Apoptosis (2007) 12:573–591 DOI 10.1007/s10495-006-0010-3
Acidic extracellular pH shifts colorectal cancer cell death from apoptosis to necrosis upon exposure to propionate and acetate, major end-products of the human probiotic propionibacteria Anna¨ıg Lan · Dominique Lagadic-Gossmann · Christophe Lemaire · Catherine Brenner · Gw´ena¨el Jan
Published online: 29 December 2006 C Springer Science + Business Media, LLC 2006
Abstract The human probiotic Propionibacterium freudenreichii kills colorectal adenocarcinoma cells through apoptosis in vitro via its metabolites, the short chain fatty acids (SCFA), acetate and propionate. However, the precise mechanisms, the kinetics of cellular events and the impact of environmental factors such as pH remained to be specified. For the first time, this study demonstrates a major impact of a shift in extracellular pH on the mode of propionibacterial SCFA-induced cell death of HT-29 cells, in the pH range 5.5 to 7.5 prevailing within the colon. Propionibacterial SCFA triggered apoptosis in the pH range 6.0 to 7.5, a lethal process lasting more than 96 h. Indeed at pH 7.5, SCFA induced cell cycle arrest in the G2/M phase, followed by a sequence of cellular events characteristic of apoptosis. By contrast, at pH 5.5, the same SCFA triggered a more rapid and drastic lethal process in less than
This work has been supported by a grant from CRITT sant´e Bretagne. A. Lan · G. Jan () UMR 1253 INRA Agrocampus, Science & Technologie du Lait et de l’Oeuf, 65, rue de St Brieuc, 35042 Rennes cedex, France e-mail:
[email protected] A. Lan Laboratoires Standa, 68 rue R. Kaskoreff, 14050 Caen cedex 4, France D. Lagadic-Gossmann INSERM U620, Universit´e Rennes1, Facult´e de Pharmacie, 2 av Prof L´eon Bernard, 35043 Rennes cedex, France C. Lemaire · C. Brenner CNRS UMR 8159, LGBC, Universit´e de Versailles, 45 av. des Etats Unis, 78035 Versailles
24 h. This was characterised by sudden mitochondrial depolarisation, inner membrane permeabilisation, drastic depletion in ATP levels and ROS accumulation, suggesting death by necrosis. Thus, in digestive cancer prophylaxis, the observed pH-mediated switch between apoptosis and necrosis has to be taken into account in strategies involving SCFA production by propionibacteria to kill colon cancer cells. Keywords Cell death . Colorectal cancer . Short-chain fatty acids Abbreviations AV annexinV-FITC m mitochondrial transmembrane potential DHE dihydroethidium DiOC6(3) dihexyloxacarbocyanine iodide Eth Ethidium Etop. etoposide FDA fluorescein diacetate IM inner membrane LND lonidamine superoxide anion O2 .− Men Menadione PARP poly-ADP-ribose polymerase PCD programmed cell death PI propidium iodide PTPC permeability transition pore complex RIP receptor-interacting protein ROS reactive oxygen species SCFA short-chain fatty acid TNF tumor necrosis factor TRAIL TNF α related apoptosis inducing ligand
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Introduction Colorectal cancer constitutes a major concern in developed countries and is linked to environmental factors such as lifestyle, particularly diet, as supported by epidemiology studies [1, 2]. Therefore, a growing interest focuses on probiotics as cancer-preventing or reducing agents. In this context, we have been interested in the ability of food-grade dairy propionibacteria to kill cancer cells. They possess a peculiar fermentative metabolism which leads to the production of carbon dioxide and short-chain fatty acids (SCFA). This characteristic may constitute a key probiotic potential, as it has been previously shown that the SCFA butyrate is a potent inducer of apoptosis in colon cancer cells in vitro [3]. Indeed, treatment with physiological concentrations of butyrate elicits pleitropic effects on cell cycle [4] and gene expression [5–7], leading to cell death through apoptosis. Regarding the other SCFA, acetate and propionate, which are the major end-products of dairy propionibacteria, were shown to kill two human adenocarcinoma cell lines by apoptosis during co-cultures with the dairy species Propionibacterium freudenreichii and Propionibacterium acidipropionici [8]. The active concentrations of acetate and propionate (in the 10 to 30 mM range) were compatible with physiological concentrations. Indeed, acetate, propionate and butyrate are present in the colon at concentrations around 60, 25 and 10 mM, respectively [3, 9]. These concentrations can be modulated by food components and depend on an equilibrium between microbial synthesis and colonic absorption [9]. Moreover, human faeces with high populations of dairy propionibacteria were shown to contain enhanced concentrations of SCFA compared with faeces without propionibacteria [10]. We thus hypothesised that these bacteria may be useful probiotics in the context of colon cancer prevention. Several modes of cell death can occur in mammalian cells including apoptosis and necrosis [11]. Apoptosis or type I programmed cell death (PCD), is mediated by a family of cystein proteases, the caspases, and requires intracellular ATP to proceed. Two main apoptotic pathways have been described: the extrinsic pathway, which is mediated by activation of death receptor and caspase-8 [12], and the intrinsic pathway, in which mitochondria play the role of integrator/coordinator [13, 14]. The apoptotic phenotype is characterized by cell shrinkage, nuclear condensation, DNA fragmentation and cell dismantling in apoptotic bodies [14]. The other death pathways are less well-defined, but caspases would not be involved in these pathways [15]. Necrosis has long been considered as an accidental and unregulated death, known to be associated with an important drop of intracellular ATP and characterized by abrupt plasma membrane rupture with consequent release of intracellular components. However, several studies have also indicated the existence Springer
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of necrosis-like PCD [16–18]. Under some circumstances, a shift between the different types of cell death can occur. For instance, an acidic extracellular pH has been recently shown to induce necrosis, instead of apoptosis, in human adenocarcinoma cell line HT-29 treated with TRAIL (TNF α related apoptosis inducing ligand [19]). In this lethal process, the kinase RIP (receptor-interacting protein) might be a key mediator (Meurette et al., unpublished data). Therefore, the cell microenvironment might be determinant for the type of cell death induced by toxic stimuli. In this context, as pH can vary in a 5.5 to 7.5 pH range within the lumen of human colon [20, 21] and as acidic extracellular pH is a common feature of solid tumors [22, 23], the impact of this factor on the cell death induced by propionibacterial SCFA has to be considered. Thus, the aim of the present study was to identify the SCFA-induced cellular and molecular mechanisms of cell death in the pH range of 5.5–7.5 using the human colon adenocarcinoma cell line HT-29.
