ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine

Abstract

δ-Valerobetaine (δVB) is a constitutive milk metabolite with antioxidant and anti-inflammatory activities. Here, we tested the antineoplastic properties of milk δVB on human colorectal cancer cells. CCD 841 CoN (non-tumorigenic), HT-29 (p53 mutant adenocarcinoma) and LoVo (APC/RAS mutant adenocarcinoma) cells were exposed to 3 kDa milk extract, δVB (2 mM) or milk+δVB up to 72 h. Results showed a time-and dose-dependent capability of δVB to inhibit cancer cell viability, with higher potency in LoVo cells. treatment with milk+δVB arrested cell cycle in G2/M and SubG1 phases by upregulating p21, cyclin A, cyclin B1 and p53 protein expressions. Noteworthy, δVB also increased necrosis (P < 0.01) and when used in combination with milk it improved its activity on live cell reduction (P < 0.05) and necrosis (P < 0.05). δVB-enriched milk activated caspase 3, caspase 9, Bax/ Bcl-2 apoptotic pathway and reactive oxygen species (ROS) production, whereas no effects on ROS generation were observed in CCD 841 CoN cells. The altered redox homeostasis induced by milk+δVB was accompanied by upregulation of sirtuin 6 (SIRT6). SIRT6 silencing by small interfering RnA blocked autophagy and apoptosis activated by milk+δVB, unveiling the role of this sirtuin in the RoS-mediated apoptotic LoVo cell death.

Synergistic Effect of Dietary Betaines on SIRT1-Mediated Apoptosis in Human Oral Squamous Cell Carcinoma Cal 27

“…Cell proliferation and viability were firstly evaluated by testing milk, an aliment of fundamental importance for humans due to the remarkable content of substances with high nutritional and functional values, including betaines. Noteworthy, recent data indicate that 3-kDa milk extracts show higher efficiency in inhibiting colon cancer cell viability compared to betaine alone [8][9][10]. Milk extracts were tested on FaDu, UM-SCC-17A, and Cal 27 cells.…”

“…A growing body of evidence shows that betaines are important human nutrients that participate in the methionine cycle acting as methyl donors. Compounds belonging to this class, such as glycine betaine, stachydrine, ergothioneine, γ-butyrobetaine (γBB), and δ-valerobetaine (δVB) show protective effects against cardiovascular disease and cancer [3][4][5][6][7][8].…”

“…δVB (N,N,N-trimethyl-5-aminovaleric acid) is produced by rumen microbiota from the transformation of N ε -trimethyllysine and is likely degraded by the intestinal host microbiota via γ-butyrobetaine hydroxylase enzyme, thus suggesting a possible metabolic connection between δVB and γBB [9,10]. In vitro biological activities of δVB include inhibition of β-oxidation of fatty acids in mice cardiomyocytes, anti-inflammatory effects in endothelia damaged by hyperglycemia, and cytotoxic effects in human colon adenocarcinoma cells [6,8,12]. In vivo and clinical evidence indicate that δVB inversely correlates with metabolic syndrome markers after the intake of whole grain-rich diets [13,14].…”

“…In vivo and clinical evidence indicate that δVB inversely correlates with metabolic syndrome markers after the intake of whole grain-rich diets [13,14]. The molecular mechanism through which δVB triggers colon cancer cell autophagy and apoptosis involves altered cellular redox homeostasis and sirtuin 6 (SIRT6) modulation [8].…”

“…The present study aimed at evaluating the possible anticancer effects of milk betaines in oral squamous cell carcinomas [25]. In particular, in light of recent evidence showing that pure δVB displays more pronounced health-promoting properties when in combination with other milk components [6,8], particular emphasis was placed on the evaluation of its possible synergistic/additive effects with γBB, given the remarkable structural resemblance of these two betaines.…”