Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells
2004; Wiley; Volume: 114; Issue: 1 Linguagem: Inglês
10.1002/ijc.20588
ISSN1097-0215
AutoresPierre-Olivier Harmand, Raphaël E. Duval, Christiane Delage, Alain Simon,
Tópico(s)NF-κB Signaling Pathways
ResumoInternational Journal of CancerVolume 114, Issue 1 p. 1-11 Cancer Cell BiologyFree Access Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells Pierre-Olivier Harmand, Pierre-Olivier Harmand Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, FranceSearch for more papers by this authorRaphaël Duval, Raphaël Duval Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, FranceSearch for more papers by this authorChristiane Delage, Christiane Delage Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, FranceSearch for more papers by this authorAlain Simon, Corresponding Author Alain Simon [email protected] Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, France Fax: +33-555-43-59-11Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, 2 Rue du Docteur Marcland, 87025 Limoges Cedex, FranceSearch for more papers by this author Pierre-Olivier Harmand, Pierre-Olivier Harmand Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, FranceSearch for more papers by this authorRaphaël Duval, Raphaël Duval Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, FranceSearch for more papers by this authorChristiane Delage, Christiane Delage Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, FranceSearch for more papers by this authorAlain Simon, Corresponding Author Alain Simon [email protected] Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, Limoges Cedex, France Fax: +33-555-43-59-11Laboratoire de Chimie-Physique, UPRES EA 1085, Faculté de Pharmacie, 2 Rue du Docteur Marcland, 87025 Limoges Cedex, FranceSearch for more papers by this author First published: 02 November 2004 https://doi.org/10.1002/ijc.20588Citations: 118 AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Over the coming years, skin cancer could become a significant public health problem. Previous results indicate that ursolic acid (UA), a pentacyclic triterpene acid, has pleiotropic biologic activities such as antiinflammatory and antiproliferative activities on cancer cells. As UA represents a promising chemical entity for the protection of human skin, in agreement with tests done by the cosmetic industry, we investigated its effects on the M4Beu human melanoma cell line. In this report, we demonstrated for the first time that UA had a significant antiproliferative effect on M4Beu, associated with the induction of an apoptotic process, characterized by caspase-3 activation, the downstream central effector of apoptosis. We demonstrated that UA-induced apoptosis was dependent on the mitochondrial intrinsic pathway, as shown by transmembrane potential collapse (ΔΨm) and by alteration of the Bax-Bcl-2 balance, with a concomitant increase in Bax expression and decrease in Bcl-2 expression. We also showed that UA-induced ΔΨm was associated with apoptosis-inducing factor leakage from mitochondria. Taken together, our results suggest that UA-induced apoptosis on M4Beu cells is accomplished via triggering of mitochondrial pathway. In conclusion, UA could be an encouraging compound in the treatment or prevention of skin cancer and may represent a new promising anticancer agent in the treatment of melanoma. © 2004 Wiley-Liss, Inc. Classically considered as rare, melanoma is the most serious form of skin cancer and its frequency is increasing in all Caucasian skin populations, particularly in the 30–50 age bracket.1 Worldwide, during the past 10 years, the number of melanoma cases and its associated mortality increased more than all other cancers.2, 3 The present epidemiologic models predict that, in the coming years, 1 person in 65 born in Western countries could be confronted with a melanoma during his or her life time.4 Skin cancers could become a significant public health problem over the coming years. Hence, studying skin cancer in vitro, particularly melanoma cell lines, and reducing this frequency by all possible means are very important. In this study, we