An Integrative Approach Using Molecular and Metabolomic Studies Reveals the Connection of Glutamic Acid with Telomerase and Oxidative Stress in Berberine-Treated Colorectal Cancer Cell Line HCT 116.

Abstract

Colorectal cancer (CRC) is one of the common deadliest cancers worldwide. In Malaysia, the numbers of new CRC cases were horrific and worrisome. Telomerase is both prognostic indicator and predictor of carcinogenesis in CRC patients. Berberine, a telomerase inhibitor, was used in clinical trials and metabolomic studies; however, the association of telomerase with metabolites and metabolic pathways was not fully understood. Colorectal cancer cell line HCT 116 was cultured and treated with 10.54 µg/mL berberine. The cells were harvested at different time points to conduct subsequent analyses. The methods used in this research were real time-polymerase chain reaction (RT-PCR) to assess RNA expressions; Western blot to determine protein levels; TELOTAGGG Telomerase PCR ELISA to determine relative telomerase activity (RTA); 4',6-diamidino-2-phenylindole (DAPI) staining to determine percentage of nuclei damage; fluorescence microscopy for cell area; spectrophotometric potassium iodide assay for intracellular hydrogen peroxide concentration [H₂O₂]; as well as liquid chromatography mass spectrometry (LCMS) and tandem mass spectrometry (MS/MS) to investigate the intracellular metabolites. Partial least square-discriminant analysis (PLS-DA) score plot exhibited an improved separation compared to principal component analysis (PCA) when metabolomic data analysis of HCT 116 at various berberine treatment durations was conducted. Time and berberine treatment had an impact on RTA in HCT 116. RTA was discovered to be positively and negatively correlated to 14 and 2 metabolites, respectively. Glutamic acid was consistently found correlated to RTA. Other four metabolites, i.e., MG(14:0), [3-[hydroxy(phosphonooxy)phosphoryl]oxyphenyl] phosphono hydrogen phosphate), (3S,6S)-6-[[(3S,6R)-6-[(2S,3S,5S)-2,5-diiodo-4-methoxy-6-methyloxan-3-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, and 1-[5-O-(5'-adenylyloxyphosphonyl)-beta-D-ribofuranosyl]-5-amino-1H-imidazole-4-carboxamide, were newly discovered to be connected to RTA in HCT 116. Four metabolic pathways that majorly affected shared glutamic acid and glutamine. Nitrogen metabolism, D-glutamine and D-glutamate metabolism, glyoxylate and dicarboxylate metabolism, and aminoacyl-tRNA biosynthesis have been identified to be associated with RTA. Network analyses hinted that glutamic acid was also associated with oxidative stress mechanism. The multiple roles glutamic acid acted in diverse metabolic pathways and interaction networks emphasized the importance of glutamic acid in HCT 116 regarding RTA. This research establishes the association between RTA and several chosen RNAs, proteins, metabolites, and oxidative stress mechanisms, consequential in morphological alteration in HCT 116, to expand the knowledge of the intricate biological relationships and telomerase mechanism in CRC.