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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

The Repression of E2F-1 Is Critical for the Activity of Minerval against Cancer

Laboratory of Molecular and Cellular Biomedicine, Institut Universitari d'Investigacions en Ciències de la Salut, Associate Unit of the Consejo Superior de Investigaciones Cientificas, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain (J.M., J.C., V.L., A.L.-B., P.V.E.); Department of Hematology, Hospital Son Dureta, Palma de Mallorca, Spain (A.G., J.B.); and Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain (A.D.)

Received April 28, 2005; accepted July 14, 2005.

	Abstract

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The recently discovered anticancer drug Minerval (2-hydroxy-9-cis-octadecenoic acid) is a synthetic fatty acid that modifies the structure of the membrane. This restructuring facilitates the recruitment of protein kinase C (PKC) to membranes and is associated with the antineoplastic activity of Minerval in cellular and animal models of cancer. Minerval is a derivative of oleic acid (OA) with an enhanced antiproliferative activity in human cancer cells and animal models of cancer, which is associated with PKC activation and p21^CIP overexpression. However, the signaling cascades involved in its pharmacological activity remain largely unknown. Here, we showed that this drug induced cell cycle arrest before entry into S phase, human lung adenocarcinoma (A549) cells accumulating in the G₀/G₁ phase. This cell cycle arrest was associated with a marked decrease in the expression of E2F-1. This transcription factor activates several cell cycle-related genes, and, accordingly, the expression of certain cyclins and cyclin-dependent kinases (cdks) was markedly lower upon exposure to Minerval. The reduced availability of these kinase heterodimers was associated with reduced phosphorylation of the retinoblastoma protein (pRb) observed after drug treatment. Significantly, hypophosphorylated pRb remains bound to E2F-1 and maintains this transcription factor inactive. The modulation of these antiproliferative mechanisms by Minerval explains its anticancer potency, through a new therapeutic strategy that can be used to develop new antitumor drugs. On the other hand, apoptosis did not seem to be involved in its pharmacological mechanism. Interestingly, whereas the changes induced by OA were only modest, they may reflect the beneficial effects of high olive oil intake against cancer.

View larger version (29K): [in this window] [in a new window]   Fig. 1. a, structural comparison of OA and Minerval. In the molecular models, the carbon atoms are shown in black, hydrogen in white, and oxygen in gray. The only difference between both molecules is the oxygen atom on the carbon of Minerval. b, regulation of the cell cycle progression by E2F-1 and the effects of Minerval. Cell division is associated with an increase in the levels of cyclin/cdk that phosphorylate, among other targets, pRb. Phosphorylated pRb (pRb-p) releases active E2F-1 (E2F-1*), which activates the transcription of several cell cycle-related genes, including E2F-1 itself. The effects induced by Minerval are shown, i.e., hypophosphorylation of pRb, E2F-1 repression by inactivation through pRb binding, and inhibition of the cell cycle progression.

	Materials and Methods

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Cell Culture. A549 human lung adenocarcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). A549 cells were maintained at 37°C in a fully humidified atmosphere of 5% CO₂ in air. Monolayer cultures were maintained in exponential growth using 2 mM glutamine-supplemented RPMI 1640 medium, containing 10% bovine calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Tissue culture medium and supplements were all purchased from Sigma (Madrid, Spain).

Cell Proliferation Assays. A549 cells were plated at a density of 1.5 x 10⁵ cells in 24-well plates with 0.5 ml of culture medium per well. After incubating overnight to allow cell attachment, the cells were treated with 25 to 100 µM Minerval or 25 to 250 µM OA for 24, 48, and 72 h. At the end of these periods, unattached cells were recovered by centrifugation for 5 min at room temperature and 600g and combined with the adherent cells that had been harvested with 0.05% trypsin in sterile phosphate-buffered saline (PBS, 137 mM NaCl, 2.6 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄, at pH 7.3) for 5 min at 37°C and centrifuged as described above. The cells were immediately counted using an automated cell counter (Advia 120; Bayer Diagnostics, Tarrytown, NY), and cell viability was determined by trypan blue exclusion method (0.2% trypan blue in PBS buffer).

