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Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2
peptides 28 (2007) 1344–1350
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2 C.L. Wang a, T.B. Ng b,*, F. Yuan a, Z.K. Liu, F. Liu a,c a
Department of Microbiology, College of Life Science, Nankai University, Tianjin, China Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China c Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin, China b
article info
abstract
Article history:
A new cyclic lipopeptide (CLP) purified from Bacillus subtilis natto T-2 dose dependently
Received 27 March 2007
inhibited growth in human leukemia K562 cells. The results of fluorescent staining indicated
Received in revised form
that CLP brought about apoptosis in K562 cells. Flow cytometric analysis also demonstrated
7 June 2007
that CLP caused dose-dependent apoptosis of K562 cells through cell arrest at G1 phase.
Accepted 7 June 2007
Western blotting revealed that CLP-induced apoptosis in K562 cells was associated with
Published on line 20 June 2007
caspase-3 and poly(ADP-ribose)polymerase (PARP) protein. It is estimated that CLP inhibited proliferation in K562 cells by inducing apoptosis.
Keywords:
# 2007 Published by Elsevier Inc.
Apoptosis Human leukemia K562 cells Cyclic lipopeptide (CLP) Anti-tumor
1.
Introduction
Apoptosis, which is a major way of programmed cell death, plays an important role in the regulation of tissue development and homeostasis [10,21,22,24]. Therefore, induction of apoptotic cell death is a promising emerging strategy for prevention and treatment of cancer. In recent years, attention has been paid to natural products with potential cancer inhibiting effect [6]. The traditional Japanese soy food, ‘‘natto’’, is produced by fermenting steamed soybeans with the Gram-positive bacterium, Bacillus subtilis (natto) (formerly designated as B. natto), B. natto cells are safe to humans under normal host conditions [19,30]. Among the Bacillus genus, B. subtilis produces a broad spectrum of bioactive lipopeptides with a great potential for biotechnological and biopharmaceutical applications * Corresponding author. Tel.: +852 2609 6872; fax: +852 2603 5123. E-mail address: [email protected] (T.B. Ng). 0196-9781/$ – see front matter # 2007 Published by Elsevier Inc. doi:10.1016/j.peptides.2007.06.014
[3,23,26,31,33]. The characteristic structural element in lipopeptides is a specific fatty acid, which is combined with an amino acid moiety. As a consequence of this amphiphilic structure, lipopeptides have various interesting biological properties. They exhibit antifungal properties, moderate antibacterial and hemolytic properties, induce the formation of ion channels in lipid bilayer membranes, and exhibit antitumor and anti-viral activities [11,15,18,28,32]. Although some studies have tested lipopeptides for potential anti-tumor effects [29,32], the mode of action of lipopeptides has not been elucidated in detail, and there is no report on the effect of CLP purified from B. natto on K562 cells. Thus, the aim of the present investigation was to evaluate the growth inhibitory and apoptosis-inducing activities of the cyclic lipopeptide (CLP) purified from B. natto in human leukemia K562 cells with special emphasis on its mode of action.
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peptides 28 (2007) 1344–1350
2.
Materials and methods
2.1.
Materials
Human leukemia K562 cell line was obtained from Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China), PI and Hoechst 33342 dimethylsulfoxide (DMSO), and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co., antibodies for PARP, caspase-3, p21, p23, and Cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
2.2.
Isolation and HPLC MS/MS analysis of CLP
B. natto T-2 was isolated from a traditional fermented food natto in Japan and identified using standard morphological and biochemical tests [4]. Crude CLP was prepared from culture supernatants of B. natto T-2 by acid precipitation, extraction with methanol, and charcoal treatment as previously described [30]. Crude CLP was applied to a Pharmadex LH 20 column (10 mm 250 mm) and eluted with sodium phosphate buffer (pH 7.4) at a flow rate 0.5 ml/min (with UV detection at 210 nm). Three peaks (Fig. 1) were detected and the cytotoxic activity of each fraction was assayed using the MTT dye method. The third peak with strong cytotoxic activity was condensed, desalted, and the product was used as CLP sample in the following cell experiments. Peak 3 was further purified by HPLC on a Hydrosphere C18 column (5 mm 250 mm) (Waters, USA), using as the mobile phase 20% acetonitrile/ 0.1% trifluoroacetic acid. The flow rate was 0.5 ml/min and the eluate was monitored by UV absorption at 210 nm. Two peaks were detected (Fig. 2), peak 5 with strong cytotoxic activity was collected, condensed, and the product was used as the purified CLP. The amino acid sequence of the purified CLP was checked by Macromass Quattro Ultima Pt (Macro, USA), the capillary voltage and collision pressure were 3000 V and 2.5 104 Pa, respectively.
2.3.
Cell culture
Human leukemia K562 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum,
Fig. 2 – HPLC chromatograms of CLPs.
100 U/ml penicillin and 100 mg/ml streptomycin in 25 cm2 culture flasks. Human lung fibroblasts were routinely grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ ml streptomycin in 25 cm2 culture flasks. All the cells were cultured in a humidified atmosphere with 5% CO2 at 37 8C.
2.4.
Cell viability studies (MTT)
Viable cells were detected using the MTT dye, which forms blue formazan crystals that are reduced by mitochondrial dehydrogenase present in living cells. K562 cells and HLF cells were suspended at a final concentration of 105 cells/ml and seeded in 96-microwell flat-bottom plates. The cells were treated with CLP at varying concentrations (2, 4, 8, 16, 32, 64 mg/ml). After treatment, MTT (5 mg/ml) was added, and the 96-well plate was vibrated in a micro-vibrator for 4 h. The precipitated formazan was dissolved in DMSO, and the optical density of each well was measured at 570 nm with an automated plate reader (Sunrise Co. Ltd.). The treated groups were compared with control groups in the absence of CLP. The growth inhibitory ratio of CLP was calculated according to the following equation:
Inhibitory ratio of growth ð%Þ ¼
AB 100 A
where A is the average OD of control group, and B is the average OD of treated group.
2.5.
Fig. 1 – Elution profile of CLPs from Pharmadex LH 20 gel filtration column.
Fluorescent staining of nuclei of K562 cells
Hoechst 33342 staining was carried out as previously described [35]. Briefly, K562 cells in an exponential growth phase were seeded in 24-well culture plates at a final concentration of 105 per well. The cells were treated with CLP for 24 h. After treatment, the cells were washed twice with PBS, and were fixed with methanol (MeOH), acetic acid (HAc) (3:1, v/v) for 10 min at 4 8C. Cells were stained with Hoechst 33342 (10 mg/ml) for 20 min in the dark, and were then observed under a fluorescence microscope (Olympus BX41, Japan) in less than 15 min.
