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Ganoderma lucidum induced apoptosis in NB4 human leukemia cells: Involvement of Akt and Erk
Journal of Ethnopharmacology 128 (2010) 71–78
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Ganoderma lucidum induced apoptosis in NB4 human leukemia cells: Involvement of Akt and Erk ˜ a , José Luis Manjón b , Pilar Sancho a , M. Cristina Tejedor a , Angel Herráez a , José C. Diez a,∗ Eva Calvino a b
Departamento de Bioquímica y Biología Molecular, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain Departamento de Biología Vegetal, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain
a r t i c l e
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Article history: Received 20 July 2009 Received in revised form 7 December 2009 Accepted 17 December 2009 Available online 29 December 2009 Keywords: Apoptosis Bax Bcl-2 Ganoderma lucidum (Curtis) P. Karst. Ganodermataceae Donk Leukemia p53
a b s t r a c t Aim of the study: The final goal of this work was to study the toxic and apoptosis effects induced by fractions from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] on NB4 human leukemia cells. Materials and methods: Two aqueous extracts and a methanol-extracted column-chromatography semipurified fraction were obtained from Ganoderma lucidum fruiting body. Flow cytometry analyses were used to measure cell viability, cell cycle and DNA fragmentation and to quantify apoptosis. Western-blot analyses were used to quantify changes in apoptosis proteins and intracellular kinases. Results: Aqueous extracts slightly reduce cell viability and induce DNA fragmentation in NB4 cells. Methanol-extracted semipurified fraction at dilutions down to 15% or 40% of the initial fraction concentration reduced significantly the viability of these leukemia cells (treated for 19 h) with induction of DNA fragmentation and induction of apoptosis. Overmore, the dilution down to 15% of the initial E3 concentration induced a reduction of p53 levels, of the Bcl2/Bax relationship as well as reduced levels of both unphosphorylated and phosphorylated Akt (Protein kinase Akt, protein kinase B) and Erk (Erk1 and 2). Conclusions: Induction of apoptosis and alterations in signal transduction kinases (Akt and Erk) are produced by active fractions from Ganoderma lucidum on human leukemia cells. These data could be of important relevance from the viewpoint of antitumor actions of compounds from Ganoderma lucidum. Eventual therapy applications in leukemia cells might be developed. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction On recent times, a search is under way for new therapeutic agents with better efficiency and with more selective action on tumor cells. The fungus Ganoderma lucidum (common names: Reishi, Lingzhi) is a well known mushroom in traditional Chinese medicine. Extracts from different parts (mycelia, spores and fruiting bodies) of this mushroom have been used for prevention and treatment of different diseases in ancient China. Particularly relevant are its claimed antitumor properties (Hu et al., 2002; Liu et al., 2002; Sliva, 2004). Several reports exist on the action of Ganoderma lucidum extracts against tumor cells: growth inhibition in human prostate and bladder cancer cell lines (Jiang et al., 2004a; Lu et al., 2004) and inhibition of cell proliferation and induction of apoptosis in human colon carcinoma and breast cancer cell lines (Hu et al., 2002; Hong et al., 2004; Jiang et al., 2004b). Different compounds are present as natural constituents of this mushroom. Among them, several nucleosides, proteins, polysaccharides, fatty
∗ Corresponding author. Tel.: +34 91 8854582/579; fax: +34 91 8854585. E-mail address: [email protected] (J.C. Diez). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.12.027
acids, sterols and triterpenes have been described as potentially responsible of the antitumor activity of Ganoderma lucidum (Li et al., 2005; Yeung et al., 2004). Some of the antitumor properties of Ganoderma lucidum can be assigned to polysaccharides, for example retarding the growth of sarcoma cells (Cao and Lin, 2004). It is also accepted that, at least in part, their antitumor activity may be a consequence of their immunomodulatory properties (Wilasrusmee et al., 2002; Williamson, 2002; Lin, 2005). Some triterpenes present in Ganoderma lucidum have also demonstrated inhibition of growth of hepatoma cells by altering intracellular phosphorylation pathways (Lin et al., 2003). Leukemic human cell line NB4 was derived from bone marrow cells of a female patient of acute promyelocytic leukemia. These cells are sensitive to camptotecin analogues (Song et al., 2005), arsenic trioxide (Ramos et al., 2005), retinoids (Cincinelli et al., 2005), UV radiation (Biggs et al., 2001) or prooxidant agents (Shen et al., 1999) that induce cell death. This cell line can hence be a useful model to study the action of extracts, fractions or compounds with tentative antitumor activity. Recently, it has been shown that Ganoderma lucidum inhibits proliferation of different hematopoietic cell lines such as K562,
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HL1-60, U937, Blin-1, Nalm-6, etc. (Müller et al., 2006). Moreover, some extracts and fractions obtained from Ganoderma lucidum have been shown to induce apoptosis (Hu et al., 2002; Lu et al., 2004; Yeung et al., 2004; Li et al., 2005). We have studied the cytotoxicity induced in human leukemia NB4 cells by different Ganoderma lucidum extracts prepared as it has been indicated by other authors (Sliva et al., 2002; Stanley et al., 2005) as well as the possible involvement of some apoptotic factors in such toxic effects. We also have focused our interest on activation and phosphorylation of intracellular kinases (MAP-kinases) which are altered in other cellular model systems (Sliva et al., 2002; Jiang et al., 2004a,b). The final aim of this work has been to demonstrate toxic activity and apoptosis induction by Ganoderma lucidum (Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk) on human leukemia cells. We have also tried to show a possible involvement of some intracellular kinases such as Akt or Erk in cell death processes. 2. Materials and methods 2.1. Source of Ganoderma lucidum Ganoderma lucidum (Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk) strain was isolated from the fruit body tissue (context) of a wood-inhabiting filamentous parasitic fungi growing in a natural habitat on Quercus ilex roots in Cáceres (Spain). A small part of the tissue context developed Ganoderma lucidum mycelium that it was cultured and subcultured in a Malt extract solid medium (MA). In order to produce fruiting bodies it was necessary to cultivate the fungus on lignocellulosic solid medium substrate (Chang and Miles, 2004), adequately hydrated and autoclaved. Once, the lignocellulosic medium was inoculated with Ganoderma lucidum mycelia, the substrate was incubated until a proper mycelial colonization. Subsequently, the bulk mycelium develops fruit bodies that reach maturity in 1–2 months. 2.2. Extracts of Ganoderma lucidum Fruiting bodies obtained from Ganoderma lucidum were resuspended in sterile water to a concentration of 50 mg/ml (Sliva et al., 2002; Stanley et al., 2005). They were then homogenized and centrifuged at 14,000 rpm (in a Beckman J2.21 centrifuge using JA20.1 rotor) for 5 min, at room temperature, rendering what was called extract 1 (E1). The pellet was resuspended in the same volume of sterile water, boiled for 5 min, sonicated for 20 s at 50% intensity, centrifuged at 14,000 rpm for 5 min and the supernatant obtained was called extract 2 (E2). Afterwards, these extracts were lyophilized and dissolved in 1/10 the initial volume, giving ten-fold concentrated extracts. Methanolic extracts of Ganoderma lucidum were also prepared as follows. Fruiting body (2 g) was disrupted with liquid nitrogen, resuspended at 50 mg/ml in 10% methanol and extracted three times with 10% methanol for 24 h with agitation. The soluble extract was evaporated in a rotary evaporator until dry, and was dissolved in absolute methanol. To purify this extract, it was adsorbed on 0.2 g of silicagel to be applied onto a silica gel column (1 cm × 15.5 cm) and subsequently eluted with 3:1:1 butanol:acetic acid:water. Column fractions (0.2 ml) with an Rf near 0.74 were combined and the solvent was evaporated and the final sample (1.6 mg) was resuspended in 0.01 ml DMSO to obtain the fraction 3 (E3). 2.3. Human acute promyelocytic leukemia (NB4) cells The human NB4 leukemia cell line was a kind gift from Dr. D. Delgado (Dpto. Biología Molecular, Universidad de Cantabria. Santander. Spain). They were kept in culture at 37 ◦ C and 5% CO2 in RPMI medium (Gibco-Life Technologies) with 10% FCS, 1% penicillin–streptomycin 1% and 0.02 mg/ml gentamycin.
