Molecular analysis and metabolite profiling of colorectal cancer cell line (HCT 116) associated with telomerase by RNAi-mediated silencing and inhibition via berberine / Muhammad Azizan Samad

Muhammad Azizan , Samad (2024) Molecular analysis and metabolite profiling of colorectal cancer cell line (HCT 116) associated with telomerase by RNAi-mediated silencing and inhibition via berberine / Muhammad Azizan Samad. PhD thesis, Universiti Malaya.

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Abstract

Colorectal cancer (CRC) is the most common cancer among male and female, globally, which is associated with the increment of telomerase expression and activity. It was hypothesised that telomerase was expressed during S phase, thus, the cell cycle distribution of colorectal cancer cell line (HCT 116) was analysed at 24, 48 and 72 hours of culture. It was revealed that 48 hours had the highest percentage of S phase and relative telomerase activity (RTA). Screening of selected telomerase inhibitors on HCT 116 at 48 hours showed that berberine was the most effective (lowest IC50 value: 10.30 ± 0.89 μg/mL) compared to boldine (IC50 value: 37.87 ± 3.12 μg/mL) and silymarin (IC50 value >200 μg/mL). Scaling up of berberine treatment in T75 flask resulted in IC50 of 10.54 μg/mL which was used in subsequent experiment. There were three strategies in this study i.e., berberine treatment at different time-points, berberine treatment at 48 hours, and berberine and RNAi treatment at 48 hours. Time and berberine treatment affected RTA of HCT 116, which could be reflected on the shifting of metabolic profile of HCT 116 with time and treatment as visualised by the Partial Least Squares-Discriminant Analysis (PLS-DA). Further analyses exhibited that berberine treatment caused G0/G1 phase arrest at 48 hours due to high cyclin D1 (CCND1) and low cyclin-dependent kinase 4 (CDK4) protein and mRNA levels, simultaneous downregulation of human telomerase reverse transcriptase (TERT) mRNA and human telomerase RNA component (TERC) levels, as well as a decrease in the TERT protein level and RTA. The effect of berberine treatment on the cell cycle was time-dependent as it resulted in a delayed cell cycle, and doubling time by 2.18-fold. RTA level was significantly decreased, and telomere erosion followed suit. Increased hydrogen peroxide concentration [H2O2] in berberine-treated HCT 116 was due to the decrement of catalase (CAT) mRNA as well as the increment of super oxide dismutase 1 and 2 (SOD1 and SOD2) mRNA levels which subsequently caused damage to the nuclei. Principal Component Analysis (PCA) showed polar separation of untreated and berberine-treated HCT 116. The significance of metabolic pathway discovery was improved by the addition of affected genes in the Joint-Pathway analysis. The mechanism of berberine effects was validated by RNAi targeting TERT and/or TERC. Knockdown of TERT decreased TERC. Knockdown of TERT and/or TERC elevated CCND1 as well as decreased CAT, xanthine oxidase (XO) mRNA, and XO protein levels. The repression of TERC was the most likely to be responsible for the upregulation of CCND1 protein and SOD2 mRNA levels. Berberine treatment showed the largest downregulation and upregulation of CDK4/CDK4 and SOD1 levels, respectively indicating additive effects of berberine. TERC knockdown resulted in the highest [H2O2] in HCT 116. The downregulation of TERT and/or TERC caused a G0/G1 phase arrest, which was primarily due to the overexpression of CCND1 protein, in addition to decreased S and G2/M phases. The reduction in HCT 116 cell size was mostly caused by TERT downregulation. Relative telomere length (RTL) was later reduced as a result of TERT, TERT and/or TERC downregulation which decreased RTA. HCT 116 with decreased RTA was closely clustered in the PCA indicating similarity of the metabolic profile. Glutamic acid was the most consistent metabolite across the three strategies, positively correlated with RTA, and potentially involved in oxidative stress mechanism.

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