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Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells
Biochimica et Biophysica Acta 1790 (2009) 840–844
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells Bo Liu 1, Bo Zhang 1, Ming-wei Min, He-jiao Bian, Long-fei Chen, Qian Liu, Jin-ku Bao ⁎ School of Life Sciences, Sichuan University, Chengdu 610064, China
a r t i c l e
i n f o
Article history: Received 4 February 2009 Received in revised form 16 April 2009 Accepted 23 April 2009 Available online 3 May 2009 Keywords: The Galanthus nivalis agglutinin (GNA)-related lectins Polygonatum odoratum Lectin (POL) Apoptosis Death-receptor Mitochondria Tumor necrosis factor α (TNFα)
a b s t r a c t Background: The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNArelated lectins induce cell death are still only rudimentarily understood. Methods: In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)induced L929 cell apoptosis. Results: Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis. General significance: These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
Plant lectins are reversible carbohydrate-binding proteins (or glycoproteins) of non-immuno origin that can agglutinate cells or precipitate polysaccharides and glycoconjugates [1,2]. Recent advances have revealed the occurrence of seven families of structurally and evolutionary related proteins that include the legume lectins, the Galanthus nivalis agglutinin (GNA)-related lectins, the chitinbinding lectins composed of hevein domains, the type II ribosome inactivating proteins (RIPs II) and relevant lectins, the jacalin related lectins (amaranthin lectin family) and cucurbitaceae phloem lectins [3]. Among them, the GNA-related lectins have been found exclusively in a subgroup of the monocotyledonous plants and consist of identical or similar subunits [4]. In the past few years, these proteins have been purified and characterized in detail with respect to their biochemical properties, mannose-binding specificities and various biological activities involving the anti-tumor activity [5,6]. Of note, the development of cancer is associated with apoptosis because it is a cell-intrinsic mechanism for suicide that can be regulated by several cellular signaling pathways [7]. There are two major apoptotic pathways in mammalian cells, the death-receptor pathway and mitochondrial pathway [8]. Several plant lectins were ⁎ Corresponding author. Tel.:/fax: +86 28 85415171. E-mail address: [email protected] (J. Bao). 1 These authors contributed equally to this work. 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.04.020
widely studied to possess markedly antiproliferative and apoptosisinducing activities in tumor cells [9]. For instance, mistletoe lectins, belonging to RIP II family, have also been adopted for alternative cancer therapy for several years [10,11]. In addition, several legume lectins have been also reported to bear the remarkable anti-tumor activity [12]. Nevertheless, hitherto, only a few studies of GNA-related lectins have been reported for their anti-tumor activity and molecular mechanisms [13]. Our previous study has reported that Polygonatum odoratum lectin (POL), a mannose-binding specific lectin from GNA-related lectin family, consists of four identical 11920.451 Da subunits that are linked by non-covalent bonds and is independent on metal ions [14]. However, the anti-tumor activity of POL and its underlying molecular mechanisms have not been discovered yet. In the current study, we report herein for the first time that POL induces apoptosis through both death-receptor and mitochondrial apoptotic pathways, as well as amplifies TNFα-induced apoptosis in murine fibrosarcoma L929 cells. 2. Materials and methods 2.1. Plant material and cell culture P. odoratum lectin (POL) was purified as described previously [14]. The murine fibrosarcoma L929 (#CRL-2148) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). They were routinely cultured in RPMI-1640 medium containing 10%
B. Liu et al. / Biochimica et Biophysica Acta 1790 (2009) 840–844
fetal bovine serum, 100 U/ml streptomycin, 100 U/ml penicillin, and 2 mm L-glutamine in a humidified cell incubator with an atmosphere of 5% CO2 at 37 °C. 2.2. Cell viability assay The murine fibrosarcoma L929 cells were dispensed in 96-well flat bottom microtiter plates at a density of 5 × 104 cells/ml. After 24 h incubation, they were treated with different concentrations of POL for the indicated time periods. Cell viability was measured by the 3-(4,5dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [15].
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cell apoptosis was observed under the electron microscope (Hitachi 7000, Japan). The collected cells were fixed with 500 μL PBS and 10 ml 70% ethanol at 4 °C overnight; then after washing twice with PBS, the cells were incubated with 1 ml PI staining solution (PI 50 mg/L and RNase A 1 g/L) for 30 min at 4 °C. The percentage of cells at different phases of the cell cycle or the Sub-G1 DNA content was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) [17]. 2.4. Lactate dehydrogenase (LDH) activity-based cytotoxicity assay Lactate dehydrogenase (LDH) activity was assessed using a standardized kinetic determination kit (Zhongsheng, LDH kit, Beijing, China) as the method previously described [18].
