cell biochemistry and function Cell Biochem Funct 2015; 33: 89–96. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cbf.3094
The proteins (12 and 15kDa) isolated from heat-killed Lactobacillus plantarum L67 induces apoptosis in HT-29 cells S. Song, S. Oh and K. T. Lim* Division of Animal Science, Chonnam National University, Gwangju, Korea
A number of scientific studies have revealed that Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. In this study, the production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl-2, Bax and the apoptosis-mediated proteins [caspase-8, caspase-3 and poly ADP ribose polymerase (PARP)] were evaluated to understand the induction of programmed cell death in HT-29 cells by Lactobacillus plantarum L67. The results obtained from this study indicated that the relative intensities of the apoptotic-related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t-Bid) increased with increasing concentrations of the membrane proteins isolated from heat-killed L. plantarum L67, whereas the relative intensities of cytochrome c, Bcl-2, caspase-8, caspase-3 and PARP decreased. This study determines whether proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 induce programmed cell death in HT-29 cells. Proteins isolated from L. plantarum L67 can stimulate the apoptotic signals and then consequently induce programmed cell death in HT-29 cells. The results in this study suggest that the proteins isolated from L. plantarum L67 could be used as an antitumoural agent in probiotics and as a component of supplements or health foods. Copyright © 2015 John Wiley & Sons, Ltd. key words—apoptosis; caspase-3; caspase-8; cytochrome c; HT-29 cells; Lactobacillus plantarum L67
INTRODUCTION Colorectal cancer is one of the leading causes of cancer death among all types of cancers in Korea because of western dietary habit.1,2 Many scientists have attempted to identify methods for killing cancer cells with either herbal treatments or by inducing cell death signalling such as apoptosis. Apoptosis, which is a programmed cell death, is the consequence of a highly complex cascade of cellular events, resulting in chromatin condensation, DNA fragmentation, swelling of cytoplasmic membrane and cell shrinkage.3 Intracellular reactive oxygen species (ROS), calcium efflux, the Bcl-2 family and cytochrome c from the mitochondrial membrane influence apoptosis.4 Lactobacilli and Bifidobacteria are well-known probiotic bacteria that can be used by children and immunecompromised individuals.5 The results of in vitro assays and animal, human, epidemiological and interventional studies have demonstrated that some strains of Lactobacillus could reduce the risk of certain types of cancer and inhibit the growth of certain tumours.6–9 In particular, Lactobacillus plantarum exhibits various biological effects including antitumour, anticoagulant, antiviral, immune modulatory and anti-inflammatory, antidiabetic and antioxidant or free radical scavenging activity.10–13 We reported *Correspondence to: Kye-Taek Lim, Division of Animal Science, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500757, Korea.E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
that L. plantarum L67 isolated from infant faeces has a high survival rate at low pH values. The aim of this study was to determine whether a protein isolated from L. plantarum L67 induces programmed cell death in HT-29 cells. The change in intracellular ROS production and the amount of intracellular calcium were measured, and cytochrome c, Bid, Bcl-2, Bax and apoptosismediated proteins [caspase-9, caspase-8, caspase-3 and poly ADP ribose polymerase (PARP)] were evaluated by western blotting.
MATERIALS AND METHODS Chemicals All plastic materials were purchased from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA). Penicillin G and streptomycin were obtained from Sigma (St. Louis, MO, USA). RPMI media 1640 and foetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Other chemicals and reagents were of the highest analytical grade available. Preparation of the whole, cytoplasmic and membrane protein from heat-killed or live L. plantarum L67 L. plantarum L67 from infant faeces was selected and stored according as described previously.14 Bacterial subcellular Received 19 October 2014 Revised 8 January 2015 Accepted 12 January 2015
90
s. song
fractions were prepared by the method of Rosenthal and Dziarski et al. with minor modifications.15 To separate bioactive proteins (12 and 15 kDa), centrifugal filter units were used (Merk Millipore, Darmstadt, Germany), and the proteins were then purified through a dialysis membrane (Fisherbrand, UK). The solute was then concentrated by freeze drying (Ilshin Lab Co., Ltd., Yangju-si, Korea). Two proteins (12 and 15 kDa) were not completely separated from each other because there is only a small difference in their molecular weight. The amount of protein was measured via the method of Lowry et al.,16 and the cellular proteins were stored at 70 °C before use. Cell culture and cytotoxicity HT-29 cells (ATCC® HTB-38™) were incubated in RPMI media 1640 containing 10% FBS, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin at 37 °C in an atmosphere containing 5% CO2. The cells (1 × 106 cells ml 1 and passage number 3) were distributed into either 35 mm culture dishes or 96-well flat bottom plates. The cytotoxicity of the membrane proteins isolated from heat-killed L. plantarum L67 was determined by the MTT assay.17 The cytotoxicity was also determined by the release of lactate dehydrogenase (LDH) into the medium according to the method of Bergmeyer and Bernt.18 The cells were treated with various concentrations of membrane proteins isolated from heat-killed L. plantarum L67 for 24 h at 37 °C in an atmosphere containing 5% CO2. Measurement of intracellular ROS Intracellular ROS were measured using the non-fluorescent dye 20, 70-dichlorodihydrofluorescein (H2DCF-DA), which is a membrane-permeable fluorigenic tracer that is oxidized by various ROS. The cells were pre-incubated with 10 mmol H2DCF-DA for 30 min at 37 °C and then washed twice with PBS to remove the extracellular H2DCF-DA. The cells were treated with membrane proteins isolated from heat-killed L. plantarum L67 (5, 20, 50 and 100 μg ml 1) for 24 h at 37 °C in an atmosphere containing 5% CO2. Finally, the fluorescence intensity was measured at an excitation of 485 nm and an emission of 530 nm using a fluorescence microplate reader (Dual Scanning SPECTRAmax, Molecular Devices Corp., Sunnyvale, CA, USA). Detection of intracellular calcium mobilization Intracellular calcium was determined as described previously.19 Cells were treated with membrane proteins from L. plantarum L67 (5, 20, 50 and 100 μg ml 1). The fluorescence intensity was measured at an emission wavelength of 510 nm and excitation wavelength of 340 and 380 nm using a fluorescent microplate reader (Dual Scanning SPECTRAmax, Molecular Devices). Preparation of the membrane, cytosolic, nucleic and whole protein extracts To detect the translocation of protein kinase C (PKC), membrane and cytosolic extracts were prepared according Copyright © 2015 John Wiley & Sons, Ltd.
ET AL.
to Patton’s method.20 Either the nucleic protein extract for immunoblotting of cytochrome c or the whole cellular protein extract for immunoblotting of Bid, Bcl-2, Bax, caspase-9, caspase-3, PARP and α-tubulin was isolated from HT-29 cells as described previously.21 The quantity of protein was measured via the method developed by Lowry et al. [16], and the cellular proteins were stored at 70 °C before use. Immunoblot analysis Cellular proteins were separated on a 10% polyacrylamide mini-gel at 100 V for 2 h at room temperature using a Mini-Protein II Electrophoresis Cell (Bio-Rad, Hercules, CA, USA). After electrophoresis, the proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were subsequently incubated for 2 h at room temperature with primary antibodies (Bid, Bcl-2, Bax, cytochrome c, caspase-9, caspase-3, PARP and α-tubulin) in Tris-buffered saline with Tween 20 (TBS-T) solution. After washing three times with TBS-T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (1:10 000; Cell Signaling Technology, Danvers, MA, USA) in TBS-T solution. The resulting protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Bristol, UK). The results of the immunoblot assay were calculated as relative intensity using Scion imaging software (Scion Image Beta 4.02, Frederick, MD, USA). Statistical analysis All experiments were performed separately in triplicate. All data are presented as means and standard deviations. Oneway analysis of variance and Duncan’s test were performed to determine significant differences in the multiple comparisons using SPSS ver. 11.0 (SPSS, Inc., Chicago, IL, USA). A P < 0.05 was considered significant. RESULTS Protein isolated from L. plantarum L67 After staining with Coomassie blue but not Schiff’s reagent, the proteins isolated from L. plantarum L67 were located in two bands on a 14% acrylamide gel. One had a molecular weight of 12 kDa, and the other was 15 kDa on the gel. The proteins were obtained from the membranes of heatkilled L. plantarum L67 but not from live cells or cytoplasm of heat-killed cells (Figure 1A). We used these 12 and 15 kDa proteins for the subsequent experiments (Figure 1B). Cytotoxic effect of the proteins isolated from heat-killed L. plantarum L67 on HT-29 cells We performed the MTT and LDH assays in membrane protein isolated from heat-killed L. plantarum L67 treated HTCell Biochem Funct 2015; 33: 89–96.