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2.6.
peptides 28 (2007) 1344–1350
Caspase-3 activity determination
K562 cells were plated in 12 well plates at a density of 5 105 cells/well and cultured for 24 h. Then cells were treated with CLP, and the 96-well plate was vibrated in a microvibrator for 12 h. About 5 106 cells were lysed in lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 10 mM Tris–HCl, pH 8.0, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin) for 30 min at 4 8C followed by centrifugation at 10,000 rpm for 30 min. Reaction mixtures (50 ml) with fluorogenic report substrate peptides specific for caspase-3 (fluorogenic report substrated: DEVD-AFC, 200 mM) was incubated at 37 8C with cytosolic extracts in reaction buffer (100 mM HEPES, 10% sucrose, 10 mM DTT, 0.1% 3-[(3-chloamidopropyl)dimethylammonio]1-propanesulfonate). Fluorescence was determined after 2 h (excitation wavelength, 400 nm; emission wavelength, 505 nm) with a fluorescence plate reader (Fluoroskan Ascent; Labsystems). In order to examine whether or not caspase-3 activation is involved in apoptosis triggered by CLP, K562 cells (5 106 cells) were treated with cell-permeable broad-spectrum caspase-3 inhibitor (Z-VAD-fmk, 200 mM) 3 h prior to CLP treatment. Then caspase-3 activity was determined as described above.
2.7.
Cell-cycle analysis by flow cytometry
K562 cells were incubated with CLP at varying concentrations (8, 16, 32 mg/ml) for different durations. After treatment, the DNA content and the cell cycle of K562 cells were determined by cytometry based on a previously described method [7]. Briefly, the cells were placed at a density of 106 cells/well in a 6well plate. Cells were collected, washed twice with ice-cold PBS buffer (pH 7.4), fixed with 70% ice ethanol at 20 8C for 2 h, and then stained with propidium iodide (PI) (100 mg/ml). The stained cells were then transferred to flow tubes by passing through a nylon mesh with a pore size of 40 mm. Flow cytometric analysis was performed using a flow cytometer (Beckman Coulter, Epic XL MCL). Apoptotic cells were determined by their hypochromic sub-diploid staining profiles. The distribution of cells in the different cell-cycle phases was analyzed from the DNA histogram with MODFIT and Cell Quest software.
2.8.
3.
Results
3.1.
Isolation and HPLC MS/MS analysis of CLP
It is known that either a single or a mixture of CLP with the defined amino acid composition can be supplied to the mass spectrometry equipped with ESI (electrospray ionization) to determine the amino acid sequence of the peptide. The target sodium-ionized molecule was selected by the first quadrupole mass spectrometry and then fragmented. The sodium-ionized fragments produced were analyzed and recorded by the second time of flight mass spectrometry. The peaks with a mass to charge ratio smaller than those of ionized molecules represent the sodium-ionized fragments. The analysis of those peaks would give important information about the connecting relationship of the peptide. The difference between any two peaks is the mass of the lost fragments. The difference of peaks can be used to determine the connection of some amino acid residues in a peptide chain, if the difference is the same as the mass of an amino acid residue [27,34]. The mass spectrum of the purified CLP is shown in Fig. 3A and B. The peaks 1071.96 ! 959.73 ! 860.82 ! 746.02 ! 634.12 ! 502.28 ! 390.02 for the purified CLP suggested the connection of amino acid residues in the form of Leu-Val-LeuAsp-Glu-Leu. Compared with others CLPs, they are different in the sequence of amino acid residues, indicating that the purified CLP is a new CLP isolated from B. natto.
Western blotting analysis
K562 cells were resuspended in cell lysis buffer (1% Triton X-100, 0.015 M NaCl, 10 mM Tris–HCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml of each of leupeptin and pepstatin A) and then incubated on ice for 20 min. The cell lysates were centrifuged at 10,000 rpm for 20 min at 4 8C, and the supernatant was mixed with a one-fourth volume of 4 SDS sample buffer, boiled for 5 min, and then separated through a 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred to nylon membranes using electrophoretic transfer. The membranes were blocked in 5% dry milk (1 h), rinsed, and incubated with secondary and primary antibodies (diluted 1:5000) in TBS for 1 h at room temperature. Finally each protein was detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, USA).
Fig. 3 – (A) ESI mass spectrum of purified CLP. (B) Mass spectrum of the purified CLP at m/z 1072.
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Table 1 – The effects of CLP on the caspase-3 activity of K562 cells (n = 3) Group
Caspase-3 activity (a.u.)
Control 8 mg/ml CLP 8 mg/ml CLP + Z-VAD-fmk 16 mg/ml CLP 16 mg/ml CLP + Z-VAD-fmk 32 mg/ml CLP 32 mg/ml CLP + Z-VAD-fmk
4.7 0.8 8.9 1.2 4.6 0.9 14.3 1.9 4.2 0.6 19.2 1.3 4.4 0.7
K562 cells were treated with CLP and with or without the cellpermeable broad-spectrum caspase inhibitor (Z-VAD-fmk, 200 mM) prior to CLP treatment. Caspase-3 activity was assayed by using fluorogenic peptide substrates. Data are means S.D. of three independent experiments.
Fig. 4 – Cytotoxic effects of CLPs on K562 and HLF cells. The cells were treated with various concentrations (2, 4, 8, 16, 32, 64 mg/ml) of CLPs for 24, 36, and 48 h. Cell viability was determined by MTT assay and was expressed as the mean W S.D. of three separate experiments n = 3 each of the three experiments (~) K562 cells treated for 48 h; (&) K562 cells treated for 36 h; (^) K562 cells treated for 24 h; (–) fibroblasts treated for 48 h; (*) fibroblasts treated for 36 h; (*) fibroblasts treated for 24 h.
microscopy was utilized to observe apoptosis by changes in cellular morphology, including cell shrinkage, condensation and fragmentation of nuclei, all of which are indicative of apoptosis. As shown in Fig. 5A, there were mainly living cells and only very few apoptotic nuclei detected in the control. However, apoptotic cells with shrinkage, condensed or fragmented nuclei were observed 24 h after exposure to CLP (Fig. 5B), which indicated that CLP-induced apoptosis in K562 cells.
3.4. 3.2.
The MTT assay determined the metabolic activity of the cells and reflects its growth potential, which is a balance between proliferation and death. In order to establish the concentration of CLP necessary to produce inhibitory effects on K562 cell proliferation and review the effect on HLF cells which come from normal tissue, K562 and HLF cells were incubated with CLP at concentrations varying from 2 to 64 mg/ml, as shown in Fig. 4. CLP inhibited growth of K562 cells in a dose- and timedependent manner. In contrast, the viability of HLF cells was not significantly affected by CLP when its concentration was less than 32 mg/ml.
3.3.
Caspase-3 activity determination
Inhibition of cell growth
Morphological changes of K562 cells induced by CLPs
Morphological changes provide the most direct criteria for recognizing the apoptotic process. Therefore, fluorescence
In order to examine whether caspase-3 activation is involved in apoptosis triggered by CLP. As shown in Table 1, CLP increased caspase-3 activity and caspase-3 inhibitor Z-VADfmk decreased caspase-3 activity. The results indicated that activation of caspase-3 contributes to CLP-induced apoptosis in K562 cells.
