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Carcinogenesis Advance Access originally published online on April 19, 2006
Carcinogenesis 2006 27(8):1699-1712; doi:10.1093/carcin/bgl044
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate

Annarica Calcabrini1, José Manuel García-Martínez2, Lorena González2, Mercedes Julián Tendero2, María Teresa Agulló Ortuño2, Pasqualina Crateri1, Abelardo Lopez-Rivas3, Giuseppe Arancia1, Pedro González-Porqué4 and Jorge Martín-Pérez2,*

1 Istituto Superiore di Sanità Roma
2 Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM) Madrid
3 Centro Andaluz de Biologia del Desarrollo (CSIC/UPO) Sevilla
4 Servicio de Inmunología, Hospital Ramón y Cajal Madrid

*To whom correspondence should be addressed at: Instituto de Investigaciones Biomédicas A. Sols (CSIC/UAM), Arturo Duperier 4, 28029 Madrid, Spain. Tel: +34915854416; Fax: +34915854401; E-mail: jmartin{at}iib.uam.es


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lauryl gallate is an antioxidant food additive showing low toxicity to normal cells. Here, its antiproliferative effect has been studied on three human breast cancer cell lines: estrogen-dependent, wild-type p53, MCF7; estrogen-independent, non-functional p53, MDA-MB-231 and MCF7 ADR, which overexpresses P-glycoprotein (P-gp) and displays a multidrug-resistant phenotype. Lauryl gallate inhibited proliferation and induced cell cycle alterations in all three cell lines without altering P-gp functionality in the drug-resistant cells. A stable arrest in G1 phase was observed in MCF7, while a slow-down of cell cycle progression was induced in the other two cell lines. Lauryl gallate increased p53 expression only in MCF7, and upregulated p21Cip1 and reduced cyclin D1 levels in all three cell lines. The induction of apoptosis, demonstrated by annexin V-FITC labeling, PARP cleavage and mitochondrial membrane depolarization and morphological alterations, were clearly detected in MCF7 ADR and MDA-MB-231 and to a minor extent in MCF7. Overexpression of Bcl-2 in MCF7 ADR cells demonstrated its protective role against morphological alterations and apoptosis. Lauryl gallate induction of p21Cip1 and apoptosis observed in all three cell lines was regulated by Erk1/2 activation. These findings suggest a potential use of lauryl gallate against tumors harboring p53 mutations and drug-resistant phenotypes.

Abbreviations: Dox, doxorubicin; Erk1/2, extracellular signal-regulated kinase 1/2; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolcarbocyanine iodide; JNK, c-jun N-terminal kinase; MAb, Monoclonal antibody; MAPK, Mitogen-activated protein kinase; MDR, Multidrug-resistance; MFC, mean fluorescence channel; Mek1/2, mitogen-activated protein kinase kinase; PARP, poly (ADP-ribose) polymerase; PI, Propidium iodide; P-gp, P-glycoprotein


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Breast cancer is the malignancy with the highest incidence and death rate for women (1), and chemotherapy is the treatment of choice at various stages of the disease. Although considered one of the most chemosensitive solid tumors (2), usually after a positive response, they often relapse and develop resistance to a broad spectrum of drugs. Consequently, metastatic breast cancer finally becomes refractory to cytotoxic drugs (3). Multidrug-resistance (MDR) represents one of the major obstacles for successful cancer treatment. Among other mechanisms, it is often associated with the overexpression of the membrane-bound drug-transporter P-glycoprotein (P-gp, ATP-dependent excretory pump), product of mdr-1 gene (4) and responsible for reducing intracellular accumulation of the drugs and/or modifying their distribution in tumor cells (5,6).

The inability to activate apoptotic responses also represents a mechanism of drug resistance: altered expression of proteins involved in apoptosis and/or cell cycle regulation (p53, Bcl-2 family proteins, cyclins, NF-{kappa}B) has been proposed to explain drug resistance in different cell lines (7,8). The involvement of multiple MDR mechanisms makes the development of new agents, less toxic to normal tissues and more effective against multidrug-resistant tumors than conventional antitumoral drugs, a pivotal task.

Apoptosis is generally triggered by a number of chemical and physical agents, such as oxidants, while antioxidant compounds have been reported to protect cells. However, gallic acid, a natural plant triphenol with well-known antioxidant properties, has been demonstrated to induce apoptosis in several human cell lines (9,10). More recent studies have demonstrated that octyl and lauryl esters of gallic acid are between 50 and 250 times more effective than gallic acid itself in inhibiting proliferation and inducing apoptosis in lymphoma and leukemia cell lines and in HT29 colon carcinoma (11).

Lauryl gallate has been used as an antioxidant food additive for over 50 years with the code E312. This compound shows very low toxicity to normal cells (12) and good specificity to tumor cells (11,13,14). Also, most of the toxicological and pharmacokinetic studies have already been performed (12). These properties support lauryl gallate as a good candidate for further studies not only to determine its mechanism of action but also for testing its efficacy against drug-resistant tumor cell lines.

Here, we report the effect that lauryl gallate has on three human breast cancer epithelial cell lines: the estrogen-dependent MCF7, expressing wild-type p53, and estrogen-independent MCF7 ADR and MDA-MB-231 cell lines that express non-functional p53 (15). MDA-MB-231 is characterized by an aggressive metastatic behavior (16), while MCF7 ADR was derived from its sensitive counterpart and displays a MDR phenotype involving P-gp overexpression (17).

Our results demonstrate that lauryl gallate reduced cell viability, induced p21Cip1 upregulation, cell cycle alterations and apoptosis in all of these breast cancer cell lines. Also, a role of extracellular signal-regulated kinase 1/2 (Erk1/2) on lauryl gallate-induced cytotoxicity was ascertained.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
Lauryl gallate was obtained from Fluka Chemie GmbH (Buchs, Switzerland). Monoclonal antibody (MAb) anti-p21Cip1 (clone SXM30) and annexin V-FITC-conjugated were from BD Pharmingen (San Diego, CA). MAbs anti-p53 (clone DO-1, which recognizes wild-type and mutant p53) and anti-Bcl-2 were obtained from BD Transduction Laboratories. MAb anti-poly (ADP-ribose) polymerase (PARP) was from Biomol (Biomolecules for Research Success, Plymouth Meeting, PA). MAb anti-P-gp (clone F4) was from Kamiya Biomedical Company (Seattle, WA). MAb UIC2 was purchased from Chemicon (Chemicon International, Temecula, CA). MAb anti-{alpha}-tubulin was from Sigma Chemical (St Louis, MO). Polyclonal antibodies, anti-cyclin D1 and anti-Erk2, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary horseradish peroxidase-conjugated antibodies were purchased from Biosource International (Camarillo, CA). Secondary rabbit anti-mouse IgG FITC-conjugated antibody was obtained from Dako (Dako A/S, Glostrup, Denmark). The enhanced chemiluminescence (ECL) kit was from Amersham Pharmacia Biotech (Buckinghamshire, UK). Propidium iodide (PI), 0.4% solution trypan blue, ribonuclease A (RNAse), verapamil and nocodazole were obtained from Sigma. Anti-phosphorylated-Erk1/2 (P-Erk1/2) and Mek1/2 inhibitor PD98059 were purchased from Cell Signaling Technology (Beverly, MA). Doxorubicin (Adriblastina) was obtained from Pharmacia & Upjohn (Milan, Italy). Vinblastine was purchased from Eli Lilly (Paris, France). JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolcarbocyanine iodide) was from Molecular Probes (Eugene, OR).

