Colorectal Cancer Apoptosis Induced by Dietary δ-Valerobetaine Involves PINK1/Parkin Dependent-Mitophagy and SIRT3

Abstract

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.

Keywords: PINK1/Parkin; colon cancer; mitochondrial dysfunction; mitophagy.

Figure 1

Effects of δVB on SW620 and SW480 cell proliferation. (a,b) CCD 841 CoN, (c,d) SW620 and (e,f) SW480 cells were treated with increasing concentrations of δVB (0–3 mM) for 24, 48 and 72 h. The IC50 in SW620 and SW480 was determined at 72 h incubation with 1.5 mM δVB and represented as red dotted arrows. Control cells were grown in medium containing the same volume of HBSS-10 mM Hepes. Cell proliferation inhibition was assessed using Cell Counting Kit-8 assay (Donjindo Molecular Technologies, Tokyo, Japan). Values represent the mean ± SD or the % of control of four independent experiments. * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr.

Figure 2

Effects of δVB cell cycle. Representative cell cycle analysis and average of (a,b) SW620 and (c,d) SW480 cell cycle distribution. Cells were treated with δVB (1.5 mM) for 24, 48 and 72 h. Cell cycle distribution was evaluated by flow cytometry collecting PI fluorescence as FL3-A (linear scale) and analyzed by ModFIT software (Verity Software House, Becton Dickinson, Topsham, ME, USA). For each sample, at least 10,000 events were acquired. Representative full-length blots of Western blotting analysis of cyclin A (e–h), cyclin B1 (i–l) and cyclin D (m–p) in SW620 and SW480 cells, respectively. Lane 1 = 0 h; lane 2= 24 h, lane 3 = 48 h, lane 4 = 72 h. Before lane 1; molecular weight markers (G266, Applied Biological Materials Inc., Richmond, BC, Canada). Protein expression was calculated, after normalization with internal control (α-tubulin or GAPDH), with ImageJ software and results expressed as arbitrary units (AU). * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr.

Figure 3

Effects of δVB on caspase-3 activation. Representative dot plots and analyses of caspase-3 activation by reporting annexin CF 594 and caspase-3 substrate on (a,b) SW620 and (c,d) SW480 cells treated with δVB (1.5 mM) at 24, 48 and 72 h. Cell population was assessed by flow cytometry and results expressed as % of live or apoptotic population with mean ± SD of n = 3 experiments. At least 10,000 events were acquired. Representative full-length blots of Western blotting analysis of caspase-3 in (e,f) SW620 and (g,h) SW480 cells. Lane 1 = 0 h; lane 2 = 24 h; lane 3 = 48 h; lane 4 = 72 h. Before lane 1, molecular weight markers (G266, Applied Biological Materials Inc., Richmond, BC, Canada). Protein expression was calculated, after normalization with α-tubulin as internal control, with ImageJ software and results expressed as arbitrary units (AU). Representative images and analysis of Nucview 488 and Mitoview 633 staining of (i,j) SW620 and (k,l) SW480 cells analyzed by fluorescence microscopy. Results expressed as red and green fluorescence intensity value. Scale bar: 100 μm. * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr; # p< 0.0001 vs. Ctr.

Figure 4

δVB effects on mitochondrial status. Representative images, dot plots and analysis of mitochondrial membrane potential assessed using JC-1 probe, which allows us to detect membrane depolarization through a concomitant decrease in the aggregate signal (red) and accumulation of monomeric species (green), of (a–c) SW620 and (d–f) SW480 cells. Cells were treated with δVB (1.5 mM) for 24, 48 and 72 h. Results, expressed as green fluorescence intensity, were reported as % of dead cells. Scale bar: 200 μm. Representative immunoblotting images and analyses of (g–j) COX-IV, (k–n) SIRT3 and (o–t) Bax/Bcl-2 protein levels in SW620 and SW480 cells. Lane 1 = 0 h; lane 2 = 24 h; lane 3 = 48 h; lane 4 = 72 h. Before lane 1, molecular weight markers (G266, Applied Biological Materials Inc., Richmond, BC, Canada). Protein expression was calculated, after normalization with internal control (α-tubulin or GAPDH), with ImageJ software and results expressed as arbitrary units (AU). * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr; # p< 0.0001 vs. Ctr.