2.3. Apoptosis assay 2.5. Caspase assay The murine fibrosarcoma L929 cells were seeded into 6-well culture plates with or without POL and cultured for 24 h. The changes in nuclear morphology of apoptotic cells were assessed by staining cells with the fluorescent, selective DNA and RNA-binding dye AO and examining them under the fluorescent microscopy (Green fluorescence for DNA, red fluorescence for RNA) [16]. The ultrastructure of
Caspase-3, -8, and -9 activities were measured by a colorimetric assay kit (Biovision) according to the manufacturer's instructions. Briefly, cell lysate from 1 × 106 cells was incubated at 37 °C for 2 h with 200 μM DEVD-pNA (caspase-3 substrate), IETD-pNA (caspase-8 substrate), or LEHD-pNA (caspase-9 substrate). Samples were read
Fig. 1. POL induces apoptosis in murine fibrosarcoma L929 cells. (A) Cell viability was measured by the MTT assay in L929 cells. (B) The cellular morphology was observed without or with POL under the fluorescent microscopy and electron microscopy. (C) Cells were stained with PI and the population of SubG1 cells was measured by flow cytometry after collection. (D) The L929 cells were treated with 25 μg/ml POL for 6, 12, 24 or 36 h, and the LDH content was measured by a assay kit (x ± S.D., n = 3).
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at 405 nm in a microplate reader (Bio-Tek Instruments) and expressed as fold increase on the basal level (DMSO-treated cells).
mannose-containing receptors on tumor surface and eventually lead to activations of caspase.
2.6. Detection of mitochondrial membrane potential
3.3. POL induces cell death through a death-receptor apoptotic pathway
After incubation with POL for the indicated time periods, the cells were stained with 1 μg/ml rhodamine 123 and incubated for 15 min at 37 °C. The fluorescence intensity of cells was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) [19]. 2.7. Western blot analysis The murine fibrosarcoma L929 cells were treated with 25 μg/ml POL for 0, 6, 12 or 24 h. Both adherent and floating cells were collected, and then Western blot analysis was carried out by the method as described previously with some modifications [20]. 2.8. Statistical analysis All the presented data and results were confirmed in at least three independent experiments. The data are expressed as means ± S.D. Statistical comparisons were made by Student's t-test. P b 0.05 was considered statistically significant.
To assess whether Fas-mediated pathway was activated in POLtreated cells, the levels of FasL, Fas-Associated protein with Death Domain (FADD) and caspase-8 were determined by Western blot analysis. The levels of FasL and FADD were markedly elevated and then there was obvious increase in the cleavage of procaspase-8 after POL administration (Fig. 3A). Therefore, Fas mediating apoptotic pathway was involved in POL-induced apoptosis. Previous studies have demonstrated that mistletoe lectin-I induces apoptosis via caspase-8/FLICE independent of death-receptor signaling [23]. Other reports have also demonstrated that mistletoe lectins induce apoptotic death involving a remarkable generation of ROS by activations of caspase as well as by activation of the SEK/JNK pathway [24]. However, our study showed that POL induced cell death through the death-receptor apoptotic pathway. Accordingly, The discrepancy may be due to their different molecular structures between mistletoe lectins and POL. 3.4. POL induces cell death through a mitochondrial apoptotic pathway
3. Results and discussion 3.1. POL induces apoptosis in murine fibrosarcoma L929 cells POL caused a remarkable antiproliferative effect on L929 cell growth in a time- and dose-dependent manner, and the treatment with 25 μg/ml POL for 24 h resulted in almost 50% inhibition (Fig. 1A). To characterize the POL-induced L929 cell growth inhibition, we observed the morphologic changes in the cells. When the cells were cultured with 25 μg/ml POL for 24 h, the marked apoptotic morphologic alterations were observed by AO staining under florescence microscopy (Fig. 1B). The apoptotic ultrastructural alterations were also observed under electron microscope (Fig. 1B). Furthermore, apoptosis was further evaluated by the measurement of cell number in SubG1 region. As shown in Fig. 1C, POL markedly induced the increase of SubG1 cells proportion in L929 cells. Since Lactic dehydrogenase (LDH) activity can be used as a marker for necrotic cell death [18], we investigated the effect of POL on LDH release in L929 cells. LDH content was measured using an assay kit according to the manufacturer's instructions. As shown in Fig. 1D, incubation of L929 cells with POL for 6 and 12 h had no significant effect on LDH release, but longer exposure for POL induced some degree of cytotoxic damage. Altogether, these results suggest that POL induces apoptotic cell death in L929 cells.
