Evaluation of L-Methioninase as a Targeted Anticancer Agent in Colorectal Cancer and Renal Cell Carcinoma

Abstract

The employment of L-Methioninase (L-Met), an enzyme degrading L-methionine, as a possible anticancer therapy has been provided that it selectively destroys cancer cells, which require methionine to grow. Many tumor tissues have a limited capacity for methionine production and depend on exogenous supplies of this amino acid. Therefore, they could be selectively attacked by methioninase-based treatment. The present study was carried out to investigate the effect of L-Met on cancer cells, especially colorectal cancer (HCT-116) and renal cell carcinoma (A498), and its potential as a therapeutic agent. Various techniques, such as ammonium sulfate precipitation, dialysis, ion-exchange chromatography, and gel filtration chromatography, were employed in this study for enzyme purification of L-Met. Cytotoxicity testing was performed against HCT-116 and A498 cells in the range of 25-200 µg/mL concentration by the MTT assay for viable cell quantification, Total Nuclear Intensity (TNI), and Cell Membrane Permeability (CMP). The statistical analysis was done using one-way ANOVA and Dunnett's multiple comparisons test to compare multiple groups. The optimal temperature for enzyme activity was 37° C, with pH 7 offering the best enzyme stability. Further investigation into incubation time revealed that a 48-hour period maximized enzyme yield. The enzyme was purified using a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration, achieving a 4.6-fold increase in purity. Characterization of the purified enzyme revealed a molecular weight of approximately 55 kDa, with optimal activity at pH 7 and 37°C. The enzyme demonstrated strong potential for therapeutic applications, showing dose-dependent cytotoxicity against colorectal cancer (HCT-116) and renal cancer (A498) cell lines, with significant inhibitory effects at concentrations as low as 25 µg/mL for colorectal cancer cells. The study highlights the potential of P. aeruginosa-derived L-Met showed strong activity in colorectal cancer, while activity in renal cell carcinoma was lower or inconclusive.