3.5.
Cell-cycle analysis by flow cytometry
Cell-cycle phase distribution was analyzed by flow cytometry with PI staining. The percentage of cells in G1, S, and G2/M phase, respectively was calculated using Multicycle software and is shown in Table 2. The results are presented in Fig. 6 and Table 2 showed a significant accumulation of cells in the G1 phase (36.5–57.6%), and that the number of apoptotic cells increased with the concentration of CLP.
Fig. 5 – Nuclear morphology of K562 cells. (A) Untreated control cells and (B) cells treated with 16 mg/ml CLPs for24 h. The representative cells were counted as apoptosis in fluorescence microscopy with 200T magnification. Results presented are representative of three independent experiments.
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Table 2 – Effect of lipopeptide on cell-cycle distribution of K562 cells (n = 3) Group A B C D
G1 (%)
S (%)
G2/M (%)
36.5 4.6 40.9 6.7 48.2 5.4 57.6 5.7
51.2 6.5 42.3 5.2 29.4 4.9* 15.1 4.0*
11.2 2.7 9.2 3.0 10.1 2.5* 9.1 2.7*
Apoptotic cells (%) 1.3 0.9 8.1 1.9 12.5 2.9 20.6 2.3*
(A) Cell-cycle analysis of control K562 cells for 24 h; (B) cell-cycle analysis of K562 cells following treatment with8 mg/ml CLP for 24 h; (C) cellcycle analysis of K562 cells following treatment with 16 mg/ml CLP for 24 h; (D) cell-cycle analysis of K562 cells following treatment with 32 mg/ ml CLP for 24 h. Data are means S.D. of three independent experiments. *P < 0.05 in groups by Student’s test.
3.6. Effect of CLPs on caspases-3 and apoptosis-related proteins The above results clearly indicated that CLP-induced apoptosis of K562 cells. Apoptosis is modulated by different proapoptotic and anti-apoptotic factors. Caspases-3 plays a central role in the apoptosis signaling pathway and contributes to the overall apoptotic morphology by cleavage of various cellular substrates [1,5]. Proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) which is a nuclear
enzyme involved in DNA repair process and its cleavage by caspase-3 during apoptosis disables DNA repair. The CDKIs regulate the progression of cells in the G1 phase of the cell cycle and Kip1/p27 and Cip/p21cause a blockade of G1 to S transition, thereby participating in cell-cycle regulation. As shown in Fig. 7, CLP increased the activity of caspases-3 in a concentration-dependent manner. In a time–course analysis, the activity of caspase-3 was significantly increased compared to control samples, and continued to increase after 6 h of exposure, and at 9 h, the activity of caspase-3 declined
Fig. 6 – Effect of CLP on cell-cycle distribution of K562 cells (n = 3). (A) Cell-cycle analysis of K562 cells following treatment in the absence of CLP for 24 h; (B) cell-cycle analysis of K562 cells following treatment with 8 mg/ml CLP for 24 h; (C) cell-cycle analysis of K562 cells following treatment with 16 mg/ml CLP for 24 h; (D) cell-cycle analysis of K562 cells following treatment with 32 mg/ml CLP for 24 h.
peptides 28 (2007) 1344–1350
Fig. 7 – Effect of CLP on caspase-3 and proteins in K562 cells. (A) Treatment with CLP at various concentrations for 9 h and (B) treatment with CLP at 32 mg/ml. The data shown are representative of three independent experiments.
compared to the activity at 6 h, but was still higher than that of the control. In a similar dose- and time-dependent response, CLP caused the proteolytic cleavage of PARP with an accumulation of 89 kDa fragments and disappearance of full-length 116 kDa protein. Moreover, CLP-induced expression of Cip1/p21 and Kip/p27, and a marked reduction in the expression of Cyclin D1 which plays a crucial role in G1 phase progression was observed in K562 cells.
4.
Discussion
In recent years, studies of the anti-tumor activities of lipoproteins have been of particular interest. Studies by Kameda’s group proved that lipoproteins have anti-tumor activity. This is the first evidence that lipoproteins extracted from B. natto 3321 can induce apoptosis of cancer cells [8]. In Nakahara’s study, exposure of B16 cells to lipoproteins resulted in the condensation of the chromatin, DNA fragmentation and sub-G1 arrest [36]. Sudo et al. [29] examined lipoproteins for their ability to inhibit growth and induce differentiation of HL60 human promyelocytic leukemia cells. In addition, Wakamatsu et al. [32] discovered that lipoproteins
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induces neuronal differentiation in PC12 cells and provided the groundwork for the use of microbial extracellular lipoproteins as novel reagents for the treatment of cancer cells. Although lipoproteins have been tested for potential anti-tumor effects, the mechanism is not clear. In this study, a new CLP was isolated as the effective compound from B. subtilis T-2 and an attempt was made to identify the mechanism of its anti-tumor activity. Apoptosis is a process of cell suicide, characterized by specific morphological changes such as condensation of chromatin, nuclear fragmentation, blebbing of the plasma membrane, and the presence of apoptotic bodies [9,14,22]. The results of this study revealed that CLP-induced apoptosis in K562 cells. The cell cycle is an orderly sequence of events that occurs throughout development, from stem cells to fully differentiated cells, alternating between dividing (mitosis) and what appears to be resting (inter-phase) states. During progression of the cell cycle, cellular processes such as cell growth, DNA replication and mitosis are all coordinated, and any failure in this coordination restricts entry into the next phase of the cell cycle. Flow cytometry assesses progression of the cell cycle and discriminates between cells in apoptosis and those in necrosis, resulting from the lethal effect of CLP. As shown in Fig. 6, it was confirmed that CLP caused dose-dependent apoptosis of K562 cells through cell arrest in G1 phase. The results in Fig. 7 suggest that CLP might be implicated in the disruption of cancer cell progression through increasing expression of CDKIs together with decreasing expression of Cyclin D1. Apoptosis is a form of cell death that is tightly regulated by a number of gene products that either promote or block cell death at different stages of the cell cycle. The activation of caspase family members is a critical component of the apoptotic machinery [12,25,27]. Caspase-3 is one of the key proteases responsible for the cleavage and inactivation of PARP [16,20]. PARP is involved in DNA repair and is important for the maintenance of cell viability [2,17]. Moreover, PARP cleavage during caspase-3 activation facilitates cellular disassembly and serves as a marker of apoptosis [13]. As shown in Fig. 7, caspase-3 and PARP were cleaved by CLP in a time- and concentration-dependent manner. These results suggest that apoptosis in K562 cells induced by CLPs is closely associated with the activation of caspase-3 and PARP cleavage. In conclusion, the present work showed that the purified CLP from B. natto exerted a cytotoxic action on K562 cells, and the cytotoxicity involves mainly induction of apoptosis which is closely associated with cell arrest at G1 phase and activation of caspase-3. It is hoped that results from these studies will permit the identification of key molecular targets, which may further assist in the elucidation of the mechanism of action of the purified CLP, along with facilitating the development of this highly effective anti-cancer therapeutic agent. Further studies are underway to elucidate the mechanistic nature of the differential apoptosis induced by the purified CLP.