APOPTOSIS
EFFECT OF PROTEIN OF
L.
PLANTARUM
L67
91
Figure 1. Identification of the protein isolated from heat-killed Lactobacillus plantarum L67. Heat-killed cells were heated for 30 min at 100 °C and then harvested. The proteins were separately isolated as live or heated cells, cytoplasm or membranes (A). The isolated proteins (12 and 15 kDa) were purified using centrifugal filter units and dialysis membrane as described in the Materials and methods (B). M, marker; lane 1, whole L67; lane 2, cytoplasm of live L67; lane 3, membrane of live L67; lane 4, cytoplasm of heat-killed L67; and lane 5, membrane of heat-killed L. plantarum L67
Figure 2. Cytotoxicity effects of the proteins isolated from heat-killed L. plantarum L67 in HT-29 cells. Survival (%) (A), LDH release (%) (B), the amount of intracellular reactive oxygen species (ROS) in HT-29 cells (C) and survival (%) in HT-116 cells (D). The white bar indicates the control, while the black bar indicates that HT-29 cells or HT-116 were treated with various concentrations of proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 for 24 h. All data represent the means ± standard deviations from triplicates. *Indicates a significant difference between the control and treatment of protein, P < 0.05. **Indicates a significant difference between the control and treatment of protein, P < 0.01 Copyright © 2015 John Wiley & Sons, Ltd.
Cell Biochem Funct 2015; 33: 89–96.
92
s. song
ET AL.
29 cells to determine whether L. plantarum L67 is cytotoxic. As shown in Figure 2A, cellular toxicity increased significantly when HT-29 cells were exposed to various concentrations (μg ml 1) of membrane protein of heat-killed L. plantarum L67. The viability values were 72.06, 66.01, 58.72 and 50.23% at 5, 20, 50 and 100 μg ml 1 after 24 h, respectively. As shown in Figure 2B, proteins (12 and 15 kDa) of L. plantarum L67 increased cytotoxicity in the LDH assay. As shown in Figure 2C, the relative amounts of intracellular ROS increased when HT-29 cells were treated with the membrane proteins of heat-killed L. plantarum L67 for 24 h, compared with the controls that did not have any treatment. For example, the values of intracellular ROS increased by 1.0-fold, 1.16-fold, 2.58-fold and 2.61-fold depending on the concentrations of membrane proteins (5–100 μg ml 1), compared with those values in the control. Also, cytotoxicity increased significantly by various concentrations (μg ml 1) of membrane protein of heatkilled L. plantarum L67 in another colonic carcinoma cell line such as HT-116 cells (Figure 2D). Effect of the proteins isolated from heat-killed L. plantarum L67 on intracellular calcium production in HT-29 cells The amount of intracellular calcium increased significantly and dose-dependently when HT-29 cells were treated with proteins from heat-killed L. plantarum L67 when compared with the control. For example, the values of intracellular calcium increased by 1.03-fold, 1.27-fold, 4.54-fold and 4.57fold (compared with the control) at 5, 20, 50 and 100 μg ml 1 of membrane proteins (Figure 3A). As shown in Figure 3B, when the HT-29 cells were treated with proteins from heat-killed L. plantarum L67, the translocation of PKCα was induced from the cytosol to the membrane. For the PKCα, when the HT-29 cells were treated with proteins of heat-killed L. plantarum L67, the relative intensities of PKCα increased in the membrane fraction, whereas they decreased in the cytosolic when compared with the control (Figure 3C). Effect of proteins isolated from heat-killed L. plantarum L67 on mitochondrial apoptotic-related signals (cytochrome c, Bid, Bcl-2 and Bax) and related proteins (caspase-3, caspase-8 and PARP) in HT-29 cells As shown in Figure 4A, cytochrome c activity decreased gradually in the mitochondrial fraction at 24 h, whereas the activity increased gradually in the cytosolic fraction when HT-29 cell were treated with the membrane protein of heat-killed L. plantarum L67 (5, 20, 50 and 100 μg ml 1). Relative band densities of cytochrome c in the mitochondrial fraction diminished by 1.0-fold, 0.87-fold, 0.42-fold and 0.2-fold for increasing proteins concentrations ( 5–100 μg ml 1) (Figure 4B). Cytochrome c activity showed the opposite effect in the cytosolic fraction. Bid activity was determined using a western blot analysis. As shown in Figure 4C, the relative Bid activity decreased dose dependently when HT-29 cells were treated with the membrane proteins of heat-killed L. plantarum L67 (5, 20, Copyright © 2015 John Wiley & Sons, Ltd.
Figure 3. Effect of the proteins isolated from heat-killed L. plantarum L67 2+ on the amount of intracellular Ca and PKC in HT-29 cells after 24 h. The white bar indicates the control, while the black bar indicates that HT-29 cells were treated with various concentrations of proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 for 24 h (A). Activity of PKC was evaluated using an immunoblot analysis (B). Relative intensity of PKC (C). Relative band intensities were calculated using Scion imaging software (Scion Image Beta 4.02, Maryland, USA). Data represent mean ±[ SD from experiments (n = 3). *Indicates a significant difference between the control and treatment of protein, P < 0.05. **Indicates a significant difference between the control and treatment of protein, P < 0.01
50 and 100 μg ml 1) for 24 h. The relative Bid band intensities increased significantly. Relative Bcl-2 activity decreased dose dependently. For example, the relative intensity of the Bcl-2 band decreased by 0.82-fold, 0.64fold, 0.51-fold and 0.43-fold at concentrations of 5, 20, 50 and 100 μg ml 1. The relative Bax band intensities increased by 1.08-fold, 1.62-fold, 1.74-fold and 1.61-fold at 5, 20, 50 and 100 μg ml 1 of membrane proteins from L. plantarum L67, respectively (Figure 4D). Caspase-3 and PARP proteins in the whole cell extract fraction were cleaved in a dose-dependent manner when the cells were exposed to the membrane proteins of heatCell Biochem Funct 2015; 33: 89–96.
APOPTOSIS
EFFECT OF PROTEIN OF
L.
PLANTARUM
L67
93
Figure 4. Effects of protein isolated from heat-killed L. plantarum L67 on the activity of cytochrome c (A), relative intensity of cytochrome c (B), the activity of Bcl-2 family (Bid, Bax and Bcl-2) (C) and relative intensity of Bax and Bcl-2 (D). HT-29 cells were treated with proteins of heat-killed L. plantarum L67 (5, 1 20, 50 and 100 μg ml ) for 24 h. Relative band intensities were calculated using Scion imaging software (Scion Image Beta 4.02, Maryland, USA). Data represent mean ± SD from experiments (n = 3). *Indicates a significant difference between the control and treatment of protein, P < 0.05. **Indicates a significant difference between the control and treatment of protein, P < 0.01
killed L. plantarum L67 for 24 h (Figure 5). The relative intensities of pro-caspase-8, pro-caspase-3 and PARP decreased significantly after treatment with the membrane protein. However, when HT-29 cells were treated with the membrane proteins (5, 20, 50 and 100 μg ml 1), the cleaved forms of apoptotic proteins were strongly detected. The
Figure 5. Effect of proteins isolated from heat-killed L. plantarum L67 on the activity of apoptosis-related proteins [caspase-3, caspase-8 and poly ADP ribose polymerase (PARP)] in HT-29 cells. HT-29 cells were treated 1 with proteins of heat-killed L. plantarum L67 (5, 20, 50 and 100 μg ml ) for 24 h Copyright © 2015 John Wiley & Sons, Ltd.
relative intensities of apoptotic proteins increased significantly and dose dependently. DISCUSSION When the L. plantarum L67 was co-cultured with HT-29 cells in preliminary experiments, we noted that the extracts from heat-killed L. plantarum L67 were cytotoxic but that the live cells were not. Therefore, we isolated the proteins from the cell membrane and cytoplasm in heat-killed L. plantarum L67. Notably, the cytoplasmic protein did not display activity, whereas the membrane protein was toxic to HT-29 cells. As shown in Figure 1A, many protein bands were detected in live cells without heat (Figure 1A, lines 2 and 3), compared with where several proteins in the heated cells disappeared (Figure 1A, lines 4 and 5). The reason why those proteins disappeared by heat might be denatured and deformed its threedimensional structure. The deformed structure of protein interacts to receptor on the cell membrane. The results of the interaction between protein and receptor stimulates to active signals at upstream in cell death pathway. This indicates the overexpression of two protein bands (12 Cell Biochem Funct 2015; 33: 89–96.