examined the effects of ursolic acid (UA), a pentacyclic triterpene, on M4Beu human melanoma cells. UA occurs naturally in a large variety of vegetarian foods, medicinal herbs and plants.5 UA is also the main component of protective waxlike coatings of apples, pears and other fruit consumed in a normal diet.6 This triterpene compound has been reported to induce pleiotropic biologic activities such as antibacterial, hepatoprotective, immunomodulatory and antiproliferative activities.7 UA inhibits invasion and metastasis by reducing the expression of matrix metalloproteinase-9 (MMP-9) in HT1080 human fibrosarcoma cells.8 MMPs are proteolytic enzymes, which degrade native collagens and other extracellular matrix components. These previous studies are in agreement with tests done by the cosmetic industry, which indicated that UA stimulated collagen production in cultured fibroblasts9 and increased the production of ceramides in human epidermal keratinocytes and human skin.10 Recently, we demonstrated that UA induced apoptosis in HaCaT keratinotic cells.11 As UA have a promising protective effect on human skin, we investigated its effects on growth and apoptosis in M4Beu human melanoma cell line. Apoptosis or programmed cell death is defined as an active physiologic process of cellular self-destruction, with specific morphologic and biochemical changes in the nucleus and cytoplasm.12 Apoptosis is a fundamental process both in the immune system, by regulating lymphocyte maturation and homeostasis, and in the development of pluricellular organisms. It reflects the operation of an intracellular death process that silently eliminates no-longer-needed cells without an inflammatory response.13 Apoptotic death is known to involve a cascade of proteolytic events accomplished mainly by a family of cysteine proteases,14 such as caspase-3, the major executioner of apoptosis,15 and caspase-1, which is both implicated in inflammation15 and in apoptosis.16 A crucial role for caspases in the execution phase of programmed cell death is supported by genetic,17 biochemical18 and physiologic19 evidence. Once activated, caspases cleave host cellular substrates, leading to morphologic hallmarks of apoptosis.18 The activation of proapoptotic signal transduction pathways is dependent on cell type and the subcellular targets of each stress. Among the different transduction pathways, extrinsic and intrinsic pathways, which are largely described in the literature, converge to a common final effector pathway of cell death.20 The extrinsic pathway is characterized by ligand fixation to death receptors present on the cell surface. This fixation recruits different adaptator molecules which themselves recruit procaspase-8 to form the death inducing-signaling complex (DISC).21 DISC releases caspase-8, which activates caspase-3. The intrinsic pathway is induced by different mitochondrial stresses and leads to both caspase-dependent15 and -independent22 pathways. The caspase-dependent pathway is characterized by caspase-3 activation via apoptosome formation and concomitantly caspase-9 activation. The caspase-independent pathway is characterized by the leakage of apoptosis-inducing factor (AIF) from mitochondria.23 Activation of both these pathways follows a drop in mitochondrial transmembrane potential (ΔΨm) and is regulated by Bcl-2 family members:24 Bax, a proapoptotic member, favors the leakage of apoptogenic factors from mitochondria, and Bcl-2, an antiapoptotic member, negatively regulates this leakage.25 AIF provides a direct biochemical link between mitochondrial membrane permeabilization and nuclear signs of apoptosis.22 In this report, we showed that UA induces an apoptotic process in M4Beu melanoma cells characterized by caspase-3 activation. We also demonstrated that caspase-3 activation is dependent on the mitochondrial pathway as shown by ΔΨm collapses and that UA-induced ΔΨm is also associated with AIF leakage. Finally, our results demonstrated that UA induces apoptosis in M4Beu melanoma cells via the mitochondrial pathway. Material and methods Chemicals UA was purchased from Sigma (St. Quentin Fallavier, France). A