Material and methods Chemicals When not specified, chemicals were purchased from Sigma (Sigma-Aldrich, Saint-Quentin Fallavier, France). Cell cultures The HT-29 human colon adenocarcinoma cell line was obtained from ATCC (American Type Culture Collection, Rockville, MD) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat inactivated foetal calf serum (PAN Biotech, D. Dutscher, Brumath, France), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin and 50 mg/ml streptomycin at 37◦ C under 5% CO2 . Bacterial cultures Propionibacterium freudenreichii subsp freudenreichii strain TL142 (UMR STLO, INRA-Agrocampus, Rennes, France) was cultivated as described previously [8]. Cell treatments For co-cultures, DMEM without antibiotics was inoculated (1%) with bacterial cultures heat-inactivated (80◦ C, 15 min) or alive. To study the impact of propionibacterial SCFA at different pH, DMEM supplemented with 30 mM propionate and 15 mM acetate, as previously quantified in the co-culture supernatant [8], was prepared. To avoid pH variations, 15 mM HEPES were added to DMEM supplemented or not with
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SCFA and pH was adjusted to 5.5, 6.0, 6.5 and 7.0 with 5N HCl.
a gate for calculating the percentage of cells producing O2 .− . Experiments were conducted in triplicate.
Viability assay
Detection of mitochondrial membrane potential (m)
One day after seeding onto flat-bottom-24-wells culture plates, non-confluent HT-29 cells were treated as described above. Two wells were kept as controls and received unmodified DMEM. After 0, 6, 24, 48 and 96 h of contact, cells were trypsinized and stained in a 0.4% buffered saline trypan blue solution, to determine surviving cells percentage.
Perturbations in m were monitored by flow cytometry using 3,3 dihexyloxacarbocyanine iodide (DiOC6(3), Molecular Probes). The cells were washed with PBS, incubated with a DiOC6(3) solution (5 nM in PBS, 20 min, 37◦ C, in dark). The decoupling agent FCCP (50 µM, 20 min) served as m loss positive control. Fluorescence emission was analysed by flow cytometry. Superposition of control and FCCP histograms allowed to define a gate for calculating the percentage of cells with a DiOC6(3) decreased incorporation. Three independent experiments were carried out.
Cytometry analysis Cell cycle analysis
Detection of inner membrane permeabilisation Following SCFA-treatment at both pH, adherent cells were harvested by trypsinization and pooled with floating ones, washed and incubated overnight with cold 70% ethanol in phosphate-buffered saline (PBS) prior to staining with a propidium iodide solution (40 µg/ml PI) in PBS with 50 µg/ml RNase A (30 min, 37◦ C, in dark). 10,000 cells were then sorted in a FACSCalibur flow cytometer (BD Biosciences, Le Pont de Claix, France) and analysis was performed using Cell Quest software (BD Biosciences). WinMDI software (version 2.8; http//:facs.Scripps.edu/software.html) was used to generate DNA content frequency histograms and quantify the amount of cells in the individual cell cycle phases including subG0/G1 population. Three independent experiments were carried out.
Inner membrane permeabilisation via the permeability transition pore complex (PTPC) opening was assessed as previously described [25]. Briefly, cells were pre-incubated for 15 min at 37◦ C with 1 µM Calcein-AM (C-3100, Molecular Probes) and 1 mM CoCl2 in Hanks’ balanced salt solution (without phenol red and without sodium bicarbonate; Invitrogen, Cergy-Pontoise, France) supplemented with 1 mM HEPES, pH 7.3. For treatment with SCFA, Hanks’ solution was replaced by complete DMEM at pH 5.5 or 7.5. After the indicated time, calcein fluorescence was analysed by flow cytometry. Lonidamine served as a positive inductor of PTPC opening [26, 27]. Three independent experiments were carried out.
Cell viability and apoptosis analysis by annexin V staining
Transmission electron microscopy
Cells were treated and harvested as described above. After washes in PBS, 0.5.106 cells were stained according to manufacturer’s instructions (annexinV-FITC kit, BD Biosciences). Cells were analyzed by flow cytometry. Cell sorting and data processing were carried out on 10,000 cells using the same device and software as above. Four independent experiments were performed.
Cells were grown and treated as described above in 25 cm2 flasks, rinsed with PBS and fixed overnight at 4◦ C in 0.1 M sodium cacodylate buffer, pH 7.2, containing 2% glutaraldehyde. Fixed cells were scraped, rinsed and stored at 4◦ C in cacodylate buffer containing 0.2 M sucrose. Cells were then postfixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate, and 2% uranyl acetate in water before gradual dehydration in ethanol (30% to 100%) end embedding in Epon. Thin sections (70 nm) were collected onto 200 mesh cooper grids and counterstained with lead citrate before examination with a Philips CM12 transmission electron microscope.
Reactive oxygen species detection Production of ROS was assessed fluorometrically using dihydroethidium (DHE, Molecular Probes, Eugene, OR) to detect superoxide anion (O2 .− ) as previously described [24]. Approximately 0.5· 106 cells were treated and harvested as described above, washed in PBS, and labelled with 5 µM DHE (15 min, 37◦ C, in dark). Fluorescence emission of oxidized DHE was analysed by flow cytometry. Menadione (100 µM, 15 min) was used as positive control. Superposition of control and menadione histograms allowed defining
Fluorescence microscopy To study cell viability and integrity of cytoplasmic membrane [28], cells were washed in PBS and double stained with fluorescein diacetate (FDA, 10 µg· mL−1 ) and PI (20 µg.mL−1 ) for 30 min. After washing, stained cells were then examSpringer
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ined with a fluorescence microscope (Olympus BX51) using the appropriate filters. For nuclear fluorescence microscopy observation, cells were cultured on glass coverslips before treatment. Following treatment and fixation with 4% paraformaldehyde in PBS, cell nuclei were stained with the Hoechst H33342 (2 µM, 15 min, 37◦ C). For immunostaining, fixed HT-29 cells were blocked and permeabilised with blocking solution (PBS, 2% Bovine Serum Albumin, 0.2% Triton X-100). Then, cells were incubated with sheep antibody raised against Cytochrome c (C 5723, Sigma) or with a rabbit antibody anti-Bax (N-20, Santa Cruz Biotechnology, Tebu, Le Perray en Yvelines, France) in blocking solution. After washes, coverslips were incubated with anti-sheep or anti-rabbit IgG FITC-linked secondary antibody diluted in blocking solution in dark. Counterstaining with Hoechst 33342 was then performed as described above and stained cells were then examined with fluorescence microscope. As an alternative, for mitochondrial localisation, r HT-29 cells were pre-stained with 100 nM Mitotracker Red 580 30 min in DMEM, fixed and permeabilised according to the manufacturer’s instructions, immunostained with anti-Bax antibody and counterstained with Hoechst 33342. ATP quantitation r ATP was quantitated with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Charbonni`eres, France) according to manufacturer’s instructions. Following SCFA treatments, 100 µL of CellTiter-Glo Assay were added to each well of flat-bottom-96-well culture. Following a 10 min incubation at room temperature, luminescence was determined using a plate spectrofluorimeter-luminometer (Spectra Max 2, Molecular devices, Sunnyvale, CA). The intracellular ATP level was calculated as the amount in exposed cells versus control cells.
