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. 2011 Apr;18(4):602-18.
doi: 10.1038/cdd.2010.117. Epub 2010 Oct 8.

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Affiliations

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Y Tang et al. Cell Death Differ. 2011 Apr.

Abstract

Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.

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Figures

Figure 1
Figure 1
Induction of autophagy by propionate in colon cancer cells. (a) A bulk population of HCT116 cells with stable GFP-LC3 expression was treated with propionate (C3, 3 mM) for 36 h or was starved within serum-free medium (positive control) for 24 h. Formation of acidic vesicular organelles was monitored by LysoTracker DND-99 or MDC staining. Chloroquine (CQ) (5 μM) was added to increase the accumulation of autophagosomes. Representative images of cells were taken with a fluorescence microscope at × 600 magnification. Note the pink color in a composite panel showing the colocalization of pucutate GFP-LC3, LysoTracker DND-99 and MDC staining. (b) Quantification of the percentage of cells with more than eight GFP-LC3 puncta per cell is represented as ‘GFP-LC3vac cells (%)'. GFP-LC3 expressing cells were treated with indicated concentrations of propionate for 36 h (i). The percentage of cells with punctate GFP-LC3 caused by 3 mM propionate was monitored at different time points (ii). A minimum of 100 GFP-LC3 expressing cells were counted. *P<0.01; **P<0.001, compared with PBS control at the corresponsding time. HCT116 cells were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for 24 h. The cells were stained with LAMP-2 antibody (c) or p62 antibody (d). Representative images of cells were taken with an Olympus Fluoview confocal microscope using a × 60 oil immersion objective lens. (e) HCT116 cells with stable GFP-LC3 expression were subjected to propionate at different concentrations (i) or propionate (3 mM) for the indicated times (ii). The cell lysates were immunoblotted against several autophagy markers. Autophagy markers were also examined in the cells treated with propionate in the presence of CQ (iii). (f) Increased acidic vesicular organelle formation was reflected by elevated punctate LysoTracker staining and increased MDC incorporation in HCT116 cells after propionate treatment. HCT116 cells were subjected to propionate at different concentrations (i) or propionate (3 mM) for the indicated times (ii). The cells were stained with the dyes and subjected to flow cytometry analysis. The bars represent the mean±S.E. (n=4). *P<0.01; **P<0.001, compared with PBS control at the corresponsding time
Figure 2
Figure 2
Propionate-induced autophagy activation is associated with mTOR pathway inhibition. HCT116 cells were treated with propionate at different concentrations (top) for 15 h or with 3 mM propionate for the indicated time (bottom). The phosphorylation status of mTOR and its major downstream substrates, 4E-BP1 and p70S6 kinase, was detected by western blot. As for 4E-BP1 phosphorylation, γ stands for the hyper-phosphorylated form of 4E-BP1, whereas β and α stand for medium- and low-phosphorylated forms of 4E-BP1. The data represent as least three independent experiments
Figure 3
Figure 3
Induction of autophagy through inhibition of mTOR pathway is associated with AMPK pathway activation. HCT116 cells were treated with propionate at the indicated concentrations for 15 h or with 3 mM propionate for the indicated times. (a) The expression level of Phospho-Akt, PTEN and Class I/III PI3 Kinase were detected by western blot. (b) The phosphorylation status of AMPK α/β subunits and an AMPK downstream effector acetyl-coA carboxylase were detected by western blot. The data represent as least three independent experiments. (c) The expression level of AMPKα in the HCT116 cells, which are infected with AMPKα in the presence or absence of doxycycline (Dox) was examined. The direct downstream target of mTOR pathway (p70S6) and several autophagy markers were examined by western blot
Figure 4
Figure 4