Cell Cycle Analysis. Analysis of the cell cycle by flow cytometry was performed on cells treated in the presence or absence (control) of Minerval or OA for 24 h. The cells were then washed twice with PBS, detached by trypsin treatment (see above) and fixed with 100% methanol for 2 h at 4°C. These cells were then centrifuged for 5 min at room temperature and 600g and resuspended in PBS. Finally, cells were incubated for 30 min in the presence of 100 µg/ml ethidium bromide and 100 µg/ml RNase A and analyzed on a Beckman Coulter Epics XL flow cytometer. Cell populations in the different phases of cell cycle (sub-G₁, G₀ /G₁, S, and G₂/M) were determined based on their DNA content.

Electrophoresis (SDS-Polyacrylamide Gel Electrophoresis), Immunoblotting, and Protein Quantification. Cells were incubated in the presence or absence of Minerval, as indicated above, in six-well culture plates. The cells were then washed twice with PBS and harvested with a rubber policeman in 300 µl of 10 mM Tris·HCl buffer, pH 7.4, containing 50 mM NaCl, 1 mM MgCl₂, 2 mM EDTA, 1% SDS, 5 mM iodoacetamide, and 1 mM phenylmethylsulfonyl fluoride. The cells were homogenized by ultrasound for 10 s at 50 W in a Braun Labsonic U sonicator (20% cycle), and aliquots of 30 µl were removed for total protein quantification. Then, 30 µl of 10x electrophoresis loading buffer (120 mM Tris·HCl buffer, pH 6.8, 4% SDS, 10% -mercaptoethanol, 50% glycerol, and 0.1% bromphenol blue) was added to the samples, and they were boiled for 3 min. For immunoblotting, 30 µg of total protein from the cell lysates (5–60 µg from control samples for standard curves) was subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Whatman Schleicher and Schuell, Dassel, Germany). The membranes were incubated with blocking solution for 1 h at room temperature, using PBS buffer containing 5% nonfat dry milk, 0.5% bovine serum albumin, and 0.1% Tween 20 for most immunoblots, or Tris-buffered saline buffer containing 5% nonfat dry milk and 0.1% Tween 20 for phosphorylated pRb detection. The membranes were then incubated overnight at 4°C in fresh blocking solution containing the specific primary antibodies: monoclonal anti-cyclin D1 (1:500 dilution), monoclonal anti-cyclin D3 (1:1000 dilution), monoclonal anti-cyclin E (1:1000 dilution), monoclonal anti-cdk2 (1:1000 dilution), monoclonal anti-cdk4 (1:1000 dilution), monoclonal anti-pRb (1:500 dilution), and monoclonal anti-E2F-1 (1:1000 dilution) (BD Transduction Laboratories, Heidelberg, Germany), and polyclonal anti-poly ADP-ribose polymerase (anti-PARP, 1:1000 dilution) (Cell Signaling Technology Inc., Beverly, MA). Subsequently, the membranes were incubated with sheep anti-mouse (for monoclonal antibodies) or donkey anti-rabbit (for the polyclonal antiserum) horseradish peroxidase-labeled antiserum (1:2000 dilution in blocking solution) for 1 h at room temperature. The immunoreactive protein bands were detected using the enhanced chemiluminescence Western blotting detection system (GE Healthcare, Piscataway, NJ). Quantification was performed by image analysis, for which four different concentrations of protein from control samples were loaded along with duplicate samples to be tested on 6 x 8-cm 15-well minigels. Four gels were usually processed in parallel. The films were scanned in the transparency mode with a resolution of 42 µm (600 dpi), using the Foto Look 32 software (Agfa Gevaert, Leverkusen, Germany). The integrated optical density of the four control samples of each gel was plotted against the amount of protein loaded. Integrated optical density values were interpolated into the standard curve of their corresponding gels to calculate the percentage of variation with respect to control cells (untreated) as follows:

(1)

where C is the cellular concentration of a given protein (100% being the control sample used for the standard curve), P_T is the theoretical amount of protein loaded on the gel (calculated from the standard curves), and P_R is the real amount of protein loaded.

Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of mRNA Expression. Quantitative RT-PCR was used to determine the transcriptional modulation of cyclins D1, D3, and E, cdk2 and cdk4, and E2F-1 by Minerval. For this purpose, total RNA was extracted from 3 x 10⁶ A549 cells using the RNeasy Midi kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Reverse transcription (RT) reactions were prepared using 1 µg of total RNA, 0.5 µl of oligo(dT) (500 µg/ml), 0.5 µl of random hexamers (500 µg/ml), and 1 µl of 10 mM dNTPs in a volume of 12 µl. This mixture was heated at 65°C for 5 min and immediately chilled on ice. Then, 4 µl of "5x First-Strand Buffer" (Invitrogen, Carlsbad, CA), 2 µl of 0.1 M dithiothreitol, and 1 µl of "RNase OUT" (recombinant ribonuclease inhibitor; Invitrogen) was added, and the mixture was incubated at 37°C for 2 min. Finally, 1 µl (200 units) of Moloney murine leukemia virus reverse transcriptase (Invitrogen) was added, and the mixture was incubated for 50 min at 37°C. The RT reaction was stopped by heating the tubes at 70°C for 15 min. PCR conditions were first determined in preliminary experiments using a gradient thermal cycler (Eppendorf-5 Prime, Inc., Boulder, CO). The sequence of the primers used, annealing temperatures, the length of the DNA segments, and their corresponding melting temperatures are shown in Table 1. After preliminary calculations of the RNA content by absorbance spectroscopy at 260 nm, a fine calculation of the RNA concentration was performed by quantifying the 18S RNA by PCR using the primers shown in Table 1. Real-time PCR amplifications were carried out in a LightCycler thermal cycler (Roche Diagnostics, Indianapolis, IN) using a LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics) according to the manufacturer's instructions. An initial denaturation step at 95°C for 5 min preceded thermal cycling. DNA amplification and fluorescence quantification was determined for 30 cycles at 95°C for 3 s, 55–60°C (Table 1) for 7 s and 72°C for 12 s. Fluorescence quantification was carried out at the end of the DNA extension step (72°C) after each temperature cycle because no primer-dimers were produced during the PCRs carried out (as determined by analyses of the melting temperatures for each PCR product after each experiment). Agarose gel electrophoresis was carried out to further characterize PCR products. Data were analyzed using the LightCycler software. For quantification purposes, we used eq. 2 (Pfaffl et al., 2002) as follows:

(2)

where R is the ratio between the expression of the gene studied (target) and a housekeeping "reference" gene (ref, 18S RNA), whose expression is not modulated by Minerval (as determined in preliminary experiments). This value (R) was used to calculate the relative expression in Minerval-treated cells with respect to untreated (control) cells (percentage of expression = 100 x R; this value was 100 for samples whose expression is identical to that of untreated cells). E_target and E_ref were the efficiencies of target and reference PCR amplifications, respectively. PCR efficiencies were calculated every experiment for each gene measured by preparing a mixture containing equivalent amounts of the RT reactions from all the samples prepared. This mixture was used as template for PCRs containing 5 pg to 5 ng (four independent reactions) of total RNA. These reactions were submitted to thermal cycling at the same time as reactions from control and Minerval-treated samples. Under our experimental conditions, real-time RT-PCR efficiencies ranked 1.5 to 2, with E = 2 being the ideal efficiency (meaning that one PCR "short-product" or amplicon produces two DNA molecules after each cycle). The CP-target(MEAN control - MEAN sample) value corresponded to the difference between the crossing point (CP, PCR cycle where the exponential curve is first detected over the background noise) of the control and the problem PCR curves (mean of three different reactions processed in parallel). Three to six independent experiments were carried out to evaluate the transcriptional regulation of each gene. This method of quantification, based on the determination of the beginning of exponential curves, has proven to be both accurate and reproducible as described previously (Pfaffl et al., 2002). The main trouble for mRNA quantification using real-time RT-PCR techniques is the formation of primer-dimers (Escribá et al., 2004). However, the primer sequences selected for the present study and the experimental conditions used here did not yield undesirable unspecific products. Under these conditions, this method is more convenient and precise than other quantitative approaches based on one-point measurements of PCR amplifications during the exponential phase of the curve or based on endpoint measurements (this latter method being even less accurate than the former).