stock solution (10−2 M) was prepared in sterilized dimethylsulfoxide (DMSO; Sigma) and stored at 4°C. Propidium iodide (PI) was purchased from Molecular Probes (Eugene, OR). A final solution (50 μg/ml) was obtained by adding 50 μl PI (1 mg/ml) to 1 ml of cell suspension. For mitochondrial staining, we used the fluorescent probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1; Molecular Probes). A final solution (1 μg/ml) was obtained by adding 5 μl JC-1 (100 μg/ml in DMSO) to each well of a slide flask. For the fluorescent probe 1,1-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO-3; Molecular probes), a final solution (1 μM) was obtained by adding 0.5 μl TOTO-3 (1 mM in DMSO) to each well of a slide flask. Primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) for Bax (B9, sc-7480) and Bcl-2 (100, sc-509) and from Sigma for human β-actin (AC-15) and AIF (A-7549). Secondary antibodies were purchased from Valbiotech (Abcys, Paris, France) for immunostaining and from Dako (Glostrup, Denmark) for Western blot. Cell line, cell culture and treatment M4Beu cells originate from a ganglionary human melanoma metastasis and are pigmented with a fibroblastic morphology.26 The established human melanoma cell line M4Beu was isolated from an achromic skin metastasis to lymph nodes.27 M4Beu cells were kindly provided by J.C. Maurizis (INSERM U 484, Clermont-Ferrand, France). Freshly trypsinized cells were seeded at 2 × 104 cells/cm2 and grown in Eagle's minimum essential medium, 25 mM HEPES (Gibco-BRL, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (FCS; Gibco-BRL), 1% nonessential amino acids (100×; Gibco-BRL), 1% vitamins (100×; Gibco-BRL), 1% L-glutamine (200 mM; Gibco-BRL) and 0.2% gentamycin (10 mg/ml; Gibco-BRL). Cultures were maintained in a humidified atmosphere with 5% CO2 at 37°C. Cell viability was determined by the Trypan blue dye exclusion method. For all experiments, cells were allowed to adhere and grow for 24 hr in culture medium prior to exposure to drug. During culture in the presence of UA, we observed that an increasing proportion of cells were detached from the adhered monolayer and floated in culture supernatant. To determine the extent of apoptosis in our assays, we pooled both fractions, attached and floating cells. Cell proliferation assay MTT assay. To evaluate the effect of UA on M4Beu cells, the MTT colorimetric assay was performed as described in the literature.28 This test is based on the selective ability of living cells to reduce the yellow salt MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide)] (Sigma) to a purple-blue insoluble formazan precipitate. MTT was dissolved in phosphate-buffered saline (PBS; Gibco-BRL) at 5 mg/ml. Experiments were performed in 100 μl medium in 96-well plates (Nunc, Nunclon, Denmark). After 24-hr incubation of M4Beu cells at an initial density of 5 × 103 cells/well, medium was removed and replaced by 10% FCS medium containing UA (5–20 μM). After each period of incubation (24 and 48 hr), stock MTT solution (10 μl per 100 μl medium) was added and plates were incubated at 37°C for 4 hr. Then, 100 μl sodium dodecyl sulfate (SDS; Sigma; 10% in 0.01 M HCl) were added to each well and the amount of formazan formed was obtained by scanning with an ELISA reader at 550 nm. Six wells per dose and time point were counted in 3 different experiments. Trypan blue dye exclusion method. Experiments were performed in 2 ml medium in 6-well plates (Nunc). M4Beu cells were plated at 105 cells/well and cultured for 24 hr to ensure total attachment. Then medium was removed and UA at specified concentrations was added. Three determinations per dose and time point were performed in 3 separate experiments. After UA treatment for 24 and 48 hr, cells were trypsinized and diluted with complete medium. Each sample was mixed with Trypan blue solution (0.14% in Hank's balanced salt solution; Gibco-BRL). Colored (nonviable) and dye-excluding (viable) cells were counted on a malassez hemocytometer. Cell cycle analysis Cells were seeded at 1.5 × 106 cells per 80 cm2 flask (Nunc), cultured in 10% FCS medium without or with UA (7.5, 10, 