94
s. song
and 15 kDa) in the membrane (Figure 1B, line 5). In the preliminary experiments, it was figured out that only the two proteins have a bioactivity in this study. Although the proteins isolated from live cells have many proteins and maintain their native structure, they cannot interact with HT-29 cell receptor proteins because of the mismatch between proteins from live cells and that of HT-29 cells. Consequently, the heat-deformed proteins may have fit with integral cell membrane proteins of HT-29 cells. We determined the cytotoxicity of membrane proteins isolated from heat-killed L. plantarum L67 in HT-29 cells using the MTT and LDH assays. The results indicated that the IC50 was 100 μg ml 1 after a 24 h treatment (Figure 2). Thus, membrane proteins of the heat-killed L. plantarum L67 displayed potential activity to kill HT-29 cells. The induction of apoptosis in cancer cells is a useful strategy for anticancer drug development from lactic acid bacteria. Lactobacillus can induce apoptosis with various anticancer effects, particularly L. plantarum.22,23 Studies have shown that several specific strains of lactobacilli can induce the production of pro-inflammatory cytokines [interleukin (IL)-1 and IL-6] and anti-inflammatory cytokines (IL-12 and IL-10). However, the responsible mechanisms remain unclear. In general, cancer therapies act to increase apoptosis through the death receptor (extrinsic) or the mitochondrial (intrinsic) pathways.24,25 ROS, which are molecules or ions formed by the incomplete one-electron reduction of oxygen, have a dual role (promoting or suppressing cancer) in tumourigenesis because they participate simultaneously in two signalling pathways that have inverse functions in tumourigenesis. When ROS are generated in excess, it causes cell damage and acts as a mediator to alter calcium homeostasis, which precedes other morphological and functional cellular alterations.26 The results of intracellular ROS in this study showed that the membrane protein of heat-killed L. plantarum L67 stimulated increased amounts of intracellular ROS in HT-29 cells (Figure 2C). Calcium homeostasis is an important requirement for cell functionality. Excessive calcium loading in the mitochondria may induce apoptosis by stimulating the release of apoptosis-promoting factors from the mitochondrial intermembrane space to the cytoplasm and by impairing mitochondrial function.27 As shown in Figure 3, the membrane proteins from heat-killed L. plantarum L67 increased the mobilized amount of intracellular calcium in HT-29 cells. Elevating calcium levels in the cytosol may act synergistically with ROS to open the mitochondrial membrane permeability transition pore in hepatocytes.28 Accumulation of calcium is one of the vital functions of mitochondria, and perturbation of intracellular calcium compartmentalization can cause cytotoxicity and trigger either apoptotic cell death.29 Calcium amount intracellular system is changed, when cells are stimulated by hormonal and certain extracellular stimuli through activation of phospholipase C.30,31 Namely, phospholipid is divided into inositol triphosphate (IP3) and diacylglycerol, and then, IP3 builds complex with membrane of endoplasmic reticulum Copyright © 2015 John Wiley & Sons, Ltd.
ET AL.
(ER) to open. Subsequently, calcium mobilizes from ER into cytoplasm, and then, the calcium amount increases. Thus, the increased calcium activates nucleic enzymes and appeared to cut 180–200 base pair in DNA fragments. Also, calcium activates several kinases that closely relates to apoptosis in downstream of signal pathway. Cell growth and proliferation are associated with activity changes in kinases such as PKC, in particular, cellular calcium levels.32,33 When cells are stimulated, cellular regulators may induce the translocation of PKC from cytosol to plasma membrane and activate its expression. The fluctuation of Ca2+ in excitable cells is relevant to the translocation and activation of PKC.34 As shown Figure 3, the results indicated that protein of L. plantarum L67 increased the mobilized amount of intracellular Ca2+ and translocation of PKC from cytosol to plasma membrane in HT-29 cells. Many studies have shown that calcium and PKC are indicators of apoptosis.27,28,35 Our results indicated that activation of Ca2+/PKC subsequently stimulates the induction of apoptotic signals downstream of the apoptosis pathway. Well-known surface receptors for apoptosis, such as TNF-α, are produced by immune system cells and Fas, which activate the cytosolic protease caspase-8. Caspase-8 activates caspase-3, caspase-6 and caspase-7, inducing apoptosis. However, caspase-8 is also associated with mitochondria by activating Bid, which subsequently induces apoptosis through the mitochondria.36 Under normal physiological conditions, Bid is located in the cytoplasm in an inactive state. However, when death receptors on the cell surface are activated, the Bid protein is cleaved into tBid fragments, which are repositioned on the mitochondrial membrane where they cooperate with Bax proteins. This cooperation promotes fusion of Bax with the mitochondria, resulting in configurational alterations of the Bax proteins. These changes increase the damage to the mitochondria, which result in the formation of membrane pores and allow large amounts of cytochrome c to be released from the mitochondria. Subsequently, caspase-9 is activated, leading to the induction of apoptosis in the cells.37,38 As shown in Figure 4C, Bid and Bcl-2 activity decreased and Bax increased in HT-29 cells following the addition of membrane proteins from heat-killed L. plantarum L67. We assumed that the membrane proteins blocked survival factors such as Bid and Bcl-2 but induced caspase-8 and caspase-3 activity. Our results show that membrane proteins from heat-killed L. plantarum L67 induced translocation of cytochrome c from the mitochondria to the cytosol (Figure 4A). Moreover, the caspase-cascade system plays a crucial role in cell apoptosis. In particular, caspase-3 plays a pivotal role in the terminal and execution phase of apoptosis induced by diverse stimuli.39 Pro-caspase-3 is a key mediator of apoptosis, and PARP is the main substrate for caspase-3; they are important in DNA damage repair and apoptosis. Our results indicate that the membrane proteins from heat-killed L. plantarum L67 have apoptotic effects by stimulating caspase-8 and caspase-3 activities. Additionally, treatment with the membrane proteins from heat-killed L. plantarum Cell Biochem Funct 2015; 33: 89–96.
APOPTOSIS
EFFECT OF PROTEIN OF
L67 led to cleaved PARP, which is a nuclear apoptotic marker (Figure 5). Mitochondria are closely related to intracellular ROS production and mobilization of calcium. Induction of the mitochondrial (intrinsic) cell death pathway was confirmed by caspase-8-mediated cleavage of Bid to the t-Bid protein. The translocation and oligomerization of t-Bid on the mitochondrial membrane cause the release of cytochrome c.40 Therefore, when intracellular ROS and mobilization of calcium are activated by extracellular stimuli, apoptosis-related proteins (Bid and cytochrome c) induced the cleavage of Bid and released cytochrome c into the mitochondria. The activated apoptosis-related proteins subsequently transferred their signals to downstream signal factors to express apoptotic genes in the nucleus via caspases and PARP to initiate programmed cell death. In conclusion, the results indicate that the membrane proteins from heat-killed L. plantarum L67 stimulated increased amounts of apoptotic-related factors (intracellular ROS and calcium). The relative intensities of Bax and tBid increased, whereas cytochrome c, Bcl-2 caspase-8, caspase-9, caspase-3 and PARP signals decreased in the presence of proteins of heat-killed L. plantarum L67. Taken together, the membrane proteins isolated from L. plantarum L67 induced apoptosis through apoptotic factors and signalling in HT-29 cells. Several problems remain that deal with elucidating biophysicochemical characterization of the two proteins (12 and 15 kDa) using several biochemical analyses such as proteomics using 2D electrophoresis, Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and sequencing. Additionally, the precise mechanism of the apoptotic receptor (either the TNF-α receptor or Fas receptor) on the cell membrane remains to be elucidated in the near future.