Next, the integrity of mitochondrial membranes was measured by rhodamine 123 staining. In Fig. 3B, POL decreased the rhodamine 123 fluorescence intensity in a time-dependent manner. Subsequently, the amount of cytochrome c in the mitochondria of the cells was decreased, but the amount of cytochrome c in the cytosol of the
3.2. POL induces apoptosis in a caspase-dependent manner To evaluate the involvement of caspase in POL-induced cell growth inhibition, the caspase inhibitors were applied as mentioned above. After 24 h incubation with POL, pancaspase, caspase-3, -8 and -9 inhibitors completely inhibited POL-induced cell growth inhibition as shown in Fig. 2A. It is indicated that POL induces cell apoptosis in a caspase-dependent manner. Moreover, we further examined the activities of caspase-3, -8 and -9 by colorimetric analysis. As shown in Fig. 2B, POL increased the activities of caspase-3, -8 and -9 in a timedependent manner. Our previous studies have reported that several legume lectins such as Concanavalin A (ConA) and Phaseolus coccineus lectin (PCL) can induce tumor cell apoptosis in a caspase-dependent pathway [12,21,22]. Consistent with these results, it is suggested that POL induces tumor cell apoptosis dependently of caspase because these plant lectins may possess some identical or similar sugarbinding activities, which can subsequently bind the sugar chains or
Fig. 2. POL-induced apoptosis is a caspase-dependent pathway. (A) The L929 cells were treated with POL for 24 h, with or without 10 μM z-VAD-fmk, z-DEVD-fmk, inhibitor z-IETD-fmk or z-LEHD-fmk pretreatment for 1 h, and then cell growth inhibition was determined (x ± S.D., n = 3), ⁎⁎p b 0.01 vs POL group. (B) Cytosolic fraction of the cells was analyzed for changes in the activities of caspase-3, -8 and -9 by colorimetric assay (x ± S.D., n = 3).
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Fig. 3. POL induces apoptosis via death-receptor and mitochondrial pathways. (A) Cell lysates were separated by 12% SDS-PAGE, and the levels of FasL, FADD and caspase-8, were detected by Western blot analysis, β-Actin was used as an equal loading control. (B) After treated with 25 μg/ml POL for various time periods, the cells were loaded with 1 μg/ml rhodamine-123 at 37 °C for 30 min, and analyzed by flow cytometry (x ± S.D., n = 3). (C) Cell lysates were separated by 12% SDS-PAGE, and the levels of cytochrome c, caspase-9 and caspase-3 were detected by Western blot analysis. β-Actin was used as an equal loading control.
cells was increased, suggesting the cytochrome c was released from mitochondria (Fig. 3C). These results clearly indicate that POLinduced apoptosis in L929 cells is mediated by a mitochondrial pathway. Since the release of cytochrome c from mitochondria can activate caspase cascade, we investigated the involvements of caspase-9 and caspase-3 in POL-induced apoptosis. In Fig. 2C, the caspase-9 activation was determined by measurement of the active forms of caspase-9. The active form of caspase-3 was observed during POL treatment. These results suggest that the treatment of POL activate both initiator and executioner caspase in a timedependent manner. Importantly, our result is in good agreement with the previous report that mistletoe lectin-II, induced apoptotic death in cancer cells involving a remarkable generation of intracellular hydrogen peroxide (H2O2) and activations of caspase9 and caspase-3 [25]. Thus, these results would provide new evidence for further understanding more apoptotic mechanisms of plant lectins.