Western blotting Following SCFA-treatment, adherent cells were harvested by trypsinization, pooled with floating ones, washed with PBS and sonicated in lysis buffer. Total proteins (30 µg) of each sample were resolved on 14% polyacrylamide gels. After transfer onto PVDF membrane (Amersham, Orsay, France) and incubation in blocking solution (PBS, 0.1% Tween 20, 5% non-fat milk), membranes were incubated with primary antibodies diluted in blocking solution (overnight, 4◦ C). Antibodies directed against caspase-8 (12F5, Alexis Biochemicals, Coger, Paris, France), caspase-9 (C 7729, Sigma), active form of caspase-3 (C 8487, Sigma), caspase-7 (p20, Santa Cruz Biotechnology, Tebu, Le Perray en Yvelines, France), RIP1 (BD Biosciences), PARP (Roche Diagnostics, Meylan, France), Cytochrome c (C 5723, Sigma), Bax (N20, Santa Cruz Biotechnology), VDAC (rabbit anti-VDAC Springer
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custom polyclonal serum, Eurogentec, Seraing, Belgium) and Actin (A 2066, Sigma) were used. After washes, blots were incubated with anti-mouse, anti-rabbit or anti-goat IgG horseradish peroxidase-linked secondary antibody in blocking solution. After additional washes, the specific bands were detected by chemiluminescence using the ECL detection system according to the manufacturer’s instructions on a Storm Phosphorimager (Amersham). Subcellular fractionation After treatment, cells were washed with PBS-EDTA, trypsined, and resuspended in mitochondrial buffer containing 10 mM KCl, 0.15 mM MgCl2 , 10 mM Tris-HCl pH 7.6, 0.4 mM PMSF and 10 µM cytochalasin B. The cell suspensions were incubated for 30 min and homogenized on ice at 4◦ C with 200 strokes each in 250 mM sucrose, using a Dounce glass homogenizer. The first centrifugation was at 800 g for 3 min at 4◦ C to yield nuclei and unbroken cells as a pellet. The supernatant was centrifuged at 6,800 g for 10 min at 4◦ C and the mitochondrial pellet was resuspended in mitochondrial buffer. The cytosolic fraction was obtained as follows: cells were resuspended in Cell Free System buffer (200 mM mannitol, 68 mM sucrose, 2 mM MgCl2 , 2 mM NaCl, 2.5 mM KH2 PO4 , 0.5 mM EGTA, 5 mM pyruvate, 1 mM DTT, 0.1 mM PMSF and 10 mM HEPES-NaOH pH 7.4) [29], subjected to 5 cycles of freezing/defrosting and centrifuged (150,000 g for 60 min at 4◦ C). The supernatant was kept. All fractions were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE, 30 µg per lane), as described above. Statistical analysis Results are expressed as the mean ± standard deviation (s.d.). Significance was determined by Student’s t test for paired samples. The significance is shown as follows: ∗ P < 0.05.
Results pH regulates the cell death of human colon adenocarcinoma HT-29 cells induced by propionibacterial SCFA In order to specify the cytotoxic potential of propionibacterial metabolites on colon cancer cells, we co-cultured the dairy species P. freudenreichii subsp. freudenreichii strain TL142 with HT-29 cells. During co-culture, bacterial growth led to a limited (0 to 6 h) and then massive (24 to 48 h) cell death of the HT-29 cells (Fig. 1(A)) concomitant with the SCFA acetate and propionate production. As a control, killed propionibacteria had no effect on cell viability while SCFA
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Percent of viable cells
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Fig. 1 Extracellular pH modulates cell death mediated by dairy propionibacteria metabolites. (A) Effect of propionibacteria or their metabolites on HT-29 colon cancer cells. HT-29 cells were co-cultured with P. freudenreichii TL142, either alive () or heat-killed (), with SCFA (acetate and propionate, at the concentrations found in P. freudenreichii supernatants) (), or left untreated (). (B) Impact of pH on cell death mediated by SCFA. HT-29 cells were treated with SCFA in buffered DMEM adjusted to pH 5.5 (), 6.0 ( • ), 6.5 (), or 7.0 (). Cell viability was determined at different times of treatment by trypan blue exclusion test. Values are represented as the proportion of live cells as compared with untreated cells analysed at the same time. (C) Effect of SCFA on HT-29 cell layers. Microscopy photographies (× 100 magnification) of the cell layers were taken after treatment in the presence ( + ) or in the absence (−) of propionibacterial SCFA, at pH 5.5 or 7.5. One representative of three experiments is shown