CONFLICT OF INTEREST The authors declare no potential conflicts of interest with respect to this research, authorship and/or publication of this article.
ACKNOWLEDGEMENTS This work was completely financed by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea in 2014.
REFERENCES 1. Stein K, Borowicki A, Scharlau D, et al. Effects of synbiotic fermentation products on primary chemoprevention in human colon cells. J Nutr Biochem 2012; 23: 777–784. 2. Aleksandrova K, Pischon T, Jenab M, et al. Combined impact of healthy lifestyle factors on colorectal cancer: a large European cohort study. BMC Med 2014; 12: 168. Copyright © 2015 John Wiley & Sons, Ltd.
L.
PLANTARUM
L67
95
3. Tastsuta T, Scharwey M, Langer T. Mitochondrial lipid trafficking. Trends Cell Boil 2014; 24: 44–52. 4. Rasola A, Bernardi P. Mitochondrial permeability transition in Ca (2+) -dependent apoptosis and necrosis. Cell Calcium 2011; 50: 222–233. 5. Borriello SP, Hammoles WP, Holzapfel W, et al. Safety of probiotics that contain Lactobacilli or Bifidobacteria. Cli Infect Dis 2003; 36: 775–780. 6. Lankaputhra WEV, Shah NP. Antimutagenic properties of probiotic bacteria and of organic acids. Mutat Res 1998; 397: 169–182. 7. Reddy BS, Rivenson A. Inhibitory effect of Bifidobacterium longum on colon, mammolary, and liver carcinogenesis induced by 2-amino3-methylimidazo[4, 5-f]quinoline, a food mutagen. Cancer Res 1993; 53: 3914–3918. 8. Brady LJ, Gallaher DD, Busta FF. The role of probiotic cultures in the prevention of colon cancer. J Nutr 2000; 130: 410–414. 9. Burns AJ, Rowland IR. Anti-carcinogenicity of probiotics and prebiotics. Curr Issues Intest Microbiol 2000; 1: 13–24. 10. Andersson U, Bränning C, Ahrné S, et al. Probiotics lower plasma glucose in the high-fat fed C57BL/6J mouse. Benefical microbes 2010; 1: 189–196. 11. Kassayová M, Bobrov N, Strojný L, Kisková T, et al. Preventive effects of probiotic bacteria Lactobacillus plantarum and dietary fiber in chemically-induced mammary carcinogenesis. Anticancer Res 2014; 34: 4969–4975. 12. Giardina S, Scilironi C, Michelotti A, et al. In vitro anti-inflammatory activity of selected oxalate-degrading probiotic bacteria: potential applications in the prevention and treatment of hyperoxaluria. J Food Sci 2014; 79: 384–390. 13. Li S, Zhao Y, Zhang L, et al. Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods. Food Chem 2012; 135: 1914–1919. 14. Song S, Bae DW, Lim K, et al. Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing. Int J Food Microbiol 2014; 18: 135–143. 15. Rosenthal RS, Dziarski R. Isolation of peptidoglycan and soluble epetidoglycan fragments. Methods Enzymol 1994; 235: 253–285. 16. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275. 17. Mossmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55–63. 18. Bergmeyer HU, Bernt E. Lactate dehydrogenase. In Methods of enzymatic analysis 2nd edition. Bergmeyer HU (ed.) Academic Press: New York, 1974; 574–579. 19. Kimata M, Shichijo M, Miura T, Serizawa I, et al. Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells. Biocheml Biophl Res Co 1999; 257: 895–900. 20. Patton WF, Dhanak MR, Jacobson BS. Differential partitioning of plasma membrane proteins into the triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution. J Cell Sci 1989; 92: 85–91. 21. Oh PS, Lim KT. Blocking of intracellular ROS production by phytoglycoprotein (30 kDa) causes anti-proliferation in bisphenol Astimulated Chang liver cells. J Appl Toxicol 2008; 28: 749–758. 22. Asha, Gayathri D. Synergistic impact of Lactobacillus fermentum, Lactobacillus plantarum and vincristine on 1,2-dimethylhydrazineinduced colorectal carcinogenesis in mice. Exp Ther Med 2012; 3: 1049–1054. 23. Hsu YL, Kuo PL, Lin CC. Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells. J Pharmacol Exp Ther 2005; 313: 333–344. 24. Mohamad N, Gutiérrez A, Núñez M, et al. Mitochondrial apoptotic pathways. Biocell 2005; 29: 149–161. 25. Brunelle JK, Chandel NS. Oxygen deprivation induced cell death: an update. Apoptosis 2002; 7: 475–482. 26. Csordás G, Várnai P, Golenár T, et al. Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface. Mol Cell 2010; 39: 121–132. Cell Biochem Funct 2015; 33: 89–96.
96
s. song
27. Festjens N, Vanden Berghe T, Vandenabeele P. Necrosis, a wellorchestrated form of cell demise: signalling cascades, important mediators and concomitant immolune response. Biochim Biophys Acta 2006; 1757: 1371–1387. 28. Gearhart TJ, Bouchard MJ. Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes. J Virol 2010; 84: 2675–2686. 29. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calciumapoptosis link. Nature reviews. Mol Cell Biol 2003; 4: 552–565. 30. Patterson RL, van Rossum DB, Nikolaidis N, Gill DL, Snyder SH. Phospholipase C-gamma: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci 2005; 30: 688–697. 31. Paradies G, Petrosillo G, Paradies V, Ruggiero FM. Role of cardiolipin peroxidation and Ca2+ in mitochondrial dysfunction and disease. Cell Calcium 2009; 45: 643–650. 32. Li-Ping Z, Da-Lei Z, Jian H, et al. Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways. Reprod Biol Endocrinol 2010; 19: 8–66. 33. Naumann I, Kappler R, von Schweinitz D, et al. Bortezomib primes neuroblastoma cells for TRAIL-induced apoptosis by linking the death receptor to the mitochondrial pathway. Clin Cancer Res 2011; 17: 3204–3218.
Copyright © 2015 John Wiley & Sons, Ltd.
ET AL.
34. Carifoli E. Calcium-mediated cellular signals: a story of failures. Trends Biochem Sci 2004; 9: 371–379. 35. Zhang S, Nie S, Huang D, Huang J, Feng Y, Xie M. Ganoderma atrum polysaccharide evokes antitumor activity via cAMP-PKA mediated ap2+ optotic pathway and down-regulation of Ca /PKC signal pathway. Food Chem Toxicol 2014; 68: 239–246. 36. Kantari C, Walczak H. Caspase-8 and bid: caught in the act between death receptors and mitochondria. Biochim Biophys Acta 1813; 2011: 558–563. 37. Song G, Chen GG, Hu T, Lai PB. Bid stands at the crossroad of stress-response pathways. Curr Cancer Drug Targets 2010; 10: 584–592. 38. Shore GC, Nguyen M. Bcl-2 proteins and apoptosis: choose your partner. Cell 2008; 135: 1004–1006. 39. Hao Z, Duncan GS, Chang CC, Elia A, et al. Specific ablation of the apoptotic functions of cytochrome c reveals a differential requirement for cytochrome c and Apaf-1 in apoptosis. Cell 2005; 121: 579–591. 40. Kim BM, Choi YJ, Han Y, et al. N, N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells. Toxicol Appl Pharmacol 2009; 239: 87–97.
Cell Biochem Funct 2015; 33: 89–96.