Acknowledgments We thank support of this project by funds from National Natural Science Foundation (grant no. 30570051) and Tianjin Natural Science Foundation (grant no. 06YFJMJC07500).
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The secretarial assistance of Ms Kathy Lau is greatly appreciated.
references
[1] Baell JB, Huang DC. Prospects for targeting the Bcl-2 family of proteins to develop novel cytotoxic drugs. Biochem Pharmacol 2002;64:851–63. [2] Boulares AH, Zoltoski AJ, Smulson ME. Roles of DNA fragmentation factor and polymerase in an amplification phase of tumor necrosis factor-induced apoptosis. J Biol Chem 2001;276:38185–92. [3] Cameotra SS, Makkar RS. Recent applications of biosurfactants as biological and immunological molecules. Ecol Ind Microbiol 2004;7:262–6. [4] Cappuccino JG, Sherman N. Microbiology—a laboratory manual, 4th ed., Harlow: Addison-Wesley; 1999. p. 183–6. [5] Corbiere C, Liagre B, Terro F, Beneytout JL. Induction of antiproliferative effect by diosgenia through activation of p53, release of apoptosis-inducing factor (AIF) and modulation of capase-3 activity in different human cancer cells. Cell Res 2004;14:188–96. [6] Igor NS, Li SM, Colby JL, Ho CT. Polymethoxylated flavonea induce Ca2+-mediated apoptosis in breast cancer cells. Life Sci 2006;80:245–63. [7] Jakopec S, Dubravicic K, Polanc S, Kosmarlj J, Osmak M. Diazene Jk-279 induces apoptosis-like cell death in human cervical carcinoma cells. Toxicol In Vitro 2006;20:217–26. [8] Kameda Y, Oira S, Matsui K, Kanatomo S, Hase T. Antitumor activity of Bacillus natto. V. Isolation and characterization of surfactin in the culture medium of Bacillus natto KMD 2311. Chem Pharm Bull (Tokyo) 1974;22:938–44. [9] Kaufmanm SH, Hengarther MO. Programmed cell death: alive and well in the new millennium. Trends Cell Biol 2001;262:526–34. [10] Kalai M, Lamkanfi M, Denecker G, Boogmans M, Lippens S, Meeus A. Regulation of the expression and processing of caspase-12. J Cell Biol 2003;162:457–67. [11] Kishore D, Ashis KM. Assessment of mosquito larvicidal potency of cyclic lipopeptides produced by Bacillus subtilis strains. Acta Trop 2006;97:168–73. [12] Klein G. Cancer, apoptosis, and non-immune surveillance. Cell Death Differ 2004;11:13–7. [13] Kolenko VM, Uzzo RG, Bukowski R, Finke JH. Caspasedependent and independent death pathways in cancer therapy. Apoptosis 2000;5:17–20. [14] Lee EO, Kwon BM, Song GY. Heyneanol A induces apoptosis via cytochrome c release and caspase activation in human leukemiac U937 cells. Life Sci 2004;74:2313–26. [15] Mohammad SR, Takashi A, Makoto S. Biofilm fermentation of iturin A by a recombinant strain of Bacillus subtilis 168. J Biotechnol 2007;127:503–7. [16] Monasterio A, Urdaci MC, Pinchuk IV, Lopez MN, Martinezirujo JJ. Flavonoids induce apoptosis in human leukemia U937 cells through caspase- and caspase-calpaindependent pathways. Nutr Cancer 2004;50:90–100. [17] Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, et al. Caspase-12 mediates end-oplasmic-reticulumspecific apoptosis and cytotoxicity by amiloid. Science 2004;3:98–103.
[18] Nielsen TH, Sorensen J. Production of cyclic lipopeptides by Pseudomonas fluorescens strains in bulk soil and in the sugar beet rhizosphere. Appl Environ Microbiol 2003;69:861–8. [19] Oggioni MR, Galieni P, Bigazzi C, Pozzi G, Valensin PE. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J Clin Microbiol 1998;36:325–6. [20] Oommen S, John AR, Srinivas G, Karunagaran D. Allicin (from garlic) induces caspase-mediated apoptosis in cancer cells. J Pharmacol 2004;485:97–103. [21] Reed J. Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med 2001;7:314–9. [22] Reed J. Apoptosis-targeted therapies for cancer. Cancer Cells 2003;3:17–25. [23] Roich DV, Pauli GR, Vater JC. Antimycoplasma properties and application in cell culture, a lipopeptide antibiotic from Bacillus subtilis. Microbiol Jpn 1997;63:44–9. [24] Sergeev IN. Calcium signaling in cancer and vitamin D. J Steroid Biochem Mol Biol 2005;97:145–51. [25] Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 2002;9:459–70. [26] Singh P, Cameotra SS. Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 2004;22:142–6. [27] Srinivas G, Anto RJ, Srinivas P, Lakshmi V, Karunagaran D. Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose)polymerase cleavage and activation of caspase-9. Eur J Pharmacol 2003;473:117–25. [28] Stanislav GB, Tatiana AR, Nkita BP. A novel lipopeptide, an inhibitor of bacterial adhesionfrom the thermophilic and halotolerant subsurface Bacillus licheniformis strain 603. Biochim Biophys Acta 2003;1634:107–15. [29] Sudo T, Zhao X, Wakamatsu Y, Shibahara M, Nakahara T. Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids. Cytotechnology 2000;33:259–64. [30] Tomohiro H, Rieko H, Shizue S, Akio A, Kan K. Cytokine responses of human intestinal epithelial-like Caco-2 cells to the nonpathogenic bacterium Bacillus subtilis. J Food Microbiol 2003;82:255–64. [31] Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 2002;68:6210–9. [32] Wakamatsu Y, Zhao X, Jin C, Day N, Shibahara M, Nomura N, et al. Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERKrelated signal cascade. Eur J Biochem 2001;268:374–83. [33] Bessler WG, Mittenbuhler K, Escher V, Huber M. Lipopeptide adjuvants in combination treatment. Int Immunopharmacol 2003;3:1217–24. [34] Yang SZ, Wei DZ, Mu BZ. Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism. J Biochem Biophys Methods 2006;68:69–74. [35] Ye CL, Qian F, Wei DZ, Lu YH, Liu JW. Induction of apoptosis in K562 human leukemia cells by 20 ,40 -dihydroxy-60 methoxy-30 ,50 -dimethylchalcone. Leukemia Res 2005;29:887–92. [36] Zhao X, Wakamatsu Y, Shibahara M, Nakahara N, Geltinger C, Nakahara T, et al. Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation in PC12 cells through an ERK-related signal cascade. Eur J Biochem 2001;268:374–83.