View larger version (92K): [in this window] [in a new window]   Fig. 2. Effects of Minerval and OA on cell proliferation. a, phase contrast microscopy of A549 cells incubated for 24 h in the presence or absence (control) of 100 µM Minerval or 100 µM OA. The magnification is indicated on the left. Number of A549 cells calculated by flow cytometry in the presence or absence (control) of 50 µM (dotted line) and 100 µM (dashed line) of Minerval (b) or OA (c).

	Results

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Minerval inhibited the proliferation of human lung adenocarcinoma (A549) cells in a time- and dose-dependent manner. After 72 h, 100 µM Minerval inhibited cell proliferation by up to 66 ± 2% (P < 0.01; Fig. 2, a and b). In contrast, 100 µM OA only exerted modest (<10%) and nonsignificant (P < 0.05) effects on A549 cell proliferation (Fig. 2c). We studied the effect of Minerval on the cell population to determine when the cells entered in cell cycle arrest. Minerval (100 µM, 24 h) induced a significant increase in the number of cells in G₀/G₁ phase, with a concomitant decrease in the cell population in S and G₂ (Fig. 3). These data suggest that Minerval treatment impaired progression through the G₁ checkpoint. In contrast, OA, which is a natural fatty acid and a precursor of Minerval, did not significantly affect cell proliferation (Fig. 3) or the proportion of cells found in different stages of the cell cycle (Fig. 3) at the concentrations of Minerval used. However, at higher concentrations of OA (250 µM), inhibition of cell proliferation could be detected (42 ± 2%, P < 0.05, at 72 h). Flow cytometry highlighted the absence of a significant sub-G₁ population, a peak that is associated with apoptosis (Fig. 3). Furthermore, no PARP fragmentation was observed (Fig. 3). These results indicate that the lower number of cells in the presence of Minerval was due to impaired cell proliferation rather than to apoptosis of A549 cells.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Effects of Minerval and OA on the cell cycle. DNA content of A549 cells was measured by flow cytometry after a 24-h exposure to Minerval and ethidium bromide staining as indicated under Materials and Methods. Histograms of untreated control cells (a) and cells treated with 100 µM of Minerval (b) or OA (c) are shown. The fluorescence values used to calculate the peaks corresponding to the G0/G1 and the G2/M phase are indicated on each histogram. Bar graphs show the percentage of A549 cells in G0/G1 (d) and S/G2/M (e) phase with respect to the total cell number after 24-h in the presence or absence (C) of 100 µM Minerval (M) or OA. The data are the mean ± S.E.M. values of six independent experiments; *, P < 0.01. f, immunoblotting analysis of PARP in A549 cells incubated in the presence or absence of Minerval (M), OA (O), and a positive control of apoptosis (C+).