2.4. Cell treatments with Ganoderma lucidum extracts and fraction E1 and E2 extracts and E3 fraction were added separately to 1 ml aliquots of cell culture (0.5 × 106 cell/ml) and allowed to act for 19 h. Liophylized E1 extract (14.9 mg dried weight) was dissolved in 0.9 ml of sterile distilled water and 60 l were added per ml of cell culture. Liophylized E2 extract (3 mg dried weight) was dissolved in 0.9 ml of sterile distilled water and 60 l were added per ml of cell culture. Fraction 3 (E3) was added at three different doses, equivalent to 40% and 15% of the initial concentration of E3, as 5, 2 and 0.7 l of E3 per ml of culture, respectively (containing 0.8, 0.32 and 0.1 mg of the dried mass contained in E3). Ganoderic acid C2 was purchased from Chromadex (http://www.chromadex.com, code ASB-00007058) and prepared at 20 mg/ml in DMSO. Cells (0.5 × 106 cells/ml) were treated with Ganoderic acid C2 at a final concentration of 0.4 mg/ml for 19 h. Cells were also treated with 100 M etoposide for 19 h as a positive control of toxicity and apoptosis induction. DMSO (5 l) was also added to cells (1 ml at 0.5 × 106 cell/ml) as a negative control since no toxic effect was observed on cell culture (i.e. no reduction of cell viability, no DNA fragmentation, etc.). 2.5. Permeability of treated cells to propidium iodide and flow cytometry analyses Cell viability of NB4 cells treated with Ganoderma lucidum extracts was determined by flow cytometry by measuring the level of impermeability to propidium iodide. 2.5 × 105 treated cells were collected and washed in PBS and centrifuged at 1,200 rpm for 5 min. The cells were resuspended in 500 l of PBS and stained with propidium iodide (0.1 mg/ml) and analyzed either in FACScan or FACScalibur (Becton Dickinson, San José, CA, USA). For each sample the acquisition was finished at 10,000 counts. Data analysis was performed using WinMDI software (Trotter, 2004). Cell fragments were discriminated from the non-viable cells in dot plots FSC/FL2-H, where FL-2-H corresponds to propidium iodide fluorescence. 2.6. Cytometric analysis of cell populations with subdiploid DNA and cell cycle Apoptotic cells were counted on the basis of DNA content per cell after permeabilization with NP40. After treatments with 100 M etoposide or Ganoderma lucidum extracts, 2.5 × 105 cells were collected and washed with PBS. The pellet was resuspended in 475 l of a solution containing 0.5 mg/ml RNase, 0.1% NP-40 in PBS and incubated for 30 min, in order to extract low molecular weight DNA from cell nuclei. The remnant DNA in cells was stained with 0.05 mg/ml propidium iodide, immediately measured in the cytometer. Cells with hypodiploid DNA (apoptotic cells) were distinguished from those containing diploid DNA (non-apoptotic cells) on the basis of a different fluorescence intensity of propidium iodide. Cell cycle progress was studied in the same samples. Histograms of the untreated cells were used to define the positions of the different phases G1, S and G2/M phases of cell cycle. 2.7. Annexin V-FITC assays: phosphatidylserine exposure on the outer side of membrane To quantify the level of apoptosis through the presence of phosphatidylserine in the outer membrane side in NB4 treated cells the
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Annexin V-FITC Apoptosis Detection Kit from Calbiochem was used. For this assay, 3 × 106 treated cells were collected and centrifuged at 1500 rpm for 3 min. They were then incubated for 15 min in the dark at room temperature in 500 l PBS containing 10 l of binding reagent and 1.25 l Annexin V-FITC. Subsequently, the cells were centrifuged at 2,300 rpm for 5 min and resuspended in 500 l binding buffer 1× diluted in PBS. 10 l propidium iodide was added and the samples were analyzed in a FACScalibur (Becton Dickinson, San José, CA, USA) cytometer. Data were analyzed using WinMDI 2.8 software (Trotter, 2004). 2.8. Western-blot analyses Cells were grown at a concentration of 2 × 105 cells/ml, treated with the extracts and collected by centrifugation at 1,200 rpm for 5 min and resuspended in 200 l lysis buffer solution containing 50 mM Tris/HCl pH 8.0, 150 mM NaCl; 5 mM EDTA; 0.5% NP-40; 1 mM PMSF. After 20 min at 4 ◦ C, cells were sonicated for 20 s (duty cycle 100%, output control 50%) and then centrifuged (14,000 rpm, 5 min, 4 ◦ C). Resulting supernatants were analyzed by electrophoresis and blotting. Proteins (20 g/well) were loaded into a 10% SDS-PAGE, electrophoretically separated and, transferred to nitrocellulose membranes as described. Membranes were blocked with 5% powder milk in TTBS (50 mM Tris pH 7.2, 140 mM NaCl, 0.06% Tween 20). Afterwards, they were washed with TTBS and incubated with diverse diluted in TTBS containing milk. All the following antibodies were obtained from Santa Cruz Biotechnology, CA, USA, and used at the dilution indicated: anti-Bax mouse monoclonal IgG2b (B-9; sc-7480), 1:100; anti-Bcl-2 mouse monoclonal IgG1 (C-2; sc7382), 1:200; anti-Akt1/2/3 (H-136, sc8312), 1:400; anti-pAkt1/2/3 (Ser 437, sc-7985-R), 1:400; anti Erk1/2 (sc-154), 1:3,000; anti p-Erk1/2 (Tyr204 , sc-7383), 1:200; anti-NFB p50 and p105 (EA0, sc-8414), 1:200; anti-NFB p65 (F-6, sc-8008), 1:200. Anti-p53 mouse monoclonal IgG (Ab-1, OP03) was purchased from Calbiochem (Oncogene Research Products, MA, USA) and diluted 1:50. Monoclonal anti--actin was purchased from Sigma A5441 and used at 1:5,000. After incubation of the nitrocellulose membranes with antibodies overnight at 4 ◦ C, the bands were revealed using enhanced chemiluminescence (ECL) detection (Amersham) with goat antimouse horse-radish peroxidase (HRP)-conjugated polyclonal antibodies (1:2,000) from Promega (Madison, WI, USA). The intensities of the bands were corrected with respect to the intensity of actin band in the blot as a control for constitutive expression and quantified relative to the intensity of the band in control cells without any treatment which were considered as 100%. 2.9. Statistical procedures All the experiments were repeated three or four times. The mean and SEM were graphically represented. In order to determine the significance of the effect, Student’s test was used. The asterisk * indicate p < 0.05. For calculations we used Microsoft Excel 2003. 3. Results 3.1. Viability of NB4 cells and induction of DNA fragmentation after treatment with Ganoderma lucidum aqueous extracts We studied the cell viability measure through permeability to propidium iodide, of human leukemia NB4 cells after independent treatments with aqueous E1 or E2 extract for 19 h. Fig. 1 shows a representative flow cytometry profile (upper part) and the statistically processed data (lower panel). As can be observed, treatment of
Fig. 1. Cell viability of human leukemia NB4 cells treated with E1 and E2 extracts. NB4 cells were treated with E1, E2 extracts from Ganoderma lucidum or with C2 ganoderic acid for 19 h (as indicated in Section 2). Viability was measured as impermeability to propidium iodide. Upper part of the figure shows flow cytometry profiles of NB4 cells treated as indicated. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
NB4 cells with Ganoderma lucidum undiluted E1 extract prepared in the conditions described by other authors (Sliva et al., 2002; Stanley et al., 2005) cells reduced cell viability to 77% with respect to control untreated cells (Fig. 1). Impermeability to propidium iodide showed a reduction after treatment with E2 extract as compared to control cells. Compounds such as ganoderic acids have been considered as immunomodulators of intracellular activities in different cell types. In the case of NB4 cells, no effect was observed (Fig. 1) after addition of 0.4 mg/ml ganoderic acid C2 (a concentration similar to that used by other authors on different cell lines Müller et al., 2006). In order to study the induction of DNA fragmentation by Ganoderma lucidum extracts on NB4 cells we checked the levels of hypodiploid DNA after cell treatments. Fig. 2 shows the levels of fragmented DNA induced by Ganoderma lucidum E1 or E2 extracts or ganoderic acid C2 measured by flow cytometry as the fraction of subdiploid DNA. It can be observed that only E1 extract produced relevant DNA fragmentation, 44%. A lower effect (14%) was produced by C2 ganoderic acid treatment.