3.5. POL amplifies TNFα-induced apoptosis
As tumor necrosis factor α (TNFα) is known as a sensitive agent in murine fibrosarcoma L929 cells [26], we investigated whether TNFαinduced cell apoptosis can be augmented by POL. The L929 cells were treated with serial concentrations of different TNFα in the presence of POL. POL, which possessed only low cytotoxic effects alone, markedly enhanced the TNFα-induced apoptosis, indicating that there is a combinatorial treatment with both POL and TNFα in L929 cells (Fig. 4). It is suggested that POL can potentiate the apoptotic effect of TNFα at low concentration. It would be, therefore, interesting to investigate whether POL can overcome the resistance of drug-refractory tumor cells in future lectin investigations. 4. Conclusion
In summary, we report for the first time that P. odoratum lectin (POL), a mannose-binding specific GNA-related lectin, induces murine fibrosarcoma L929 cell apoptosis in a caspase-dependent manner. Subsequently, we demonstrate that POL induces cell death through both death-receptor and mitochondrial apoptotic pathways, as well as amplifies TNFα-induced L929 cell apoptosis. These inspiring findings would provide new molecular basis for further exploring apoptotic mechanisms of the GNA-related lectins in future cancer therapeutics.
Acknowledgments
Fig. 4. POL treatment can enhance TNFα-induced apoptosis in L929 cells. The L929 cells were incubated with various doses of TNFα. Meanwhile, the L929 cells were coincubated with the indicated concentrations of POL.
We apologize to those authors whose work we could not cite owing to space limitations. We are grateful to Dr. Yan Cheng for her critical review of this manuscript. This work was supported in part by grants from the National Natural Science Foundation of China (General Programs: no. 30270331 and no. 30670469).
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References [1] I. Goldstein, R.C. Hughes, M. Monsigny, T. Osawa, N. Sharon, What should be called a lectin? Nature 285 (1980) 66. [2] N. Sharon, H. Lis, Lectins as cell recognition molecules, Science 246 (1989) 227–234. [3] E.J. Van Damme, W.J. Peumans, A. Barre, P. Rougé, Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles, Crit. Rev. Plant Sci. 17 (1998) 645–662. [4] E.J.M. Van Damme, S.N. Tsuruta, D.F. Smith, M. Ongenaert, C.H. Winter, P. Rougé, I. Goldstein, et al., Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of mutispecificity through domain duplication and devergent evolution, Biochem. J. 404 (2007) 51–61. [5] N. Sharon, Carbohydrate-specific reagents and biological recognition molecules, J. Biol Chem. 282 (2007) 2753–2764. [6] B. Liu, Y. Cheng, H.J. Bian, J.K. Bao, Molecular mechanisms of Polygonatum cyrtonema lectin induced apoptosis and autophagy in cancer cells, Autophagy 5 (2009) 253–255. [7] O.H. Michael, The biochemistry of apoptosis, Nature 407 (2000) 770–777. [8] T. Andrew, Apoptosis and autophagy: regulatory connections between two supposedly different processes, Apoptosis 13 (2008) 1–9. [9] E.G. De Mejía, V. Prisecaru, Lectins as bioactive plant proteins: a potential in cancer treatment, Crit. Rev. Food Sci. Nutr. 45 (2005) 425–445. [10] G. Seifert, P. Jesse, A. Laengler, T. Reindl, M. Lüth, S. Lobitz, et al., Molecular mechanisms of mistletoe plant extract-induced apoptosis in acute lymphoblastic leukemia in vivo and in vitro, Cancer Lett. 264 (2008) 218–228. [11] H. Stauder, E.D. Kreuser, Mistletoe extracts standardized in terms of mistletoe lectins (ML1) in oncology: current state of clinical research, Oncology 25 (2002) 374–380. [12] B. Liu, C.Y. Li, H.J. Bian, M.W. Min, L.F. Chen, J.K. Bao, Antiproliferative and apoptosis-inducing mechanism of Concanavalin A in human melanoma A375 cells, Arch. Biochem. Biophys. 482 (2009) 1–6. [13] B. Liu, Y. Cheng, B. Zhang, H.J. Bian, J.K. Bao, Polygonatum cyrtonema lectin induces apoptosis and autophagy in human melanoma A375 cells through a mitochondria-mediated ROS-p38-p53 pathway, Cancer Lett. 275 (2009) 54–60. [14] H.Z. Lv, C. Liu, J.K. Bao, The fluorescence quenching of Polygonatum odoratum (Mill.) Druce lectin, J. Sichuan University (Nat. Sci. Ed.) 43 (2006) 1389–1393.