Minerval induced a marked and significant decrease in the expression of the transcription factor E2F-1. After exposure to Minerval, the levels of E2F-1 protein decreased by 90 ± 4% (P < 0.001; Fig. 4a) and the mRNA levels by 55 ± 5% (P < 0.01; Fig. 4b) in A549 cells. In contrast, OA produced only a modest but significant decrease in the accumulation of E2F-1 protein (20 ± 1%, P < 0.05; Fig. 4a). This effect may partially explain the antiproliferative activity of Minerval.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Effect of Minerval and OA on E2F-1 expression. a, E2F-1 protein levels in cells incubated in the presence of 100 µM Minerval (M) or OA (O) for 24 h. Control cells (C) not exposed to additional fatty acid are also shown. The inset shows representative immunoreactive bands in control (C), Minerval- (M), and OA (O)-treated cells. b, E2F-1 mRNA levels in A549 cells determined by real-time RT-PCR and incubated in the presence or absence (control) of 100 µM Minerval or OA for 24 h. Data shown are the mean ± S.E.M. values of six independent experiments. *, P < 0.01; **, P < 0.001.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Effect of Minerval and OA on pRb phosphorylation. A549 cells were incubated in the presence or absence (control, C) of 100 µM Minerval (M) or OA (O) for 24 h. Bars correspond to the ratio between hyperphosphorylated (pRb-p) and hypophosphorylated (pRb) forms of pRb. Data shown are the mean ± S.E.M. values of four independent experiments. The inset shows a representative immunoblot of pRb and pRb-p from cells incubated in the absence (control, C) or presence of Minerval (M) or OA (O). *, P < 0.05; **, P < 0.01.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effect of Minerval and OA on the expression of cyclin and cdk proteins. Protein concentrations were quantified by immunoblot analysis in cells treated for 24 h with vehicle (open columns), 100 µM Minerval (closed columns, a) or 100 µM OA (closed columns, b). Representative immunoreactive bands are also shown. The data shown are the mean ± S.E.M. values of five independent experiments. The insets show representative immunoblots of the various cyclins and cdks from control (C), Minerval- (M), and oleic acid (O)-treated A549 cells. For further details, see Materials and Methods. *, P < 0.05; **, P < 0.01.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Effect of Minerval on the relative expression of cyclin and cdk mRNA transcripts. A549 cells were incubated in the presence or absence (control, 100%, dotted line) of 100 µM Minerval for 24 h. Transcriptional rates were quantified by real-time RT-PCR, as described under Materials and Methods. The results are the mean ± S.E.M. values of at least three independent experiments; *, P < 0.01.

	Discussion

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Previous studies have shown that long-term olive oil consumption is associated with a dose-dependent reduction in the incidence of various types of cancer (Martin-Moreno et al., 1994; Llor et al., 2003). The main component of olive oil is OA (about 80%). There is also growing evidence that certain fatty acids exert a protective effect against some types of cancer (Begin and Ells, 1987; Tronstad et al., 2001, 2002; Llor et al., 2003; Akihisa et al., 2004). In line with these hypotheses, the lipid molecules Edelfosine (Et-18-OCH₃) and Miltefosine (hexadecylphosphocholine) are potent antineoplastic drugs targeted to the membrane (Jendrossek and Handrick, 2003). Similarly, polyunsaturated fatty acid (e.g., docosahexaenoic acid and eicosapentaenoic acid) derivatives have anticancer activity (Siddiqui et al., 2005, and references therein). The signal pathways by which these compounds act are not fully understood, so they may share common molecular mechanisms with Minerval.

Recently, we showed that exposure to Minerval inhibits the growth of cancers in both animal models and cultured cells (Martínez et al., 2005). Here, we sought to gain more information about the molecular mechanisms involved in this pharmacological action. We found that Minerval induced cell cycle arrest in G₀/G₁ and marked and significant decreases of E2F-1, both in terms of protein and mRNA. This transcription factor is involved in the control of a large number of cell cycle-related genes (Young et al., 2003). Thus, an increase in E2F-1 expression is associated with an elevated rate of carcinoma cell growth (Gorgoulis et al., 2002). In turn, its reduction is associated with a lack of proliferation, senescence, and cell differentiation (Saunders et al., 1993; Dimri et al., 1994; Fajas et al., 2002). Therefore, the decrease of E2F-1 levels is probably a key event in the antiproliferative activity of Minerval. To our knowledge, this is the first time that a free fatty acid has been shown to regulate E2F-1 expression. Likewise, the modest decrease of E2F-1 levels and pRb phosphorylation induced by OA might be involved in the protective effects of olive oil against cancer (Martin-Moreno et al., 1994).