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We also evaluated apoptosis induction by Ganoderma lucidum E3 fraction on NB4 cells. We measured the fluorescence due to subdiploid DNA as a measure of DNA fragmentation what can be correlated with either necrosis or apoptosis (Fig. 4). In addition, the high levels of annexin V-FITC showed the fraction of cell population in each stage of apoptosis induced by the action Ganoderma lucidum compounds present in E3 fraction (Fig. 3). Figs. 3 and 4 reveal that in control cells there is only 8% of the total cell population that is positive for annexin V-FITC. Higher values of apoptosis measured as positive annexin V-FITC fluorescence (31% and 46%) were obtained after treatments with Ganoderma lucidum E3 fraction. As it can be observed in Fig. 4, most of the toxic effect (DNA fragmentation) produced by Ganoderma lucidum E3 fraction (at 15% and 40% of the initial concentration) correlated with an apoptosis process (compare the values of subdiploid DNA and annexin V-FITC in Fig. 4). Thus, we can assume an apoptosis process in NB4 cells as a result of the action of Ganoderma lucidum compounds present in E3 fraction. 3.3. Expression of apoptosis related proteins in NB4 human leukemia cells treated with an active semipurified fraction from Ganoderma lucidum
Fig. 2. Induction of subdiploid DNA in treated NB4 cells. The levels of DNA fragmentation produced by Ganoderma lucidum extracts (E1 and E2) and ganoderic acid C2 in NB4 cells as measured by the induction of subdiploid DNA are shown. Upper part of the figure shows flow cytometry profiles of the cell cycle of NB4 cells treated as indicated. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
3.2. Action of Ganoderma lucidum active methanolic fraction In the present work, we have also studied the effect of a chromatography fraction isolated from the methanolic extract of Ganoderma lucidum fruiting bodies. This fraction, named E3, showed also cytotoxic effects on NB4 cells. Fig. 3 shows the cell viability in control (untreated) human leukemia NB4 cells and in cells treated with E3 fraction diluted down to 40%, 15% or 5% (of the initial E3 fraction concentration). We evaluated both permeability to propidium iodide and annexin V-FITC fluorescence. Both parameters showed reductions in cell viability induced by E3 fraction and that the effect is concentration-dependent (Fig. 3); results for 5% dilution of E3 fraction (not shown) were not significantly different from the control. E3 fraction diluted down to 40% renders cell viability values of 68% and 51% as determined, respectively, by propidium iodide permeability and by annexin V-FITC fluorescence negative population.
The apoptosis induced on human leukemia NB4 cells by this fraction obtained from Ganoderma lucidum was studied. The changes in some proteins involved in the apoptosis response were analyzed by Western-blot analysis on NB4 cells treated for 19 h with Ganoderma lucidum E3 fraction 15%. Fig. 5 shows the profiles obtained for p53, Bax and Bcl2 proteins in control and treated human leukemia NB4 cells. p53 levels showed a reduction to 41% after Ganoderma lucidum E3 fraction 15% treatment. Bcl-2 showed also a reduction to 85% of the level in control NB4 cells. On the contrary, Bax increased its expression to 112% of control cells. Thus, we can observe a reduction of Bcl2/Bax ratio as a result of E3 fraction. We also studied the levels of intracellular kinases such as Akt or Erk using specific antibodies against the unphosphorylated and the phosphorylated forms. As it can be observed in Fig. 5, both Akt and Erk levels were reduced as a consequence of the action of active compounds present in E3 fraction. The phosphorylated forms were also reduced with respect to control cells. Anyhow, the level of the unphosphorylated form of Akt is higher than its phosphorylated counterpart. In contrast, pERK1 shows a higher level than the unphosphorylated ERK and pERK2 reduced clearly its proportion in treated cells. In order to know if the level of transcription factors such as NFB can influence the activation of the apoptosis cascade or the intracellular kinases signaling we studied NFB changes produced by Ganoderma lucidum E3 fraction diluted down to 15%. All NFB subunits showed reduced levels after the treatment in the conditions above indicated (Fig. 5). 4. Discussion 4.1. Effect of the treatment of acute promyelocytic leukemia NB4 cells with isolated fractions from Ganoderma lucidum: cell viability Several extracts from Ganoderma lucidum have already been used to treat tumor cells (Hu et al., 2002; Jiang et al., 2004a,b; Lu et al., 2004). These effects could be partially associated to triterpenes (Min et al., 2000), polysaccharides (Miyazaki and Nishijima, 1981; Sone et al., 1985) or proteins with immunomodulatory properties (Lin et al., 1997) mainly due to their action on the inhibition of DNA synthesis (Mizushina et al., 1998). We have tried to eluci-
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Fig. 3. Cell viability of human leukemia NB4 cells treated with E3 fraction. NB4 cells were treated for 19 h with Ganoderma lucidum E3 fraction (diluted down to 15% and 40%, striped and crosshatched bars, respectively). Cell viability was measured as the preservation of cell impermeability to propidium iodide and the counting of cells with low inner propidium fluorescence and low binding to annexin V-FITC. Upper part of the figure shows flow cytometry profiles of NB4 cells treated as indicated. Middle part of the figure shows flow cytometry profiles of cell cycle of treated NB4 cells. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
date the effect of Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] compounds on cellular activities of human leukemia cells (NB4) used as a model. We have used aqueous extracts E1 and E2 prepared as described by other authors. It is highly probable that sugars are present in these extracts. Several polysaccharides present in Ganoderma lucidum have been shown to have immunomodulatory and antitumor activities (Sliva et al., 2002; Stanley et al., 2005; Cao and Lin, 2006). We have also pre-
pared an E3 fraction isolated in column-chromatography from a methanol extract of Ganoderma lucidum. Based on the solvents used it may be possible that compounds with structure similar to terpenoid molecules could be present in this fraction (Min et al., 2000; Lin et al., 2003; Li et al., 2005). As it can be observed in Figs. 1 and 3, the treatment with aqueous extracts E1, E2 or the E3 fraction reduced viability of NB4 human leukemia cells. Ganoderma lucidum extracts have been
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Fig. 4. Apoptosis induction in human leukemia NB4 cells treated with E3 fraction. NB4 cells were treated for 19 h with Ganoderma lucidum E3 fraction (diluted down to 15% and 40%, striped and crosshatched bars, respectively). Cell apoptosis was measured as the induction of subdiploid DNA and the counting of cells with high binding to annexin V-FITC. Upper part of the figure shows flow cytometry profiles of the cell cycle of treated NB4 cells. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
shown to diminish the viability of HL-60 cells (Kim et al., 2007). Other authors reported inhibition of the growth induced by Ganoderma lucidum extracts on different leukemia cells such as acute myeloblastic leukemia (HL-60), chronic myeloid leukemia and acute lymphoblastic leukemia (Blin-1 and Nalm-6) (Müller et al., 2006). 4.2. Induction of apoptosis in human leukemia NB-4 cells by E1, E2 extracts and E3 fraction from Ganoderma lucidum We have also determined the appearance of subdiploid DNA after treatment with Ganoderma lucidum extracts on NB4 human leukemia cells. Cell death induction was produced after treatment with Ganoderma lucidum extract E1, ganoderic acid C2 and the E3 fraction as reflected by the release of subdiploid DNA (Figs. 2 and 4).
