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.

Figure 1

Inhibition of colorectal adenocarcinoma cell viability by milk-δVB. HT-29 and LoVo cells were treated with (a) increasing concentrations of δVB (up to 2 mM) or (b) increasing volumes of milk (up to 40% v/v) for 72 h. (c) Cell viability was determined after treatment with milk (40% v/v) enriched with serial concentrations of δVB (0.1, 0.5, 1, 1.5, 1.8 and 2 mM). After 72 h incubation, the IC₅₀ was reached at the concentration of δVB 1.972 mM. IC₅₀ values were calculated using GraphPad. (d) Colon cells were incubated for 72 h with 40% v/v milk, δVB (2 mM), or milk supplemented with δVB (milk + δVB). Control cells were grown in medium containing the same volume (% v/v) of HBSS-10 mM Hepes. Cell growth inhibition was assessed using MTT assay. Values represent the mean ± SD of three independent experiments. *P < 0.05 vs Ctr, **P < 0.01 vs Ctr, ^†P < 0.05 vs milk, ^††P < 0.01 vs milk, ^#P < 0.05 vs δVB, °P < 0.01 vs δVB ^●P <  0.05 vs 0.1 mM δVB, and ^●●P  <  0.01 vs 0.1 mM δVB.

Figure 2

LoVo cell cycle alteration induced by milk-δVB. (a,b) Representative cell cycle analysis and average of LoVo cell cycle distribution. LoVo cells were treated with milk (40% v/v), δVB (2 mM), milk enriched with δVB (milk + δVB), or HBSS-10 mM Hepes (40% v/v) (Ctr) for 72 h. Cell cycle distribution was assessed by flow cytometry collecting PI fluorescence as FL3-A (linear scale) and analysis by ModFIT software (Verity Software House, USA, Becton Dickinson) (www.vsh.com/products/mflt/). For each sample at least 10.000 events were analyzed. (c–f) Representative images of Western blotting analysis of cyclin A, cyclin B1, p21 and p53 in LoVo cells after 72 h of treatment. Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. Protein determination was performed with Image J software 1.52n version and quantified using α-tubulin or β-actin. Values are expressed as arbitrary units (AU). *P < 0.05 vs Ctr; **P < 0.01 vs Ctr; ^†P < 0.05 vs milk, ^††P < 0.01 vs milk, ^#P < 0.05 vs δVB, °P < 0.01 vs δVB. The full-length blots are showed in the supplementary information (Fig. S2).

Figure 3

Autophagy induced by milk-δVB. (a) Representative images of green detection reagent imaged by confocal laser microscopy after 72 h in LoVo cells incubated with 40% v/v buffalo milk (milk), δVB (2 mM) and milk supplemented with δVB (milk + δVB). Scale bar, 50 μm. (b) As for flow cytometry analysis, at least 10.000 events were acquired in log mode. For the quantitative evaluation of green detection reagent, FlowJo V10 software was used to calculate median fluorescence intensities. (c) Bar graph of the fluorescence intensity values of green detection reagent-labeled vesicles and expressed as fold change of the control (Ctr). Analysis was carried out by determinations in triplicate of n = 3 experiments **P < 0.01 vs Ctr, ^†P < 0.05 vs milk, ^#P < 0.05 vs δVB. (d–g) Protein expression levels of LC3BII/ LC3BI, p62, Atg7 and Beclin 1, measured by Western blot in control LoVo cells (Ctr) or cells treated for 72 h with milk (40% v/v), δVB (2 mM) and milk+δVB. Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. Protein content was calculated with Image J software 1.52n version and expressed as arbitrary units (AU). *P < 0.05 vs Ctr; **P < 0.01 vs Ctr; ^††P < 0.01 vs milk. The full-length blots are showed in the supplementary information (Fig. S3).

Figure 4

Upregulation of SIRT6 protein expression. (a,b) SIRT6 protein expression levels expressed as arbitrary units (AU) with *P < 0.05 vs Ctr, ^†P < 0.05 vs milk. Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. (c,d) Representative confocal images of SIRT6 expression (red) and α-actin (green) in control cells (Ctr) and cells exposed to milk+ δVB for 72 h. Nuclei were counterstained with DAPI (blue). Scale Bar, insert = 200 μm; Enlarged = 50 μm.