Control of the cell cycle involves a number of revision checkpoints. Analysis of the DNA content in A549 cells by flow cytometry showed an accumulation of cells in the G₀/G₁ phase after exposure to Minerval. This result suggests that Minerval-treated cells exit from the cell cycle before they enter S phase, possibly due to their incapacity to overcome the G₁ checkpoint. Cyclins D and E as well as cdk2 and cdk4 are involved in the progression from G₁ to S phase (Sherr, 1996). These cyclins and cdks form heterodimers that regulate cell cycle progression through phosphorylation of target proteins. Interestingly, the genes encoding cdk and cyclins are regulated by E2F-1 and other members of the E2F family (Gorgoulis et al., 2002; Ma et al., 2003). Therefore, we studied what effect Minerval had on their cellular concentrations. We found that Minerval induced marked reductions of cyclin D3, cdk2, and cdk4, which might affect the G₁/S phase transition. In most systems, the regulation of cell cycle progression is mainly due to changes in the levels of cyclins, whereas the cellular concentrations of cdks are maintained constant. However, in certain cancer cells, cell cycle arrest is associated with reductions of both cyclins and cdks (Strobeck et al., 2000). Minerval clearly modulated the expression of cdk2 and cdk4 (protein and mRNA levels). The reduction in the levels of these kinases probably amplified the antiproliferative effects of down-regulating cyclin D3. In agreement with the present results, inhibition of these kinases impairs cell proliferation (Keshamouni et al., 2004). In contrast, the levels of cyclin D1 mRNA were higher in the presence of the drug. However, the cellular protein concentration of cyclin D1 was not significantly altered by Minerval treatments. This result supports the involvement of proteasome-mediated degradation, possibly also involved in the above-mentioned down-regulation of cyclins and cdks as suggested previously (Martínez et al., 2005). Finally, cyclin E remained unaffected by exposure to Minerval, indicating that this protein was not involved in its activity.

Hypophosphorylated pRb acts as a switch for cell cycle progression, holding cells in G₁ until they are ready to progress into S phase. It binds to several key regulators of cell cycle progression, including members of the E2F family of transcription factors (Mundle and Saberwal, 2003). When pRb is bound to E2F-1, it not only inhibits E2F-1-mediated transcriptional activation but also represses the transcription of a large number of cell cycle-related genes (Mundle and Saberwal, 2003; Young et al., 2003). Under the appropriate conditions, pRb phosphorylation at the G₁/S phase transition induces the release of active E2F-1 (Slansky et al., 1993; Ohtani et al., 1995; Ishida et al., 2001; Muller et al., 2001). Because cyclin/cdk heterodimers regulate pRb phosphorylation (Lundberg and Weinberg, 1998), the hypophosphorylated state of pRb induced by Minerval could be due to their down-regulation.

The changes here reported in E2F-1, cyclins, cdks, and pRb are consistent with the reduced proliferation in the presence of Minerval. In this context, Minerval would impair cell proliferation through at least two feedback mechanisms. On one hand, E2F-1 regulates its own expression, such that the low levels of E2F-1 protein are associated with low transcriptional rates of E2F-1 and cell cycle-related genes (Fig. 1b). In contrast, E2F-1 reduction results in down-regulation of cyclins and cdks (Fig. 1b). This event would produce a decrease in pRb phosphorylation, which would promote the accumulation of high levels of the cell cycle inhibitory complex E2F-1/pRb. The binding of pRb to E2F-1 further reduces its capacity to activate the transcription of cell cycle-related genes (Dyson, 1998) and represses the expression of certain genes required for cell cycle progression (Young et al., 2003). By contrast to cell proliferation, Minerval did not induce apoptosis in A549 cells as deduced by the lack of caspase activation, DNA laddering (data not shown), poly(ADP-ribose) polymerase degradation, sub-G₀ cell cytometry peaks, or apoptotic bodies.