Fig. 5. Levels of apoptosis factors, intracellular kinases and NFB in human leukemia NB4 cells treated with E3 fraction. Representative Western blot analyses of expression of apoptosis proteins in NB4 human leukemia cells after treatment for 19 h with E3 fraction diluted to 15%. The intensities of the bands were determined in treated cells and their values were corrected to the intensity of actin band used as a control. These values were also corrected to the level of the respective band in untreated cells.
Furthermore, E3 fraction induced apoptosis which was also demonstrated by an increase in the number of annexin V-FITC positive cells. Lucidenic acid B from Ganoderma lucidum increased the number of apoptotic cells with a loss in the mitochondrial membrane potential (Hsu et al., 2008). An extract containing polysacharides from Ganoderma lucidum showed also a similar effect with mitochondrial damage in HL-60 cells (Kim et al., 2007). These authors demonstrated that cleaved DNA content increase from 3% in control cells to 15% in treated cells with reduction in the mitochondrial membrane potential after a 24 h treatment of HL-60 cells with a Ganoderma lucidum extract (Kim et al., 2007) showing an implication of the mitochondria in the toxic effect of Ganoderma on these leukemia cells (Kim et al., 2007). These effects were also observed in HL-60, Blin-1 and U937 cells (Müller et al., 2006). Thus, the hypothesis that induction of apoptosis by E3 fraction could be mediated by the mitochondria should be investigated. The use of a mixture of compounds may be useful since it can result in reduction of individual toxicity of the mixed compounds and additionally the interaction among them may be responsible for the therapeutic effects “in vivo” (Wilasrusmee et al., 2002). Furthermore, different compounds may modulate intracellular signalling and produce synergistic effect (Williamson, 2002). Triterpenoid compounds from Ganoderma lucidum abolish cell growth and invasive role of cancer cells and polysaccharides produce a stimulation of the immunological system giving rise to the release of cytokines and to an activation of immune cells (Lin, 2005). Other authors showed an increase in DNA cleavage when using extracts from Ganoderma lucidum and from Duchesnea chrysantha (Kim et al., 2007) with an enhanced induction of mitochondrial damage and apoptosis. These authors showed a correlation of these effects with increased mitochondrial accumulation of Bax, release of cytochrome c and activation of caspase 3. These data could
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explain that cell death induction by aqueous extracts was higher than that induced by ganoderic acid C2 (see Fig. 1) on the basis of a synergistic effects of the compounds present in these aqueous extracts from Ganoderma lucidum. 4.3. Expression of apoptosis proteins in human leukemia NB4 cells treated with semipurified E3 fraction from Ganoderma lucidum In NB4 cells treated with E3 fraction diluted to 15% we observed a reduced level of p53 with respect to control, as well as an increase in Bax levels and decrease in Bcl-2 levels (Fig. 5). These results are in accordance with those previously shown by several authors in different cell lines after treatments with Ganoderma lucidum extracts. Changes in the proportion of the expression levels of members of Bcl-2 family have been shown in HL-60 leukemia cells treated with lucidenic acid B (Hsu et al., 2008). Other authors have hypothesized a relationship between apoptosis induced by extracts from Ganoderma lucidum and D. chrysantha in HL-60 cells and changes in the mitochondria with reduction of Bcl-2, increase of Bax, release of cytochrome c and activation of caspase 3 (Kim et al., 2007). Our results show that NB4 cells treated with E3 seem to pass apoptosis with a reduction of p53 and Bcl-2 and increase of the levels of Bax. Several observations show that activation of Akt may induce anti-apoptotic effects in culture. Nevertheless the mechanisms involved in such a process are not completely understood and may vary depending of the type of cell studied. Anyhow, other factors must also be considered, i.e., signaling by NFB or the phosphorylation of apoptotic proteins such as Bad or procaspase-9 (Datta et al., 1997; Cardone et al., 1998). Treating U937 human leukemia cells with two different antitumor compounds rendered a relevant inhibition on Akt phosphorylation with a possible involvement of Akt in apoptosis processes (Jia et al., 2003). In NB4 leukemia cells treated with the methanolic fraction E3 of Ganoderma lucidum diluted to 15% we also observed a reduction of the levels of Akt and p-Akt (Fig. 5) similar to the results described in other cell types treated with Ganoderma lucidum extracts. The inhibition of phosphorylation of Erk1/2 may contribute to apoptosis induction in leukemia cells as a response to drug treatment (Wu et al., 2004). Other authors have described that treatment with extracts from Ganoderma lucidum on neuronal PC12 cells induced differentiation and neuroprotection with activation of Ras/ERK pathway (Cheung et al., 2000). Furthermore, a glycoproteic fraction from Ganoderma lucidum has been shown to activate several proteins such as Erk, Jnk y p38 when added to macrophages (Hsu et al., 2004). Moreover, a polysaccharide fraction from Ganoderma lucidum inhibited the proliferation of breast cancer cells reducing the expression of Erk (Xie et al., 2006). NB4 cells treated with the methanolic E3 fraction suffer a reduction in Erk and pErk2 (Fig. 5) claiming in favour of an involvement of this kinase in the cell death process induced by Ganoderma lucidum in these leukemia cells. Since other authors have described similar effects of some compounds from Ganoderma lucidum such as lucidenic acid B on other cancer cells (HepG-2 human liver cells) (Weng et al., 2008) it may be possible that some compounds present in the methanolic fraction from Ganoderma lucidum could be implicated in the inhibition of Erk in these leukemia cells. We have also observed that the levels of transcription factors such as NFB become reduced after treatment with the E3 fraction used in this study (Fig. 5). Some authors have shown similar results in breast cancer cells (Yeung et al., 2004) and prostate cancer cells (Lu et al., 2004) as a result of the treatment with Ganoderma lucidum aqueous extracts containing polysaccharides and triterpenoid compounds. A similar result was described in cortical neurons (Zhao et al., 2004) treated with a polysaccharidic fraction from Ganoderma
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lucidum and in human liver cancer cells treated with lucidenic acid B from Ganoderma lucidum (Weng et al., 2008). Our results are in accordance with those shown by these above mentioned authors. Thus, reduction of transcription activity of this factor seems to be a very common phenomenon in toxicity and cell death induced by Ganoderma lucidum in cancer cells. In conclusion, we demonstrate a toxic activity of the above shown extracts and fraction from Ganoderma lucidum on human leukemia cells probably mediated by apoptosis. We also propose that the active compounds present in these fractions from Ganoderma lucidum might be particularly useful in human leukemia therapy. Acknowledgements This work was supported in part by Grants from UAH PI2004/025, CAM-UAH 2005-040 and CCG06-UAH/SAL-0672 and ˜ was supported by a Miguel de Cervantes F.I.S. PI060119. E. Calvino fellowship from Universidad de Alcalá. 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culture of mycelium of Ganoderma lucidum. Agricultural Biological Chemistry 49, 2641–2653. Song, M.G., Gao, S.M., Du, K.M., Xu, M., Yu, Y., Zhou, Y.H., Wang, W., Chen, Z., Zhu, Y.S., Chen, G.Q., 2005. Nanomolar concentration of NSC606985, a camptothecin analog, induces leukemic-cell apoptosis through protein kinase C delta-dependent mechanisms. Blood 105, 3714–3721. Stanley, G., Harvey, K., Slivova, V., Jiang, J., Sliva, D., 2005. Ganoderma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-b1 from prostate cancer cells. Biochemical Biophysical Research Communication 330, 46–52. Trotter, J., 2004. WinMDI (Windows Multiple Document Interface for Flow Cytometry), version 2.8. Available from http://facs.scripps.edu/software.html. Weng, C.J., Chau, C.F., Hsieh, Y.S., Yang, S.F., Yen, G.C., 2008. Lucidenic acid inhibits PMA-induced invasion of human hepatoma cells through inactivating MAPK/ERK signal transduction pathway and reducing binding activities of NF-kappaB and AP-1. Carcinogenesis 29, 147–156. Wilasrusmee, C., Kittur, S., Siddiqui, J., Bruch, D., Wilasrumee, S., Kittur, D.S., 2002. In vitro immunomodulatory effects of ten commonly used herbs on murine lymphocytes. Journal of Alternative and Complementary Medicine 8, 467–475. Williamson, E.M., 2002. Synergy and other interactions in phytomedicines. Phytomedicine 8, 401–409. Wu, J., Wong, W.W., Khosravi, F., Minden, M.D., Penn, L.Z., 2004. Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells to lovastatin-induced apoptosis. Cancer Research 64, 6461–6468. Xie, Y.Z., Li, S.Z., Yee, A., La Pierre, D.P., Deng, Z., Lee, D.Y., 2006. Ganoderma lucidum inhibits tumour cell proliferation and induces tumour cell death. Enzyme and Microbial Technology 40, 177–185. Yeung, W.H., Lu, Q., Zhang, Q., Go, V.L., 2004. Chemical and biochemical basis of the potential anti-tumor properties of Ganoderma lucidum. Current Topics in Nutraceutical Research 2, 67–77. Zhao, H.B., Lin, S.Q., Liu, J.H., Lin, Z.B., 2004. Polysaccharide extract isolated from Ganoderma lucidum protects rat cerebral cortical neurons from hypoxia/reoxygenation injury. Journal Pharmacological Science 95, 294–298.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ganoderma lucidum induced apoptosis in NB4 human leukemia cells: Involvement of Akt and Erk ˜ a , José Luis Manjón b , Pilar Sancho a , M. Cristina Tejedor a , Angel Herráez a , José C. Diez a,∗ Eva Calvino a b
