ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk \u03b4-Valerobetaine

Nunzia D’Onofrio,Assunta Lombardi,Francesca Borrelli,Gianluca Neglia,Elisa Martino,Maria Luisa Balestrieri,Giuseppe Campanile,Ferdinando Fiorino,Nunzio Antonio Cacciola

doi:10.1038/s41598-020-65865-6

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.

Highlights

In human and mouse heart tissues, δVB regulates energy metabolism by inhibiting fatty acids β-oxidation with an effect similar to that of meldonium, a drug known to improve cardiac mitochondrial function after ischemia[10,11]
Despite the δVB content in buffalo milk is lower than 2 mM, 40% (v/v) of milk extract induced cytotoxicity in HT-29 and LoVo cell lines
The present study provides the first evidence of the cytotoxic effect of δVB in HT-29 and LoVo colon cancer cells, with a highest potency displayed in LoVo cells

Summary

Introduction

In human and mouse heart tissues, δVB regulates energy metabolism by inhibiting fatty acids β-oxidation with an effect similar to that of meldonium, a drug known to improve cardiac mitochondrial function after ischemia[10,11]. Dietary phenolics are the most promising as possible future adjuvant in CRC management, the gap between preclinical and clinical research still exists since the amounts needed to exert some effects largely exceed common dietary doses In this contest, exploring the anticancer properties of compounds occurring in highly consumed foods, such as milk, could represent a promising avenue in the search of naturally occurring biomolecules. The present study was designed to investigate the anti-neoplastic activity of a milk extract enriched with δVB in human colorectal adenocarcinoma To this end, this study was conducted on HT-29 and LoVo cell lines showing APC/RAS (LoVo) and p53 (HT-29) mutations, known to be critical in the development of CRC via increasing adenomatous dysplasia

Methods

Results

Conclusion