Cell cultures
Human breast cancer cell line MCF7 and its multidrug-resistant derivative MCF7 ADR were kindly provided by Dr K. Cowan (Nebraska Medical Center, Omaha, NE). MDA-MB-231 cell line was from American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS), 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. MCF7 ADR cells were maintained in medium supplemented with 10 µM doxorubicin. Prior to each experiment, cells were grown in drug-free medium for 1 week. Bcl-2 overexpressing MCF7 ADR clone (MAB25) and its control transfectant clone (MAN9) were provided by Dr Del Bufalo (Regina Elena Cancer Institute, Rome, Italy) (18). MCF7-E6 cell line (expressing the human papillomavirus type 16 E6 protein) and MCF7-C4 (control) have been described previously (19). To generate MDA-MB-231 cells expressing a mouse mutant temperature-sensitive p53 (Val135), cells were cotransfected with the plasmid pBabe-puro (2 µg), which confers resistance to puromycin, and 10 µg of either empty vector or pLTRcGp53 (Val135) (20). Resistant clones were selected in 1 µg/ml puromycin (Sigma). Clones 1 and 8 were chosen after analysis of p53 and p21 expression by western blot.

Cell proliferation assay
MCF7, MCF7 ADR and MDA-MB-231 cells were seeded (5 x 105 per 60-mm plate) in complete medium. After 24 h, cultures were treated with different concentrations of lauryl gallate (0.5, 1, 5 and 10 µM) for 24, 48 and 72 h. MCF7 C4 and E6, Bcl-2 overexpressing MCF7 ADR clone (MAB25) and its control transfectant clone (MAN9), MDA-MB-231 clones 1 and 8, expressing temperature-sensitive p53, were treated with 5 and 10 µM lauryl gallate for 72 h. Lauryl gallate stock solution (10 mM) was prepared in dimethyl sulfoxide (DMSO) and added to the medium to achieve the desired final concentration. Control samples were treated with DMSO vehicle alone. To assess cell viability, after incubation for specified time at 37°C, aliquots from both floating and adherent cells were mixed with a 0.4% trypan blue solution (1:1) and loaded on to a hemocytometer. The percentage of viable and dead cells per sample was calculated to determine the effect of lauryl gallate on cell growth.

Analysis of P-gp functionality
MCF7 and MCF7 ADR cells were seeded (5 x 105 per 60-mm plate) in complete medium. After 24 h, cultures were treated with 5 µM doxorubicin (Dox) for 1 h in the presence or in absence of 50 µM verapamil or 5 µM lauryl gallate. At the end of treatment, samples were washed with cold phosphate-buffered saline (PBS), harvested and submitted to P-gp labeling. To this purpose, cells were fixed with 3.7% paraformaldehyde in PBS for 10 min at 4°C, washed twice with cold PBS and incubated with anti-P-gp monoclonal antibody (clone F4, 2 µg/ml in PBS containing 1% BSA) for 30 min at 4°C. After two washes in cold PBS, cells were incubated with a secondary rabbit anti-mouse IgG FITC conjugate antibody for 30 min at 4°C, washed twice in cold PBS and analyzed on the FACScan flow cytometer (Becton Dickinson, Mountain View, CA). For UIC2 reactivity shift assay, MCF7 ADR cells (106 cells per tube) were harvested and resuspended in 1 ml of PBS supplemented with 2% FCS. The cells were warmed up to 37°C in a water bath for 10 min. Then samples were treated with lauryl gallate (final concentrations 5 and 10 µM) or vinblastine (10 µg/ml) and incubated at 37°C for 10 min with periodic agitation. Vinblastine was used, as it is a well-known UIC2 shift agent (21). MAb UIC2 (final concentration 12.5 µg/ml) was added to cell suspensions and, after mixing, incubated for an additional 15 min at 37°C. Cells were then washed twice in ice-cold PBS supplemented with 2% FCS and 0.01% sodium azide (Shift Stop Buffer, SSB), stained on ice in SSB for an additional 15 min with a secondary rabbit anti-mouse IgG FITC-conjugated antibody. Dead cells were excluded from the analysis by adding PI to cell suspensions immediately before acquisition. Samples were analyzed on a FACScan flow cytometer equipped with a 15 mW, 488 nm, air-cooled argon ion laser. The fluorescence emissions were collected through a 530 nm band-pass filter for fluorescein and a 575 nm band-pass filter for doxorubicin and PI. At least 10 000 events per sample were acquired in log mode.

Cell cycle analysis
MCF7, MCF7 ADR and MDA-MB-231 cells were seeded in complete medium (5 x 105 per 60-mm plate). After 24 h, they were treated for 16 h with 0.5 µg/ml nocodazole to synchronize them in G2/M phase. Then samples were washed and placed in complete medium with DMSO (control) or lauryl gallate (5 µM) for 24 and 48 h to assess the effects on cell cycle progression. For experiments of mitogen-activated protein kinase kinase (Mek1/2) activity inhibition, cells were treated with 10 µM PD98059 for 2 h prior to addition of 5 µM lauryl gallate for 48 h. Both floating and adherent cells were collected, washed twice with cold PBS and centrifuged. The pellet was fixed in 70% ethanol in PBS at 4°C for 1 h, washed twice with cold PBS and then resuspended in PBS containing 40 µg/ml PI and 100 µg/ml RNAse, at 37°C for 30 min. Samples were then analyzed on the FACScan flow cytometer. After activation of the 'doublet discrimination module' and exclusion of cell debris defining a gate in the side and forward scatter dot-plot, at least 10 000 events per sample were acquired in linear mode. Percentage of cells in subG1, G1, S and G2/M phases were calculated using the CellQuest software (Becton Dickinson).