Departamento de Bioquímica y Biología Molecular, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain Departamento de Biología Vegetal, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 20 July 2009 Received in revised form 7 December 2009 Accepted 17 December 2009 Available online 29 December 2009 Keywords: Apoptosis Bax Bcl-2 Ganoderma lucidum (Curtis) P. Karst. Ganodermataceae Donk Leukemia p53
a b s t r a c t Aim of the study: The final goal of this work was to study the toxic and apoptosis effects induced by fractions from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] on NB4 human leukemia cells. Materials and methods: Two aqueous extracts and a methanol-extracted column-chromatography semipurified fraction were obtained from Ganoderma lucidum fruiting body. Flow cytometry analyses were used to measure cell viability, cell cycle and DNA fragmentation and to quantify apoptosis. Western-blot analyses were used to quantify changes in apoptosis proteins and intracellular kinases. Results: Aqueous extracts slightly reduce cell viability and induce DNA fragmentation in NB4 cells. Methanol-extracted semipurified fraction at dilutions down to 15% or 40% of the initial fraction concentration reduced significantly the viability of these leukemia cells (treated for 19 h) with induction of DNA fragmentation and induction of apoptosis. Overmore, the dilution down to 15% of the initial E3 concentration induced a reduction of p53 levels, of the Bcl2/Bax relationship as well as reduced levels of both unphosphorylated and phosphorylated Akt (Protein kinase Akt, protein kinase B) and Erk (Erk1 and 2). Conclusions: Induction of apoptosis and alterations in signal transduction kinases (Akt and Erk) are produced by active fractions from Ganoderma lucidum on human leukemia cells. These data could be of important relevance from the viewpoint of antitumor actions of compounds from Ganoderma lucidum. Eventual therapy applications in leukemia cells might be developed. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction On recent times, a search is under way for new therapeutic agents with better efficiency and with more selective action on tumor cells. The fungus Ganoderma lucidum (common names: Reishi, Lingzhi) is a well known mushroom in traditional Chinese medicine. Extracts from different parts (mycelia, spores and fruiting bodies) of this mushroom have been used for prevention and treatment of different diseases in ancient China. Particularly relevant are its claimed antitumor properties (Hu et al., 2002; Liu et al., 2002; Sliva, 2004). Several reports exist on the action of Ganoderma lucidum extracts against tumor cells: growth inhibition in human prostate and bladder cancer cell lines (Jiang et al., 2004a; Lu et al., 2004) and inhibition of cell proliferation and induction of apoptosis in human colon carcinoma and breast cancer cell lines (Hu et al., 2002; Hong et al., 2004; Jiang et al., 2004b). Different compounds are present as natural constituents of this mushroom. Among them, several nucleosides, proteins, polysaccharides, fatty
∗ Corresponding author. Tel.: +34 91 8854582/579; fax: +34 91 8854585. E-mail address: [email protected] (J.C. Diez). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.12.027
acids, sterols and triterpenes have been described as potentially responsible of the antitumor activity of Ganoderma lucidum (Li et al., 2005; Yeung et al., 2004). Some of the antitumor properties of Ganoderma lucidum can be assigned to polysaccharides, for example retarding the growth of sarcoma cells (Cao and Lin, 2004). It is also accepted that, at least in part, their antitumor activity may be a consequence of their immunomodulatory properties (Wilasrusmee et al., 2002; Williamson, 2002; Lin, 2005). Some triterpenes present in Ganoderma lucidum have also demonstrated inhibition of growth of hepatoma cells by altering intracellular phosphorylation pathways (Lin et al., 2003). Leukemic human cell line NB4 was derived from bone marrow cells of a female patient of acute promyelocytic leukemia. These cells are sensitive to camptotecin analogues (Song et al., 2005), arsenic trioxide (Ramos et al., 2005), retinoids (Cincinelli et al., 2005), UV radiation (Biggs et al., 2001) or prooxidant agents (Shen et al., 1999) that induce cell death. This cell line can hence be a useful model to study the action of extracts, fractions or compounds with tentative antitumor activity. Recently, it has been shown that Ganoderma lucidum inhibits proliferation of different hematopoietic cell lines such as K562,
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HL1-60, U937, Blin-1, Nalm-6, etc. (Müller et al., 2006). Moreover, some extracts and fractions obtained from Ganoderma lucidum have been shown to induce apoptosis (Hu et al., 2002; Lu et al., 2004; Yeung et al., 2004; Li et al., 2005). We have studied the cytotoxicity induced in human leukemia NB4 cells by different Ganoderma lucidum extracts prepared as it has been indicated by other authors (Sliva et al., 2002; Stanley et al., 2005) as well as the possible involvement of some apoptotic factors in such toxic effects. We also have focused our interest on activation and phosphorylation of intracellular kinases (MAP-kinases) which are altered in other cellular model systems (Sliva et al., 2002; Jiang et al., 2004a,b). The final aim of this work has been to demonstrate toxic activity and apoptosis induction by Ganoderma lucidum (Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk) on human leukemia cells. We have also tried to show a possible involvement of some intracellular kinases such as Akt or Erk in cell death processes. 2. Materials and methods 2.1. Source of Ganoderma lucidum Ganoderma lucidum (Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk) strain was isolated from the fruit body tissue (context) of a wood-inhabiting filamentous parasitic fungi growing in a natural habitat on Quercus ilex roots in Cáceres (Spain). A small part of the tissue context developed Ganoderma lucidum mycelium that it was cultured and subcultured in a Malt extract solid medium (MA). In order to produce fruiting bodies it was necessary to cultivate the fungus on lignocellulosic solid medium substrate (Chang and Miles, 2004), adequately hydrated and autoclaved. Once, the lignocellulosic medium was inoculated with Ganoderma lucidum mycelia, the substrate was incubated until a proper mycelial colonization. Subsequently, the bulk mycelium develops fruit bodies that reach maturity in 1–2 months. 2.2. Extracts of Ganoderma lucidum Fruiting bodies obtained from Ganoderma lucidum were resuspended in sterile water to a concentration of 50 mg/ml (Sliva et al., 2002; Stanley et al., 2005). They were then homogenized and centrifuged at 14,000 rpm (in a Beckman J2.21 centrifuge using JA20.1 rotor) for 5 min, at room temperature, rendering what was called extract 1 (E1). The pellet was resuspended in the same volume of sterile water, boiled for 5 min, sonicated for 20 s at 50% intensity, centrifuged at 14,000 rpm for 5 min and the supernatant obtained was called extract 2 (E2). Afterwards, these extracts were lyophilized and dissolved in 1/10 the initial volume, giving ten-fold concentrated extracts. Methanolic extracts of Ganoderma lucidum were also prepared as follows. Fruiting body (2 g) was disrupted with liquid nitrogen, resuspended at 50 mg/ml in 10% methanol and extracted three times with 10% methanol for 24 h with agitation. The soluble extract was evaporated in a rotary evaporator until dry, and was dissolved in absolute methanol. To purify this extract, it was adsorbed on 0.2 g of silicagel to be applied onto a silica gel column (1 cm × 15.5 cm) and subsequently eluted with 3:1:1 butanol:acetic acid:water. Column fractions (0.2 ml) with an Rf near 0.74 were combined and the solvent was evaporated and the final sample (1.6 mg) was resuspended in 0.01 ml DMSO to obtain the fraction 3 (E3). 2.3. Human acute promyelocytic leukemia (NB4) cells The human NB4 leukemia cell line was a kind gift from Dr. D. Delgado (Dpto. Biología Molecular, Universidad de Cantabria. Santander. Spain). They were kept in culture at 37 ◦ C and 5% CO2 in RPMI medium (Gibco-Life Technologies) with 10% FCS, 1% penicillin–streptomycin 1% and 0.02 mg/ml gentamycin.
