention of thyroid cancer development focusing on the microRNAs regulation
pathway should be further investigated.
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