12.5, 15 and 20 μM). At 24 and 48 hr posttreatment, adherent and floating cell populations were combined and cell viability was determined by the Trypan blue dye exclusion method. For DNA content analysis, 106 cells were fixed in 1 ml ethanol (70% in PBS), washed in PBS and stained with PI (50 μg/ml in final concentration). Flow cytometric analyses were performed as previously described11 using a FACS Vantage cell sorter (Becton-Dickinson, San Jose, CA) equipped with a 488 nm argon laser. For each sample, the forward vs. right-angle scatter cytogram was used to exclude debris and aggregates. A minimum of 2 × 104 cells was analyzed with linear amplification for PI fluorescence that was collected with a 600 nm long pass filter. Cell distribution in the different phases of the cell cycle was estimated using ModFit LT™, Verity Software House, Inc., Topsham, ME. RNA extraction and semiquantitative RT-PCR analysis of M4Beu culture extracts Total RNA was extracted from cells cultured in 10% FCS medium without or with UA (10, 12.5 and 15 μM) at 24 and 48 hr by a single-step guanidium thiocyanate-phenol chloroform method using TRIzol reagent (Gibco-BRL). Two μg total RNA were transcribed into cDNA using to the Omniscript RT Kit (Qiagen), and 2 μl of the reverse-transcribed cDNA were used for PCR according to the HotStart Taq DNA Polymerase Mix Kit (Qiagen) protocol with 20 pmol of human sense and antisense primers (Table I). Reactions were incubated in a thermal cycler (Tpersonal Biometra, Biolabo, Archamps, France) and resulting fragments were visualized by electrophoresis on a 1.2% agarose gel containing ethidium bromide. Quantification of each band was performed using Low DNA Mass Ladder (Invitrogen, Cergy Pontoise, France) by densitometry analysis software (Kodak 1D image analysis software). Table I. Oligonucleotides and PCR Product Size cDNA species Genbank accession number Corresponding 5′-primer nucleotide Corresponding 3′-primer nucleotide Size of PCR product (bp) Human caspase-1 4502572 252–275 688–711 460 Human caspase-3 475911 68–69 499–521 454 Human caspase-8 15718703 651–671 879–899 249 Human caspase-9 AB020979 378–400 676–695 318 Human β-actin XM_00414 590–611 1132–1158 569 Human p53 AH002918 129–151 609–632 504 Human p21WAFI/Cip1 AF265443 430–454 849–873 444 Human Bax L22473 90–110 541–563 474 Human Bcl-2 M14745 1386–1405 1829–1848 463 Protein extraction and Western blot analysis of M4Beu cells Preparation of total protein extracts from UA-treated M4Beu. For total protein extraction, UA-treated M4Beu cells were cultured in 80 cm2 culture flasks for 24 or 48 hr. Cells were collected and centrifuged at 2,000g for 10 min at 4°C. The pellet was resuspended in 500 μl lysis buffer (50 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), pH 7.5, 1% deoxycholate, 1% Nonidet P40, 0.1% SDS, 150 mM NaCl, 20 μg/ml aprotinine, 40 μl/ml protease cocktail inhibitor; Sigma) and incubated on ice for 10 min, then centrifuged at 15,000g for 20 min at 4°C and the supernatant was collected. Western blot. For Western blot analysis, a 50 μg sample of total proteins was separated on SDS-15% polyacrylamide gels (Bio-Rad Laboratories, Richmond, CA) for the detection of Bcl-2 and Bax proteins, SDS-10% for β-actin proteins, and transferred to a cellulose nitrate transfer membrane BioBond-NC (Whatman International, Maidstone-Kent, UK) for 3 hr at 20 V, 100 mA, using transfer buffer (48 mM Tris, 39 mM glycine, pH 8.3, 20% methanol, 0.037% SDS). The membranes were blocked by incubation in PBS buffer containing 4% milk and 0.1% Tween for 1 hr at room temperature and washed 3 times with PBS buffer-0.1% Tween. Then membranes were blotted overnight at 4°C with various dilutions of primary antibodies, specifically, mouse monoclonal IgG anti-Bax (1:200), mouse monoclonal IgG anti-Bcl-2 (1:200) and mouse monoclonal anti-β-actin (1:5,000). The blots were washed 6 times with PBS-0.1% Tween and developed with horseradish peroxidase (HRP)-linked secondary antibodies, rabbit antimouse IgG (1:1,000). All blots were developed by ECL Western Blotting Detection Kit Reagent (Amersham Pharmacia Biotech, Piscataway, NJ), a