l-Carnitine and Acetyl-l-Carnitine Induce Metabolism Alteration and Mitophagy-Related Cell Death in Colorectal Cancer Cells

<b>Background/Objectives</b>: Colorectal cancer (CRC) remains one of the most common and deadly malignancies worldwide, driven by metabolic reprogramming and mitochondrial dysfunction, which support tumor growth and progression. Several studies showed that nutrition is a contributing factor in the prevention and management of CRC. In this context, carnitines, amino acid derivatives abundant in food of animal origin, such as meat and milk, are crucial for mitochondrial function. Recently, l-carnitine and acetyl-l-carnitine have received particular attention due to their antioxidant, anti-inflammatory, and antitumor properties. However, to date, there is no conclusive evidence on the effects of l-carnitine and acetyl-l-carnitine in CRC or the underlying molecular mechanism. <b>Methods</b>: In this study, we investigated in HCT 116 and HT-29 CRC cells the effects of l-carnitine and acetyl-l-carnitine on mitochondrial homeostasis by XF HS Seahorse Bioanalyzer and cell death pathways by flow cytometry and western blot assays. <b>Results</b>: Data showed that l-carnitine and acetyl-l-carnitine reduced cell viability (<i>p</i> < 0.001), modulated cellular bioenergetics, and induced oxidative stress (<i>p</i> < 0.001). These phenomena promoted autophagic flux and the mitophagy process via PINK1 and Parkin modulation after 72 h of treatment. Of note, the combined treatment with l-carnitine and acetyl-l-carnitine showed a synergistic effect and enhanced the effect of single carnitines on tumor cell growth and metabolic dysfunction (<i>p</i> < 0.05). Moreover, exposure to l-carnitine and acetyl-l-carnitine promoted CRC cell apoptosis, suggesting a mechanism involving mitophagy-related cell death. These data were associated with increased SIRT4 expression levels (<i>p</i> < 0.01) and the activation of AMPK signaling (<i>p</i> < 0.01). <b>Conclusions</b>: Overall, the results, by supporting the importance of nutritional factors in CRC management, highlight l-carnitine and acetyl-l-carnitine as promising agents to target CRC metabolic vulnerabilities.