2.4. Cell treatments with Ganoderma lucidum extracts and fraction E1 and E2 extracts and E3 fraction were added separately to 1 ml aliquots of cell culture (0.5 × 106 cell/ml) and allowed to act for 19 h. Liophylized E1 extract (14.9 mg dried weight) was dissolved in 0.9 ml of sterile distilled water and 60 l were added per ml of cell culture. Liophylized E2 extract (3 mg dried weight) was dissolved in 0.9 ml of sterile distilled water and 60 l were added per ml of cell culture. Fraction 3 (E3) was added at three different doses, equivalent to 40% and 15% of the initial concentration of E3, as 5, 2 and 0.7 l of E3 per ml of culture, respectively (containing 0.8, 0.32 and 0.1 mg of the dried mass contained in E3). Ganoderic acid C2 was purchased from Chromadex (http://www.chromadex.com, code ASB-00007058) and prepared at 20 mg/ml in DMSO. Cells (0.5 × 106 cells/ml) were treated with Ganoderic acid C2 at a final concentration of 0.4 mg/ml for 19 h. Cells were also treated with 100 M etoposide for 19 h as a positive control of toxicity and apoptosis induction. DMSO (5 l) was also added to cells (1 ml at 0.5 × 106 cell/ml) as a negative control since no toxic effect was observed on cell culture (i.e. no reduction of cell viability, no DNA fragmentation, etc.). 2.5. Permeability of treated cells to propidium iodide and flow cytometry analyses Cell viability of NB4 cells treated with Ganoderma lucidum extracts was determined by flow cytometry by measuring the level of impermeability to propidium iodide. 2.5 × 105 treated cells were collected and washed in PBS and centrifuged at 1,200 rpm for 5 min. The cells were resuspended in 500 l of PBS and stained with propidium iodide (0.1 mg/ml) and analyzed either in FACScan or FACScalibur (Becton Dickinson, San José, CA, USA). For each sample the acquisition was finished at 10,000 counts. Data analysis was performed using WinMDI software (Trotter, 2004). Cell fragments were discriminated from the non-viable cells in dot plots FSC/FL2-H, where FL-2-H corresponds to propidium iodide fluorescence. 2.6. Cytometric analysis of cell populations with subdiploid DNA and cell cycle Apoptotic cells were counted on the basis of DNA content per cell after permeabilization with NP40. After treatments with 100 M etoposide or Ganoderma lucidum extracts, 2.5 × 105 cells were collected and washed with PBS. The pellet was resuspended in 475 l of a solution containing 0.5 mg/ml RNase, 0.1% NP-40 in PBS and incubated for 30 min, in order to extract low molecular weight DNA from cell nuclei. The remnant DNA in cells was stained with 0.05 mg/ml propidium iodide, immediately measured in the cytometer. Cells with hypodiploid DNA (apoptotic cells) were distinguished from those containing diploid DNA (non-apoptotic cells) on the basis of a different fluorescence intensity of propidium iodide. Cell cycle progress was studied in the same samples. Histograms of the untreated cells were used to define the positions of the different phases G1, S and G2/M phases of cell cycle. 2.7. Annexin V-FITC assays: phosphatidylserine exposure on the outer side of membrane To quantify the level of apoptosis through the presence of phosphatidylserine in the outer membrane side in NB4 treated cells the
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Annexin V-FITC Apoptosis Detection Kit from Calbiochem was used. For this assay, 3 × 106 treated cells were collected and centrifuged at 1500 rpm for 3 min. They were then incubated for 15 min in the dark at room temperature in 500 l PBS containing 10 l of binding reagent and 1.25 l Annexin V-FITC. Subsequently, the cells were centrifuged at 2,300 rpm for 5 min and resuspended in 500 l binding buffer 1× diluted in PBS. 10 l propidium iodide was added and the samples were analyzed in a FACScalibur (Becton Dickinson, San José, CA, USA) cytometer. Data were analyzed using WinMDI 2.8 software (Trotter, 2004). 2.8. Western-blot analyses Cells were grown at a concentration of 2 × 105 cells/ml, treated with the extracts and collected by centrifugation at 1,200 rpm for 5 min and resuspended in 200 l lysis buffer solution containing 50 mM Tris/HCl pH 8.0, 150 mM NaCl; 5 mM EDTA; 0.5% NP-40; 1 mM PMSF. After 20 min at 4 ◦ C, cells were sonicated for 20 s (duty cycle 100%, output control 50%) and then centrifuged (14,000 rpm, 5 min, 4 ◦ C). Resulting supernatants were analyzed by electrophoresis and blotting. Proteins (20 g/well) were loaded into a 10% SDS-PAGE, electrophoretically separated and, transferred to nitrocellulose membranes as described. Membranes were blocked with 5% powder milk in TTBS (50 mM Tris pH 7.2, 140 mM NaCl, 0.06% Tween 20). Afterwards, they were washed with TTBS and incubated with diverse diluted in TTBS containing milk. All the following antibodies were obtained from Santa Cruz Biotechnology, CA, USA, and used at the dilution indicated: anti-Bax mouse monoclonal IgG2b (B-9; sc-7480), 1:100; anti-Bcl-2 mouse monoclonal IgG1 (C-2; sc7382), 1:200; anti-Akt1/2/3 (H-136, sc8312), 1:400; anti-pAkt1/2/3 (Ser 437, sc-7985-R), 1:400; anti Erk1/2 (sc-154), 1:3,000; anti p-Erk1/2 (Tyr204 , sc-7383), 1:200; anti-NFB p50 and p105 (EA0, sc-8414), 1:200; anti-NFB p65 (F-6, sc-8008), 1:200. Anti-p53 mouse monoclonal IgG (Ab-1, OP03) was purchased from Calbiochem (Oncogene Research Products, MA, USA) and diluted 1:50. Monoclonal anti--actin was purchased from Sigma A5441 and used at 1:5,000. After incubation of the nitrocellulose membranes with antibodies overnight at 4 ◦ C, the bands were revealed using enhanced chemiluminescence (ECL) detection (Amersham) with goat antimouse horse-radish peroxidase (HRP)-conjugated polyclonal antibodies (1:2,000) from Promega (Madison, WI, USA). The intensities of the bands were corrected with respect to the intensity of actin band in the blot as a control for constitutive expression and quantified relative to the intensity of the band in control cells without any treatment which were considered as 100%. 2.9. Statistical procedures All the experiments were repeated three or four times. The mean and SEM were graphically represented. In order to determine the significance of the effect, Student’s test was used. The asterisk * indicate p < 0.05. For calculations we used Microsoft Excel 2003. 3. Results 3.1. Viability of NB4 cells and induction of DNA fragmentation after treatment with Ganoderma lucidum aqueous extracts We studied the cell viability measure through permeability to propidium iodide, of human leukemia NB4 cells after independent treatments with aqueous E1 or E2 extract for 19 h. Fig. 1 shows a representative flow cytometry profile (upper part) and the statistically processed data (lower panel). As can be observed, treatment of
Fig. 1. Cell viability of human leukemia NB4 cells treated with E1 and E2 extracts. NB4 cells were treated with E1, E2 extracts from Ganoderma lucidum or with C2 ganoderic acid for 19 h (as indicated in Section 2). Viability was measured as impermeability to propidium iodide. Upper part of the figure shows flow cytometry profiles of NB4 cells treated as indicated. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
NB4 cells with Ganoderma lucidum undiluted E1 extract prepared in the conditions described by other authors (Sliva et al., 2002; Stanley et al., 2005) cells reduced cell viability to 77% with respect to control untreated cells (Fig. 1). Impermeability to propidium iodide showed a reduction after treatment with E2 extract as compared to control cells. Compounds such as ganoderic acids have been considered as immunomodulators of intracellular activities in different cell types. In the case of NB4 cells, no effect was observed (Fig. 1) after addition of 0.4 mg/ml ganoderic acid C2 (a concentration similar to that used by other authors on different cell lines Müller et al., 2006). In order to study the induction of DNA fragmentation by Ganoderma lucidum extracts on NB4 cells we checked the levels of hypodiploid DNA after cell treatments. Fig. 2 shows the levels of fragmented DNA induced by Ganoderma lucidum E1 or E2 extracts or ganoderic acid C2 measured by flow cytometry as the fraction of subdiploid DNA. It can be observed that only E1 extract produced relevant DNA fragmentation, 44%. A lower effect (14%) was produced by C2 ganoderic acid treatment.
