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Emerging strategies to improve healthy aging include dietary interventions as a tool to promote health benefits and reduce the incidence of aging-related comorbidities. The health benefits of milk are also linked to its richness in betaines and short-chain acylcarnitines, which act synergistically in conferring anticancer, anti-inflammatory, and antioxidant properties. Whey, despite being a dairy by-product, still has a considerable content of bioactive betaines and acylcarnitines. Here, we investigated the anticancer properties of whey from Mediterranean water buffalo ( Bubalus bubalis ) milk by testing its antiproliferative effects in colorectal cancer (CRC) cells HT-29, HCT 116, LoVo and SW480. Results indicated that treatment with whey for 72 h inhibited cell proliferation ( p &lt; 0.001), induced cell cycle arrest, and apoptosis via caspase-3 activation, and modulated cell metabolism by limiting glucose uptake and interfering with mitochondrial energy metabolism with the highest effects observed in HT-29 and HCT 116 cells. At molecular level, these effects were accompanied by upregulation of sirtuin 3 (SIRT3) ( p &lt; 0.01) and peroxisome proliferator-activated receptor (PPAR)-γ expression ( p &lt; 0.001), and downregulation of lactate dehydrogenase A (LDHA) ( p &lt; 0.01), sterol regulatory-element binding protein 1 (SREBP1) ( p &lt; 0.05), and PPAR-α ( p &lt; 0.01). Transient SIRT3 gene silencing blocked the effects of whey on the LDHA, PPAR-γ, and PPAR-α protein expressions ( p &lt; 0.01) suggesting that the whey capacity of perturbating the metabolic homeostasis in CRC cell lines is mediated by SIRT3.

SIRT3 and Metabolic Reprogramming Mediate the Antiproliferative Effects of Whey in Human Colon Cancer Cells