at the concentrations found in propionibacterial supernatant (15 mM and 30 mM respectively [8]) were clearly cytotoxic. To investigate the impact of pH on this lethal effect, this last treatment was repeated in a 5.5 to 7.5 pH range. The SCFAinduced cell death time course was similar in the 6.0 to 7.5 pH range (Fig. 1(B)), but much faster at pH 5.5. Indeed, at pH 5.5 in the presence of SCFA, cell death reached 88% and 97% at 6 h and 24 h, respectively. As a control, cells treated at pH 5.5 in the absence of SCFA underwent a much limited cell death reaching 20.7% and 44.3% at 6 h and 24 h, respectively, while no cell death was observed in the 6.0 to 7.0 pH range in the absence of SCFA (data not shown). By contrast, 96 h of SCFA treatment at pH 7.5 were necessary to reach 83% cell death. Acidic pH therefore potentiated the effects of SCFA. Accordingly, modifications of cell morphology and decrease in the number of attached cells upon exposure during 24 h to SCFA were more drastic at pH 5.5 than at pH 7.5 (Fig. 1(C)). pH 5.5 alone killed cells without comparable effect on cell morphology and attachment. As these data suggested differences in the type of cell death induced at the two pH, the following sets of experiments were carried out during 24 h at pH 5.5 and during 96 h at pH 7.5. SCFA at pH 7.5 induce cell cycle arrest The impact of propionibacterial SCFA on HT-29 cell cycle distribution was studied at pH 7.5 and 5.5 by flow cytometry measurement of DNA content of cells stained with propidium iodide (PI). Treatment with SCFA at pH 7.5 caused an accumulation of cells in G2/M phase from 12 h of treatment (39.4 ± 4.5%) with a maximum of 53.0 ± 15.9% at 24 h, compared to control cells (22.7 ± 6.7%) (Fig. 2(A)). Concomitant with cell cycle arrest, proliferation inhibition was also observed, as shown by the decreased proportion of cells in S phase. By contrast, no effect on cell cycle repartition was seen at pH 5.5, compared to control cells, independently of the treatment time (Fig. 2(B)). Cells in subG1 phase, indicative of an apoptotic process, appeared from 24 h and then increased significantly from 48 h of treatment at pH 7.5 (Fig. 2(C)). Such hypodiploid cells were never observed when cells were treated with SCFA at acidic pH whatever the time of treatment. SCFA induce pH-dependent differential cell death processes Apoptosis versus necrosis induction In order to analyse the kinetics and the type of cell death, we analysed SCFA-treated cells by flow cytometry after double labelling with annexinV-FITC (AV) and PI (Fig. 3). A quantitative analysis, based on 4 independent experiments, evidenced distinct mechanisms. Indeed, cells treated with Springer
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Fig. 2 SCFA produced by propionibacteria trigger cell cycle arrest at pH 7.5 but not 5.5. (A and B) Effect of SCFA on the repartition of cycle phases in HT-29 cells. DNA content of HT-29 cells treated with propionibacterial SCFA, either at pH 7.5 (A) or 5.5 (B) was analysed by flow cytometry. This analysis was performed until 100% of cell death was reached. The distribution of cell cycle phases of control untreated cells, both at pH 7.5 and pH 5.5, remained unchanged during the whole experiment. Presented values correspond to the proportion of each subset of cells (sub-G1, G0/G1, S, G2/M), within the total cell population, for each treatment. ∗ P < 0.05, treated versus control, Student’s t test. Data represent the mean (n = 3); bars ± s.d. (C) Impact of pH on the occurrence of sub-G1 cells. Representative histograms corresponding to analysis of HT-29 cells treated with SCFA at pH 7.5 or pH 5.5 are shown. The vertical bar indicates the position of cells with sub-G1 DNA content, indicative of apoptosis. The percentage of the population of cells containing less than 2n of DNA in each condition is indicated
SCFA at pH 7.5 underwent modifications characteristic of apoptosis, including first AV positive staining, indicating phosphatidylserine exposure, and then both AV and PI positive staining, indicating a later loss of membrane integrity (Fig. 3(A) and (C)). In the presence of SCFA at pH 7.5, the
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percentage of apoptotic cells increased in a time-dependent manner to reach 65.7 ± 9.8% after 96 h of treatment (Fig. 3(A)). At this stage, 38.5 ± 2.8% of the cells were late apoptotic (AV + /PI + ), 27.2 ± 9.5% early apoptotic (AV + /PI−) and 23.8 ± 11.0% viable (AV−/PI−), consis-
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Fig. 3 SCFA induce different patterns of cell death depending on pH. FACS analysis of annexin V-FITC (AV) binding to HT-29 cells was performed after counterstaining with propidium iodide (PI). (A and B) Quantitative analysis of independent experiments of AV/PI staining of HT-29 cells treated with SCFA at pH 7.5 (A) or 5.5 (B). Presented values correspond to the proportion of each subset of cells (AV-PI−, AV-PI + , AV + PI−, AV + PI + ), within the total cell population, for each treatment. Each value is given as the mean (n = 4) ± s.d. ∗ P < 0.05, treated versus control, Student’s t test. (C) Representative experiment of AV/PI staining of HT-29 cells treated with SCFA at pH 7.5 or pH 5.5
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tent with the cell death kinetics described above (Fig. 1). By contrast, cells treated with SCFA at pH 5.5 underwent a much more rapid and drastic permeabilisation towards PI, indicating a rapid loss of membrane integrity, prior to AV positive staining, and appeared as necrotic (Fig. 3(B) and (C)). The percentage of necrotic cells swiftly increased. Indeed, after 6 h of treatment, 76.6% of the cells were necrotic (PI positive) whereas pH 5.5 alone during the same time had
no effect. Almost all cells were killed after 12 h of treatment with SCFA at pH 5.5.