Figure 5

Apoptotic mechanism. (a,b) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry. Data are expressed as mean ± SD of n = 4 experiments. At least 10.000 events were acquired. (c–h) Protein expression levels of caspase 3, caspase 9, PARP, Bax and Bcl-2 from LoVo cells treated for 72 h with milk (40% v/v), δVB (2 mM), milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. Analysis of densitometric intensity was calculated with Image J 1.52n version software. Arbitrary units of protein expression (AU) were quantified using α-tubulin or β-actin. Antibodies against Bax, Bcl-2 and SIRT6 (reported in Fig. 4) were blotted on the same filter and quantified by using the same loading control (α-tubulin). (i,j) Flow cytometric analysis and representative dot plots of annexin V-FITC and PI double staining LoVo cells treated with caspase 9 inhibitor Z-LEHD-FMK (40 μM) or chloroquine (50 μM). Data are expressed as mean ± SD of n = 3 experiments. At least 10.000 events were acquired. (k) Cleaved caspase 3 protein expression level from LoVo cells treated for 72 h with milk+δVB, Z-LEHD-FMK + milk+δVB or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, lane 3 = Z-LEHD-FMK + milk+δVB. (l) Protein expression levels of caspase 8 in LoVo cells after 72 h of treatment with milk (40% v/v), δVB (2 mM), milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1=Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. *P < 0.05 vs Ctr, **P < 0.01 vs Ctr, ^†P < 0.05 vs milk, ^††P < 0.01 vs milk, ⁺P < 0.05 vs milk+δVB, ⁺⁺P < 0.01 vs milk+δVB. The full-length blots are included in the supplementary information (Fig. S4).

Figure 6

Evaluation of redox homeostasis. (a,b) Flow cytometry analysis of intracellular ROS levels after incubation of LoVo with 10 µM DCFH-DA was performed as described under Method section. (c) Time-line (12, 24, 48, and 72 h) of extracellular H₂O₂ production was determined with Amplex Red H₂O₂/peroxidase assay. Data are shown as mean ± SD of three independent experiments. (d) Time-dependent mitochondrial superoxide levels assessed in LoVo cells by MitoSOX-confocal microscopy analysis after 12, 24, 48, and 72 h of treatment with milk (40% v/v), δVB (2 mM), milk + δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). (e) Representative confocal images of MitoSOX (red) and phalloidin (green) in control cells (Ctr), milk (40% v/v), δVB (2 mM) or milk + δVB for 72 h. Nuclei were counterstained with DAPI. Scale bar, 50μm. (f,g) MitoSOX-based flow cytometry in cells treated with vehicle (Ctr), milk (40% v/v), δVB (2 mM) or milk + δVB, for 72 h in serum-free medium. Results are expressed as mean fluorescence intensity (MFI). (h,i) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry in LoVo cells treated for 72 h with HBSS-10 mM Hepes (40% v/v) (Ctr) milk+ δVB, or NAC + milk+ δVB. Data are expressed as mean ± SD of n = 3 experiments. At least 10.000 events were acquired. (j) The effect of 72 h treatment with δVB (2 mM) on mitochondrial function was determined by assaying cytochrome oxidase activity in whole LoVo lysates. ^*P < 0.05 vs Ctr, **P < 0.01 vs Ctr, ^††P < 0.01 vs milk, °P < 0.01 vs δVB, ^§P < 0.001 vs Ctr; ^§§P < 0.0001 vs Ctr, ^¶P < 0.001 vs milk, ⁺P < 0.05 vs milk+δVB, ⁺⁺P < 0.01 vs milk+δVB.

Figure 7

Suppression of ROS production. (a,b) Flow cytometry analysis of green detection reagent staining following incubation with δVB (2 mM) and δVB + NAC. **P < 0.01 vs Ctr, °P < 0.01 vs δVB. (c, d) Arbitrary units (AU) of SIRT6 protein expression in cells treated with δVB and milk+δVB in the presence of NAC. Lane 1 = Ctr, lane 2 = NAC + δVB, lane 3 = NAC + milk + δVB, *P < 0.05 vs Ctr. (e) SIRT6 protein expression in cells treated with RNAifectin transfection reagent (Vehicle), scramble siRNA (50 nM) (Scramble), SIRT6-siRNA (50 nM) or medium only (Ctr). Lane 1 = Ctr, lane 2 = Vehicle, lane 3 = Scramble, lane 4 = SIRT6-siRNA. (f,g) Flow cytometric autophagic activity performed by green detection reagent in LoVo cells treated for 72 h with milk+δVB, SIRT6-siRNA+milk+ δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Rapamycin (1 μM) was used as positive control. FlowJo V10 software was used to calculate median fluorescence intensities. (h) Protein expression levels of LC3BI/ LC3BII from LoVo cells treated for 72 h with milk + δVB, SIRT6-siRNA + milk + δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, lane 3 = SIRT6-siRNA + milk + δVB. (i,j) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry in LoVo cells after 72 h of treatment with milk+δVB, SIRT6-siRNA+milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Data are expressed as mean ± SD of n = 4 experiments. At least 10.000 events were acquired. (k) Protein expression levels of PARP from LoVo cells treated for 72 h with milk + δVB, SIRT6-siRNA + milk + δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, Lane 3 = SIRT6 siRNA + milk + δVB. *P < 0.05 vs Ctr, **P < 0.01 vs Ctr, ⁺P < 0.05 vs milk + δVB, ⁺⁺P < 0.01 vs milk + δVB.