Figure 5

δVB effects on mitophagy. Representative confocal images of mitophagy (red) and lysosome (green) dyes and analysis of (a,b) SW620 and (c,d) SW480 cells treated with δVB (1.5 mM) for 24, 48 and 72 h. Nuclei were counterstained with DAPI (blue). Fluorescence intensity analysis was performed by ImageJ software and expressed as arbitrary fluorescence units (AFU) of dye signal ± SD of n = 3 replicates. Scale bar: 5 μm. Representative immunoblotting images and analyses of (e–h) PINK1, (i–l) Parkin and (m–p) LC3B protein levels in SW620 and SW480 cells. Lane 1 = 0 h; lane 2 = 24 h; lane 3 = 48 h; lane 4 = 72 h. Before lane 1, molecular weight markers (G266, Applied Biological Materials Inc., Richmond, BC, Canada). Protein expression was calculated, after normalization with internal control (α-tubulin or GAPDH), with ImageJ software and results expressed as arbitrary units (AU). * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr; # p < 0.0001 vs. Ctr.

Figure 6

Effects of δVB on colorectal adenocarcinoma cell death. Representative dot plots and analyses of annexin V-FITC and PI-stained (a,b) SW620 and (c,d) SW480 cells treated with 1.5 mM δVB for 24, 48 and 72 h. Cell viability/death was assessed by flow cytometry where at least 10,000 events were acquired. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. Data are expressed as mean ± SD of n = 3 experiments. Representative full-length blots of Western blotting analysis of procaspase-9 (e–h) and PARP (i–l) in SW620 and SW480 cells. Lane 1 = 0 h; lane 2 = 24 h; lane 3 = 48 h; lane 4 = 72 h. Before lane 1, molecular weight markers (G266, Applied Biological Materials Inc., Richmond, BC, Canada). Protein expression was calculated, after normalization with internal control (α-tubulin or GAPDH), with ImageJ software and results expressed as arbitrary units (AU). * p < 0.05 vs. Ctr; ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr.

Figure 7

Mitophagy inhibition reduced δVB-mediated apoptosis. Representative images and analysis of Mitoview 633 (red) and Nucview 488 (green) staining of (a,b) SW620 and (c,d) SW480 cells. Cells were treated with mitophagy inhibitor 3-MA, δVB (1.5 mM) or combined 3-MA + δVB (1.5 mM) for 72 h, as described under Materials and Methods. Results, expressed as red and green fluorescence intensity, were reported as fluorescence intensity value. Scale bar: 100 μm. Representative dot plots and analyses of annexin V-FITC and PI-stained (e,f) SW620 and (g,h) SW480 cells. Cell viability/death was assessed by flow cytometry where at least 10,000 events were acquired. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. Data are expressed as mean ± SD of n = 3 experiments. Representative full-length blots of Western blotting analysis of procaspase-9 (i–l), procaspase-3 (m–p) and (q–t) SIRT3 protein expression levels in SW620 and SW480 cells. Lane 1 = Ctr; lane 2 = 3-MA; lane 3 = δVB; lane 4 = 3-MA + δVB. Before lane 1, molecular weight markers (G266, Applied Biological Materials Inc., Richmond, BC, Canada). Protein expression was calculated, after normalization with α-tubulin as internal control, with ImageJ software and results expressed as arbitrary units (AU). ** p < 0.01 vs. Ctr; *** p < 0.001 vs. Ctr; # p < 0.0001 vs. Ctr; § p < 0.05 vs. δVB; §§ p < 0.01 vs. δVB; † p < 0.001 vs. δVB.

Figure 8

Proposed cellular events leading to colorectal cancer apoptosis induced by δVB. Evidence from the present study suggest that mitochondrial dysfunction and downregulation of SIRT3 induced by δVB fueled the mechanism of mitophagy, which in turn triggered cell death by apoptosis, as evidenced by the inhibition of mitophagy with 3-AM which resulted in the attenuation of intrinsic apoptosis.