Cytotoxic and anti-migration activities of protein extracts from Trametes scopulosa (BERK.) Bres. mycelium against HCT 116 colon cancer cell line / Amal A. Rhaffor
Colon cancer is one of the leading causes of cancer death. It is the third most common cancer in men and the second among women globally. Metastasis is a spread of cancer cells to distant locations in the body and usually occurs at stage IV of colon cancer. Distant metastasis is the principle cau...
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| Format: | Thesis |
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2017
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| Online Access: | http://studentsrepo.um.edu.my/9324/ http://studentsrepo.um.edu.my/9324/1/Amal_A_Rhaffor.pdf http://studentsrepo.um.edu.my/9324/5/amal.pdf |
| Summary: | Colon cancer is one of the leading causes of cancer death. It is the third most
common cancer in men and the second among women globally. Metastasis is a spread
of cancer cells to distant locations in the body and usually occurs at stage IV of colon
cancer. Distant metastasis is the principle cause of colon cancer deaths. It involves 6
main steps; local invasion, intravasation, circulation, extravasation, proliferation and
angiogenesis. Polypore mushrooms have been investigated for its medicinal properties
especially for anti-tumour activity. Trametes scopulosa is one of the polypore
mushroom. However to date, no research concerning the anti-tumour effect of
Trametes scopulosa has been published. This study is therefore aims to evaluate the
anti-tumour potentials by looking at the cytotoxic and anti-migration activities of
solvent and protein extracts from T. scopulosa (KUM70034) against colon cancer cell
line (HCT 116). Pre-screening for cytotoxic activity against HCT 116 using MTT assay
showed that all solvent extracts of T. scopulosa (methanol, hexane, dichloromethane
and ethyl acetate) were deemed not actively cytotoxic against HCT 116 cells. However,
all protein fractions obtained by ammonium sulphate precipitation at 30%, 60% and
90% (F30, F60 and F90) were actively cytotoxic against HCT 116 cells with fraction 60
exhibiting the most potent cytotoxicity (IC50 0.84±0.05 μg/mL). However, the fractions
were not cytotoxic against normal lung fibroblast cell line (MRC-5). F60 that contained
the highest protein level of 459.745±17.55 μg/mL was separated into a few single bands
corresponding to a molecular weight ranging from 12 kDa to 64 kDa. Subsequently,
F60 was evaluated for cytotoxicity against HCT 116 and MRC-5 cells at 20 μg/mL
protein concentration. Results showed that the F60 of T. scopulosa when tested by
protein content was indeed cytotoxic against HCT 116 cells with IC50 value of
0.57±0.06 μg/mL protein but showed no cytotoxic effect against MRC-5 cells. The in vitro scratch wound assay was used to measure inhibition of cellular migration activity.
The HCT 116 cells treated with F60 slowed down the migration activity of the cells as
compared to the control group (non-treated cell line). The fraction was further purified
by anion exchange chromatography and collected fractions (P1, P2 and P3) were
retested for cytotoxic and anti-migration activities. Results showed that P2 and P3
fractions had better cytotoxic and anti-migration effects against HCT 116 cell line as
compared to P1. However, the cytotoxic effects of both fractions were lower than the
partially purified protein (F60). The P2 and P3 fractions were then further evaluated for
synergism study. Results showed that, the cytotoxic effect of the combine mixture at a
ratio of 1:1 was greater than F60. This suggests that P2 and P3 fractions have
synergistic cytotoxic effect. Both fractions were then subjected to protein analysis using
LCMS Q-TOF. Two proteins were characterized as pyranose-2-oxidase and carboxylic
ester hydrolase. Two uncharacterized proteins were also found. However, only
pyranose-2-oxidase has potential as anti-tumour effect against HCT 116 cancer cells.
As a conclusion, this study suggests that proteins from mycelial extract of T. scopulosa
could potentially to be used as an anti-tumour agent against colon cancer cells. |
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