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We also evaluated apoptosis induction by Ganoderma lucidum E3 fraction on NB4 cells. We measured the fluorescence due to subdiploid DNA as a measure of DNA fragmentation what can be correlated with either necrosis or apoptosis (Fig. 4). In addition, the high levels of annexin V-FITC showed the fraction of cell population in each stage of apoptosis induced by the action Ganoderma lucidum compounds present in E3 fraction (Fig. 3). Figs. 3 and 4 reveal that in control cells there is only 8% of the total cell population that is positive for annexin V-FITC. Higher values of apoptosis measured as positive annexin V-FITC fluorescence (31% and 46%) were obtained after treatments with Ganoderma lucidum E3 fraction. As it can be observed in Fig. 4, most of the toxic effect (DNA fragmentation) produced by Ganoderma lucidum E3 fraction (at 15% and 40% of the initial concentration) correlated with an apoptosis process (compare the values of subdiploid DNA and annexin V-FITC in Fig. 4). Thus, we can assume an apoptosis process in NB4 cells as a result of the action of Ganoderma lucidum compounds present in E3 fraction. 3.3. Expression of apoptosis related proteins in NB4 human leukemia cells treated with an active semipurified fraction from Ganoderma lucidum
Fig. 2. Induction of subdiploid DNA in treated NB4 cells. The levels of DNA fragmentation produced by Ganoderma lucidum extracts (E1 and E2) and ganoderic acid C2 in NB4 cells as measured by the induction of subdiploid DNA are shown. Upper part of the figure shows flow cytometry profiles of the cell cycle of NB4 cells treated as indicated. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
3.2. Action of Ganoderma lucidum active methanolic fraction In the present work, we have also studied the effect of a chromatography fraction isolated from the methanolic extract of Ganoderma lucidum fruiting bodies. This fraction, named E3, showed also cytotoxic effects on NB4 cells. Fig. 3 shows the cell viability in control (untreated) human leukemia NB4 cells and in cells treated with E3 fraction diluted down to 40%, 15% or 5% (of the initial E3 fraction concentration). We evaluated both permeability to propidium iodide and annexin V-FITC fluorescence. Both parameters showed reductions in cell viability induced by E3 fraction and that the effect is concentration-dependent (Fig. 3); results for 5% dilution of E3 fraction (not shown) were not significantly different from the control. E3 fraction diluted down to 40% renders cell viability values of 68% and 51% as determined, respectively, by propidium iodide permeability and by annexin V-FITC fluorescence negative population.
The apoptosis induced on human leukemia NB4 cells by this fraction obtained from Ganoderma lucidum was studied. The changes in some proteins involved in the apoptosis response were analyzed by Western-blot analysis on NB4 cells treated for 19 h with Ganoderma lucidum E3 fraction 15%. Fig. 5 shows the profiles obtained for p53, Bax and Bcl2 proteins in control and treated human leukemia NB4 cells. p53 levels showed a reduction to 41% after Ganoderma lucidum E3 fraction 15% treatment. Bcl-2 showed also a reduction to 85% of the level in control NB4 cells. On the contrary, Bax increased its expression to 112% of control cells. Thus, we can observe a reduction of Bcl2/Bax ratio as a result of E3 fraction. We also studied the levels of intracellular kinases such as Akt or Erk using specific antibodies against the unphosphorylated and the phosphorylated forms. As it can be observed in Fig. 5, both Akt and Erk levels were reduced as a consequence of the action of active compounds present in E3 fraction. The phosphorylated forms were also reduced with respect to control cells. Anyhow, the level of the unphosphorylated form of Akt is higher than its phosphorylated counterpart. In contrast, pERK1 shows a higher level than the unphosphorylated ERK and pERK2 reduced clearly its proportion in treated cells. In order to know if the level of transcription factors such as NFB can influence the activation of the apoptosis cascade or the intracellular kinases signaling we studied NFB changes produced by Ganoderma lucidum E3 fraction diluted down to 15%. All NFB subunits showed reduced levels after the treatment in the conditions above indicated (Fig. 5). 4. Discussion 4.1. Effect of the treatment of acute promyelocytic leukemia NB4 cells with isolated fractions from Ganoderma lucidum: cell viability Several extracts from Ganoderma lucidum have already been used to treat tumor cells (Hu et al., 2002; Jiang et al., 2004a,b; Lu et al., 2004). These effects could be partially associated to triterpenes (Min et al., 2000), polysaccharides (Miyazaki and Nishijima, 1981; Sone et al., 1985) or proteins with immunomodulatory properties (Lin et al., 1997) mainly due to their action on the inhibition of DNA synthesis (Mizushina et al., 1998). We have tried to eluci-
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Fig. 3. Cell viability of human leukemia NB4 cells treated with E3 fraction. NB4 cells were treated for 19 h with Ganoderma lucidum E3 fraction (diluted down to 15% and 40%, striped and crosshatched bars, respectively). Cell viability was measured as the preservation of cell impermeability to propidium iodide and the counting of cells with low inner propidium fluorescence and low binding to annexin V-FITC. Upper part of the figure shows flow cytometry profiles of NB4 cells treated as indicated. Middle part of the figure shows flow cytometry profiles of cell cycle of treated NB4 cells. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
date the effect of Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] compounds on cellular activities of human leukemia cells (NB4) used as a model. We have used aqueous extracts E1 and E2 prepared as described by other authors. It is highly probable that sugars are present in these extracts. Several polysaccharides present in Ganoderma lucidum have been shown to have immunomodulatory and antitumor activities (Sliva et al., 2002; Stanley et al., 2005; Cao and Lin, 2006). We have also pre-
pared an E3 fraction isolated in column-chromatography from a methanol extract of Ganoderma lucidum. Based on the solvents used it may be possible that compounds with structure similar to terpenoid molecules could be present in this fraction (Min et al., 2000; Lin et al., 2003; Li et al., 2005). As it can be observed in Figs. 1 and 3, the treatment with aqueous extracts E1, E2 or the E3 fraction reduced viability of NB4 human leukemia cells. Ganoderma lucidum extracts have been
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Fig. 4. Apoptosis induction in human leukemia NB4 cells treated with E3 fraction. NB4 cells were treated for 19 h with Ganoderma lucidum E3 fraction (diluted down to 15% and 40%, striped and crosshatched bars, respectively). Cell apoptosis was measured as the induction of subdiploid DNA and the counting of cells with high binding to annexin V-FITC. Upper part of the figure shows flow cytometry profiles of the cell cycle of treated NB4 cells. Lower part of the figure shows the statistical representation of the results. These values represent the mean of three different experiments. The standard errors of the mean are shown as errors bars, *p < 0.05.