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journal homepage: www.elsevier.com/locate/peptides
Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2 C.L. Wang a, T.B. Ng b,*, F. Yuan a, Z.K. Liu, F. Liu a,c a
Department of Microbiology, College of Life Science, Nankai University, Tianjin, China Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China c Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin, China b
article info
abstract
Article history:
A new cyclic lipopeptide (CLP) purified from Bacillus subtilis natto T-2 dose dependently
Received 27 March 2007
inhibited growth in human leukemia K562 cells. The results of fluorescent staining indicated
Received in revised form
that CLP brought about apoptosis in K562 cells. Flow cytometric analysis also demonstrated
7 June 2007
that CLP caused dose-dependent apoptosis of K562 cells through cell arrest at G1 phase.
Accepted 7 June 2007
Western blotting revealed that CLP-induced apoptosis in K562 cells was associated with
Published on line 20 June 2007
caspase-3 and poly(ADP-ribose)polymerase (PARP) protein. It is estimated that CLP inhibited proliferation in K562 cells by inducing apoptosis.
Keywords:
# 2007 Published by Elsevier Inc.
Apoptosis Human leukemia K562 cells Cyclic lipopeptide (CLP) Anti-tumor
1.
Introduction
Apoptosis, which is a major way of programmed cell death, plays an important role in the regulation of tissue development and homeostasis [10,21,22,24]. Therefore, induction of apoptotic cell death is a promising emerging strategy for prevention and treatment of cancer. In recent years, attention has been paid to natural products with potential cancer inhibiting effect [6]. The traditional Japanese soy food, ‘‘natto’’, is produced by fermenting steamed soybeans with the Gram-positive bacterium, Bacillus subtilis (natto) (formerly designated as B. natto), B. natto cells are safe to humans under normal host conditions [19,30]. Among the Bacillus genus, B. subtilis produces a broad spectrum of bioactive lipopeptides with a great potential for biotechnological and biopharmaceutical applications * Corresponding author. Tel.: +852 2609 6872; fax: +852 2603 5123. E-mail address: [email protected] (T.B. Ng). 0196-9781/$ – see front matter # 2007 Published by Elsevier Inc. doi:10.1016/j.peptides.2007.06.014
[3,23,26,31,33]. The characteristic structural element in lipopeptides is a specific fatty acid, which is combined with an amino acid moiety. As a consequence of this amphiphilic structure, lipopeptides have various interesting biological properties. They exhibit antifungal properties, moderate antibacterial and hemolytic properties, induce the formation of ion channels in lipid bilayer membranes, and exhibit antitumor and anti-viral activities [11,15,18,28,32]. Although some studies have tested lipopeptides for potential anti-tumor effects [29,32], the mode of action of lipopeptides has not been elucidated in detail, and there is no report on the effect of CLP purified from B. natto on K562 cells. Thus, the aim of the present investigation was to evaluate the growth inhibitory and apoptosis-inducing activities of the cyclic lipopeptide (CLP) purified from B. natto in human leukemia K562 cells with special emphasis on its mode of action.
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2.
Materials and methods
2.1.
Materials
Human leukemia K562 cell line was obtained from Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China), PI and Hoechst 33342 dimethylsulfoxide (DMSO), and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co., antibodies for PARP, caspase-3, p21, p23, and Cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
2.2.
Isolation and HPLC MS/MS analysis of CLP
B. natto T-2 was isolated from a traditional fermented food natto in Japan and identified using standard morphological and biochemical tests [4]. Crude CLP was prepared from culture supernatants of B. natto T-2 by acid precipitation, extraction with methanol, and charcoal treatment as previously described [30]. Crude CLP was applied to a Pharmadex LH 20 column (10 mm 250 mm) and eluted with sodium phosphate buffer (pH 7.4) at a flow rate 0.5 ml/min (with UV detection at 210 nm). Three peaks (Fig. 1) were detected and the cytotoxic activity of each fraction was assayed using the MTT dye method. The third peak with strong cytotoxic activity was condensed, desalted, and the product was used as CLP sample in the following cell experiments. Peak 3 was further purified by HPLC on a Hydrosphere C18 column (5 mm 250 mm) (Waters, USA), using as the mobile phase 20% acetonitrile/ 0.1% trifluoroacetic acid. The flow rate was 0.5 ml/min and the eluate was monitored by UV absorption at 210 nm. Two peaks were detected (Fig. 2), peak 5 with strong cytotoxic activity was collected, condensed, and the product was used as the purified CLP. The amino acid sequence of the purified CLP was checked by Macromass Quattro Ultima Pt (Macro, USA), the capillary voltage and collision pressure were 3000 V and 2.5 104 Pa, respectively.
2.3.
Cell culture
Human leukemia K562 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum,
Fig. 2 – HPLC chromatograms of CLPs.
100 U/ml penicillin and 100 mg/ml streptomycin in 25 cm2 culture flasks. Human lung fibroblasts were routinely grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ ml streptomycin in 25 cm2 culture flasks. All the cells were cultured in a humidified atmosphere with 5% CO2 at 37 8C.
2.4.
Cell viability studies (MTT)
Viable cells were detected using the MTT dye, which forms blue formazan crystals that are reduced by mitochondrial dehydrogenase present in living cells. K562 cells and HLF cells were suspended at a final concentration of 105 cells/ml and seeded in 96-microwell flat-bottom plates. The cells were treated with CLP at varying concentrations (2, 4, 8, 16, 32, 64 mg/ml). After treatment, MTT (5 mg/ml) was added, and the 96-well plate was vibrated in a micro-vibrator for 4 h. The precipitated formazan was dissolved in DMSO, and the optical density of each well was measured at 570 nm with an automated plate reader (Sunrise Co. Ltd.). The treated groups were compared with control groups in the absence of CLP. The growth inhibitory ratio of CLP was calculated according to the following equation:
Inhibitory ratio of growth ð%Þ ¼
AB 100 A
where A is the average OD of control group, and B is the average OD of treated group.
2.5.
Fig. 1 – Elution profile of CLPs from Pharmadex LH 20 gel filtration column.
Fluorescent staining of nuclei of K562 cells
Hoechst 33342 staining was carried out as previously described [35]. Briefly, K562 cells in an exponential growth phase were seeded in 24-well culture plates at a final concentration of 105 per well. The cells were treated with CLP for 24 h. After treatment, the cells were washed twice with PBS, and were fixed with methanol (MeOH), acetic acid (HAc) (3:1, v/v) for 10 min at 4 8C. Cells were stained with Hoechst 33342 (10 mg/ml) for 20 min in the dark, and were then observed under a fluorescence microscope (Olympus BX41, Japan) in less than 15 min.
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2.6.