Western blotting
Cells (1–2 x 106 cells per dish) were washed twice in ice-cold Tris-buffered saline [TBS; 20 mM Tris–HCl (pH 7.6) and 140 mM NaCl] and lysed at 4°C in 200 µl of lysis buffer [10 mM Tris–HCl (pH 7.6), 50 mM NaCl, 30 mM sodium pyrophosphate, 5 mM EDTA, 0.5% Nonidet P40, 1% Triton X-100, 50 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM benzamidine, 1 mM iodoacetamide and 1 mM phenantroline]. Cell lysates were obtained by centrifugation at 17 000x g for 30 min at 4°C; protein concentration in the supernatant was determined by BCA protein assay (Pierce, Rockford, IL), and lysates were adjusted to equivalent concentrations with lysis buffer. Aliquots of 10–40 µg of total cell lysate were then separated on SDS–PAGE. Proteins were transferred to PVDF membranes that were blocked overnight at 4°C with 5% non-fat milk in TTBS (TBS with 0.05% Tween-20). Incubation with primary specific antibodies and horseradish peroxidase-conjugated secondary antibodies was performed in blocking solution for 1 h at room temperature. Immunoreactive bands were visualized by ECL kit. Membrane stripping was performed incubating for 30 min at 65°C in 62.5 mM Tris–HCl (pH 6.8) containing 2% SDS and 100 mM 2-mercaptoethanol, and extensively washed with TTBS at room temperature. Stripping was checked by re-exposure to enhanced chemiluminescence (ECL), and membranes were subsequently blocked and reproved as described above.

Quantification of apoptosis by annexin V-FITC labeling
To study cell surface phosphatidylserine exposure, an early marker of apoptotic cell death, MCF7, MCF7 ADR and MDA-MB-231 cells were treated with 5 µM lauryl gallate for 0.5, 1, 6 and 24 h. MCF7 C4 and E6, Bcl-2overexpressing MCF7 ADR clone (MAB25) and its control transfectant clone (MAN9), MDA-MB-231 clones 1 and 8, expressing temperature-sensitive p53, were treated with 5 µM lauryl gallate for 6 and 24 h. For Mek1/2 activity inhibition experiments, MCF7, MCF7 ADR and MDA-MB-231 cells were treated with 10 µM PD98059 for 2 h prior to addition of 5 µM lauryl gallate for 48 h. Detached and adherent cells were collected and labeled for 10 min at room temperature with annexin V-FITC-conjugated (1 µg/ml, final concentration) and with PI (40 µg/ml, final concentration) and immediately acquired on flow cytometer.

Ultrastructural analysis by transmission electron microscopy
Cells were seeded in complete medium (1.5 x 106 per 90-mm plate). After 24 h, samples were treated with 5 µM lauryl gallate for 48 h. After this treatment, both detached and adherent cells were collected and washed with PBS. The pellet was fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature for 1 h. After post-fixation with 1% OsO4 in cacodylate buffer (room temperature, 1 h), cells were dehydrated through graded ethanol concentrations with a final propylene oxide dehydration. Samples were embedded in Epon 812 resin (Electron Microscopy Science, Fort Washington, PA). Ultrathin sections, obtained with a LKB ultramicrotome (LKB, Bromma, Sweden), were stained with uranyl acetate and lead citrate and examined with a Philips 208S electron microscope (Philips Electron Optics B.V., Eindhoven, The Netherlands). To evaluate cellular and mitochondrial morphological alterations, ~50 cells per sample were observed from two independent experiments.

Analysis of mitochondrial membrane potential (MMP)
MMP was evaluated using the lipophilic cationic probe JC-1, stocked as 1 mg/ml in DMSO and freshly diluted in complete medium. Cells were treated with 5 µM lauryl gallate for 6 and 24 h. Detached and adherent cells were collected and incubated at 37°C with medium containing JC-1 (10 ng/ml) for 10 min. Finally, cells were washed and resuspended in cold PBS for flow cytometric analysis. JC-1 accumulates into the mitochondria as both monomers (responsible for green fluorescence emission, FL1) and aggregates (red fluorescence emission, FL2) depending on MMP. Depolarization of the mitochondrial membrane is represented by an increase of FL1 and a decrease of FL2 signals, collected through a 530 and 575 nm band-pass filters, respectively. Results are presented as mean fluorescence channel (MFC) values, calculated by the CellQuest software.

Statistical analysis
Data were expressed as means ± SD. For comparison between two groups, a t-test for independent samples was used and considered significant at *P < 0.05; **P < 0.01; or ***P < 0.001 levels.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cell proliferation
The effect of lauryl gallate treatment on the growth of three different human breast cancer cell lines (MCF7, MCF7 ADR and MDA-MB-231) is shown in Figure 1. Concentrations between 0.5 and 10 µM lauryl gallate reduced the number of viable cells in all three cell lines (Figure 1A). Lauryl gallate was more effective in MDA-MB-231 than in the other two cell lines, since 50% inhibition of cell proliferation was induced with 1 µM and complete inhibition attained with 5 µM. As shown in Figure 1B, MCF7 ADR and MDA-MB-231 were more sensitive to lauryl gallate than MCF7, since a higher percentage of trypan blue stained cells were found after treatment with increasing concentrations of this compound. Higher sensitivity to lauryl gallate, versus MCF7, was confirmed by counting detached cells or direct observation by phase-contrast microscopy (data not shown).


Figure 1
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  		Fig. 1 Effect of lauryl gallate on MCF7, MCF7 ADR and MDA-MB-231 cell proliferation. Cells were treated with different concentrations of lauryl gallate for 24, 48 and 72 h. At each time, both floating and adherent cells were labeled with a 0.4% trypan blue solution and loaded onto a hemocytometer and negative and positive trypan blue stained cells were counted. (A) Number of trypan blue negative cells. (B) Percentage of trypan blue stained cells. Reported values represent the mean ± SD from three independent experiments carried out in triplicates. Asterisks indicate a significant difference between control and treated cells in each cell line (*P< 0.05; **P< 0.01; ***P< 0.001).