Fig. 4 SCFA induce different cell morphology depending on pH. Transmission electron micrographs of HT-29 cells treated in the absence (A and D) or in the presence (B and C) of propionibacterial SCFA. Cells were treated at pH 7.5 (a and b) or pH 5.5 (c and d) during 6 h (Bc and Bd), 12 h (Cc and Cd), 48 h (Ba and Bb) or 60 h (Ca and Cb). Early and late apoptotic stages are observed with chromatin condensation and
fragmentation (Ba and Ca). Swollen mitochondria are enlarged (Bb and Cb). Alteration of the chromatin without fragmentation is observed at pH 5.5 (Bc and Dc). Rupture of the plasma membrane is observed (Bd and Cd). H2 O2 (15 mM, 6 h, Da and Db) was used as necrosis control. No effect on cellular morphology was observed in control cells left untreated during 6h at pH 5.5 (Ac and Ad)
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Cell morphology To investigate the morphological features of cell death upon exposure to the different treatments, HT-29 cells were analysed at different stages by transmission electron microscopy
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(Fig. 4). Proliferating control HT-29 cells displayed the characteristic morphology of cultured intestine epithelial cells. The nucleoplasmic ratio was high, the cytoplasmic membrane displayed the typical aspect corresponding to microvilli of the intestine epithelium (Fig. 4(Aa)). Nuclei contained finely granular and regularly dispersed chromatin. Mitochondria were numerous, principally located in the perinuclear space, and displayed a homogenous morphology (Fig. 4(Ab)). After 48 h of treatment with SCFA at pH 7.5, HT-29 cells showed unequivocal signs of apoptosis. Cells shrank and became rounded (Fig. 4(Ba)). Chromatin condensation and margination, near to the nuclear membrane, was observed. Some mitochondria showed a swollen matrix (Fig. 4(Bb)). After 60 h of treatment, cells were more shrunk, the nuclear chromatin condensation was more pronounced and fragmentation started; moreover the membrane integrity was lost (Fig. 4(Ca)). Most of the mitochondria were swollen and distorted (Fig. 4(Cb)). At pH 5.5, control cells exposed to this pH in the absence of SCFA showed no morphological characteristic of cell death, neither apoptosis nor necrosis (Fig. 4(Ac)). Treatment with SCFA at pH 5.5 for 6 h (Fig. 4(Bc) and 4(Bd)) and for 12 h (Fig. 4(Cc) and 4(Cd)), leading to a much more rapid loss in viability, triggered distinct morphological changes. Vacuolisation of the cytoplasm and
Fig. 5 SCFA induce differential changes in chromatin and cell integrity depending on pH. (A and B) HT-29 cells were stained with Hoechst 33342 to visualize nucleus morphology using fluorescence microscopy (magnification, × 800). (A) Arrows indicate chromatin margination or condensation (b), nuclear fragmentation (c) and formation of apoptotic bodies (d and e). (B) Chromatin clumping without fragmentation is
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swollen mitochondria were not observed (Fig. 4(Bc)). The chromatin appeared clumped, but without marginalisation (Fig. 4(Cc)). By contrast with the other treatments, early loss of the cytoplasmic membrane integrity occurred from 6 h, leading to a leak of the cellular content (Fig. 4(Bb)). Similar morphological characteristics were observed after 6 h of treatment with H2 O2 (15 mM), used as a positive control of necrosis induction (Fig. 4(Da) and 4(Db)). Chromatin organization and plasma membrane integrity Nucleus morphology of HT-29 cells incubated in the presence of SCFA at pH 5.5 or 7.5 was analysed using Hoechst 33342. After exposure to SCFA at pH 7.5 during 24 h, cells with the characteristic condensed chromatin structure of apoptotic nuclei were observed (Fig. 5(Ab)). After 48 h, fragmented nuclei appeared (Fig. 5(Ac)), followed by the appearance of apoptotic bodies increasing between 72 and 96 h of treatment (Fig. 5(Ad) and (Ae)). During cell death induced by SCFA at pH 5.5, none of these nuclear apoptotic features was observed (Fig. 5(B)) in accordance with the morphology of necrotic cells observed in Fig. 4. Moreover, double staining with fluorescein diacetate and PI revealed membrane blebbing of SCFA-treated cells at pH 7.5 during 24 h without
observed at pH 5.5. (C) FDA/PI staining evidences typical apoptotic signs at the level of plasma membrane at pH 7.5 but not 5.5. At pH 7.5, cells appeared viable with sites of membrane blebbing (a). At pH 5.5, loss of plasma membrane integrity was evidenced by PI positive staining (b)
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DNA staining by PI, indicative of plasma membrane integrity and early apoptotic features (Fig. 5(Ca)). By contrast, cells treated during 24 h with SCFA at pH 5.5 appeared only redstained without any nuclear apoptotic events (e.g. chromatin fragmentation), indicative of a loss of plasma membrane permeability characteristic of necrosis (Fig. 5(Cb)).
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We tested the SCFA effects on the production of superoxide anion (O2 .− ) using the fluoroprobe dihydroethidium (DHE). As shown in Fig. 6, production of O2 − in SCFA-treated HT29 cells increased with time whatever extracellular pH. Indeed, a significant increase (3.5-fold) was observed in SCFA
treated cells compared to untreated counterparts after 60 h of treatment at pH 7.5 (Fig. 6(A)). This effect was more drastic (7-fold) when cells were treated with SCFA at pH 5.5 during 12 h, nearing the value of positive control (Menadione, Men.) (Fig. 6(B)). Thus, SCFA clearly triggered an increased amount of ROS in HT-29 cells, such an accumulation being much more rapid and dramatic at pH 5.5. Changes in mitochondrial membrane potential (m), were studied by flow cytometry after mitochondria staining with DiOC6(3) (Fig. 7). In both SCFA treatments, a decreased incorporation of DiOC6(3), indicative of a m dissipation and hence mitochondrial membrane depolarisation, was observed. This m loss was progressive when cells were treated at pH 7.5. Cells with a decreased m
Fig. 6 SCFA induce a more rapid superoxide anion generation at pH 5.5 compared to pH 7.5. Representative experiments and quantitative analysis of independent experiments (n = 3) of ROS detection in HT-29 cells treated with SCFA at pH 7.5 (A) or at pH 5.5 (B). Cells were stained with dihydroethidium and analysed by flow cytometry.
The prooxidant Menadione (Men., 100 µM, 15 min, pH 7.5) was used as positive control. Values are represented as a proportion of cells with increased ROS (increase of fluorescence intensity), within the total cell population, for each treatment. Each value is presented as the mean ± s.d. ∗ P < 0.05, treated versus control, Student’s t Test
SCFA induce mitochondrial alterations
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Fig. 7 SCFA induce a more rapid mitochondria permeabilisation at pH 5.5 compared to pH 7.5. SCFA induce mitochondial membrane potential (m) dissipation of HT-29 cells treated with SCFA at pH 7.5 (A) or at pH 5.5 (B). A representative experiment of m dissipation at each pH is shown. The decoupling FCCP (50 µM, 20 min, pH 7.5) was used as positive control. Quantitative analysis of m loss of independent experiments (n = 3) are shown. Values are represented as a proportion of cells with decreased m (decrease of fluorescence in-
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tensity), within the total cell population, for each treatment. Each value is given as the mean ± s.d. ∗ P < 0.05, treated versus control, Student’s t Test. (C) Inner membrane permeabilization via the permeability transition pore complex opening is analysed at both pH by flow cytometry using a method based on calcein fluorescence quenching. Lonidamine (LND, 15 h, 250 µM, pH 7.5) was used as a PTPC-dependent apoptosis inducer. Values are represented as a proportion of calcein negative cells, within the total cell population, for each treatment