The proteins (12 and 15kDa) isolated from heat-killed Lactobacillus plantarum L67 induces apoptosis in HT-29 cells S. Song, S. Oh and K. T. Lim* Division of Animal Science, Chonnam National University, Gwangju, Korea
A number of scientific studies have revealed that Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. In this study, the production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl-2, Bax and the apoptosis-mediated proteins [caspase-8, caspase-3 and poly ADP ribose polymerase (PARP)] were evaluated to understand the induction of programmed cell death in HT-29 cells by Lactobacillus plantarum L67. The results obtained from this study indicated that the relative intensities of the apoptotic-related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t-Bid) increased with increasing concentrations of the membrane proteins isolated from heat-killed L. plantarum L67, whereas the relative intensities of cytochrome c, Bcl-2, caspase-8, caspase-3 and PARP decreased. This study determines whether proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 induce programmed cell death in HT-29 cells. Proteins isolated from L. plantarum L67 can stimulate the apoptotic signals and then consequently induce programmed cell death in HT-29 cells. The results in this study suggest that the proteins isolated from L. plantarum L67 could be used as an antitumoural agent in probiotics and as a component of supplements or health foods. Copyright © 2015 John Wiley & Sons, Ltd. key words—apoptosis; caspase-3; caspase-8; cytochrome c; HT-29 cells; Lactobacillus plantarum L67
INTRODUCTION Colorectal cancer is one of the leading causes of cancer death among all types of cancers in Korea because of western dietary habit.1,2 Many scientists have attempted to identify methods for killing cancer cells with either herbal treatments or by inducing cell death signalling such as apoptosis. Apoptosis, which is a programmed cell death, is the consequence of a highly complex cascade of cellular events, resulting in chromatin condensation, DNA fragmentation, swelling of cytoplasmic membrane and cell shrinkage.3 Intracellular reactive oxygen species (ROS), calcium efflux, the Bcl-2 family and cytochrome c from the mitochondrial membrane influence apoptosis.4 Lactobacilli and Bifidobacteria are well-known probiotic bacteria that can be used by children and immunecompromised individuals.5 The results of in vitro assays and animal, human, epidemiological and interventional studies have demonstrated that some strains of Lactobacillus could reduce the risk of certain types of cancer and inhibit the growth of certain tumours.6–9 In particular, Lactobacillus plantarum exhibits various biological effects including antitumour, anticoagulant, antiviral, immune modulatory and anti-inflammatory, antidiabetic and antioxidant or free radical scavenging activity.10–13 We reported *Correspondence to: Kye-Taek Lim, Division of Animal Science, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju 500757, Korea.E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
that L. plantarum L67 isolated from infant faeces has a high survival rate at low pH values. The aim of this study was to determine whether a protein isolated from L. plantarum L67 induces programmed cell death in HT-29 cells. The change in intracellular ROS production and the amount of intracellular calcium were measured, and cytochrome c, Bid, Bcl-2, Bax and apoptosismediated proteins [caspase-9, caspase-8, caspase-3 and poly ADP ribose polymerase (PARP)] were evaluated by western blotting.
MATERIALS AND METHODS Chemicals All plastic materials were purchased from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA). Penicillin G and streptomycin were obtained from Sigma (St. Louis, MO, USA). RPMI media 1640 and foetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Other chemicals and reagents were of the highest analytical grade available. Preparation of the whole, cytoplasmic and membrane protein from heat-killed or live L. plantarum L67 L. plantarum L67 from infant faeces was selected and stored according as described previously.14 Bacterial subcellular Received 19 October 2014 Revised 8 January 2015 Accepted 12 January 2015
90
s. song
fractions were prepared by the method of Rosenthal and Dziarski et al. with minor modifications.15 To separate bioactive proteins (12 and 15 kDa), centrifugal filter units were used (Merk Millipore, Darmstadt, Germany), and the proteins were then purified through a dialysis membrane (Fisherbrand, UK). The solute was then concentrated by freeze drying (Ilshin Lab Co., Ltd., Yangju-si, Korea). Two proteins (12 and 15 kDa) were not completely separated from each other because there is only a small difference in their molecular weight. The amount of protein was measured via the method of Lowry et al.,16 and the cellular proteins were stored at 70 °C before use. Cell culture and cytotoxicity HT-29 cells (ATCC® HTB-38™) were incubated in RPMI media 1640 containing 10% FBS, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin at 37 °C in an atmosphere containing 5% CO2. The cells (1 × 106 cells ml 1 and passage number 3) were distributed into either 35 mm culture dishes or 96-well flat bottom plates. The cytotoxicity of the membrane proteins isolated from heat-killed L. plantarum L67 was determined by the MTT assay.17 The cytotoxicity was also determined by the release of lactate dehydrogenase (LDH) into the medium according to the method of Bergmeyer and Bernt.18 The cells were treated with various concentrations of membrane proteins isolated from heat-killed L. plantarum L67 for 24 h at 37 °C in an atmosphere containing 5% CO2. Measurement of intracellular ROS Intracellular ROS were measured using the non-fluorescent dye 20, 70-dichlorodihydrofluorescein (H2DCF-DA), which is a membrane-permeable fluorigenic tracer that is oxidized by various ROS. The cells were pre-incubated with 10 mmol H2DCF-DA for 30 min at 37 °C and then washed twice with PBS to remove the extracellular H2DCF-DA. The cells were treated with membrane proteins isolated from heat-killed L. plantarum L67 (5, 20, 50 and 100 μg ml 1) for 24 h at 37 °C in an atmosphere containing 5% CO2. Finally, the fluorescence intensity was measured at an excitation of 485 nm and an emission of 530 nm using a fluorescence microplate reader (Dual Scanning SPECTRAmax, Molecular Devices Corp., Sunnyvale, CA, USA). Detection of intracellular calcium mobilization Intracellular calcium was determined as described previously.19 Cells were treated with membrane proteins from L. plantarum L67 (5, 20, 50 and 100 μg ml 1). The fluorescence intensity was measured at an emission wavelength of 510 nm and excitation wavelength of 340 and 380 nm using a fluorescent microplate reader (Dual Scanning SPECTRAmax, Molecular Devices). Preparation of the membrane, cytosolic, nucleic and whole protein extracts To detect the translocation of protein kinase C (PKC), membrane and cytosolic extracts were prepared according Copyright © 2015 John Wiley & Sons, Ltd.
ET AL.
to Patton’s method.20 Either the nucleic protein extract for immunoblotting of cytochrome c or the whole cellular protein extract for immunoblotting of Bid, Bcl-2, Bax, caspase-9, caspase-3, PARP and α-tubulin was isolated from HT-29 cells as described previously.21 The quantity of protein was measured via the method developed by Lowry et al. [16], and the cellular proteins were stored at 70 °C before use. Immunoblot analysis Cellular proteins were separated on a 10% polyacrylamide mini-gel at 100 V for 2 h at room temperature using a Mini-Protein II Electrophoresis Cell (Bio-Rad, Hercules, CA, USA). After electrophoresis, the proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were subsequently incubated for 2 h at room temperature with primary antibodies (Bid, Bcl-2, Bax, cytochrome c, caspase-9, caspase-3, PARP and α-tubulin) in Tris-buffered saline with Tween 20 (TBS-T) solution. After washing three times with TBS-T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (1:10 000; Cell Signaling Technology, Danvers, MA, USA) in TBS-T solution. The resulting protein bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Bristol, UK). The results of the immunoblot assay were calculated as relative intensity using Scion imaging software (Scion Image Beta 4.02, Frederick, MD, USA). Statistical analysis All experiments were performed separately in triplicate. All data are presented as means and standard deviations. Oneway analysis of variance and Duncan’s test were performed to determine significant differences in the multiple comparisons using SPSS ver. 11.0 (SPSS, Inc., Chicago, IL, USA). A P < 0.05 was considered significant. RESULTS Protein isolated from L. plantarum L67 After staining with Coomassie blue but not Schiff’s reagent, the proteins isolated from L. plantarum L67 were located in two bands on a 14% acrylamide gel. One had a molecular weight of 12 kDa, and the other was 15 kDa on the gel. The proteins were obtained from the membranes of heatkilled L. plantarum L67 but not from live cells or cytoplasm of heat-killed cells (Figure 1A). We used these 12 and 15 kDa proteins for the subsequent experiments (Figure 1B). Cytotoxic effect of the proteins isolated from heat-killed L. plantarum L67 on HT-29 cells We performed the MTT and LDH assays in membrane protein isolated from heat-killed L. plantarum L67 treated HTCell Biochem Funct 2015; 33: 89–96.