 
Functionality and expression of P-gp in MCF7 ADR cell line
Interestingly, as observed above, lauryl gallate was very effective against the multidrug-resistant MCF7 ADR cell line. To analyze whether this compound affected the functionality of P-gp, cells were loaded with doxorubicin (Dox), in absence (control) or in presence of verapamil (a known inhibitor of P-gp activity) or lauryl gallate. Cells were then incubated with anti-P-gp antibody to assess the resistant phenotype (Figure 2A). MCF7 cells were used as negative control. MCF7 ADR cells resulted to be positive for P-gp labeling and accumulated very low amount of Dox. The presence of verapamil significantly increased the Dox fluorescent signal. On the contrary, lauryl gallate did not affect the amount of accumulated drug, indicating that resistant cells were able to extrude Dox in the presence of this compound. Sensitive MCF7 cells, negative for P-gp expression, accumulated higher amount of Dox, both in the absence and in the presence of verapamil or lauryl gallate. To confirm this result, UIC2 reactivity shift assay was performed (21). As shown in Figure 2B, UIC2 labeling was increased in the presence of vinblastine (a known P-gp substrate), while lauryl gallate treatment produced no shift in UIC2 reactivity even when used at higher concentration (10 µM), indicating that it was unable to bind to P-gp molecules and alter their conformation.


Figure 2
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  		Fig. 2 Functionality of P-gp. (A) Analysis of doxorubicin accumulation and P-gp expression. MCF7 and MCF7 ADR cells were treated with Dox in absence or in presence of 50 µM verapamil or 5 µM lauryl gallate for 1 h. Samples were then fixed with 3.7% paraformaldehyde in PBS for 10 min at 4°C, labeled with anti-P-gp monoclonal antibody and secondary rabbit anti-mouse IgG FITC-conjugated antibody (see Materials and methods section for details). x-axis represents the level of expression of P-gp, y-axis the signal from the fluorescent molecule of doxorubicin. (B) UIC2 reactivity shift assay in MCF7 ADR cells. In each graph, the fluorescent profile of MAb UIC2 staining of cell surface in presence of vinblastine (P-gp substrate) or lauryl gallate (5 or 10 µM) (solid line) is compared with profile of untreated cells (dotted line). x-axis represents fluorescence intensity due to UIC2 labeling, y-axis, cell number. The results shown are representative of two independent experiments.

 
Cell cycle analysis and expression of signaling molecules
As observed above, lauryl gallate caused cell growth inhibition. Therefore, we studied the effect of lauryl gallate on cell cycle progression and on expression of proteins involved in its regulation (Figure 3). Cell cycle studies were performed on synchronized cells to emphasize the effect of lauryl gallate on cycle progression. Samples were treated for 16 h with 0.5 µg/ml nocodazole to synchronize cells in G2/M. Then, they were washed and placed in complete medium with DMSO (control) or 5 µM lauryl gallate for 24 and 48 h. As shown in Figure 3A, treatment with nocodazole induced G2/M accumulation in all three cell lines, and recovered progressively to a normal cycle after washing-out nocodazole. In contrast, in the presence of lauryl gallate (5 µM), MCF7 accumulated in G1 phase. Also MCF7 ADR seemed to accumulate in G1 in the presence of lauryl gallate at 24 h but an evident shift to G2/M phase was observed at 48 h. Similarly, in MDA-MB-231 5 µM lauryl gallate increased the percentage of cells in G1–S phases at 24 h and S–G2/M phases at 48 h. These findings suggested that lauryl gallate induced cell cycle alterations consisting of blocking MCF7 in G1 and delaying cell cycle progression in MDA-MB-231 and MCF7 ADR cells.


Figure 3
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  		Fig. 3 Analysis of cell cycle by flow cytometry and p53, p21 and cyclin D1 expression by western blot. (A) MCF7, MCF7 ADR and MDA-MB-231 cells were treated for 16 h with 0.5 µg/ml nocodazole (N). Then samples were washed and placed in complete medium with DMSO (control, C) or 5 µM lauryl gallate (LG) for 24 and 48 h to assess the effects on cell cycle progression. At each time, both floating and adherent cells were collected, processed for PI staining and analyzed on FACScan. The percentage of cells in each phase (subG1, G1, S, G2/M) was calculated using the CellQuest software (Becton Dickinson) and represented graphically. Reported values represent the mean ± SD of three independent experiments. (B) Comparative analysis of p53 levels in untreated MCF7, MCF7 ADR and MDA-MB-231 cells. Values of p53 levels after being normalized to the levels of {alpha}-tubulin are shown below each blot. (C) Effect of lauryl gallate on p53, p21 and cyclin D1 expression. Cells were cultured with 5 µM lauryl gallate for 24, 48 and 72 h (+), or with DMSO as a control for each time point (–). After lysis, 10 (p53) or 30 µg (p21 and cyclin D1) of proteins from total cell lysate were separated on SDS–PAGE, transferred to a membrane that was incubated with the specific antibodies for p53 (1:5000), p21 (1:1000) and cyclin D1 (1:1000). Lower panels show stripped membrane reprobed with an anti-{alpha}-tubulin antibody for loading control. Changes in the levels of p53, p21 and cyclin D1 after being normalized to the levels of {alpha}-tubulin are shown below each blot. Control samples at 24, 48 and 72 h are considered as the unit. The results shown are representative of four independent experiments.

 
To characterize the molecular mechanisms underlying these different behaviors, we determined p53 expression, which plays an important role in cell cycle regulation and sensitivity to cytotoxic drugs (22). Most p53 mutations result in protein stabilization (15,23). Therefore, as expected, the levels of p53 detected in MCF7 ADR and MDA-MB-231 are higher than in p53 wild-type expressing MCF7 cells (Figure 3B). As shown in Figure 3C, treatment of MCF7 with 5 µM lauryl gallate for 24, 48 and 72 h increased p53 protein, while in MCF7 ADR and MDA-MB-231 cells, lauryl gallate did not modify p53 expression.

As p53 activates expression of several endogenous genes, including p21Cip1 (termed p21 hereafter), a known inhibitor of cell cycle progression (24), we analyzed its expression in both control and lauryl gallate-treated cells (5 µM up to 72 h). Interestingly, lauryl gallate caused a time-dependent induction of p21 in all cell lines (Figure 3C). As MCF7 ADR and MDA-MB-231 are p53 mutant cell lines, the upregulation of p21 expression was likely p53-independent. Since lauryl gallate induced alterations in cell cycle progression, we studied the expression of cyclin D1, involved in G1 to S transition (Figure 3C). Treatment with 5 µM lauryl gallate reduced the expression of cyclin D1, when compared with controls.

Apoptosis induction
Cell cycle modifications and alteration of its regulatory proteins are frequently associated with induction of apoptosis. To determine whether lauryl gallate activated apoptosis, some biochemical and morphological parameters were evaluated (Figure 4). First, surface exposure of phosphatidylserine, an early event of apoptotic program, was analyzed. Cells were treated with 5 µM lauryl gallate for 0.5, 1, 6 and 24 h and then labeled with annexin V-FITC-conjugated. As shown in Figure 4A, lauryl gallate significantly induced apoptosis. Early appearance (0.5 h) of apoptotic cells (defined as annexin V-positive/PI-negative) increased until 6 h in MCF7 and MCF7 ADR, while in MDA-MB-231 reached maximum after 1 h, as compared with control (DMSO-treated) cells. MCF7 cells appeared to be more resistant to apoptosis than the other cell lines, even though lauryl gallate was able to inhibit cell proliferation in this cell line. These results were also confirmed by analysis of chromatin condensation and fragmented nuclei after Hoechst labeling (data not shown).