chemiluminescence method, following the manufacturer's protocol. Blots were quantified using Kodak 1D analysis software. Detection of caspase catalytic activities The activities of caspase-1 and -3 were studied using the CaspACE Assay System Flurometric (Promega, Charbonnières, France). The activities of caspase-8 and -9 were studied using specific substrate/inhibitor peptides (Bachem biochimie, Voisins-le-Bretonneux, France). Assays are based on fluorometric measurement of fluorescent 7-amino-4-methylcoumarin (AMC) after cleavage from the AMC-labeled peptide substrates, Ac-DEVD-AMC for caspase-3 activity, Ac-YVAD-AMC for caspase-1 activity,29 Ac-IETD-AMC substrate for caspase-8 activity and Ac-LEHD-AMC substrate for caspase-9 activity.30 After 10, 12.5, or 15 μM UA treatment for 24 and 48 hr, cells were collected and homogenized in lysis buffer according to the manufacturer's protocol. Fluorometric assays were conducted in white opaque tissue culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) and all measurements were carried out in duplicate. First, 100 μl assay buffer (10 mM DTT, 2% DMSO, caspase buffer; Promega, Madison, WI) were added to each well. Peptide substrates for caspase-1, -3, -8 and -9 were added to each well to 5 × 10−5 M final concentration. Caspase-3 inhibitor (Ac-DEVD-CHO), caspase-1 inhibitor (Ac-YVAD-CHO), caspase-8 inhibitor (Ac-IETD-CHO; Bachem Biochimie) and caspase-9 inhibitor (Ac-LEHD-CHO; Bachem Biochimie) were also used at 5 × 10−5 M immediately before the addition of substrate. Cell lysates were added to the reaction mixture to start the reaction. Background fluorescence was determined in wells containing assay buffer and substrate without cell lysate. Assay plates were incubated at 37°C for 1 hr for the measurement of caspase-1, -3, -8 and -9 activities. Fluorescence was measured with a microplate reader (Fluorolite 1000; Dynatech Laboratories, Helsinki, Finland) using 360 nm excitation and 460 nm emission filters. Raw data (relative unit of fluorescence, or RUF) corresponded to the concentrations of AMC released. Staining of ΔΨm with JC-1 JC-1 is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green to orange. High mitochondrial polarization is indicated by orange fluorescence due to JC-1 aggregate formation. Depolarized regions are indicated by the green fluorescence of JC-1 monomers. M4Beu cells were seeded in 8-well slide flasks coated with permanox cover plastic and treated with UA or medium alone for controls. After incubation with UA, 5 μl JC-1 was added to each well and incubated for 30 min at 37°C. After 3 washes with PBS, the coverslips were mounted and the fluorescence was visualized under fluorescent microscopy (Leica, Vienna, Austria). Detection of ΔΨm drop by flow cytometry in M4Beu cells Changes in mitochondrial membrane potential. Mitochondrial membrane potential was estimated using the fluorescent probes JC-1 and TOTO-3. Briefly, after cell death induction by UA, suspensions were adjusted to 1 × 106 cells/ml, stained 35 min with 1 μg/ml JC-1 at 37°C, then 5 min with 1 μM TOTO-3 at 37°C.31 Double labeling of ΔΨm and plasma membrane integrity. TOTO-3 is a membrane-impermeant acid nucleic fluorescent probe. When plasma membrane integrity is altered, this probe enters cells, binds to nucleic acids and exhibits a strong fluorescence. Hence, TOTO-3 discriminates between living cells with maintained plasma membrane integrity and dead cells without plasma membrane integrity. Briefly, cells were resuspended in 500 μl for mitochondrial staining with 5 μl JC-1, then 0.5 μl TOTO-3 was added and cells were immediately analyzed by flow cytometry. After double staining with TOTO-3/JC-1, a minimum of 10,000 cells (after debris and aggregate elimination) was analyzed by a FACS Vantage equipped with helium/neon and argon lasers. TOTO-3 red fluorescence was excited with 633 nm helium/neon laser and was measured on FL4 (660 nm long pass filter). Green fluorescence of JC-1 was recorded on FL1 (530 ± 15 nm band pass filter), whereas JC-1 orange fluorescence was collected on FL2 (575 ± 13 nm band pass filter). Cytometric data were analyzed with Win MDI 