APOPTOSIS
EFFECT OF PROTEIN OF
L.
PLANTARUM
L67
91
Figure 1. Identification of the protein isolated from heat-killed Lactobacillus plantarum L67. Heat-killed cells were heated for 30 min at 100 °C and then harvested. The proteins were separately isolated as live or heated cells, cytoplasm or membranes (A). The isolated proteins (12 and 15 kDa) were purified using centrifugal filter units and dialysis membrane as described in the Materials and methods (B). M, marker; lane 1, whole L67; lane 2, cytoplasm of live L67; lane 3, membrane of live L67; lane 4, cytoplasm of heat-killed L67; and lane 5, membrane of heat-killed L. plantarum L67
Figure 2. Cytotoxicity effects of the proteins isolated from heat-killed L. plantarum L67 in HT-29 cells. Survival (%) (A), LDH release (%) (B), the amount of intracellular reactive oxygen species (ROS) in HT-29 cells (C) and survival (%) in HT-116 cells (D). The white bar indicates the control, while the black bar indicates that HT-29 cells or HT-116 were treated with various concentrations of proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 for 24 h. All data represent the means ± standard deviations from triplicates. *Indicates a significant difference between the control and treatment of protein, P < 0.05. **Indicates a significant difference between the control and treatment of protein, P < 0.01 Copyright © 2015 John Wiley & Sons, Ltd.
Cell Biochem Funct 2015; 33: 89–96.
92
s. song
ET AL.
29 cells to determine whether L. plantarum L67 is cytotoxic. As shown in Figure 2A, cellular toxicity increased significantly when HT-29 cells were exposed to various concentrations (μg ml 1) of membrane protein of heat-killed L. plantarum L67. The viability values were 72.06, 66.01, 58.72 and 50.23% at 5, 20, 50 and 100 μg ml 1 after 24 h, respectively. As shown in Figure 2B, proteins (12 and 15 kDa) of L. plantarum L67 increased cytotoxicity in the LDH assay. As shown in Figure 2C, the relative amounts of intracellular ROS increased when HT-29 cells were treated with the membrane proteins of heat-killed L. plantarum L67 for 24 h, compared with the controls that did not have any treatment. For example, the values of intracellular ROS increased by 1.0-fold, 1.16-fold, 2.58-fold and 2.61-fold depending on the concentrations of membrane proteins (5–100 μg ml 1), compared with those values in the control. Also, cytotoxicity increased significantly by various concentrations (μg ml 1) of membrane protein of heatkilled L. plantarum L67 in another colonic carcinoma cell line such as HT-116 cells (Figure 2D). Effect of the proteins isolated from heat-killed L. plantarum L67 on intracellular calcium production in HT-29 cells The amount of intracellular calcium increased significantly and dose-dependently when HT-29 cells were treated with proteins from heat-killed L. plantarum L67 when compared with the control. For example, the values of intracellular calcium increased by 1.03-fold, 1.27-fold, 4.54-fold and 4.57fold (compared with the control) at 5, 20, 50 and 100 μg ml 1 of membrane proteins (Figure 3A). As shown in Figure 3B, when the HT-29 cells were treated with proteins from heat-killed L. plantarum L67, the translocation of PKCα was induced from the cytosol to the membrane. For the PKCα, when the HT-29 cells were treated with proteins of heat-killed L. plantarum L67, the relative intensities of PKCα increased in the membrane fraction, whereas they decreased in the cytosolic when compared with the control (Figure 3C). Effect of proteins isolated from heat-killed L. plantarum L67 on mitochondrial apoptotic-related signals (cytochrome c, Bid, Bcl-2 and Bax) and related proteins (caspase-3, caspase-8 and PARP) in HT-29 cells As shown in Figure 4A, cytochrome c activity decreased gradually in the mitochondrial fraction at 24 h, whereas the activity increased gradually in the cytosolic fraction when HT-29 cell were treated with the membrane protein of heat-killed L. plantarum L67 (5, 20, 50 and 100 μg ml 1). Relative band densities of cytochrome c in the mitochondrial fraction diminished by 1.0-fold, 0.87-fold, 0.42-fold and 0.2-fold for increasing proteins concentrations ( 5–100 μg ml 1) (Figure 4B). Cytochrome c activity showed the opposite effect in the cytosolic fraction. Bid activity was determined using a western blot analysis. As shown in Figure 4C, the relative Bid activity decreased dose dependently when HT-29 cells were treated with the membrane proteins of heat-killed L. plantarum L67 (5, 20, Copyright © 2015 John Wiley & Sons, Ltd.
Figure 3. Effect of the proteins isolated from heat-killed L. plantarum L67 2+ on the amount of intracellular Ca and PKC in HT-29 cells after 24 h. The white bar indicates the control, while the black bar indicates that HT-29 cells were treated with various concentrations of proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 for 24 h (A). Activity of PKC was evaluated using an immunoblot analysis (B). Relative intensity of PKC (C). Relative band intensities were calculated using Scion imaging software (Scion Image Beta 4.02, Maryland, USA). Data represent mean ±[ SD from experiments (n = 3). *Indicates a significant difference between the control and treatment of protein, P < 0.05. **Indicates a significant difference between the control and treatment of protein, P < 0.01
50 and 100 μg ml 1) for 24 h. The relative Bid band intensities increased significantly. Relative Bcl-2 activity decreased dose dependently. For example, the relative intensity of the Bcl-2 band decreased by 0.82-fold, 0.64fold, 0.51-fold and 0.43-fold at concentrations of 5, 20, 50 and 100 μg ml 1. The relative Bax band intensities increased by 1.08-fold, 1.62-fold, 1.74-fold and 1.61-fold at 5, 20, 50 and 100 μg ml 1 of membrane proteins from L. plantarum L67, respectively (Figure 4D). Caspase-3 and PARP proteins in the whole cell extract fraction were cleaved in a dose-dependent manner when the cells were exposed to the membrane proteins of heatCell Biochem Funct 2015; 33: 89–96.
APOPTOSIS
EFFECT OF PROTEIN OF
L.
PLANTARUM
L67
93
Figure 4. Effects of protein isolated from heat-killed L. plantarum L67 on the activity of cytochrome c (A), relative intensity of cytochrome c (B), the activity of Bcl-2 family (Bid, Bax and Bcl-2) (C) and relative intensity of Bax and Bcl-2 (D). HT-29 cells were treated with proteins of heat-killed L. plantarum L67 (5, 1 20, 50 and 100 μg ml ) for 24 h. Relative band intensities were calculated using Scion imaging software (Scion Image Beta 4.02, Maryland, USA). Data represent mean ± SD from experiments (n = 3). *Indicates a significant difference between the control and treatment of protein, P < 0.05. **Indicates a significant difference between the control and treatment of protein, P < 0.01
killed L. plantarum L67 for 24 h (Figure 5). The relative intensities of pro-caspase-8, pro-caspase-3 and PARP decreased significantly after treatment with the membrane protein. However, when HT-29 cells were treated with the membrane proteins (5, 20, 50 and 100 μg ml 1), the cleaved forms of apoptotic proteins were strongly detected. The
Figure 5. Effect of proteins isolated from heat-killed L. plantarum L67 on the activity of apoptosis-related proteins [caspase-3, caspase-8 and poly ADP ribose polymerase (PARP)] in HT-29 cells. HT-29 cells were treated 1 with proteins of heat-killed L. plantarum L67 (5, 20, 50 and 100 μg ml ) for 24 h Copyright © 2015 John Wiley & Sons, Ltd.
relative intensities of apoptotic proteins increased significantly and dose dependently. DISCUSSION When the L. plantarum L67 was co-cultured with HT-29 cells in preliminary experiments, we noted that the extracts from heat-killed L. plantarum L67 were cytotoxic but that the live cells were not. Therefore, we isolated the proteins from the cell membrane and cytoplasm in heat-killed L. plantarum L67. Notably, the cytoplasmic protein did not display activity, whereas the membrane protein was toxic to HT-29 cells. As shown in Figure 1A, many protein bands were detected in live cells without heat (Figure 1A, lines 2 and 3), compared with where several proteins in the heated cells disappeared (Figure 1A, lines 4 and 5). The reason why those proteins disappeared by heat might be denatured and deformed its threedimensional structure. The deformed structure of protein interacts to receptor on the cell membrane. The results of the interaction between protein and receptor stimulates to active signals at upstream in cell death pathway. This indicates the overexpression of two protein bands (12 Cell Biochem Funct 2015; 33: 89–96.