Figure 4
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  		Fig. 4 Analysis of apoptosis induction by lauryl gallate. (A) Cell surface annexin V binding was studied by flow cytometry. MCF7, MCF7 ADR and MDA-MB-231 cells were treated with 5 µM lauryl gallate for 0.5, 1, 6 and 24 h. At each time detached and adherent cells were collected and labeled with annexin V-FITC-conjugated (1 µg/ml). Staining with PI immediately before cell acquisition allowed differentiating between apoptotic and necrotic cells. The percentage of apoptotic cells refers to annexin V-positive/PI-negative cells. Reported values represent the mean ± SD of three independent experiments. Asterisks indicate a significant difference between control and treated cells in each cell line (*P < 0.05; **P < 0.01; ***P < 0.001). (B) Analysis of PARP cleavage. MCF7, MCF7 ADR and MDA-MB-231 cells were cultured with lauryl gallate (10 µM for MCF7, and 5 µM for MCF7 ADR and MDA-MB-231) for 24, 48 and 72 h (+). Cells treated with DMSO were used as a control for each time point (–). After lysis, 40 µg of total cell lysate were separated on SDS–PAGE transferred to a membrane, and immunodetected with monoclonal antibody anti-PARP (1:1000). Lower panels show stripped membranes reprobed with an anti-{alpha}-tubulin antibody for loading control. The results shown are representative of three independent experiments. (C) Transmission electron microscopy analysis of MDA-MB-231 cell line. Cells were treated with 5 µM lauryl gallate for 48 h. Both detached and adherent cells were collected, fixed with 2.5% glutaraldehyde, post-fixed with 1% OsO4 and processed for electron microscopy analysis (see Materials and methods). Electron micrographs of control (1) and lauryl gallate-treated MDA-MB-231 cells (2 and 3). Arrows indicate dilation of nuclear envelope. Arrowheads indicate dilation of rough endoplasmic reticulum (RER). N, nucleus; M, highly condensed mitochondria. The results shown are representative of two independent experiments. Approximately 50 cells per sample (control and lauryl gallate-treated cells) were observed. Bars = 1 µm.

 
Induction of apoptosis by lauryl gallate was also demonstrated by PARP cleavage, a commonly late marker of apoptosis (25). In MCF7 cells, only after treatment with 10 µM lauryl gallate for 72 h the 85 kDa fragment was detected (Figure 4B), indicating that the lower dose produced very low percentage of apoptotic cells. Since MCF7 cell line does not express a functional caspase 3 (26), other caspases must be responsible for PARP cleavage (27). In contrast, treatment with 5 µM lauryl gallate clearly induced the 85 kDa fragment in MCF7 ADR and MDA-MB-231 at 48 and 72 h (Figure 4B). Similar results were found at 10 µM (data not shown). Consistent with these observations, lauryl gallate induced internucleosomal DNA fragmentation in MDA-MB-231 and MCF7 ADR cells after 72 h treatment, as they showed the typical DNA ladder even at 5 µM, while in MCF7 cells this effect was not observed at any concentration (data not shown).

To gain insight into morphological alterations caused by lauryl gallate, we performed transmission electron microscopy studies (Figure 4C). Exposure to 5 µM lauryl gallate for 48 h caused ultrastructural alterations typical of apoptotic cell death that resulted more evident in MDA-MB-231 and MCF7 ADR than in MCF7 cell line, as previously demonstrated by different techniques. Electron micrographs in Figure 4C refer to morphological features of MDA-MB-231 cell line. Untreated cells showed a nucleus characterized by a homogeneously distributed chromatin, a cytoplasm with numerous mitochondria and lysosomes and a cell surface covered by short microvilli (Figure 4C1). Lauryl gallate exposure led to dramatic alterations of the normal cellular architecture. These included an evident condensation and marginalization of chromatin, a diffuse vacuolization of cytoplasm and loss of microvilli with subsequent smoothing of cell surface (Figure 4C2). When observed at higher magnification, other typical markers of cell damage, such as the highly condensed mitochondria, the dilation of the nuclear envelope and the rough endoplasmic reticulum could be observed (Figure 4C3).

It has been demonstrated that Bcl-2 protein protects from apoptosis (28). Since lauryl gallate treatment induced apoptosis, but to a different extent, in MCF7, MCF7 ADR and MDA-MB-231, we analyzed possible alterations of Bcl-2 expression. As shown in Figure 5A, MCF7 expressed higher Bcl-2 levels than the other two cell lines. Lauryl gallate decreased Bcl-2 expression in MCF7 ADR and MDA-MB-231 cells, but did not affect expression of Bcl-2 in MCF7 (Figure 5B). Among different mechanisms through which Bcl-2 inhibits apoptosis, prevention of mitochondrial membrane depolarization due to a cytotoxic insult plays a central role (29). To verify whether lauryl gallate induced alteration of mitochondrial functionality and that differential Bcl-2 expression could affect this response, a flow cytometric study was performed after loading with JC-1 probe. In MCF7 only light alterations in FL1 and FL2 emissions were observed in the presence of lauryl gallate for 6 and 24 h (Figure 5C). In MCF7 ADR and MDA-MB-231 the treatment induced an evident increase of FL1 and a parallel decrease of FL2 signals, indicating mitochondrial membrane depolarization. As compared to sensitive MCF7 and MDA-MB-231 cells, MCF7 ADR cells showed a lower labeling pattern. This finding could be ascribed to P-gp activity responsible for JC-1 extrusion from cell (30). Nevertheless, modifications of membrane potential due to treatment were clearly detectable. Transmission electron microscopy observations of mitochondrial ultrastructure were in agreement with higher alterations of mitochondrial functionality observed in MCF7 ADR and in MDA-MB-231 than in MCF7 (Figure 5D). Control MCF7 cells showed typical mitochondrial morphology, characterized by a double membrane containing a homogeneous matrix and a system of parallel cristae (Figure 5D1). Mitochondria of untreated MCF7 ADR and MDA-MB-231 cells displayed very similar features (data not shown). Treatment with 5 µM lauryl gallate for 48 h induced in MCF7 cells only modest structural alterations consisting in a reduced number of cristae (Figure 5D2). However, in MCF7 ADR, swollen mitochondria showing an altered morphology, a shortening and reduction in number of cristae were clearly detected (Figure 5D3). Also in MDA-MB-231 mitochondrial structural modifications were more evident than in MCF7 ADR. In fact, numerous organelles with increased matrix density and severe vacuolization of the cristae were detected (Figure 5D4).