[15] B. Liu, X.C. Xu, Y. Cheng, J. Huang, Y.H. Liu, J.K. Bao, et al., Apoptosis-inducing effect and structural basis of Polygonatum cyrtonema lectin and chemical modification properties on its mannose-binding sites, BMB Rep. 41 (2008) 369–375. [16] Y. Cheng, F. Qiu, S. Tashiro, S. Onodera, T. Ikejima, ERK and JNK mediate TNFαinduced p53 activation in apoptotic and autophagic L929 cell death, Biochem. Biophys. Res. Commun. 376 (2008) 483–488. [17] B. Liu, H. Peng, Q. Yao, J. Li, E.J. Van Damme, J. Balzarini, J.K. Bao, Bioinformatics analyses of the mannose-binding lectins from Polygonatum cyrtonema, Ophiopogon japonicus and Liparis noversa with antiproliferative and apoptosis-inducing activities, Phytomedicine 16 (2009) 601–608. [18] J.R. Maria, S.D. Miranda, A.M. Thomas, Apoptosis is a major mechanism of deoxycholate-induced gastric mucosal cell death, Am. J. Physiol. Gastrointest. Liver Physiol. 285 (2003) 870–879. [19] N.E. Saris, V.V. Teplova, I.V. Odinokova, T.S. Azarashvily, Interference of calmidazolium with measurement of mitochondrial membrane potential using the tetraphenylphosphonium electrode or the fluorescent probe rhodamine 123, Anal. Biochem. 328 (2004) 109–112. [20] Y. Cheng, F. Qiu, J. Huang, S.I. Tashiro, S. Onodera, T. Ikejima, Apoptosissuppressing and autophagy-promoting effects of calpain on oridonin-induced L929 cell death, Arch. Biochem. Biophys 475 (2008) 148–155. [21] B. Liu, M.W. Min, J.K. Bao, Induction of apoptosis by concanavalin A and its molecular mechanisms in cancer cells, Autophagy 5 (2009) 432–433. [22] J. Chen, B. Liu, N. Ji, J. Zhou, J.K. Bao, et al., A novel sialic acid-specific lectin from Phaseolus coccineus seeds with potent antineoplastic and antifungal activities, Phytomedicine 16 (2009) 352–360. [23] B. Heike, H.E. Ingo, V. Wolfgang, S.O. Klaus, W. Sebastian, Mistletoe lectin activates caspase-8/FLICE independently of death receptor signaling and enhances anticancer drug-induced apoptosis, Cancer Res. 59 (1999) 2083–2090. [24] W.H. Kim, W.B. Park, B. Gao, M.H. Jung, Critical role of reactive oxygen species and mitochondrial membrane potential in Korean mistletoe lectin-induced apoptosis in human hepatocarcinoma cells, Mol. Pharmacol. 66 (2004) 1383–1396. [25] M.S. Kima, J. Leeb, K.M. Leec, S.H. Yanga, S. Choid, S.Y. Chungd, T.Y. Kima, W.H. Jeongf, R. Parka, Involvement of hydrogen peroxide in mistletoe lectin-II-induced apoptosis of myeloleukemic U937 cells, Life Sci. 73 (2003) 1231–1243. [26] Y. Cheng, F. Qiu, Y.C. Ye, Z.M. Guo, S.I. Tashiro, S. Onodera, T. Ikejima, Autophagy inhibits reactive oxygen species-mediated apoptosis via activating p38-nuclear factor-kappaB survival pathways in oridonin-treated murine fibrosarcoma L929 cells, FEBS J. 276 (2009) 1291–1306.