2.8 software (developed by Dr. J. Trotter, Scripps Institute, La Jolla, CA). Gates were set using FL1 vs. FL2 cytograms and an FL2 vs. FL4 cytogram for control cells. Protein immunostaining M4Beu cells were seeded in 8-well slide flasks (Lab Tek, Nunc) coated with permanox cover plastic and treated with UA or medium alone for controls. After incubation with UA, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X100 (v/v) in 0.1% sodium citrate (w/v) in PBS for 20 min. Nonspecific binding was blocked by PBS supplemented with 0.1% bovine serum albumin (BSA) for 45 min at room temperature. M4Beu cells were then incubated overnight at 4°C with rabbit anti-AIF (1:500) in PBS-0.1% BSA, mouse anti-Bax (1:200) in PBS-0.1% BSA or mouse anti-Bcl-2 (1:200) in PBS-0.1% BSA. After washing with PBS, FITC-conjugated secondary antibody (rabbit antimouse IgG 1:500 for Bax and Bcl-2, mouse antirabbit IgG 1:500 for AIF from Valbiotech) was added and incubated in the dark at 37°C for 1 hr. After 3 washes, coverslips were mounted and the fluorescence was visualized under fluorescent microscopy (Leica). Statistical analyses Data are expressed as the means ± SD of 3 or more separate experiments. Statistical analysis of the data included an overall analysis of variance (ANOVA) followed by posthoc PLSD Fisher's test using the statistical functions in the Statview program (SAS Institute, Cary, NC). A probability of p < 0.05 was considered significant. Results Effect of ursolic acid on cell growth Cells were cultured in 10% FCS-containing medium without or with UA (5, 7.5, 10, 12.5, 15 and 20 μM) for 2 days and cell proliferation was evaluated by MTT assay and by Trypan blue dye exclusion method. With the MTT assay, we observed a minor increase in proliferation compared to controls until 24-hr treatment, where it remained stable at 106% (5–12.5 μM UA; Fig. 1a, histograms). Then it slightly decreased from 15 to 20 μM UA, where the percentage of cell viability fell from 103% (15 μM UA) to 95% (20 μM UA). At 48 hr, the MTT assay did not show a significant difference in cell proliferation between 5 and 12.5 μM UA (109% to 104%, respectively). Afterward, we observed a decrease in proliferation for 15–20 μM UA (99% and 85%, respectively; Fig. 1b, histograms). IC50 could not be determined by MTT assay either at 24 or at 48 hr. At 24 hr, using the Trypan blue test, we observed decreased viability, which was significantly UA dose-dependent, from 99% (5 μM UA) to 49% (20 μM UA; Fig. 1a, curve). At 48 hr, the percentage of viability dropped from 86% to 19% at 5 and 20 μM UA, respectively (Fig. 1b, curve). IC50 was observed between 15 and 20 μM UA for 24 hr and between 12.5 and 15 μM UA for 48 hr. We used 10, 12.5 and 15 μM UA for the following experiments. Figure 1Open in figure viewerPowerPoint Effect of UA on M4Beu cell viability. Cells were treated with various concentrations of UA and relative cell viability was assessed by Trypan blue method and MTT assay for 24 hr (a) and 48 hr (b). Results are expressed as percentage of untreated controls ± SD obtained from 3 separate experiments. Cell cycle analysis A representative cell cycle analysis of M4Beu cells cultured without or with UA (7.5, 10, 12.5 and 15 μM) for 24 and 48 hr is shown in Figure 2(a) and (b), respectively. At 24 hr as well as at 48 hr, UA did not induce cell cycle arrest at any phases of the cell cycle (Fig. 2). Nevertheless, at 24 hr, a sub-G1 cell population appeared with 20 μM UA (8.89%; Fig. 2a, see arrows). Moreover, at 48 hr, we observed the presence of this sub-G1 cell population peak starting at 10 until 20 μM UA (7.24%, 12.64%, 20.11% and 31.48%, respectively; Fig. 2b, see arrows). This sub-G1 cell population is normally associated with the presence of apoptotic cells. Figure 2Open in figure viewerPowerPoint Cell cycle analysis. M4Beu cells were cultured with various concentrations of UA for 24 hr (a) and 48 hr (b). Cell phase distribution was determined by PI staining and FACS analysis as described in text. The cell cycle phase distribution in control and UA-treated M4Beu cells was as follows (control and 7.5, 10, 12.5, 15, 20 μM UA). G1: 