Figure 5
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  		Fig. 5 Biochemical and morphological alterations induced in mitochondria by lauryl gallate. (A) Comparative analysis of Bcl-2 levels in untreated MCF7, MCF7 ADR and MDA-MB-231 cells. Values of Bcl-2 levels after being normalized to the levels of {alpha}-tubulin are shown below each blot. (B) Effect of lauryl gallate treatment on Bcl-2 expression. MCF7, MCF7 ADR and MDA-MB-231 cells were cultured with 5 µM lauryl gallate for 48 h (+), or with DMSO as a control for each time point (–). After lysis, 30 µg of total cell lysate were separated on SDS–PAGE, and Bcl-2 was detected by western blot with monoclonal antibody anti-Bcl-2 (1:1000). Lower panels show the stripped membranes reprobed with an anti-{alpha}-tubulin antibody for loading control. Changes in the levels of Bcl-2 after being normalized to the levels of {alpha}-tubulin are shown below each blot. Control samples at 48 h are considered as the unit. The results shown are representative of three independent experiments. (C) Flow cytometric analyses of mitochondrial functionality. After treatment with 5 µM lauryl gallate for 6 and 24 h, cells were collected and incubated with 10 ng/ml JC-1, as described in Materials and methods. Mitochondrial membrane depolarization is detected as an increase of FL1 and a decrease of FL2 fluorescence emissions. Data are presented as MFC values and represent the mean ± SD of three independent experiments. (D) Transmission electron microscopy analysis of mitochondria of MCF7, MCF7 ADR and MDA-MB-231 cells. Cells were treated with 5 µM lauryl gallate for 48 h. Both detached and adherent cells were collected, and electron microscopy analysis was performed as in Figure 4C. Mitochondria of a control MCF7 cell (1), arrows indicate parallel cristae. Mitochondria of a lauryl gallate-treated MCF7 cell (2), arrows indicate two organelles with a reduced number of the cristae. Mitochondria of a lauryl gallate-treated MCF7 ADR cell (3), arrow indicates a swollen mitochondrion showing loss of cristae. Mitochondria of a lauryl gallate-treated MDA-MB-231 cell (4), arrow indicates a highly condensed mitochondrion showing vacuolization of the cristae. The results shown are representative of two independent experiments. Approximately 50 cells per sample (control and lauryl gallate-treated cells) were observed. Bars = 0.5 µm.

 
Effect of Bcl-2 and p53 expression on apoptosis induction by lauryl gallate
As described above, lauryl gallate-induced apoptosis involved reduction of Bcl-2 expression and mitochondrial membrane depolarization and structural modifications. Furthermore, Bcl-2 expression was higher in MCF7 than in the other two cell lines, suggesting that Bcl-2 could account for the lower sensitivity of MCF7 to lauryl gallate. To examine this hypothesis, a Bcl-2 overexpressing clone (MAB25) derived from transfected MCF7 ADR cells was used (18). Cell viability and annexin V-FITC labeling analyses showed that overexpression of Bcl-2 protected cells from apoptosis. Treatment with lauryl gallate significantly reduced cell viability (Figure 6A) and induced apoptosis (Figure 6B) in MAN9 (control transfectant clone) when compared to MAB25 (Bcl-2 overexpressing clone). When analyzed by transmission electron microscopy, MAN9 showed evident morphological alterations (condensed chromatin, vacuolization of cytoplasm) after treatment with lauryl gallate (5 µM for 48 h, Figure 6C2) as compared to control cells (Figure 6C1). At higher magnification, dilation of endoplasmic reticulum and nuclear envelope were clearly detectable and mitochondria appeared strongly altered, showing swelling and loss of cristae (Figure 6C3). On the contrary, few treated MAB25 cells showed morphological features of apoptosis, as most of them displayed nuclear and cytoplasmic morphology (Figure 6C5) similar to control (Figure 6C4). Furthermore, most of mitochondria preserved their ultrastructure despite 48 h of treatment (Figure 6C6).


Figure 6
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  		Fig. 6 Effect of Bcl-2 and p53 expression on lauryl gallate-induced cytotoxicity. (A) Analysis of cell viability of Bcl-2 overexpressing clone (MAB25) and of control transfectant clone MAN9. Cells were treated with 5 or 10 µM lauryl gallate (LG) for 72 h and counted as in Figure 1. (B) Analysis of annexin V binding. MAN9 and MAB25 clones were treated with 5 µM lauryl gallate for 6 and 24 h and labeled with annexin V-FITC-conjugated (1 µg/ml) as in Figure 4A. (C) Transmission electron microscopy analysis of MAN9 and MAB25 clones. Cells were treated with 5 µM lauryl gallate for 48 h and processed for electron microscopy analysis as in Figure 4C. Electron micrographs of MAN9 cells (1, control; 2 and 3, lauryl gallate-treated cells), and MAB25 cells (4, control; 5 and 6, lauryl gallate-treated cells). Arrows point to dilation of the nuclear envelope. Arrowheads indicate dilation of endoplasmic reticulum (ER). N, nucleus; M, mitochondria. Bars = 1 µm. (D) Analysis of cell viability (left panel) and apoptosis (right panel) in MCF7-C4 and E6 cell lines, in MDA-MB-231 clones 1 and 8, expressing temperature-sensitive p53. Analyses were performed as described in panels A and B, respectively. Reported values represent the mean ± SD of three independent experiments. Asterisks indicate significant difference (**P < 0.01; ***P < 0.001).

 
Since the most sensitive cells were those expressing mutant inactive forms of p53, MCF7 ADR and MDA-MB-231, we next analyzed the possible contribution of p53 as a target for lauryl gallate effects by two independent approaches. First, we used a MCF7 clone (MCF7-E6) expressing E6 protein from human papilloma virus type 16, which induces p53 degradation, therefore expressing lower levels of wild-type p53 than control clone C4 (data not shown) (19). Second, we generated clones of MDA-MB-231 expressing temperature-sensitive mutant p53Val135, which acquires active wild-type conformation at 32°C (20). Clones were tested for increased expression of p21 at 32°C versus 37°C by western blot (data not shown), and clones 1 and 8 were selected. MCF7 and MDA-MB-231 clones were treated as described above to study cytotoxic responses (cell viability and apoptosis) to lauryl gallate. If wild-type p53 confers a certain degree of resistance, decreasing its expression in MCF7-E6 should make them more sensitive to lauryl gallate than control MCF7-C4. Although we observed a slightly higher sensitivity to lauryl gallate (decreased cell viability and increased apoptosis), when calculations were made adjusting basal values, there were not significant differences between control MCF7-C4 clone and MCF7-E6 (Figure 6D). Similarly, the expression of functional p53 in MDA-MB-231 was supposed to reduce sensitivity to lauryl gallate. However, no significant differences were found between clones expressing p53-Val135 at 32 and 37°C (Figure 6D).