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significantly appeared from 24 h, reaching a maximum at 60 h, concomitantly with a progressive entry into apoptosis (Fig. 7(A)). By contrast, an abrupt and early m dissipation, as soon as 4 h of treatment, was observed at pH 5.5, reaching a maximum at 12 h (Fig. 7(B)). This drastic loss of mitochondrial integrity was concomitant with massive cell death (Fig. 3). Interestingly, using the fluorescent dye calcein (MM 809 Da), that allows to monitor the inner membrane (IM) permeabilisation in cellula via its release from the mitochondrial matrix and its quenching by cobalt ions [25], we observed that SCFA favored the opening of large inner membrane channels such as the permeability transition pore complex (PTPC) whatever the pH tested (Fig. 7(C), (D)), as compared to the positive control Lonidamine (LND). Nevertheless, as observed for the loss of m, an earlier and more drastic IM permeabilisation process was induced at pH 5.5. Indeed, as soon as 2 h after SCFA treatment at pH 5.5 about 50% of cells were calcein− (Fig. 7(D)), while at pH 7.5 this percentage was reached only after 36 h of incubation (Fig. 7(C)). Moreover, whatever the pH studied, the comparison between the percentage of calcein− versus DiOC6(3)− cells suggests that IM permeabilisation precedes and thus appears, at least in part, responsible for the loss of m by promoting the mitochondrial permeability transition. ATP depletion is early and drastic at pH 5.5 Treatment of HT-29 cells with SCFA triggered a reduction in the cellular content of ATP at both pH (Fig. 8). However, this depletion was sudden and drastic at pH 5.5. By contrast, it occurred in a much more delayed and progressive manner at pH 7.5. Exposure to SCFA treatment at pH 7.5 thus resulted in a progressive reduction of ATP levels, compatible with apoptotic pathway, from 86.8 ± 6.7% to 28.1 ± 5.2% of the control level after 24 to 96 h of treatment with SCFA (Fig. 8(A)). Conversely, treatment with SCFA at pH 5.5 caused a marked decrease in ATP content to 30.5 ± 6.5% of the control level after 30 min of treatment, and to 5.5 ± 4.5% after 6 h of treatment (Fig. 8(B)), in association with a massive loss of viability as described in Fig. 1. Induction of hallmarks of the mitochondrial apoptotic pathway Bax and Cytochrome c relocation. Translocation of Bax from cytosol to mitochondria constitutes a key cellular event of the apoptotic program that precedes Cytochrome c release. While Bax immunostaining was homogenous throughout control cells (Fig. 9(A)), it exhibited a more punctuate cellular distribution, suggesting re-
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distribution of Bax from the cytosolic to the mitochondrial compartment, in apoptotic cells during SCFA treatment at pH 7.5. Such a relocation was detected in a limited number of cells at 24 h of treatment, and in half of the cells after 60 h. By contrast, no such translocation was observed during the lethal process at pH 5.5 in the presence of SCFA (data not shown). To further analyse the subcellular localisation of Bax, r Red 580 was used as a mitochondrial marker Mitotracker that is independent of the level of the mitochondrial inner membrane potential (Fig. 9(B)). According to a cytosolic Bax localisation, there was no colocalisation of Bax and Mitotracker in control cells. By contrast, upon 48 h SCFA treatment at pH 7.5, Bax localised in mitochondria, as shown by the yellow fluorescence that corresponds to the merge of the red and green fluorescence, indicating a colocalisation of Bax and Mitotracker in SCFA-treated cells (Fig. 9(B)). After 24 h of treatment with SCFA at pH 7.5 (Fig. 9(A)), immunostaining also suggested a redistribution of Cytochrome c from mitochondria to the cytosol in a limited fraction of cells, also exhibiting typical chromatin condensation. To confirm these relocations, mitochondrial fractions as well as cytosolic fractions were separated by differential centrifugation following SCFA treatment at pH 7.5. Immunoblotting examination of these subcellular fractions for the presence of Bax and Cytochrome c (Fig. 9(D)) confirmed changes in their respective subcellular localisation. The amount of Bax detected decreased in the cytoplasm and slightly increased in mitochondria during the time course of treatment. Concomitantly, Cytochrome c accumulated in the cytoplasm while released from mitochondria. The fraction of apoptotic cells then increased between 24 and 72 h of treatment, in accordance with the slow death kinetics established in Fig. 1. SCFA at pH 5.5 also triggered the release of Cytochrome c from mitochondria into cytosol. However, this leakage was detected very early following the onset of treatment and occurred in the whole population (Fig. 9(C)). Moreover, no typical sign of chromatin condensation was observed in these cells, compared to control. SCFA treatment at pH 7.5, but not at pH 5.5, is associated with caspase activation Cells treated with these SCFA, either at pH 7.5 or 5.5, were then analysed by western blotting for caspase activation and for the cleavage of the caspase-3 substrate PARP (Fig. 10). Treatments by 100 µM etoposide and by 15 mM H2 O2 were used as positive controls of apoptosis and necrosis, respectively. SCFA at pH 7.5 induced cleavage of procaspase-8, -9, -7 and -3 as shown by the decreased amount of initiator procaspases and the accumulation of active forms of initiator
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Intracellular ATP level (%)
A 100 90 80 70 60 50 40 30 20 10 0
*
* *
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*
*
*
*
*
1.5h 2h 2.5h 3h 4h 5h Time of SCFA treatment, pH 5.5
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* H2O2 6h pH 7.5
Fig. 8 Drastic ATP depletion is induced by SCFA at pH 5.5. Cellular content of ATP of HT-29 cells treated with SCFA at pH 7.5 (A) or at pH 5.5 (B) was measured by luminometry. The intracellular ATP level was calculated relative to the amount detected at the same time in control
cells left untreated (pH 7.5). The necrosis inducer H2 O2 (15 mM, 6 h, pH 7.5) was used as an ATP depletion positive control. Each value is given as the mean of relative ATP amount ± s.d. of 4 independent experiments. ∗ P < 0.05, treated versus control, Student’s t Test
and effector caspases: the subunits p41 and p18 of caspase-8, the subunit p35 of caspase-9 and subunits p20 of caspase-3 and caspase-7 (Fig. 10(A)). For all 4 caspases, cleavage was detected from 48 h of treatment. Maximal accumulation was achieved after 72 h for cleaved caspase-3, -7 and -9 while it was maximal after 96 h for cleaved caspase-8. Accordingly, cleavage of the caspase substrate PARP occurred after 48 h of treatment and gave rise to the characteristic apoptotic 89 kDa fragment (Fig. 10(A)). SCFA at pH 5.5 clearly failed to trigger these effects in HT-29 cells. No characteristic 89 kDa PARP apoptotic fragment was observed, neither in the presence of SCFA at pH 5.5, nor in the presence of H2 O2 .(Fig. 10(B)). PARP remained uncleaved during SCFA treatment at pH 5.5. Finally, in order to test the possible involvement of RIP1, in SCFA-induced cell death, we studied
its expression at both pH (Fig. 10). RIP1 appeared cleaved under treatment with SCFA at pH 7.5 or with the apoptotic inducer, but neither at pH 5.5 nor by the necrosis inducer.