94
s. song
and 15 kDa) in the membrane (Figure 1B, line 5). In the preliminary experiments, it was figured out that only the two proteins have a bioactivity in this study. Although the proteins isolated from live cells have many proteins and maintain their native structure, they cannot interact with HT-29 cell receptor proteins because of the mismatch between proteins from live cells and that of HT-29 cells. Consequently, the heat-deformed proteins may have fit with integral cell membrane proteins of HT-29 cells. We determined the cytotoxicity of membrane proteins isolated from heat-killed L. plantarum L67 in HT-29 cells using the MTT and LDH assays. The results indicated that the IC50 was 100 μg ml 1 after a 24 h treatment (Figure 2). Thus, membrane proteins of the heat-killed L. plantarum L67 displayed potential activity to kill HT-29 cells. The induction of apoptosis in cancer cells is a useful strategy for anticancer drug development from lactic acid bacteria. Lactobacillus can induce apoptosis with various anticancer effects, particularly L. plantarum.22,23 Studies have shown that several specific strains of lactobacilli can induce the production of pro-inflammatory cytokines [interleukin (IL)-1 and IL-6] and anti-inflammatory cytokines (IL-12 and IL-10). However, the responsible mechanisms remain unclear. In general, cancer therapies act to increase apoptosis through the death receptor (extrinsic) or the mitochondrial (intrinsic) pathways.24,25 ROS, which are molecules or ions formed by the incomplete one-electron reduction of oxygen, have a dual role (promoting or suppressing cancer) in tumourigenesis because they participate simultaneously in two signalling pathways that have inverse functions in tumourigenesis. When ROS are generated in excess, it causes cell damage and acts as a mediator to alter calcium homeostasis, which precedes other morphological and functional cellular alterations.26 The results of intracellular ROS in this study showed that the membrane protein of heat-killed L. plantarum L67 stimulated increased amounts of intracellular ROS in HT-29 cells (Figure 2C). Calcium homeostasis is an important requirement for cell functionality. Excessive calcium loading in the mitochondria may induce apoptosis by stimulating the release of apoptosis-promoting factors from the mitochondrial intermembrane space to the cytoplasm and by impairing mitochondrial function.27 As shown in Figure 3, the membrane proteins from heat-killed L. plantarum L67 increased the mobilized amount of intracellular calcium in HT-29 cells. Elevating calcium levels in the cytosol may act synergistically with ROS to open the mitochondrial membrane permeability transition pore in hepatocytes.28 Accumulation of calcium is one of the vital functions of mitochondria, and perturbation of intracellular calcium compartmentalization can cause cytotoxicity and trigger either apoptotic cell death.29 Calcium amount intracellular system is changed, when cells are stimulated by hormonal and certain extracellular stimuli through activation of phospholipase C.30,31 Namely, phospholipid is divided into inositol triphosphate (IP3) and diacylglycerol, and then, IP3 builds complex with membrane of endoplasmic reticulum Copyright © 2015 John Wiley & Sons, Ltd.
ET AL.
(ER) to open. Subsequently, calcium mobilizes from ER into cytoplasm, and then, the calcium amount increases. Thus, the increased calcium activates nucleic enzymes and appeared to cut 180–200 base pair in DNA fragments. Also, calcium activates several kinases that closely relates to apoptosis in downstream of signal pathway. Cell growth and proliferation are associated with activity changes in kinases such as PKC, in particular, cellular calcium levels.32,33 When cells are stimulated, cellular regulators may induce the translocation of PKC from cytosol to plasma membrane and activate its expression. The fluctuation of Ca2+ in excitable cells is relevant to the translocation and activation of PKC.34 As shown Figure 3, the results indicated that protein of L. plantarum L67 increased the mobilized amount of intracellular Ca2+ and translocation of PKC from cytosol to plasma membrane in HT-29 cells. Many studies have shown that calcium and PKC are indicators of apoptosis.27,28,35 Our results indicated that activation of Ca2+/PKC subsequently stimulates the induction of apoptotic signals downstream of the apoptosis pathway. Well-known surface receptors for apoptosis, such as TNF-α, are produced by immune system cells and Fas, which activate the cytosolic protease caspase-8. Caspase-8 activates caspase-3, caspase-6 and caspase-7, inducing apoptosis. However, caspase-8 is also associated with mitochondria by activating Bid, which subsequently induces apoptosis through the mitochondria.36 Under normal physiological conditions, Bid is located in the cytoplasm in an inactive state. However, when death receptors on the cell surface are activated, the Bid protein is cleaved into tBid fragments, which are repositioned on the mitochondrial membrane where they cooperate with Bax proteins. This cooperation promotes fusion of Bax with the mitochondria, resulting in configurational alterations of the Bax proteins. These changes increase the damage to the mitochondria, which result in the formation of membrane pores and allow large amounts of cytochrome c to be released from the mitochondria. Subsequently, caspase-9 is activated, leading to the induction of apoptosis in the cells.37,38 As shown in Figure 4C, Bid and Bcl-2 activity decreased and Bax increased in HT-29 cells following the addition of membrane proteins from heat-killed L. plantarum L67. We assumed that the membrane proteins blocked survival factors such as Bid and Bcl-2 but induced caspase-8 and caspase-3 activity. Our results show that membrane proteins from heat-killed L. plantarum L67 induced translocation of cytochrome c from the mitochondria to the cytosol (Figure 4A). Moreover, the caspase-cascade system plays a crucial role in cell apoptosis. In particular, caspase-3 plays a pivotal role in the terminal and execution phase of apoptosis induced by diverse stimuli.39 Pro-caspase-3 is a key mediator of apoptosis, and PARP is the main substrate for caspase-3; they are important in DNA damage repair and apoptosis. Our results indicate that the membrane proteins from heat-killed L. plantarum L67 have apoptotic effects by stimulating caspase-8 and caspase-3 activities. Additionally, treatment with the membrane proteins from heat-killed L. plantarum Cell Biochem Funct 2015; 33: 89–96.
APOPTOSIS
EFFECT OF PROTEIN OF
L67 led to cleaved PARP, which is a nuclear apoptotic marker (Figure 5). Mitochondria are closely related to intracellular ROS production and mobilization of calcium. Induction of the mitochondrial (intrinsic) cell death pathway was confirmed by caspase-8-mediated cleavage of Bid to the t-Bid protein. The translocation and oligomerization of t-Bid on the mitochondrial membrane cause the release of cytochrome c.40 Therefore, when intracellular ROS and mobilization of calcium are activated by extracellular stimuli, apoptosis-related proteins (Bid and cytochrome c) induced the cleavage of Bid and released cytochrome c into the mitochondria. The activated apoptosis-related proteins subsequently transferred their signals to downstream signal factors to express apoptotic genes in the nucleus via caspases and PARP to initiate programmed cell death. In conclusion, the results indicate that the membrane proteins from heat-killed L. plantarum L67 stimulated increased amounts of apoptotic-related factors (intracellular ROS and calcium). The relative intensities of Bax and tBid increased, whereas cytochrome c, Bcl-2 caspase-8, caspase-9, caspase-3 and PARP signals decreased in the presence of proteins of heat-killed L. plantarum L67. Taken together, the membrane proteins isolated from L. plantarum L67 induced apoptosis through apoptotic factors and signalling in HT-29 cells. Several problems remain that deal with elucidating biophysicochemical characterization of the two proteins (12 and 15 kDa) using several biochemical analyses such as proteomics using 2D electrophoresis, Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and sequencing. Additionally, the precise mechanism of the apoptotic receptor (either the TNF-α receptor or Fas receptor) on the cell membrane remains to be elucidated in the near future.