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Caspase-3 activity determination
K562 cells were plated in 12 well plates at a density of 5 105 cells/well and cultured for 24 h. Then cells were treated with CLP, and the 96-well plate was vibrated in a microvibrator for 12 h. About 5 106 cells were lysed in lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 10 mM Tris–HCl, pH 8.0, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin) for 30 min at 4 8C followed by centrifugation at 10,000 rpm for 30 min. Reaction mixtures (50 ml) with fluorogenic report substrate peptides specific for caspase-3 (fluorogenic report substrated: DEVD-AFC, 200 mM) was incubated at 37 8C with cytosolic extracts in reaction buffer (100 mM HEPES, 10% sucrose, 10 mM DTT, 0.1% 3-[(3-chloamidopropyl)dimethylammonio]1-propanesulfonate). Fluorescence was determined after 2 h (excitation wavelength, 400 nm; emission wavelength, 505 nm) with a fluorescence plate reader (Fluoroskan Ascent; Labsystems). In order to examine whether or not caspase-3 activation is involved in apoptosis triggered by CLP, K562 cells (5 106 cells) were treated with cell-permeable broad-spectrum caspase-3 inhibitor (Z-VAD-fmk, 200 mM) 3 h prior to CLP treatment. Then caspase-3 activity was determined as described above.
2.7.
Cell-cycle analysis by flow cytometry
K562 cells were incubated with CLP at varying concentrations (8, 16, 32 mg/ml) for different durations. After treatment, the DNA content and the cell cycle of K562 cells were determined by cytometry based on a previously described method [7]. Briefly, the cells were placed at a density of 106 cells/well in a 6well plate. Cells were collected, washed twice with ice-cold PBS buffer (pH 7.4), fixed with 70% ice ethanol at 20 8C for 2 h, and then stained with propidium iodide (PI) (100 mg/ml). The stained cells were then transferred to flow tubes by passing through a nylon mesh with a pore size of 40 mm. Flow cytometric analysis was performed using a flow cytometer (Beckman Coulter, Epic XL MCL). Apoptotic cells were determined by their hypochromic sub-diploid staining profiles. The distribution of cells in the different cell-cycle phases was analyzed from the DNA histogram with MODFIT and Cell Quest software.
2.8.
3.
Results
3.1.
Isolation and HPLC MS/MS analysis of CLP
It is known that either a single or a mixture of CLP with the defined amino acid composition can be supplied to the mass spectrometry equipped with ESI (electrospray ionization) to determine the amino acid sequence of the peptide. The target sodium-ionized molecule was selected by the first quadrupole mass spectrometry and then fragmented. The sodium-ionized fragments produced were analyzed and recorded by the second time of flight mass spectrometry. The peaks with a mass to charge ratio smaller than those of ionized molecules represent the sodium-ionized fragments. The analysis of those peaks would give important information about the connecting relationship of the peptide. The difference between any two peaks is the mass of the lost fragments. The difference of peaks can be used to determine the connection of some amino acid residues in a peptide chain, if the difference is the same as the mass of an amino acid residue [27,34]. The mass spectrum of the purified CLP is shown in Fig. 3A and B. The peaks 1071.96 ! 959.73 ! 860.82 ! 746.02 ! 634.12 ! 502.28 ! 390.02 for the purified CLP suggested the connection of amino acid residues in the form of Leu-Val-LeuAsp-Glu-Leu. Compared with others CLPs, they are different in the sequence of amino acid residues, indicating that the purified CLP is a new CLP isolated from B. natto.
Western blotting analysis
K562 cells were resuspended in cell lysis buffer (1% Triton X-100, 0.015 M NaCl, 10 mM Tris–HCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml of each of leupeptin and pepstatin A) and then incubated on ice for 20 min. The cell lysates were centrifuged at 10,000 rpm for 20 min at 4 8C, and the supernatant was mixed with a one-fourth volume of 4 SDS sample buffer, boiled for 5 min, and then separated through a 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred to nylon membranes using electrophoretic transfer. The membranes were blocked in 5% dry milk (1 h), rinsed, and incubated with secondary and primary antibodies (diluted 1:5000) in TBS for 1 h at room temperature. Finally each protein was detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, USA).
Fig. 3 – (A) ESI mass spectrum of purified CLP. (B) Mass spectrum of the purified CLP at m/z 1072.
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Table 1 – The effects of CLP on the caspase-3 activity of K562 cells (n = 3) Group
Caspase-3 activity (a.u.)
Control 8 mg/ml CLP 8 mg/ml CLP + Z-VAD-fmk 16 mg/ml CLP 16 mg/ml CLP + Z-VAD-fmk 32 mg/ml CLP 32 mg/ml CLP + Z-VAD-fmk
4.7 0.8 8.9 1.2 4.6 0.9 14.3 1.9 4.2 0.6 19.2 1.3 4.4 0.7
K562 cells were treated with CLP and with or without the cellpermeable broad-spectrum caspase inhibitor (Z-VAD-fmk, 200 mM) prior to CLP treatment. Caspase-3 activity was assayed by using fluorogenic peptide substrates. Data are means S.D. of three independent experiments.
Fig. 4 – Cytotoxic effects of CLPs on K562 and HLF cells. The cells were treated with various concentrations (2, 4, 8, 16, 32, 64 mg/ml) of CLPs for 24, 36, and 48 h. Cell viability was determined by MTT assay and was expressed as the mean W S.D. of three separate experiments n = 3 each of the three experiments (~) K562 cells treated for 48 h; (&) K562 cells treated for 36 h; (^) K562 cells treated for 24 h; (–) fibroblasts treated for 48 h; (*) fibroblasts treated for 36 h; (*) fibroblasts treated for 24 h.
microscopy was utilized to observe apoptosis by changes in cellular morphology, including cell shrinkage, condensation and fragmentation of nuclei, all of which are indicative of apoptosis. As shown in Fig. 5A, there were mainly living cells and only very few apoptotic nuclei detected in the control. However, apoptotic cells with shrinkage, condensed or fragmented nuclei were observed 24 h after exposure to CLP (Fig. 5B), which indicated that CLP-induced apoptosis in K562 cells.
3.4. 3.2.
The MTT assay determined the metabolic activity of the cells and reflects its growth potential, which is a balance between proliferation and death. In order to establish the concentration of CLP necessary to produce inhibitory effects on K562 cell proliferation and review the effect on HLF cells which come from normal tissue, K562 and HLF cells were incubated with CLP at concentrations varying from 2 to 64 mg/ml, as shown in Fig. 4. CLP inhibited growth of K562 cells in a dose- and timedependent manner. In contrast, the viability of HLF cells was not significantly affected by CLP when its concentration was less than 32 mg/ml.
3.3.
Caspase-3 activity determination
Inhibition of cell growth
Morphological changes of K562 cells induced by CLPs
Morphological changes provide the most direct criteria for recognizing the apoptotic process. Therefore, fluorescence
In order to examine whether caspase-3 activation is involved in apoptosis triggered by CLP. As shown in Table 1, CLP increased caspase-3 activity and caspase-3 inhibitor Z-VADfmk decreased caspase-3 activity. The results indicated that activation of caspase-3 contributes to CLP-induced apoptosis in K562 cells.
3.5.