Role of Erk1/2 on lauryl gallate induction of apoptosis
Mitogen-activated protein kinase (MAPK) signaling pathways are activated in response to several cytotoxic compounds (31). To explore whether lauryl gallate could induce MAPK activation, lysates from cells treated with 5 µM lauryl gallate for 24, 48 and 72 h were subjected to western blot analysis using anti-phospho-MAPKs antibodies to detect their activation. As shown in Figure 7A, lauryl gallate induced phosphorylation of Erk1/2 at 48 and 72 h in all three cell lines. Concerning other members of the MAPK family, no phosphorylation of p38 kinase was observed, while only a light c-jun N-terminal kinase (JNK) activation was detected (data not shown). A number of studies have demonstrated a correlation between Erk1/2 activation and p21 induction following cellular stress (32–34). To verify whether Erk1/2 activation was required for lauryl gallate-induced p21 upregulation, we inhibited their activation with PD98059. Treatment with 10 µM PD98059 for 2 h prior to addition of 5 µM lauryl gallate for 48 h partially blocked both activation of Erk1/2 and upregulation of p21 (Figure 7B) in all three cell lines. These results suggest that Erk1/2 activation was required for p21 upregulation. As this protein plays an important role in cell cycle, we then analyzed the effects of Erk1/2 activation on cell cycle progression. As previously shown (Figure 3) lauryl gallate caused MCF7 accumulation in G1, while in MCF7 ADR and MDA-MB-231 it induced slow-down of cell cycle progression. Pretreatment with 10 µM PD98059 for 2 h partially diminished G1 accumulation in MCF7 cells and also modified the temporary accumulation in G2/M observed in the other two cell lines, where an increased G1 was clearly detected (Figure 7C, upper panel). These results suggested that Erk1/2 activation may be involved in cell cycle alterations induced by lauryl gallate.


Figure 7
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  		Fig. 7 Role of Erk1/2 in lauryl gallate-induced cytotoxicity. (A) Effect of lauryl gallate on Erk1/2 activation. MCF7, MCF7 ADR and MDA-MB-231 cells were cultured with 5 µM lauryl gallate (LG) or DMSO, as a control, for 24, 48 and 72 h. After lysis, 30 µg of total cell lysate were separated on SDS–PAGE, transferred to a membrane that was then incubated with polyclonal antibody anti-phosphorylated-Erk1/2 (P-Erk1/2) (1:2000). Lower panels show the stripped membranes reprobed with anti-Erk2 antibody for loading control. (B) Role of lauryl gallate on Erk1/2 induction of p21 expression. Cells were treated with 10 µM PD98059 for 2 h prior to addition of 5 µM lauryl gallate (LG) for 48 h. Analysis of P-Erk1/2 and Erk2 proteins were performed as above. For p21 detection, 40 µg of total cell lysate were separated on SDS–PAGE, transferred to a membrane that was then incubated with monoclonal antibody anti-p21 (1:1000). Lower panels show stripped membranes reprobed with an anti-{alpha}-tubulin antibody for loading control. Changes in the levels of P-Erk1/2 and p21 after being normalized to the levels of Erk2 and {alpha}-tubulin, respectively, are shown below each blot. Control samples are considered as the unit. Results shown are representative of three independent experiments. (C) Role of Erk1/2 on cell cycle alterations (upper panel) and apoptosis (lower panel) induced by lauryl gallate. Cells were treated with 10 µM PD98059 for 2 h prior to addition of 5 µM lauryl gallate (LG) for 48 h. Cell cycle analyses were performed as in Figure 3A. Apoptosis was determined with annexin V-FITC-conjugated (1 µg/ml) as in Figure 4A. Reported values represent the mean ± SD of three independent experiments. Asterisks indicate a significant difference between samples treated with PD98059+LG and those treated with LG alone in each cell line (*P < 0.05; **P < 0.01).

 
Since it has been previously shown regarding the involvement of Erk1/2 pathway in apoptosis (35–38), we analyzed the role of Erk1/2 activation in lauryl gallate-induced apoptosis. As observed in Figure 7C, lower panel, cells treated with 10 µM PD98059 for 2 h prior to addition of 5 µM lauryl gallate for 48 h, showed a significant reduction of the percentage of apoptotic cells as compared with lauryl gallate-treated cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The effect of lauryl gallate, an antioxidant gallic acid derivative, has been studied on three human breast cancer cell lines: wild-type p53 MCF7, and two non-functional p53 cell lines, MCF7 ADR (multidrug-resistant) and the highly proliferative and invasive MDA-MB-231. Our results show that lauryl gallate behaved as a potent antiproliferative agent inducing cell cycle alterations and apoptosis in all cell lines tested.

A wide variety of cytotoxic insults result in activation of mechanisms that arrest cell cycle progression at specific checkpoints, presumably to allow cells to repair DNA damage. This is consistent with G1 arrest of MCF7 induced by lauryl gallate. However, in MCF7 ADR and MDA-MB-231, a stable block in G1 phase was not achieved and cell cycle progressed allowing cells to accumulate damages.

Cell cycle-mediated apoptosis is a common way for cell death upon treatment with anti-tumor agents and inability to activate the apoptotic program confers drug resistance. Another mechanism to evade the action of cytotoxic agents is to express MDR genes, such as mdr-1 encoding for P-gp (4). For that reason, one of the strategies employed to overcome drug resistance is to inhibit the function of P-gp, allowing resistant cells to accumulate drugs to levels similar to those found in sensitive cells (39). Therefore, it is of an utmost importance to obtain compounds that will not be affected by P-gp excretory action. Our results demonstrate that lauryl gallate induces strong effects in the multidrug-resistant MCF7 ADR cells, characterized by P-gp overexpression. Similarly, cytofluorimetric assays by doxorubicin accumulation and UIC2 shift demonstrated that lauryl gallate did not interfere with the proper functionality of P-gp when analyzed towards other compounds. This result is in contrast with recent studies demonstrating that polyphenols from green tea, such as epigallocatechin gallate, inhibited activity of P-gp, increasing intracellular accumulation of P-gp substrates, acting as potential MDR reversal agents when used in combination with antitumoral compounds (40,41). The higher complexity of these polyphenols may explain a different behavior from the one we report for lauryl gallate.