64.7% and 49.0%, 62.1%, 62.0%, 64.6%, 57.8% for 24 hr; 73.0% and 72.6%, 73.9%, 74.7%, 73.2%, 77.4% for 48 hr. S: 24.7% and 26.5%, 22.7%, 23.7%, 15.6%, 26.1% for 24 hr; 19.1% and 16.5%, 15.3%, 12.6%, 15.9%, 15.6% for 48 hr. G2-M: 10.5% and 24.4%, 15.2%, 14.2%, 19.8%, 16.1% for 24 hr; 7.8% and 10.84%, 10.7%, 12.7%, 10.9%, 19.7% for 48 hr. Sub-G1: 1.4% and 1.2%, 1.4%, 1.9%, 1.73%, 8.9% for 24 hr; 1.6% and 3.2%, 7.5%, 12.6%, 20.1%, 31.5% for 48 hr. p21 and p53 mRNA expression Since a time- and UA dose-dependent sub-G1 cell population appeared, we decided to study p21 and p53 mRNA expression. RT-PCR analysis of p53 mRNA expression of treated or untreated M4Beu cells showed no significant difference in p53 mRNA expression at 24 hr (Fig. 3, left). At 48 hr, p53 mRNA expression increased at 10 μM (1.34-fold vs. control) and then declined significantly at 12.5 and 15 μM (3.13- and 3.12-fold less than controls, respectively; Fig. 3, right). Moreover, we observed no significant difference between assays and control for p21 mRNA expression at 24 and 48 hr (Fig. 3). UA did not affect p21 mRNA expression, in agreement with results obtained for cell cycle analysis, but altered p53 mRNA expression. Figure 3Open in figure viewerPowerPoint Analysis of p53 and p21 mRNA expression in UA-treated M4Beu cells. Cells were incubated with different concentrations of UA for 24 and 48 hr. p53 and p21 transcripts were quantified using β-actin as an internal control. Quantification of each band was performed by densitometry analysis software (1D image analysis software, Kodak) and results were expressed as the ratio p53/β-actin or p21/β-actin in relative arbitrary units. Results are expressed as mean ± SD (asterisk, p < 0.05) of 3 separate experiments. Effect of UA on caspases We first explored if the apparent proapoptotic effect of UA could be linked to a variation in caspase-3 expression and activity (Fig. 4). M4Beu cells were cultured in 10% FCS medium in the absence or presence of 10, 12.5 and 15 μM UA at 24 and 48 hr. Caspase-3 mRNA expression was significantly increased after 24-hr treatment (1.39-, 1.48- and 1.62-fold vs. control for 10, 12.5 and 15 μM UA, respectively; Fig. 4a, left). At 48-hr treatment, after an increase in caspase-3 mRNA expression (1.52-fold vs. control for 10 μM UA), we observed a significant decrease in caspase-3 mRNA expression (1.66- and 2.77-fold less than control for 12.5 and 15 μM UA, respectively; Fig. 4a, right). We then considered caspase-3 activity. Our results showed that, at 24-hr treatment with UA, there was an excellent correlation between increasing caspase-3 mRNA expression and caspase-3 activity (Fig. 4b, left). However, after 48-hr treatment, caspase-3 activity was significantly increased between 10 for 20 μM UA (1.08-, 1.27-, 1.97- and 2.86-fold vs. control for 10, 12.5, 15 and 20 μM UA, respectively), in contrast to caspase-3 mRNA expression (Fig. 4b, right). These results indicate that UA-induced apoptosis might be mediated in part through caspase-3 activation at 24- and 48-hr treatment. Figure 4Open in figure viewerPowerPoint (a) Effect of UA on caspase-3 mRNA expression. M4Beu cells were cultured with various concentrations of UA for 24 and 48 hr. Total RNA was immediately extracted for RT-PCR experiments. Caspase-3 transcripts were quantified using β-actin as an internal control. Quantification of each band was performed by densitometry analysis software and results were expressed as ratio caspase-3/β-actin in relative arbitrary units (asterisk, p < 0.05). (b) Effect of UA on caspase-3 activity in M4Beu cells for 24 and 48 hr. The measurement of caspase-3 activity is expressed in relative units of fluorescence (RUF). Results are expressed as mean ± SD (asterisk, p < 0.05; double asterisk, p < 0.01) of 3 separate experiments. Secondly, we studied caspase-1 mRNA expression and activity to ensure that the proapoptotic effect of UA could not be due to induction of an inflammatory response. At 24 hr, we observed no significant changes in caspase-1 mRNA expression (Fig. 5a, left). At 48 hr, a slight decrease is notewo
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