Discussion Upregulation or facilitation of apoptosis by dietary components might protect against colon cancer development, which is often linked with an impairment of apoptosis. SCFA are known to have pro-apoptotic effects [3]. A diet efficient in enhancing the colonic content of pro-apoptotic SCFA may thus help in the prevention or in the treatment of colon cancer, as a preventive diet or as a complement to cancer therapy, respectively. Springer
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Control 0h pH 7.5
SCFA 24 h pH 7.5
SCFA 48 h pH 7.5
SCFA 60 h pH 7.5
Hoechst 33342
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Mitotracker
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Control 6h pH 5.5
SCFA 2 h pH 5.5
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SCFA 6 h pH 5.5
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Hoechst 33342 Cytochrome c
Hoechst 33342
Bax
A
C
Hoechst 33342 Cytochrome c
SCFA 48h pH 7.5
Control 0h pH 7.5
B
Fig. 9 SCFA trigger different protein relocation depending on pH. (A and B) Time course of Bax and Cytochrome c subcellular relocation upon SCFA treatment at pH 7.5 (A and B) or 5.5 (C). Cells were immunostained with antibodies directed against Bax or Cytochrome c, prior to counter-staining with Hoechst 33342 and fluorescence microscopy observation. (A) Relocation of Bax and of Cytochrome c in apoptotic cells is indicated by arrows. (B) Bax co-localisation with mitochondria upon SCFA treatment at pH 7.5. Cells were stained r Red 580 prior to Bax with the mitochondrial marker Mitotracker Springer
SCFA 12 h pH 5.5
immunostaining. In the merged panel, Bax and Mitotracker colocalisation results in yellow fluorescence. (D) Western blot analysis of Cytochrome c and of Bax relocation upon SCFA treatment at pH 7.5. Following cell treatment and subcellular fraction, cytosolic and mitochondrial fractions were analysed by western blotting and revealed using the same anti-Cytochrome c and anti-Bax antibodies. Antibodies directed against VDAC and Actin were used as loading control for mitochondrial and cytoplasmic fractions, respectively (Continued on next page.)
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D
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Cytosolic fraction SCFA pH 7.5 0h
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Actin VDAC
Fig. 9 (Continued.)
In a previous report, we have shown that propionate and acetate, major end-products of the human probiotic propionibacteria, used at physiological concentrations, kill HT-29 cells by apoptosis [8]. However, several questions remained with regard to the early effects of propionibacterial SCFA on cell cycle and on cell energetic status, the precise kinetics of key events in cellula and the impact of environmental pH. The present study reports for the first time that the mode of cell death can be modulated by the extracellular pH in a 5.5 to 7.5 physiological range met within the lumen of the normal and tumoral human colon [21, 30]. Indeed, propionibacterial SCFA trigger apoptosis at pH 7.5 while at pH 5.5 a necrotic process was observed. Note that pH 5.5 alone did not provoke such drastic effects. Interestingly, SCFA treatment in the pH range of 6.0 to 7.0 induced apoptosis rather than necrosis (data not shown), suggesting a breakpoint between pH 5.5 and 6.0. Moreover, the death kinetics was faster at acidic pH than at pH 7.5. Treatment with SCFA at pH 7.5 caused all morphological characteristics of type I PCD including membrane blebbing, chromatin condensation and fragmentation, phosphatidyl serine exposure and apoptotic bodies formation (Figs. 3, 4 and 5). Conversely, we have clearly identified morphological features of necrosis in cells treated with SCFA at pH 5.5, manifested by rapid swelling and disruption of internal organelles and plasma membrane lysis without chromatin fragmentation (Figs. 3, 4 and 5). Moreover, necrosis induced by SCFA at pH 5.5 was not secondary to apoptosis induction because no apoptotic cells were detected before appearance of necrotic cells (Fig. 3). Such an extracellular pH-dependent shift has been previously observed upon TRAIL or chlorine p6 treatment [19, 31]. We demonstrate here that treatment with SCFA induced cell cycle arrest in G2/M phase prior to apoptosis at pH 7.5 but not 5.5 (Fig. 2). Bernhard et al. demonstrated such an effect under butyrate treatment, another SCFA clearly recognized as apoptosis inducer in cancer cells [32]. However
butyrate has also been shown to inhibit cell proliferation with accumulation of cells in the G0/G1 phase of the cell cycle in HT-29 cells [33] and in SW620 human colonic carcinoma cells in which a concomitant cell cycle arrest can also occur in G2/M [4, 33]. Mitochondria have been described as the central integrator/coordinator of apoptosis, necrosis and autophagy [13, 34, 35]. Among various plausible mechanisms, mitochondrial membrane permeability can be promoted by opening of the mitochondrial PTPC [36]. Here, we demonstrate that SCFA treatment caused an IM permeabilisation, presumably via the opening of PTPC, which correlates with the dissipation of the m (Fig. 7) and with the swelling of mitochondrial matrix (Fig. 4). Therefore, PTPC is an early target of SCFA in pro-apoptotic conditions [8], but also in necrotic conditions as shown in the present work. As proposed by Halestrap et al. ROS could be responsible for the opening of PTPC [37] or the consequence of mitochondrial alteration caused by leakiness of the electron transport chain [38]. Here, we observed a marked production of superoxide anions at the different pH but we could not conclude if ROS are the cause or the consequence of PTPC opening since these events seemed to be concomitant at both pH (Figs. 6 and 7). Loss of m is considered as a major determinant in the cellular commitment to death because it leads to a bioenergetic catastrophy. Thus, the dissipation of the proton gradient across the inner membrane blocks the oxidative phosphorylation, thus altering production of ATP [39]. It has been reported that intracellular concentration of ATP can switch apoptosis to necrosis [40–42]. Indeed, whereas necrosis is accompanied by an ATP depletion to a level incompatible with cell survival [43], apoptosis is an active process requiring energy under the form of ATP. An ATP loss above 70% would promote necrosis [44], whereas at least 25% of basal ATP level would be requisite to promote apoptosis [45]. Our data are consistent with the literature. We demonstrate here that treatment with SCFA at pH 5.5 caused an ATP drop below 5% of initial concentration in 6 h of treatment, leading Springer
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Fig. 10 SCFA treatment at pH 7.5, but not 5.5, is associated with caspase activation. Processing of initiator caspases (caspase -9 and -8), effector caspases (caspase-3 and -7), PARP and RIP-1 was followed by western blot in HT-29 cells treated with SCFA at pH 7.5 (A) and at pH 5.5 (B). (A) SCFA at pH 7.5 induced caspases processing. The cleaved subunits of caspase-8, -9, -3, -7 and apoptotic fragment of PARP were detected by immunoblot in SCFA-treated HT-29 cells at pH 7.5. Note