Propionate-induced AMPK signaling activation is associated with mitochondrial defect-induced cellular ATP depletion and oxidative stress. (a) HCT116 cells were treated with propionate (3 mM) for the indicated times. The intracellular ATP level was measured using a CellTiter-Glo Luminescent Cell Viability Assay kit. The bars represent the mean±S.E. (n=4). (b) HCT116 cells were treated with propionate (0, 1, 3, 10 mM) for the indicated times and were stained against JC-1 for flow cytometry. There was a significant increase in the number of cells with lowered red fluorescence (FL-2 (R3)), indicating a change in the Δψ (i). The percentage of cells with polarized versus depolarized Δψ was presented as bar graph (ii). The bars represent the mean±S.E. (n=3). (c) Mitochondria were stained with MitoTracker Deep Red and MitoTracker Green FM simultaneously, and the staining intensities were determined by flow cytometry (i and ii), with the mean fluorescence presented in the bar graph (iii). The bars represent the mean±S.E. (n=3). (d) HCT116 cells were treated with propionate (3 mM) in the absence or presence of cyclosporin A (CsA, 2 μM) for 24 h. Then the cells were stained with calcein-AM, CoCl2 and MitoTracker Red. Representative images were obtained by fluorescence microscopy. Magnification, × 600. (e) Propionate treated cells were stained with MitoTracker Deep Red and calcein-AM. The staining intensities were analyzed by flow cytometry (i) and were presented as bar graph (ii). The bars represent the mean±S.E. (n=3). (f) HCT116 cells were treated with propionate (3 mM) in the presence or absence of N-acetylcysteine (NAC) (1 mM) for 24 h and the cells were stained with Carboxy-H2DCFDA for ROS detection and representative images were obtained by fluorescence microscopy. Magnification, × 100. (g) Carboxy-DCF fluorescence was analyzed by flow cytometry. (h) HCT116 cells were treated with propionate (0, 1, 3, 10 mM) for 48 h. The cells were stained with Carboxy-H2DCFDA and MitoTracker Deep Red. The fluorescence intensities were analyzed by flow cytometry. The percentage of cells with enhanced Carboxy-H2DCFDA and reduced MitoTracker Deep Red staining were presented as bar graph. The bars represent the mean±S.E. (n=3)
Figure 5
Figure 5
Defective mitochondria are targeted for autophagic degradation. (a) HCT116 cells with stable GFP-LC3 expression were treated with propionate (3 mM) or vehicle control (PBS) for 24 h. Mitochondria were stained by MitoTracker Deep Red, and representative images were obtained by fluorescence microscopy. Magnification, × 600. Arrows indicate the cells with autophagy induction by propionate. Note the smeared mitochondria staining in the cells with autophagy activation, which is reflected by the formation of GFP-LC3 dots. Yellow in the merged image indicates colocalization of GFP-LC3 with mitochondria. The treated cells were also stained with COXIV antibody (b) for mitophagy detection with a confocal microscope. HCT116 cells were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for 48 h. The cells were stained with COXIV/LAMP2 (c) or COXIV/p62 (d) antibodies. Representative images were obtained with an Olympus Fluoview confocal microscope using a × 60 oil immersion objective lens. HCT116 cells with stable GFP-LC3 expression were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for the indicated times. The GFP-LC3 and MitoTracker Deep Red fluorescence were analyzed by flow cytometry (ei) and the proportion of cells with increased GFP fluorescence and reduced MitoTracker Deep Red staining was presented as bar graph (eii). The bars represent the mean±S.E. (n=4). Arrows indicate the cells with autophagy induction by propionate
Figure 5
Figure 5
Defective mitochondria are targeted for autophagic degradation. (a) HCT116 cells with stable GFP-LC3 expression were treated with propionate (3 mM) or vehicle control (PBS) for 24 h. Mitochondria were stained by MitoTracker Deep Red, and representative images were obtained by fluorescence microscopy. Magnification, × 600. Arrows indicate the cells with autophagy induction by propionate. Note the smeared mitochondria staining in the cells with autophagy activation, which is reflected by the formation of GFP-LC3 dots. Yellow in the merged image indicates colocalization of GFP-LC3 with mitochondria. The treated cells were also stained with COXIV antibody (b) for mitophagy detection with a confocal microscope. HCT116 cells were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for 48 h. The cells were stained with COXIV/LAMP2 (c) or COXIV/p62 (d) antibodies. Representative images were obtained with an Olympus Fluoview confocal microscope using a × 60 oil immersion objective lens. HCT116 cells with stable GFP-LC3 expression were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for the indicated times. The GFP-LC3 and MitoTracker Deep Red fluorescence were analyzed by flow cytometry (ei) and the proportion of cells with increased GFP fluorescence and reduced MitoTracker Deep Red staining was presented as bar graph (eii). The bars represent the mean±S.E. (n=4). Arrows indicate the cells with autophagy induction by propionate