Cell-cycle analysis by flow cytometry
Cell-cycle phase distribution was analyzed by flow cytometry with PI staining. The percentage of cells in G1, S, and G2/M phase, respectively was calculated using Multicycle software and is shown in Table 2. The results are presented in Fig. 6 and Table 2 showed a significant accumulation of cells in the G1 phase (36.5–57.6%), and that the number of apoptotic cells increased with the concentration of CLP.
Fig. 5 – Nuclear morphology of K562 cells. (A) Untreated control cells and (B) cells treated with 16 mg/ml CLPs for24 h. The representative cells were counted as apoptosis in fluorescence microscopy with 200T magnification. Results presented are representative of three independent experiments.
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Table 2 – Effect of lipopeptide on cell-cycle distribution of K562 cells (n = 3) Group A B C D
G1 (%)
S (%)
G2/M (%)
36.5 4.6 40.9 6.7 48.2 5.4 57.6 5.7
51.2 6.5 42.3 5.2 29.4 4.9* 15.1 4.0*
11.2 2.7 9.2 3.0 10.1 2.5* 9.1 2.7*
Apoptotic cells (%) 1.3 0.9 8.1 1.9 12.5 2.9 20.6 2.3*
(A) Cell-cycle analysis of control K562 cells for 24 h; (B) cell-cycle analysis of K562 cells following treatment with8 mg/ml CLP for 24 h; (C) cellcycle analysis of K562 cells following treatment with 16 mg/ml CLP for 24 h; (D) cell-cycle analysis of K562 cells following treatment with 32 mg/ ml CLP for 24 h. Data are means S.D. of three independent experiments. *P < 0.05 in groups by Student’s test.
3.6. Effect of CLPs on caspases-3 and apoptosis-related proteins The above results clearly indicated that CLP-induced apoptosis of K562 cells. Apoptosis is modulated by different proapoptotic and anti-apoptotic factors. Caspases-3 plays a central role in the apoptosis signaling pathway and contributes to the overall apoptotic morphology by cleavage of various cellular substrates [1,5]. Proteolytic cleavage of poly(ADP-ribose)polymerase (PARP) which is a nuclear
enzyme involved in DNA repair process and its cleavage by caspase-3 during apoptosis disables DNA repair. The CDKIs regulate the progression of cells in the G1 phase of the cell cycle and Kip1/p27 and Cip/p21cause a blockade of G1 to S transition, thereby participating in cell-cycle regulation. As shown in Fig. 7, CLP increased the activity of caspases-3 in a concentration-dependent manner. In a time–course analysis, the activity of caspase-3 was significantly increased compared to control samples, and continued to increase after 6 h of exposure, and at 9 h, the activity of caspase-3 declined
Fig. 6 – Effect of CLP on cell-cycle distribution of K562 cells (n = 3). (A) Cell-cycle analysis of K562 cells following treatment in the absence of CLP for 24 h; (B) cell-cycle analysis of K562 cells following treatment with 8 mg/ml CLP for 24 h; (C) cell-cycle analysis of K562 cells following treatment with 16 mg/ml CLP for 24 h; (D) cell-cycle analysis of K562 cells following treatment with 32 mg/ml CLP for 24 h.
peptides 28 (2007) 1344–1350
Fig. 7 – Effect of CLP on caspase-3 and proteins in K562 cells. (A) Treatment with CLP at various concentrations for 9 h and (B) treatment with CLP at 32 mg/ml. The data shown are representative of three independent experiments.
compared to the activity at 6 h, but was still higher than that of the control. In a similar dose- and time-dependent response, CLP caused the proteolytic cleavage of PARP with an accumulation of 89 kDa fragments and disappearance of full-length 116 kDa protein. Moreover, CLP-induced expression of Cip1/p21 and Kip/p27, and a marked reduction in the expression of Cyclin D1 which plays a crucial role in G1 phase progression was observed in K562 cells.
4.
Discussion
In recent years, studies of the anti-tumor activities of lipoproteins have been of particular interest. Studies by Kameda’s group proved that lipoproteins have anti-tumor activity. This is the first evidence that lipoproteins extracted from B. natto 3321 can induce apoptosis of cancer cells [8]. In Nakahara’s study, exposure of B16 cells to lipoproteins resulted in the condensation of the chromatin, DNA fragmentation and sub-G1 arrest [36]. Sudo et al. [29] examined lipoproteins for their ability to inhibit growth and induce differentiation of HL60 human promyelocytic leukemia cells. In addition, Wakamatsu et al. [32] discovered that lipoproteins
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induces neuronal differentiation in PC12 cells and provided the groundwork for the use of microbial extracellular lipoproteins as novel reagents for the treatment of cancer cells. Although lipoproteins have been tested for potential anti-tumor effects, the mechanism is not clear. In this study, a new CLP was isolated as the effective compound from B. subtilis T-2 and an attempt was made to identify the mechanism of its anti-tumor activity. Apoptosis is a process of cell suicide, characterized by specific morphological changes such as condensation of chromatin, nuclear fragmentation, blebbing of the plasma membrane, and the presence of apoptotic bodies [9,14,22]. The results of this study revealed that CLP-induced apoptosis in K562 cells. The cell cycle is an orderly sequence of events that occurs throughout development, from stem cells to fully differentiated cells, alternating between dividing (mitosis) and what appears to be resting (inter-phase) states. During progression of the cell cycle, cellular processes such as cell growth, DNA replication and mitosis are all coordinated, and any failure in this coordination restricts entry into the next phase of the cell cycle. Flow cytometry assesses progression of the cell cycle and discriminates between cells in apoptosis and those in necrosis, resulting from the lethal effect of CLP. As shown in Fig. 6, it was confirmed that CLP caused dose-dependent apoptosis of K562 cells through cell arrest in G1 phase. The results in Fig. 7 suggest that CLP might be implicated in the disruption of cancer cell progression through increasing expression of CDKIs together with decreasing expression of Cyclin D1. Apoptosis is a form of cell death that is tightly regulated by a number of gene products that either promote or block cell death at different stages of the cell cycle. The activation of caspase family members is a critical component of the apoptotic machinery [12,25,27]. Caspase-3 is one of the key proteases responsible for the cleavage and inactivation of PARP [16,20]. PARP is involved in DNA repair and is important for the maintenance of cell viability [2,17]. Moreover, PARP cleavage during caspase-3 activation facilitates cellular disassembly and serves as a marker of apoptosis [13]. As shown in Fig. 7, caspase-3 and PARP were cleaved by CLP in a time- and concentration-dependent manner. These results suggest that apoptosis in K562 cells induced by CLPs is closely associated with the activation of caspase-3 and PARP cleavage. In conclusion, the present work showed that the purified CLP from B. natto exerted a cytotoxic action on K562 cells, and the cytotoxicity involves mainly induction of apoptosis which is closely associated with cell arrest at G1 phase and activation of caspase-3. It is hoped that results from these studies will permit the identification of key molecular targets, which may further assist in the elucidation of the mechanism of action of the purified CLP, along with facilitating the development of this highly effective anti-cancer therapeutic agent. Further studies are underway to elucidate the mechanistic nature of the differential apoptosis induced by the purified CLP.