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that anti-caspase-3 antibody only recognizes the cleaved form. Moreover, the kinase RIP-1 was cleaved and thus inactivated with SCFA at pH 7.5. (B) Neither processing of caspases, nor cleavage of RIP-1 was detected upon treatment with SCFA at pH 5.5. Etoposide (100 µM, 36 h, pH 7.5) and H2 O2 (15 mM, 6 h, pH 7.5) were used as apoptosis and necrosis inducing controls, respectively
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to irreversible cellular energy failure. The lack of ATP could also directly cause necrosis by preventing the functioning of a plasma membrane associated Na+ /K+ ATPase normally active during apoptosis, with the consequence of ionic unbalance, water uptake, cell swelling and lysis [46]. The intracellular ATP drop caused by SCFA at pH 5.5 occurred before any biochemical (ROS generation and m loss) or morphological evidence of necrosis could be detected, suggesting that this drop is an early event in this cell death pathway. A possible explanation of such an energetic catastrophy could be due to a higher diffusion of SCFA across the apical membrane. With an average pKa of 4.8, SCFA are under anionic form in extracellular compartment. SCFA diffusion across the apical membrane depends on a constant supply of protons to allow protonation of SCFA anions and subsequent formation of undissociated SCFA, that permits SCFA absorption by non ionic diffusion in the colon [47]. Reynolds et al. demonstrated that uptake of butyrate was significantly higher at acidic pH (5.5) than at pH 7.5 [48]. It was also reported that SCFA absorption is coupled to the activity of an ouabain-sensitive electroneutral H+ /K+ /ATPase in the rodent colon [49]. A massive SCFA entry might disturb plasma membrane pumps, leading to rapid ATP depletion. Such an entry would certainly have major effects on intracellular pH (leading to acidification) with drastic consequences on mitochondrial electron respiratory chain, and hence ATP level [50]. Another possibility could be related to PARP hyperactivation. Indeed it has been shown that such an event, notably triggered by DNA damage, leads to cellular ATP pool depletion and hence necrosis [51]. By contrast, SCFA treatment at pH 7.5 maintained an intracellular ATP level sufficient to allow apoptotic mechanisms, especially the apoptosome formation which is determinant for caspase activation [52]. Furthermore, inhibition of caspases induced a switch from apoptosis to necrosis in B lymphocyte [53]. PTPC opening and subsequent m loss allowed the release of apoptogenic factors into the cytosolic compartment such as Cytochrome c, procaspases, Smac/Diablo, Apoptosis-Inducing Factor and EndoG [54]. We showed here that SCFA-treatment at pH 7.5 provoked a progressive Cytochrome c release, indicative of mitochondrial apoptotic pathway implication. Interestingly, release of Cytochrome c also occurred at pH 5.5. Brooks et al. reported that neither Cytochrome c release nor Bax translocation were blocked by acidic pH (6.0) during ATP depletion [55]. By contrast, we did not observe any Bax relocation at pH 5.5. It has been shown that intracellular alkalinization caused conformational change of Bax leading to its translocation [56]. In this context, one might suppose medium acidification to induce Bax conformational change that could impede its translocation. We report here that SCFA at pH 7.5 induced activation of initiator caspases (caspase-8 and -9) known to activate the
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effector caspase-3 and -7, which, in turn, cleaved apoptotic substrates such as PARP. In contrast, caspase activation did not occur during SCFA treatment at pH 5.5 and might be due to the rapid drop of intracellular ATP. Regarding that point, it is worth noting that the acidic pH (6.0) has been reported to induce ATP depletion that could prevent caspase-9 activity at the level of apoptosome [55]. The PARP cleavage characteristic of apoptosis did not occur here under SCFA-treatment at pH 5.5. Surprisingly, the cleaved fragments observed in necrotic processes [57] were also not detected. The kinase RIP is considered as a mediator of “programmed necrosis”, even though its mechanisms of action are poorly understood [58]. RIP was identified as a crucial component of Fas-mediated, TNF and TRAIL-triggeredcaspase-independent cell death [59]. Furthermore, it has been reported that caspase-mediated inactivation of RIP induced a proapoptotic amplification loop during death receptorinduced apoptosis [60]. Moreover, PARP hyperactivationinduced necrosis required RIP1 [61]. Interestingly, a recent study showed that RIP is required for inhibition of Adenine Nucleotide Translocator activity, a major component of PTPC, leading to necrosis by inhibiting ADP/ATP exchange [62]. We demonstrated here that RIP1 was cleaved under treatment with SCFA at pH 7.5, probably by caspases, resulting in its inactivation. By contrast, no cleavage of RIP1 was detected during SCFA treatment at pH 5.5. It can thus be hypothesized that RIP1 may be involved in SCFA-induced necrosis at pH 5.5, but these data need further investigation.
Conclusion In conclusion, our study clearly shows that the environmental pH, a physicochemical parameter which varies within the colon, is an important factor for determining the type of cell death induced by propionibacteria. As necrosis could potentiate the anti-tumor innate immune response [35], the present apoptosis-necrosis switch induced by SCFA might be of importance for cancer therapy, especially for the treatment of solid tumors known to be related to an acidic microenvironment. Thus, in addition to the possibility of using live propionibacteria in diet as preventive anti-cancer agents, it remains to be determined first the in vivo relevance of a SCFA-based therapeutic strategy, and second how to efficiently convey SCFA to tumors. Regarding that point, it is noteworthy that a phase I study has recently been performed with an esterified form of another SCFA, namely butyrate, with encouraging results [63]. The use of nanoparticles might also be an alternative way of targeting such agents to disease location [64]. Interestingly, in this latter study, the authors described more than additive in vitro apoptotic effects by combining nanoparticles with butyrate and Springer
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another anticancer drug. Such a potentiation of the effects of classical chemotherapy upon association with propionate and acetate will clearly deserve further investigation. Finally, the observation of the apoptosis to necrosis switch upon these SCFA raises new questions about effects of the various organic acids present in the colon. If this switch occurs for other cell types or for other acids remains to be elucidated. Acknowledgments The authors wish thank Dr. O. Meurette and Dr. L. Huc for their helpful advice on cytometric analysis, and C. Longin for technical assistance in TEM assay.
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