Alterations in membrane structure regulate the localization and activity of peripheral proteins involved in cell proliferation (Escribá et al., 1995, 1997; Vögler et al., 2004; Martínez et al., 2005; Yang et al., 2005). An increase of the membrane nonlamellar phase propensity favors recruitment and subsequent activation of PKC. Minerval binds to membranes and increases their nonlamellar phase propensity, inducing recruitment and moderate activation of PKC in model (liposome) and A549 cell membranes (Barceló et al., 2004; Martínez et al., 2005). PKC activates the cell cycle inhibitor p21^CIP (Kashiwagi et al., 2000) and also inhibits E2F during G₁ phase (Nakaigawa et al., 1996), being therefore upstream E2F-1. This effect of Minerval on the membrane lipid organization is of structural nature. Indeed, the structural analog of Minerval, OA, induces similar modifications on the membrane structure, whereas the structurally unrelated analogs, elaidic and stearic acids, do not significantly influence the membrane lipid organization (Funari et al., 2003; Prades et al., 2003; Barceló et al., 2004). Moreover, OA but not elaidic and stearic acids modulate cell signaling upon membrane structure regulation (Yang et al., 2005). The lesser anticancer activity of OA might be due to its use as energy source, as discussed above. The antiproliferative activity of Minerval is associated with a moderate activation of PKC. A huge PKC activation, such as the one provided by phorbol esters, has opposite effects on cell proliferation. Indeed, in certain types of tumors there have been described overexpressions of PKC isozymes (Kamimura et al., 2004). Therefore, in these tumors Minerval would possibly lack of antiproliferative activity. However, other noncytotoxic mechanisms could be involved in its anticancer action, which may justify the broad range of tumors that respond to treatments with this drug. The present data also explain in part how the modulation of membrane structure by Minerval or other anticancer lipid drugs may affect the regulation of gene expression (Escribá et al., 1995, 2002; Jendrossek and Handrick, 2003; Siddiqui et al., 2005). Other possible mechanisms for the pharmacological activity of Minerval, such as the modification of fatty acid metabolism, the stimulation of peroxidative cell damage, or the modification of the lipid composition of the membrane, should not be discarded (Gonzalez et al., 1993; Belury, 2002; Prades et al., 2003). However, under the experimental conditions used, apoptosis did not seem to be involved in the antineoplastic activity of Minerval in A549 cells.

In summary, the impairment of the cell cycle progression induced by Minerval is associated with the down-regulation of E2F-1, cyclins, and cdks and a reduction in the phosphorylation of pRb. The present results are relevant because they 1) characterize relevant molecular mechanisms involved in the anticancer activity of Minerval, and they 2) show molecular events regulated upon membrane structure modulation. In this context, Minerval does not belong to any of the known groups of anticancer drugs and establishes a novel therapeutic approach that can be applied to develop anticancer drugs with low toxicity.

	Footnotes

 
This work was supported by Grants SAF2001-0839, SAF2003-00232, and SAF2004-05249 (Ministerio de Educación y Ciencia). "Fundacion Marathon", "Conselleria de Sanitat", and "Conselleria d'Economia, Hisenda i Innovació (Grants PRDIB-2002GC2-11 and PRID2004-10131) del Govern Balear" and "Centre Coordinador de Drogodependències" also provided funds for this work. J.C. was supported by a fellowship from the Spanish Ministerio de Educación y Ciencia. J.M. received the "Elsevier Young Scientist Award" for communication in the 2004 International Society for the Study of Fatty Acids and Lipids Meeting (Brighton, UK), showing some of the results included here.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.088716.

ABBREVIATIONS: OA, oleic acid; PKC, protein kinase C; pRb, retinoblastoma protein; cdk, cyclin-dependent kinase; PBS, phosphate-buffered saline; PARP, poly ADP-ribose polymerase; RT-PCR, reverse transcription-polymerase chain reaction; RT, reverse transcription; PCR, polymerase chain reaction; CP, crossing point.

¹ These authors contributed equally to this work.

Address correspondence to: Dr. Pablo V. Escribá, Laboratory of Molecular and Cellular Biomedicine, Associate Unit of the "Instituto de la Grasa" (CSIC), IUNICS, Department of Biology, University of the Balearic Islands, Ctra. de Valldemossa Km 7.5, E-07122 Palma de Mallorca, Spain. E-mail: pablo.escriba{at}uib.es

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