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a g e n
Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells Bo Liu 1, Bo Zhang 1, Ming-wei Min, He-jiao Bian, Long-fei Chen, Qian Liu, Jin-ku Bao ⁎ School of Life Sciences, Sichuan University, Chengdu 610064, China
a r t i c l e
i n f o
Article history: Received 4 February 2009 Received in revised form 16 April 2009 Accepted 23 April 2009 Available online 3 May 2009 Keywords: The Galanthus nivalis agglutinin (GNA)-related lectins Polygonatum odoratum Lectin (POL) Apoptosis Death-receptor Mitochondria Tumor necrosis factor α (TNFα)
a b s t r a c t Background: The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNArelated lectins induce cell death are still only rudimentarily understood. Methods: In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)induced L929 cell apoptosis. Results: Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis. General significance: These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
Plant lectins are reversible carbohydrate-binding proteins (or glycoproteins) of non-immuno origin that can agglutinate cells or precipitate polysaccharides and glycoconjugates [1,2]. Recent advances have revealed the occurrence of seven families of structurally and evolutionary related proteins that include the legume lectins, the Galanthus nivalis agglutinin (GNA)-related lectins, the chitinbinding lectins composed of hevein domains, the type II ribosome inactivating proteins (RIPs II) and relevant lectins, the jacalin related lectins (amaranthin lectin family) and cucurbitaceae phloem lectins [3]. Among them, the GNA-related lectins have been found exclusively in a subgroup of the monocotyledonous plants and consist of identical or similar subunits [4]. In the past few years, these proteins have been purified and characterized in detail with respect to their biochemical properties, mannose-binding specificities and various biological activities involving the anti-tumor activity [5,6]. Of note, the development of cancer is associated with apoptosis because it is a cell-intrinsic mechanism for suicide that can be regulated by several cellular signaling pathways [7]. There are two major apoptotic pathways in mammalian cells, the death-receptor pathway and mitochondrial pathway [8]. Several plant lectins were ⁎ Corresponding author. Tel.:/fax: +86 28 85415171. E-mail address: [email protected] (J. Bao). 1 These authors contributed equally to this work. 0304-4165/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2009.04.020
widely studied to possess markedly antiproliferative and apoptosisinducing activities in tumor cells [9]. For instance, mistletoe lectins, belonging to RIP II family, have also been adopted for alternative cancer therapy for several years [10,11]. In addition, several legume lectins have been also reported to bear the remarkable anti-tumor activity [12]. Nevertheless, hitherto, only a few studies of GNA-related lectins have been reported for their anti-tumor activity and molecular mechanisms [13]. Our previous study has reported that Polygonatum odoratum lectin (POL), a mannose-binding specific lectin from GNA-related lectin family, consists of four identical 11920.451 Da subunits that are linked by non-covalent bonds and is independent on metal ions [14]. However, the anti-tumor activity of POL and its underlying molecular mechanisms have not been discovered yet. In the current study, we report herein for the first time that POL induces apoptosis through both death-receptor and mitochondrial apoptotic pathways, as well as amplifies TNFα-induced apoptosis in murine fibrosarcoma L929 cells. 2. Materials and methods 2.1. Plant material and cell culture P. odoratum lectin (POL) was purified as described previously [14]. The murine fibrosarcoma L929 (#CRL-2148) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). They were routinely cultured in RPMI-1640 medium containing 10%
B. Liu et al. / Biochimica et Biophysica Acta 1790 (2009) 840–844
fetal bovine serum, 100 U/ml streptomycin, 100 U/ml penicillin, and 2 mm L-glutamine in a humidified cell incubator with an atmosphere of 5% CO2 at 37 °C. 2.2. Cell viability assay The murine fibrosarcoma L929 cells were dispensed in 96-well flat bottom microtiter plates at a density of 5 × 104 cells/ml. After 24 h incubation, they were treated with different concentrations of POL for the indicated time periods. Cell viability was measured by the 3-(4,5dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [15].
841
cell apoptosis was observed under the electron microscope (Hitachi 7000, Japan). The collected cells were fixed with 500 μL PBS and 10 ml 70% ethanol at 4 °C overnight; then after washing twice with PBS, the cells were incubated with 1 ml PI staining solution (PI 50 mg/L and RNase A 1 g/L) for 30 min at 4 °C. The percentage of cells at different phases of the cell cycle or the Sub-G1 DNA content was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) [17]. 2.4. Lactate dehydrogenase (LDH) activity-based cytotoxicity assay Lactate dehydrogenase (LDH) activity was assessed using a standardized kinetic determination kit (Zhongsheng, LDH kit, Beijing, China) as the method previously described [18].
2.3. Apoptosis assay 2.5. Caspase assay The murine fibrosarcoma L929 cells were seeded into 6-well culture plates with or without POL and cultured for 24 h. The changes in nuclear morphology of apoptotic cells were assessed by staining cells with the fluorescent, selective DNA and RNA-binding dye AO and examining them under the fluorescent microscopy (Green fluorescence for DNA, red fluorescence for RNA) [16]. The ultrastructure of
Caspase-3, -8, and -9 activities were measured by a colorimetric assay kit (Biovision) according to the manufacturer's instructions. Briefly, cell lysate from 1 × 106 cells was incubated at 37 °C for 2 h with 200 μM DEVD-pNA (caspase-3 substrate), IETD-pNA (caspase-8 substrate), or LEHD-pNA (caspase-9 substrate). Samples were read
Fig. 1. POL induces apoptosis in murine fibrosarcoma L929 cells. (A) Cell viability was measured by the MTT assay in L929 cells. (B) The cellular morphology was observed without or with POL under the fluorescent microscopy and electron microscopy. (C) Cells were stained with PI and the population of SubG1 cells was measured by flow cytometry after collection. (D) The L929 cells were treated with 25 μg/ml POL for 6, 12, 24 or 36 h, and the LDH content was measured by a assay kit (x ± S.D., n = 3).