Figure 5
Figure 5
Defective mitochondria are targeted for autophagic degradation. (a) HCT116 cells with stable GFP-LC3 expression were treated with propionate (3 mM) or vehicle control (PBS) for 24 h. Mitochondria were stained by MitoTracker Deep Red, and representative images were obtained by fluorescence microscopy. Magnification, × 600. Arrows indicate the cells with autophagy induction by propionate. Note the smeared mitochondria staining in the cells with autophagy activation, which is reflected by the formation of GFP-LC3 dots. Yellow in the merged image indicates colocalization of GFP-LC3 with mitochondria. The treated cells were also stained with COXIV antibody (b) for mitophagy detection with a confocal microscope. HCT116 cells were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for 48 h. The cells were stained with COXIV/LAMP2 (c) or COXIV/p62 (d) antibodies. Representative images were obtained with an Olympus Fluoview confocal microscope using a × 60 oil immersion objective lens. HCT116 cells with stable GFP-LC3 expression were treated with propionate (3 mM) in the presence or absence of CQ (5 μM) for the indicated times. The GFP-LC3 and MitoTracker Deep Red fluorescence were analyzed by flow cytometry (ei) and the proportion of cells with increased GFP fluorescence and reduced MitoTracker Deep Red staining was presented as bar graph (eii). The bars represent the mean±S.E. (n=4). Arrows indicate the cells with autophagy induction by propionate
Figure 6
Figure 6
Rescue of cellular energy crisis through mitochondria biogenesis and alteration of glycogen and lipid metabolic pathways. (a) HCT116 cells were treated with propionate at different concentrations for 24 h (top) or with 3 mM propionate for the indicated times (bottom). The expression level of fatty acid synthase and GSK-3β was determined by western blot. The expression profile of genes regulating mitochondrial biogenesis in HCT116 cells after propionate treatment was evaluated by both semiquantitative reverse transcriptase PCR (RT-PCR) (b) and Real-time PCR (c). The bars represent the mean±S.E. (n=4). Nuclear gene mitochondrial transcription factor A (Tfam); mitochondrial transcription factor B (mtTFB); nuclear respiratory factors-1 and 2 (NRF-1 and -2); DNA polymerase gamma (Pol-γ); and peroxisome proliferator activated receptor-γ co-activator 1 (PGC-1). (d) The protein level of mitochondrial redox carriers' subunits was examined by western blot
Figure 7
Figure 7
Inhibition of autophagy potentiates propionate-induced apoptotic cell death. (a) HCT116 cells were treated with propionate (3 mM) for 36 h in the absence or presence of 3-MA (2 mM). The same treatment was applied to the cells infected with ATG5 shRNA. Acidic vesicular organelle formation was detected by LysoTracker Red DND-99 and MDC staining. Representative images are all taken at × 600 magnification with a florescence microscope. (b) Punctate GFP-LC3 marked autophagosome formation was quantified (i). Data were presented as ‘GFP-LC3vac cells (%)' (bi). *P< 0.05; **P<0.001, compared with a PBS control. Several autophagy markers were evaluated by western blot (ii). HCT116 cells pretreated with 3-MA (2 mM) or infected with ATG5 shRNA were subjected to propionate (3 mM) for the indicated times. The cell viability was measured by trypan blue exclusion-based cell staining (c), and the apoptotic cell death was evaluated by phosphatidylserine (PS)-based annexin V staining (d, g). (e) Western blot analyses of caspase-7 and caspase-3 cleavage in HCT116 cells treated with propionate (3 mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA and CQ) or AMPK inhibitor (Compound C). (f) HCT116 and SW480 cells were infected with ATG5 shRNA, and the protein knockdown was evaluated by western blot
Figure 7
Figure 7
Inhibition of autophagy potentiates propionate-induced apoptotic cell death. (a) HCT116 cells were treated with propionate (3 mM) for 36 h in the absence or presence of 3-MA (2 mM). The same treatment was applied to the cells infected with ATG5 shRNA. Acidic vesicular organelle formation was detected by LysoTracker Red DND-99 and MDC staining. Representative images are all taken at × 600 magnification with a florescence microscope. (b) Punctate GFP-LC3 marked autophagosome formation was quantified (i). Data were presented as ‘GFP-LC3vac cells (%)' (bi). *P< 0.05; **P<0.001, compared with a PBS control. Several autophagy markers were evaluated by western blot (ii). HCT116 cells pretreated with 3-MA (2 mM) or infected with ATG5 shRNA were subjected to propionate (3 mM) for the indicated times. The cell viability was measured by trypan blue exclusion-based cell staining (c), and the apoptotic cell death was evaluated by phosphatidylserine (PS)-based annexin V staining (d, g). (e) Western blot analyses of caspase-7 and caspase-3 cleavage in HCT116 cells treated with propionate (3 mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA and CQ) or AMPK inhibitor (Compound C). (f) HCT116 and SW480 cells were infected with ATG5 shRNA, and the protein knockdown was evaluated by western blot