shown to diminish the viability of HL-60 cells (Kim et al., 2007). Other authors reported inhibition of the growth induced by Ganoderma lucidum extracts on different leukemia cells such as acute myeloblastic leukemia (HL-60), chronic myeloid leukemia and acute lymphoblastic leukemia (Blin-1 and Nalm-6) (Müller et al., 2006). 4.2. Induction of apoptosis in human leukemia NB-4 cells by E1, E2 extracts and E3 fraction from Ganoderma lucidum We have also determined the appearance of subdiploid DNA after treatment with Ganoderma lucidum extracts on NB4 human leukemia cells. Cell death induction was produced after treatment with Ganoderma lucidum extract E1, ganoderic acid C2 and the E3 fraction as reflected by the release of subdiploid DNA (Figs. 2 and 4).
Fig. 5. Levels of apoptosis factors, intracellular kinases and NFB in human leukemia NB4 cells treated with E3 fraction. Representative Western blot analyses of expression of apoptosis proteins in NB4 human leukemia cells after treatment for 19 h with E3 fraction diluted to 15%. The intensities of the bands were determined in treated cells and their values were corrected to the intensity of actin band used as a control. These values were also corrected to the level of the respective band in untreated cells.
Furthermore, E3 fraction induced apoptosis which was also demonstrated by an increase in the number of annexin V-FITC positive cells. Lucidenic acid B from Ganoderma lucidum increased the number of apoptotic cells with a loss in the mitochondrial membrane potential (Hsu et al., 2008). An extract containing polysacharides from Ganoderma lucidum showed also a similar effect with mitochondrial damage in HL-60 cells (Kim et al., 2007). These authors demonstrated that cleaved DNA content increase from 3% in control cells to 15% in treated cells with reduction in the mitochondrial membrane potential after a 24 h treatment of HL-60 cells with a Ganoderma lucidum extract (Kim et al., 2007) showing an implication of the mitochondria in the toxic effect of Ganoderma on these leukemia cells (Kim et al., 2007). These effects were also observed in HL-60, Blin-1 and U937 cells (Müller et al., 2006). Thus, the hypothesis that induction of apoptosis by E3 fraction could be mediated by the mitochondria should be investigated. The use of a mixture of compounds may be useful since it can result in reduction of individual toxicity of the mixed compounds and additionally the interaction among them may be responsible for the therapeutic effects “in vivo” (Wilasrusmee et al., 2002). Furthermore, different compounds may modulate intracellular signalling and produce synergistic effect (Williamson, 2002). Triterpenoid compounds from Ganoderma lucidum abolish cell growth and invasive role of cancer cells and polysaccharides produce a stimulation of the immunological system giving rise to the release of cytokines and to an activation of immune cells (Lin, 2005). Other authors showed an increase in DNA cleavage when using extracts from Ganoderma lucidum and from Duchesnea chrysantha (Kim et al., 2007) with an enhanced induction of mitochondrial damage and apoptosis. These authors showed a correlation of these effects with increased mitochondrial accumulation of Bax, release of cytochrome c and activation of caspase 3. These data could
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explain that cell death induction by aqueous extracts was higher than that induced by ganoderic acid C2 (see Fig. 1) on the basis of a synergistic effects of the compounds present in these aqueous extracts from Ganoderma lucidum. 4.3. Expression of apoptosis proteins in human leukemia NB4 cells treated with semipurified E3 fraction from Ganoderma lucidum In NB4 cells treated with E3 fraction diluted to 15% we observed a reduced level of p53 with respect to control, as well as an increase in Bax levels and decrease in Bcl-2 levels (Fig. 5). These results are in accordance with those previously shown by several authors in different cell lines after treatments with Ganoderma lucidum extracts. Changes in the proportion of the expression levels of members of Bcl-2 family have been shown in HL-60 leukemia cells treated with lucidenic acid B (Hsu et al., 2008). Other authors have hypothesized a relationship between apoptosis induced by extracts from Ganoderma lucidum and D. chrysantha in HL-60 cells and changes in the mitochondria with reduction of Bcl-2, increase of Bax, release of cytochrome c and activation of caspase 3 (Kim et al., 2007). Our results show that NB4 cells treated with E3 seem to pass apoptosis with a reduction of p53 and Bcl-2 and increase of the levels of Bax. Several observations show that activation of Akt may induce anti-apoptotic effects in culture. Nevertheless the mechanisms involved in such a process are not completely understood and may vary depending of the type of cell studied. Anyhow, other factors must also be considered, i.e., signaling by NFB or the phosphorylation of apoptotic proteins such as Bad or procaspase-9 (Datta et al., 1997; Cardone et al., 1998). Treating U937 human leukemia cells with two different antitumor compounds rendered a relevant inhibition on Akt phosphorylation with a possible involvement of Akt in apoptosis processes (Jia et al., 2003). In NB4 leukemia cells treated with the methanolic fraction E3 of Ganoderma lucidum diluted to 15% we also observed a reduction of the levels of Akt and p-Akt (Fig. 5) similar to the results described in other cell types treated with Ganoderma lucidum extracts. The inhibition of phosphorylation of Erk1/2 may contribute to apoptosis induction in leukemia cells as a response to drug treatment (Wu et al., 2004). Other authors have described that treatment with extracts from Ganoderma lucidum on neuronal PC12 cells induced differentiation and neuroprotection with activation of Ras/ERK pathway (Cheung et al., 2000). Furthermore, a glycoproteic fraction from Ganoderma lucidum has been shown to activate several proteins such as Erk, Jnk y p38 when added to macrophages (Hsu et al., 2004). Moreover, a polysaccharide fraction from Ganoderma lucidum inhibited the proliferation of breast cancer cells reducing the expression of Erk (Xie et al., 2006). NB4 cells treated with the methanolic E3 fraction suffer a reduction in Erk and pErk2 (Fig. 5) claiming in favour of an involvement of this kinase in the cell death process induced by Ganoderma lucidum in these leukemia cells. Since other authors have described similar effects of some compounds from Ganoderma lucidum such as lucidenic acid B on other cancer cells (HepG-2 human liver cells) (Weng et al., 2008) it may be possible that some compounds present in the methanolic fraction from Ganoderma lucidum could be implicated in the inhibition of Erk in these leukemia cells. We have also observed that the levels of transcription factors such as NFB become reduced after treatment with the E3 fraction used in this study (Fig. 5). Some authors have shown similar results in breast cancer cells (Yeung et al., 2004) and prostate cancer cells (Lu et al., 2004) as a result of the treatment with Ganoderma lucidum aqueous extracts containing polysaccharides and triterpenoid compounds. A similar result was described in cortical neurons (Zhao et al., 2004) treated with a polysaccharidic fraction from Ganoderma
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lucidum and in human liver cancer cells treated with lucidenic acid B from Ganoderma lucidum (Weng et al., 2008). Our results are in accordance with those shown by these above mentioned authors. Thus, reduction of transcription activity of this factor seems to be a very common phenomenon in toxicity and cell death induced by Ganoderma lucidum in cancer cells. In conclusion, we demonstrate a toxic activity of the above shown extracts and fraction from Ganoderma lucidum on human leukemia cells probably mediated by apoptosis. We also propose that the active compounds present in these fractions from Ganoderma lucidum might be particularly useful in human leukemia therapy. Acknowledgements This work was supported in part by Grants from UAH PI2004/025, CAM-UAH 2005-040 and CCG06-UAH/SAL-0672 and ˜ was supported by a Miguel de Cervantes F.I.S. PI060119. E. Calvino fellowship from Universidad de Alcalá. 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