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B. Liu et al. / Biochimica et Biophysica Acta 1790 (2009) 840–844
at 405 nm in a microplate reader (Bio-Tek Instruments) and expressed as fold increase on the basal level (DMSO-treated cells).
mannose-containing receptors on tumor surface and eventually lead to activations of caspase.
2.6. Detection of mitochondrial membrane potential
3.3. POL induces cell death through a death-receptor apoptotic pathway
After incubation with POL for the indicated time periods, the cells were stained with 1 μg/ml rhodamine 123 and incubated for 15 min at 37 °C. The fluorescence intensity of cells was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) [19]. 2.7. Western blot analysis The murine fibrosarcoma L929 cells were treated with 25 μg/ml POL for 0, 6, 12 or 24 h. Both adherent and floating cells were collected, and then Western blot analysis was carried out by the method as described previously with some modifications [20]. 2.8. Statistical analysis All the presented data and results were confirmed in at least three independent experiments. The data are expressed as means ± S.D. Statistical comparisons were made by Student's t-test. P b 0.05 was considered statistically significant.
To assess whether Fas-mediated pathway was activated in POLtreated cells, the levels of FasL, Fas-Associated protein with Death Domain (FADD) and caspase-8 were determined by Western blot analysis. The levels of FasL and FADD were markedly elevated and then there was obvious increase in the cleavage of procaspase-8 after POL administration (Fig. 3A). Therefore, Fas mediating apoptotic pathway was involved in POL-induced apoptosis. Previous studies have demonstrated that mistletoe lectin-I induces apoptosis via caspase-8/FLICE independent of death-receptor signaling [23]. Other reports have also demonstrated that mistletoe lectins induce apoptotic death involving a remarkable generation of ROS by activations of caspase as well as by activation of the SEK/JNK pathway [24]. However, our study showed that POL induced cell death through the death-receptor apoptotic pathway. Accordingly, The discrepancy may be due to their different molecular structures between mistletoe lectins and POL. 3.4. POL induces cell death through a mitochondrial apoptotic pathway
3. Results and discussion 3.1. POL induces apoptosis in murine fibrosarcoma L929 cells POL caused a remarkable antiproliferative effect on L929 cell growth in a time- and dose-dependent manner, and the treatment with 25 μg/ml POL for 24 h resulted in almost 50% inhibition (Fig. 1A). To characterize the POL-induced L929 cell growth inhibition, we observed the morphologic changes in the cells. When the cells were cultured with 25 μg/ml POL for 24 h, the marked apoptotic morphologic alterations were observed by AO staining under florescence microscopy (Fig. 1B). The apoptotic ultrastructural alterations were also observed under electron microscope (Fig. 1B). Furthermore, apoptosis was further evaluated by the measurement of cell number in SubG1 region. As shown in Fig. 1C, POL markedly induced the increase of SubG1 cells proportion in L929 cells. Since Lactic dehydrogenase (LDH) activity can be used as a marker for necrotic cell death [18], we investigated the effect of POL on LDH release in L929 cells. LDH content was measured using an assay kit according to the manufacturer's instructions. As shown in Fig. 1D, incubation of L929 cells with POL for 6 and 12 h had no significant effect on LDH release, but longer exposure for POL induced some degree of cytotoxic damage. Altogether, these results suggest that POL induces apoptotic cell death in L929 cells.