Figure 7
Figure 7
Inhibition of autophagy potentiates propionate-induced apoptotic cell death. (a) HCT116 cells were treated with propionate (3 mM) for 36 h in the absence or presence of 3-MA (2 mM). The same treatment was applied to the cells infected with ATG5 shRNA. Acidic vesicular organelle formation was detected by LysoTracker Red DND-99 and MDC staining. Representative images are all taken at × 600 magnification with a florescence microscope. (b) Punctate GFP-LC3 marked autophagosome formation was quantified (i). Data were presented as ‘GFP-LC3vac cells (%)' (bi). *P< 0.05; **P<0.001, compared with a PBS control. Several autophagy markers were evaluated by western blot (ii). HCT116 cells pretreated with 3-MA (2 mM) or infected with ATG5 shRNA were subjected to propionate (3 mM) for the indicated times. The cell viability was measured by trypan blue exclusion-based cell staining (c), and the apoptotic cell death was evaluated by phosphatidylserine (PS)-based annexin V staining (d, g). (e) Western blot analyses of caspase-7 and caspase-3 cleavage in HCT116 cells treated with propionate (3 mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA and CQ) or AMPK inhibitor (Compound C). (f) HCT116 and SW480 cells were infected with ATG5 shRNA, and the protein knockdown was evaluated by western blot
Figure 7
Figure 7
Inhibition of autophagy potentiates propionate-induced apoptotic cell death. (a) HCT116 cells were treated with propionate (3 mM) for 36 h in the absence or presence of 3-MA (2 mM). The same treatment was applied to the cells infected with ATG5 shRNA. Acidic vesicular organelle formation was detected by LysoTracker Red DND-99 and MDC staining. Representative images are all taken at × 600 magnification with a florescence microscope. (b) Punctate GFP-LC3 marked autophagosome formation was quantified (i). Data were presented as ‘GFP-LC3vac cells (%)' (bi). *P< 0.05; **P<0.001, compared with a PBS control. Several autophagy markers were evaluated by western blot (ii). HCT116 cells pretreated with 3-MA (2 mM) or infected with ATG5 shRNA were subjected to propionate (3 mM) for the indicated times. The cell viability was measured by trypan blue exclusion-based cell staining (c), and the apoptotic cell death was evaluated by phosphatidylserine (PS)-based annexin V staining (d, g). (e) Western blot analyses of caspase-7 and caspase-3 cleavage in HCT116 cells treated with propionate (3 mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA and CQ) or AMPK inhibitor (Compound C). (f) HCT116 and SW480 cells were infected with ATG5 shRNA, and the protein knockdown was evaluated by western blot
Figure 7
Figure 7
Inhibition of autophagy potentiates propionate-induced apoptotic cell death. (a) HCT116 cells were treated with propionate (3 mM) for 36 h in the absence or presence of 3-MA (2 mM). The same treatment was applied to the cells infected with ATG5 shRNA. Acidic vesicular organelle formation was detected by LysoTracker Red DND-99 and MDC staining. Representative images are all taken at × 600 magnification with a florescence microscope. (b) Punctate GFP-LC3 marked autophagosome formation was quantified (i). Data were presented as ‘GFP-LC3vac cells (%)' (bi). *P< 0.05; **P<0.001, compared with a PBS control. Several autophagy markers were evaluated by western blot (ii). HCT116 cells pretreated with 3-MA (2 mM) or infected with ATG5 shRNA were subjected to propionate (3 mM) for the indicated times. The cell viability was measured by trypan blue exclusion-based cell staining (c), and the apoptotic cell death was evaluated by phosphatidylserine (PS)-based annexin V staining (d, g). (e) Western blot analyses of caspase-7 and caspase-3 cleavage in HCT116 cells treated with propionate (3 mM) for the indicated times in the absence or presence of autophagy inhibitors (3-MA and CQ) or AMPK inhibitor (Compound C). (f) HCT116 and SW480 cells were infected with ATG5 shRNA, and the protein knockdown was evaluated by western blot

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