Among proteins involved in control of apoptotic pathway, Bcl-2 has been demonstrated to protect cells from both normal and experimentally induced apoptosis (28). Bcl-2 is located in the outer mitochondrial membrane and its apoptosis-regulatory effects are mainly related with mitochondrial structure and function. This protein prevents opening of mitochondrial permeability transition pores and, subsequently, inhibits mitochondrial membrane depolarization and release of apoptogenic factors to cytosol (29,42).

The higher Bcl-2 protein levels detected in MCF7 as compared to the other two cell lines could result in a lower sensitivity to lauryl gallate. This hypothesis was confirmed by electron microscopy and flow cytometry analyses, showing lower functional and morphological alterations in mitochondria of MCF7 cells as compared to those observed in MDA-MB-231 and MCF7 ADR cells. Furthermore, the protective role of Bcl-2 against lauryl gallate-induced apoptosis was clearly demonstrated by higher viability and lower number of cells with morphological apoptotic features shown by MAB25 (Bcl-2 overexpressing MCF7 ADR clone) as compared to MAN9 control clone. Similar protective effects of Bcl-2 have been reported in murine B cell lymphoma line Wehi 231 (13). Other studies have demonstrated that a partial resistance of MCF7 against camptothecin and UCN-01 is related to Bcl-2 expression (23).

Activation of G1 checkpoint requires functional p53, which transcriptionally activates downstream genes to induce G1 cell cycle arrest and, in some cases, apoptosis (22). Mutation of p53 tumor suppressor gene is one of the most common genetic alterations in human cancers, including breast cancer, which induces uncontrolled cell proliferation (43). Wild-type p53 protein has been shown to mediate apoptosis, while mutations or deletions within p53 gene have been associated, in vivo and in vitro, both with resistance to apoptosis by different chemotherapeutic agents in some cell lines and with increased sensitivity in others (44,45). Results obtained in MCF7-E6 cells and in MDA-MB-231 p53-Val135 suggest that p53 is not a major target for lauryl gallate-induced cytotoxicity.

Among p53-activated genes, p21 is a potent inhibitor of cyclin-dependent kinase activities required for progression from G1 to S phase of the cell cycle (24). Surprisingly, lauryl gallate induced p21 expression in all three cell lines tested. Increased p21 expression in MCF7 correlated with G1 arrest, and a delayed cell cycle progression in the other two cell lines. As MCF7 ADR and MDA-MB-231 cells express a mutated p53, induction of p21 by lauryl gallate could be attributed to activation of a p53-independent pathway.

A number of studies demonstrated p53-independent mechanisms able to regulate p21 expression through Erk1/2 activation (32,46). Cell cycle perturbations and p21 upregulation by synthetic compounds occurred through activation of ATM/ATR-Erk1/2 pathway (33). Also, inhibition of Mek1/2 activity clearly reduced the induction of p21 promoter, mRNA and protein expression (34). In our cell model, lauryl gallate caused sustained activation of Erk1/2, clearly detected in both wild-type and mutated p53 cell lines, indicating a p53-independent mechanism. Abrogation of Erk1/2 activation by Mek1/2 inhibitor PD98059 reduced p21 induction and cell cycle alterations (G1 block in MCF7 and delayed progression in MCF7 ADR and MDA-MB-231) due to lauryl gallate. Furthermore, apoptosis induction was significantly reduced by Erk1/2 inhibition. The involvement of Mek1/2–Erk1/2pathway in the induction of apoptosis by different cytotoxic compounds has been recently demonstrated (32,35,37,38). Also, its contribution to cell proliferation and survival is well established (47). Erk1/2 has been identified as kinase responsible for Bcl-2 phosphorylation at Ser87 residue (48). Phosphorylation of Bcl-2, usually distributed in hypophosphorylated form on the mitochondrial membrane, results in reduction of its anti-apoptotic activity and redistribution from mitochondria to cytoplasm. We could hypothesize that lauryl gallate induced apoptosis by activation of Erk1/2 and subsequent phosphorylation of Bcl-2. The ability of Mek1/2 inhibitor PD98059 to reduce apoptosis could account for this hypothesis. In addition, the lower cytotoxicity observed in MCF7 and MAB25 could be in agreement with this theory. Given that these cells expressed elevated levels of Bcl-2, a higher lauryl gallate concentration may be required to obtain the same effect as those observed in MCF7 ADR and MDA-MB-231 with lower levels of Bcl-2. As for activation of other MAPKs, lauryl gallate did not induce p38 phosphorylation and only slightly activated JNK (data not shown). Studies on the mechanism of action of other molecules with anticancer properties showed that, in addition to Erk1/2, p38 and/or JNK were also activated (35,37). The fact that the Mek1/2–Erk1/2 pathway has been shown to contribute to both cell proliferation and survival and also to mediate cell cycle alteration and apoptosis (47), agrees with the common belief that no general mechanism of action for cytotoxic compounds can be postulated.

In conclusion, our data indicate that lauryl gallate induced reduction of cell survival and cell cycle alterations in MCF7, MCF7 ADR and MDA-MB-231 cells. An evident p21 upregulation was observed in all three breast cancer cell lines, followed by apoptotic cell death. Both p21 upregulation and apoptosis induction were regulated, at least in part, by Erk1/2 activation. Interestingly, a stronger cytotoxic effect was observed on multidrug-resistant MCF7 ADR cells as well as on MDA-MB-231 carrying mutations in the p53 gene, which, in theory, should be more resistant to cytotoxic treatments and apoptosis. These findings suggest that lauryl gallate might be a good candidate for innovative therapeutic strategies against tumors carrying p53 mutations and resistant to conventional chemotherapeutic agents.


   Acknowledgments
 
We thank J. Pérez and L. Camilli for the artwork. Authors are grateful to D. Del Bulafo and D. Trisciuoglio for donation of MCF7 ADR MAN9 and MAB25 clones, and together with I. Lazaro-Trueba for their constructive comments. This work was supported by grants to JM-P from MCyT (SAF2003-02188), and FIS (01/1316, 03C03/10 and PI040682) and a grant from MCyT to AL-R (SAF2003-00402). JMGM was supported by a fellowship from FIS, LG was supported by a fellowship from Fundación Carolina, MTAO was supported by the FIS 03C03/10 grant, and MJT by a FINNOVA fellowship. JMP is a member of GEICAM.

Conflict of Interest Statement: None declared.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
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Received December 19, 2005; revised March 24, 2006; accepted March 31, 2006.


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