CONFLICT OF INTEREST The authors declare no potential conflicts of interest with respect to this research, authorship and/or publication of this article.
ACKNOWLEDGEMENTS This work was completely financed by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea in 2014.
REFERENCES 1. Stein K, Borowicki A, Scharlau D, et al. Effects of synbiotic fermentation products on primary chemoprevention in human colon cells. J Nutr Biochem 2012; 23: 777–784. 2. Aleksandrova K, Pischon T, Jenab M, et al. Combined impact of healthy lifestyle factors on colorectal cancer: a large European cohort study. BMC Med 2014; 12: 168. Copyright © 2015 John Wiley & Sons, Ltd.
L.
PLANTARUM
L67
95
3. Tastsuta T, Scharwey M, Langer T. Mitochondrial lipid trafficking. Trends Cell Boil 2014; 24: 44–52. 4. Rasola A, Bernardi P. Mitochondrial permeability transition in Ca (2+) -dependent apoptosis and necrosis. Cell Calcium 2011; 50: 222–233. 5. Borriello SP, Hammoles WP, Holzapfel W, et al. Safety of probiotics that contain Lactobacilli or Bifidobacteria. Cli Infect Dis 2003; 36: 775–780. 6. Lankaputhra WEV, Shah NP. Antimutagenic properties of probiotic bacteria and of organic acids. Mutat Res 1998; 397: 169–182. 7. Reddy BS, Rivenson A. Inhibitory effect of Bifidobacterium longum on colon, mammolary, and liver carcinogenesis induced by 2-amino3-methylimidazo[4, 5-f]quinoline, a food mutagen. Cancer Res 1993; 53: 3914–3918. 8. Brady LJ, Gallaher DD, Busta FF. The role of probiotic cultures in the prevention of colon cancer. J Nutr 2000; 130: 410–414. 9. Burns AJ, Rowland IR. Anti-carcinogenicity of probiotics and prebiotics. Curr Issues Intest Microbiol 2000; 1: 13–24. 10. Andersson U, Bränning C, Ahrné S, et al. Probiotics lower plasma glucose in the high-fat fed C57BL/6J mouse. Benefical microbes 2010; 1: 189–196. 11. Kassayová M, Bobrov N, Strojný L, Kisková T, et al. Preventive effects of probiotic bacteria Lactobacillus plantarum and dietary fiber in chemically-induced mammary carcinogenesis. Anticancer Res 2014; 34: 4969–4975. 12. Giardina S, Scilironi C, Michelotti A, et al. In vitro anti-inflammatory activity of selected oxalate-degrading probiotic bacteria: potential applications in the prevention and treatment of hyperoxaluria. J Food Sci 2014; 79: 384–390. 13. Li S, Zhao Y, Zhang L, et al. Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods. Food Chem 2012; 135: 1914–1919. 14. Song S, Bae DW, Lim K, et al. Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing. Int J Food Microbiol 2014; 18: 135–143. 15. Rosenthal RS, Dziarski R. Isolation of peptidoglycan and soluble epetidoglycan fragments. Methods Enzymol 1994; 235: 253–285. 16. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275. 17. Mossmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55–63. 18. Bergmeyer HU, Bernt E. Lactate dehydrogenase. In Methods of enzymatic analysis 2nd edition. Bergmeyer HU (ed.) Academic Press: New York, 1974; 574–579. 19. Kimata M, Shichijo M, Miura T, Serizawa I, et al. Ca2+ and protein kinase C signaling for histamine and sulfidoleukotrienes released from human cultured mast cells. Biocheml Biophl Res Co 1999; 257: 895–900. 20. Patton WF, Dhanak MR, Jacobson BS. Differential partitioning of plasma membrane proteins into the triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution. J Cell Sci 1989; 92: 85–91. 21. Oh PS, Lim KT. Blocking of intracellular ROS production by phytoglycoprotein (30 kDa) causes anti-proliferation in bisphenol Astimulated Chang liver cells. J Appl Toxicol 2008; 28: 749–758. 22. Asha, Gayathri D. Synergistic impact of Lactobacillus fermentum, Lactobacillus plantarum and vincristine on 1,2-dimethylhydrazineinduced colorectal carcinogenesis in mice. Exp Ther Med 2012; 3: 1049–1054. 23. Hsu YL, Kuo PL, Lin CC. Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells. J Pharmacol Exp Ther 2005; 313: 333–344. 24. Mohamad N, Gutiérrez A, Núñez M, et al. Mitochondrial apoptotic pathways. Biocell 2005; 29: 149–161. 25. Brunelle JK, Chandel NS. Oxygen deprivation induced cell death: an update. Apoptosis 2002; 7: 475–482. 26. Csordás G, Várnai P, Golenár T, et al. Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface. Mol Cell 2010; 39: 121–132. Cell Biochem Funct 2015; 33: 89–96.
96
s. song
27. Festjens N, Vanden Berghe T, Vandenabeele P. Necrosis, a wellorchestrated form of cell demise: signalling cascades, important mediators and concomitant immolune response. Biochim Biophys Acta 2006; 1757: 1371–1387. 28. Gearhart TJ, Bouchard MJ. Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes. J Virol 2010; 84: 2675–2686. 29. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calciumapoptosis link. Nature reviews. Mol Cell Biol 2003; 4: 552–565. 30. Patterson RL, van Rossum DB, Nikolaidis N, Gill DL, Snyder SH. Phospholipase C-gamma: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci 2005; 30: 688–697. 31. Paradies G, Petrosillo G, Paradies V, Ruggiero FM. Role of cardiolipin peroxidation and Ca2+ in mitochondrial dysfunction and disease. Cell Calcium 2009; 45: 643–650. 32. Li-Ping Z, Da-Lei Z, Jian H, et al. Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways. Reprod Biol Endocrinol 2010; 19: 8–66. 33. Naumann I, Kappler R, von Schweinitz D, et al. Bortezomib primes neuroblastoma cells for TRAIL-induced apoptosis by linking the death receptor to the mitochondrial pathway. Clin Cancer Res 2011; 17: 3204–3218.
Copyright © 2015 John Wiley & Sons, Ltd.
ET AL.
34. Carifoli E. Calcium-mediated cellular signals: a story of failures. Trends Biochem Sci 2004; 9: 371–379. 35. Zhang S, Nie S, Huang D, Huang J, Feng Y, Xie M. Ganoderma atrum polysaccharide evokes antitumor activity via cAMP-PKA mediated ap2+ optotic pathway and down-regulation of Ca /PKC signal pathway. Food Chem Toxicol 2014; 68: 239–246. 36. Kantari C, Walczak H. Caspase-8 and bid: caught in the act between death receptors and mitochondria. Biochim Biophys Acta 1813; 2011: 558–563. 37. Song G, Chen GG, Hu T, Lai PB. Bid stands at the crossroad of stress-response pathways. Curr Cancer Drug Targets 2010; 10: 584–592. 38. Shore GC, Nguyen M. Bcl-2 proteins and apoptosis: choose your partner. Cell 2008; 135: 1004–1006. 39. Hao Z, Duncan GS, Chang CC, Elia A, et al. Specific ablation of the apoptotic functions of cytochrome c reveals a differential requirement for cytochrome c and Apaf-1 in apoptosis. Cell 2005; 121: 579–591. 40. Kim BM, Choi YJ, Han Y, et al. N, N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells. Toxicol Appl Pharmacol 2009; 239: 87–97.
Cell Biochem Funct 2015; 33: 89–96.