Next, the integrity of mitochondrial membranes was measured by rhodamine 123 staining. In Fig. 3B, POL decreased the rhodamine 123 fluorescence intensity in a time-dependent manner. Subsequently, the amount of cytochrome c in the mitochondria of the cells was decreased, but the amount of cytochrome c in the cytosol of the
3.2. POL induces apoptosis in a caspase-dependent manner To evaluate the involvement of caspase in POL-induced cell growth inhibition, the caspase inhibitors were applied as mentioned above. After 24 h incubation with POL, pancaspase, caspase-3, -8 and -9 inhibitors completely inhibited POL-induced cell growth inhibition as shown in Fig. 2A. It is indicated that POL induces cell apoptosis in a caspase-dependent manner. Moreover, we further examined the activities of caspase-3, -8 and -9 by colorimetric analysis. As shown in Fig. 2B, POL increased the activities of caspase-3, -8 and -9 in a timedependent manner. Our previous studies have reported that several legume lectins such as Concanavalin A (ConA) and Phaseolus coccineus lectin (PCL) can induce tumor cell apoptosis in a caspase-dependent pathway [12,21,22]. Consistent with these results, it is suggested that POL induces tumor cell apoptosis dependently of caspase because these plant lectins may possess some identical or similar sugarbinding activities, which can subsequently bind the sugar chains or
Fig. 2. POL-induced apoptosis is a caspase-dependent pathway. (A) The L929 cells were treated with POL for 24 h, with or without 10 μM z-VAD-fmk, z-DEVD-fmk, inhibitor z-IETD-fmk or z-LEHD-fmk pretreatment for 1 h, and then cell growth inhibition was determined (x ± S.D., n = 3), ⁎⁎p b 0.01 vs POL group. (B) Cytosolic fraction of the cells was analyzed for changes in the activities of caspase-3, -8 and -9 by colorimetric assay (x ± S.D., n = 3).
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Fig. 3. POL induces apoptosis via death-receptor and mitochondrial pathways. (A) Cell lysates were separated by 12% SDS-PAGE, and the levels of FasL, FADD and caspase-8, were detected by Western blot analysis, β-Actin was used as an equal loading control. (B) After treated with 25 μg/ml POL for various time periods, the cells were loaded with 1 μg/ml rhodamine-123 at 37 °C for 30 min, and analyzed by flow cytometry (x ± S.D., n = 3). (C) Cell lysates were separated by 12% SDS-PAGE, and the levels of cytochrome c, caspase-9 and caspase-3 were detected by Western blot analysis. β-Actin was used as an equal loading control.
cells was increased, suggesting the cytochrome c was released from mitochondria (Fig. 3C). These results clearly indicate that POLinduced apoptosis in L929 cells is mediated by a mitochondrial pathway. Since the release of cytochrome c from mitochondria can activate caspase cascade, we investigated the involvements of caspase-9 and caspase-3 in POL-induced apoptosis. In Fig. 2C, the caspase-9 activation was determined by measurement of the active forms of caspase-9. The active form of caspase-3 was observed during POL treatment. These results suggest that the treatment of POL activate both initiator and executioner caspase in a timedependent manner. Importantly, our result is in good agreement with the previous report that mistletoe lectin-II, induced apoptotic death in cancer cells involving a remarkable generation of intracellular hydrogen peroxide (H2O2) and activations of caspase9 and caspase-3 [25]. Thus, these results would provide new evidence for further understanding more apoptotic mechanisms of plant lectins.
3.5. POL amplifies TNFα-induced apoptosis
As tumor necrosis factor α (TNFα) is known as a sensitive agent in murine fibrosarcoma L929 cells [26], we investigated whether TNFαinduced cell apoptosis can be augmented by POL. The L929 cells were treated with serial concentrations of different TNFα in the presence of POL. POL, which possessed only low cytotoxic effects alone, markedly enhanced the TNFα-induced apoptosis, indicating that there is a combinatorial treatment with both POL and TNFα in L929 cells (Fig. 4). It is suggested that POL can potentiate the apoptotic effect of TNFα at low concentration. It would be, therefore, interesting to investigate whether POL can overcome the resistance of drug-refractory tumor cells in future lectin investigations. 4. Conclusion
In summary, we report for the first time that P. odoratum lectin (POL), a mannose-binding specific GNA-related lectin, induces murine fibrosarcoma L929 cell apoptosis in a caspase-dependent manner. Subsequently, we demonstrate that POL induces cell death through both death-receptor and mitochondrial apoptotic pathways, as well as amplifies TNFα-induced L929 cell apoptosis. These inspiring findings would provide new molecular basis for further exploring apoptotic mechanisms of the GNA-related lectins in future cancer therapeutics.
Acknowledgments
Fig. 4. POL treatment can enhance TNFα-induced apoptosis in L929 cells. The L929 cells were incubated with various doses of TNFα. Meanwhile, the L929 cells were coincubated with the indicated concentrations of POL.
We apologize to those authors whose work we could not cite owing to space limitations. We are grateful to Dr. Yan Cheng for her critical review of this manuscript. This work was supported in part by grants from the National Natural Science Foundation of China (General Programs: no. 30270331 and no. 30670469).
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