Acknowledgments We thank support of this project by funds from National Natural Science Foundation (grant no. 30570051) and Tianjin Natural Science Foundation (grant no. 06YFJMJC07500).
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The secretarial assistance of Ms Kathy Lau is greatly appreciated.
references
[1] Baell JB, Huang DC. Prospects for targeting the Bcl-2 family of proteins to develop novel cytotoxic drugs. Biochem Pharmacol 2002;64:851–63. [2] Boulares AH, Zoltoski AJ, Smulson ME. Roles of DNA fragmentation factor and polymerase in an amplification phase of tumor necrosis factor-induced apoptosis. J Biol Chem 2001;276:38185–92. [3] Cameotra SS, Makkar RS. Recent applications of biosurfactants as biological and immunological molecules. Ecol Ind Microbiol 2004;7:262–6. [4] Cappuccino JG, Sherman N. Microbiology—a laboratory manual, 4th ed., Harlow: Addison-Wesley; 1999. p. 183–6. [5] Corbiere C, Liagre B, Terro F, Beneytout JL. Induction of antiproliferative effect by diosgenia through activation of p53, release of apoptosis-inducing factor (AIF) and modulation of capase-3 activity in different human cancer cells. Cell Res 2004;14:188–96. [6] Igor NS, Li SM, Colby JL, Ho CT. Polymethoxylated flavonea induce Ca2+-mediated apoptosis in breast cancer cells. Life Sci 2006;80:245–63. [7] Jakopec S, Dubravicic K, Polanc S, Kosmarlj J, Osmak M. Diazene Jk-279 induces apoptosis-like cell death in human cervical carcinoma cells. Toxicol In Vitro 2006;20:217–26. [8] Kameda Y, Oira S, Matsui K, Kanatomo S, Hase T. Antitumor activity of Bacillus natto. V. Isolation and characterization of surfactin in the culture medium of Bacillus natto KMD 2311. Chem Pharm Bull (Tokyo) 1974;22:938–44. [9] Kaufmanm SH, Hengarther MO. Programmed cell death: alive and well in the new millennium. Trends Cell Biol 2001;262:526–34. [10] Kalai M, Lamkanfi M, Denecker G, Boogmans M, Lippens S, Meeus A. Regulation of the expression and processing of caspase-12. J Cell Biol 2003;162:457–67. [11] Kishore D, Ashis KM. Assessment of mosquito larvicidal potency of cyclic lipopeptides produced by Bacillus subtilis strains. Acta Trop 2006;97:168–73. [12] Klein G. Cancer, apoptosis, and non-immune surveillance. Cell Death Differ 2004;11:13–7. [13] Kolenko VM, Uzzo RG, Bukowski R, Finke JH. Caspasedependent and independent death pathways in cancer therapy. Apoptosis 2000;5:17–20. [14] Lee EO, Kwon BM, Song GY. Heyneanol A induces apoptosis via cytochrome c release and caspase activation in human leukemiac U937 cells. Life Sci 2004;74:2313–26. [15] Mohammad SR, Takashi A, Makoto S. Biofilm fermentation of iturin A by a recombinant strain of Bacillus subtilis 168. J Biotechnol 2007;127:503–7. [16] Monasterio A, Urdaci MC, Pinchuk IV, Lopez MN, Martinezirujo JJ. Flavonoids induce apoptosis in human leukemia U937 cells through caspase- and caspase-calpaindependent pathways. Nutr Cancer 2004;50:90–100. [17] Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, et al. Caspase-12 mediates end-oplasmic-reticulumspecific apoptosis and cytotoxicity by amiloid. Science 2004;3:98–103.
[18] Nielsen TH, Sorensen J. Production of cyclic lipopeptides by Pseudomonas fluorescens strains in bulk soil and in the sugar beet rhizosphere. Appl Environ Microbiol 2003;69:861–8. [19] Oggioni MR, Galieni P, Bigazzi C, Pozzi G, Valensin PE. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J Clin Microbiol 1998;36:325–6. [20] Oommen S, John AR, Srinivas G, Karunagaran D. Allicin (from garlic) induces caspase-mediated apoptosis in cancer cells. J Pharmacol 2004;485:97–103. [21] Reed J. Apoptosis-regulating proteins as targets for drug discovery. Trends Mol Med 2001;7:314–9. [22] Reed J. Apoptosis-targeted therapies for cancer. Cancer Cells 2003;3:17–25. [23] Roich DV, Pauli GR, Vater JC. Antimycoplasma properties and application in cell culture, a lipopeptide antibiotic from Bacillus subtilis. Microbiol Jpn 1997;63:44–9. [24] Sergeev IN. Calcium signaling in cancer and vitamin D. J Steroid Biochem Mol Biol 2005;97:145–51. [25] Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 2002;9:459–70. [26] Singh P, Cameotra SS. Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 2004;22:142–6. [27] Srinivas G, Anto RJ, Srinivas P, Lakshmi V, Karunagaran D. Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose)polymerase cleavage and activation of caspase-9. Eur J Pharmacol 2003;473:117–25. [28] Stanislav GB, Tatiana AR, Nkita BP. A novel lipopeptide, an inhibitor of bacterial adhesionfrom the thermophilic and halotolerant subsurface Bacillus licheniformis strain 603. Biochim Biophys Acta 2003;1634:107–15. [29] Sudo T, Zhao X, Wakamatsu Y, Shibahara M, Nakahara T. Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids. Cytotechnology 2000;33:259–64. [30] Tomohiro H, Rieko H, Shizue S, Akio A, Kan K. Cytokine responses of human intestinal epithelial-like Caco-2 cells to the nonpathogenic bacterium Bacillus subtilis. J Food Microbiol 2003;82:255–64. [31] Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 2002;68:6210–9. [32] Wakamatsu Y, Zhao X, Jin C, Day N, Shibahara M, Nomura N, et al. Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERKrelated signal cascade. Eur J Biochem 2001;268:374–83. [33] Bessler WG, Mittenbuhler K, Escher V, Huber M. Lipopeptide adjuvants in combination treatment. Int Immunopharmacol 2003;3:1217–24. [34] Yang SZ, Wei DZ, Mu BZ. Determination of the amino acid sequence in a cyclic lipopeptide using MS with DHT mechanism. J Biochem Biophys Methods 2006;68:69–74. [35] Ye CL, Qian F, Wei DZ, Lu YH, Liu JW. Induction of apoptosis in K562 human leukemia cells by 20 ,40 -dihydroxy-60 methoxy-30 ,50 -dimethylchalcone. Leukemia Res 2005;29:887–92. [36] Zhao X, Wakamatsu Y, Shibahara M, Nakahara N, Geltinger C, Nakahara T, et al. Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation in PC12 cells through an ERK-related signal cascade. Eur J Biochem 2001;268:374–83.