Characterisation of metabolic changes in colorectal cancer cells of different stages / Hazwani Mohd Yusof

Accurate diagnosis and staging of colorectal cancer (CRC) lead to better prognosis.
Identification of molecular biomarkers may prove useful, but there is a lack of data on
the molecular pathophysiology of CRC progression. Characterisation of metabolic
changes in CRC cells may provide useful tools for study of potential therapeutic drug
actions. This study was aimed to characterise the metabolites profiles in CRC cells of
different stages and the metabolic pathways affected. In this study, normal colon cell
lines; CCD 840 CoN and CRC cells lines of different stages; SW 1116 (stage A), HT
29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D) were
used. The global metabolomics profiling was performed using liquid chromatographymass
spectrometry (LC/MS). Mass Profiler Professional and Metaboanalyst software
were used for statistical and pathway analysis. METLIN database was used for
identification of metabolites. MS/MS was used for validation of detected metabolites.
Validation of the affected metabolic pathways in CRC was performed in which
expression of SLC6A14 (amino acid transporter), riboflavin kinase and FAD synthetase
(enzymes in riboflavin metabolism pathway) were determined using Western blot. Lmethionine
and FAD levels, cells viability and rate of apoptosis activity were
determined with and without the presence of alpha-methyltryptophan, and lumichrome
which are inhibitors of SLC6A14, and riboflavin uptake, respectively. The results
showed that there were differential metabolites identified in CRC cells of different
stages, which could be used as potential metabolite biomarkers for staging. (R)-1-O-
[b-D-glucopyranosyl-(1->6)-b-D-glucopyranoside]-1,3-octanediol, lauroyl
diethanolamide, lysoPE(0:0/18:1(11Z)), lysoPE(0:0/22:5(4Z,7Z,10Z,13Z,16Z)),
phosphocholine, S-(formylmethyl)glutathione, and N-acetyl-DL-methionine were the
most important differential metabolites identified in stage A. In stage B, 1,2,4-
nonadecanetriol, dodecanoylcarnitine, hericene B, l-hexanoylcarnitine, pyroglutamic
acid and S-furanopetasitin were the differential metabolites while in stage C, the
differential metabolites were bis-γ-glutamylcystine, hippuric acid, lumichrome,
lysoPE(16:1(9Z)/0:0), PE(22:5(7Z,10Z,13Z,16Z,19Z)/15:0), and purine. FAD,
methyhiomethyl butyrate, muzanzagenim, 3b-hydroxy-5-cholenoic acid, ATP, Lglutamate
and lysoPE(0:0/20:0) were the differential metabolites identified in CRC
cells stage D. The results showed that L-methionine and riboflavin metabolisms were
the most prominent pathways affected in CRC cells as the cancer advances. The results
also showed that increased level of methionine observed in metabolomics study is
probably due to increased expression of SLC6A14. However, expressions of riboflavin
kinase and FAD synthetase in CRC were similar to normal cells and hence could not
account for the increased level of FMN and FAD as observed in the metabolomics study.
The results also showed that metabolites profiles identified in intracellular and secreted
CRC cells were different. In conclusion, this study identified several novel differential
metabolites which can be used as potential biomarkers for staging. In this study, for the
first time, profiles of the intracellular as well as the secreted metabolites were
determined which provides a more complete picture of the metabolic derangement that
occurs in the development and progression of CRC. The affected metabolic pathways
identified in this study may lead to further understanding of the pathophysiology of
CRC and hence, its application for treatment strategies.