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Minerval is a potent anticancer drug whose molecular mechanism of action is still not fully understood. While Minerval induces cell cycle exit in human lung cancer (A549) cells (1), it induces apoptosis in human leukemia cells (2). The IC50 of this drug for most cancer cells studied is in the range of 30-150 M, whereas its IC50 in normal cells is over 5,000 M (2). Thus, this drug does not produce toxicity at therapeutic doses despite acting efficiently in cell and animal models of human cancers (2, 3). Hence, it is important to define the mode of action of this compound and whether common molecular events underlie its therapeutic effects and the regression of different types of cancer.
Minerval was developed on the basis that the lipid composition and structure of the plasma membrane can be altered by certain antitumor drugs and that these modifications are involved in their action against cancer (4, 5). In this context, anthracyclines that are unable to enter cancer cells or bind to DNA still have strong antitumor activity (6). Indeed, those used in human therapy regulate plasma membrane structure, and they induce changes in the localization and activity of important peripheral signaling proteins, such as G proteins and PKC (4, 7, 8). This mode of action also appears to be responsible for the activity of hexamethylene bisacetamide against cancers (9, 10).
In the search for molecules capable of inducing similar regulatory effects on membrane lipid structure and peripheral protein function, oleic acid was found to be more potent than anthracyclines (8) and thus, a non-hydrolyzable analogue of this molecule was designed, Minerval (1, 11). This drug interacts with cell membranes, inducing changes in their composition and structure (1, 11, 12), and it triggers a series of events that ultimately result in the dramatic knockdown of the proliferation-inducing transcription factor E2F-1 (3).
Folates play a key role in one-carbon metabolism, which is essential for the biosynthesis of purines and hence, for DNA replication. Antifolates are classic antitumor agents that inhibit key enzymes in DNA synthesis, such as thymidylate synthase and DHFR, whose expression is regulated by E2F-1 . One of the first clinically useful blockers of DHFR was methotrexate (13, 14), which is frequently used in the treatment of acute leukemias and a number of solid tumors (15). Although this and other antifolate drugs are relevant in the treatment of cancer, drug resistance poses a major obstacle to their effectiveness. Since inhibition of DNA synthesis can induce cell cycle arrest or the induction of apoptosis, we studied whether DHFR might be involved in the mechanism by which two cancer cell lines respond differently to Minerval.
Materials and Methods
Cell Culture. Human non-small lung adenocarcinoma (A549) and Jurkat T-lymphoblastic leukemia cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained at 37°C in 5% CO2 in air and in RPMI 1640 medium supplemented with 2 mM glutamine, containing 10% bovine calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B. Tissue culture medium and supplements were purchased from Sigma (Madrid, Spain)
Cell Proliferation Assays. Cells were plated at a density of 1 x 105 (A549) cells or 1.5 x 105 (Jurkat) cells in 24-well plates with 0.5 ml of culture medium per well, and they were incubated overnight. To analyze the effect of lipids on cell proliferation, A549 cells were exposed for 24 h to between 0 and 200 M of Minerval, phosphatidylcholine (Avanti Polar Lipids, Alabaster, Alabama, USA), cholesterol (Sigma) or elaidic acid (Sigma). Jurkat cells were treated with the same lipids at concentrations between 0 and 150 M for 24 h. The time and dose to which the cell lines were exposed was based on their different sensitivities to lipid treatments.
To determine how DHFR influences the effects of Minerval, cells were incubated in the presence or absence of Minerval (100 M or 75 M Minerval for A549 and Jurkat cells respectively), in the presence or absence of 200 M folinic acid (Acros Organics, Barcelona, Spain) added every 12 h for 72 h. Unattached dividing cells were recovered by centrifugation for 5 min at room temperature (RT) and 600 Ã- g, and they were combined with the adherent cells that were harvested by centrifugation after treatment for 5 min at 37°C 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 [pH7.3]). Viable cells were counted immediately using 0.2% trypan blue in PBS.
Cell Cycle Analysis. The cell cycle was analyzed by flow cytometry in cells maintained in the presence or absence of Minerval for 72 h. After treatment, A549 cells were washed twice with PBS and detached using trypsin, while Jurkat cells were washed with PBS and collected by centrifugation. The cells were fixed with 100% methanol for 2 h at 4°C, centrifuged for 5 min at RT and 600 Ã- g, and then resuspended in PBS. Finally, cells were incubated for 30 min in the presence of 100 g/ml ethidium bromide and 100 g/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 on the basis of their DNA content.
Electrophoresis (SDS-PAGE), Immunoblotting and Protein Quantification. Cells were incubated in the presence or absence of Minerval (100 M for A549 and 75 M for Jurkat cells), in six-well culture plates under the experimental conditions indicated above. The cells were then washed twice with PBS and harvested using a rubber policeman in 300 l of 10 mM Tris-HCl buffer [pH 7.4] containing 50 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 1% SDS, 5 mM iodoacetamide, 1 mM phenylmethylsulfonylfluoride, 1 M sodium orthovanadate and 1 M cantharidin. Jurkat cells were washed twice with PBS and collected by centrifugation at 600 Ã- g for 5 min at RT. The cells were homogenized by ultrasound for 10 s at 50 W in a Braun Labsonic U sonicator (20% cycle) and 30 l aliquots were removed for total protein quantification. Subsequently, 30 l of 10 Ã- 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, 25 g of total protein from Minerval-treated samples or 5-60 g from control samples (for standard curves) was resolved on the same SDS-polyacrylamide gel (9% polyacrylamide) and transferred to nitrocellulose membranes (Whatman Schleicher and Schuell, Dassel, Germany). The membranes were incubated with blocking solution (PBS containing 5% non-fat dry milk, 0.5% BSA, and 0.1% Tween 20) for 1 h at room temperature and then overnight at 4°C in fresh blocking solution containing the specific primary antibodies: mouse anti-cyclin D3 (diluted 1:1000); mouse anti-cdk2 (diluted 1:1000); mouse anti-DHFR (diluted 1:500) from BD Biosciences Transduction Laboratories (Heidelberg, Germany); and a rabbit anti-poly ADP-ribose polymerase (anti-PARP, diluted 1:2000) from Santa Cruz Biotechnology (Santa Cruz, USA). Subsequently, the membranes were incubated with horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG (diluted 1:2000 in blocking solution) for 1 h at room temperature. The immunoreactive protein bands were visualized using the ECL Western blotting detection system (Amersham Pharmacia, UK) followed by exposure to ECL hyperfilm, and the films were scanned at a resolution of 600 dpi using the Foto 32 software (Agfa Gevaert, Germany).
Quantitative Reverse Transcription-Polymerase Chain Reaction (QRT-PCR). QRT-PCR was used to determine the regulatory effects of Minerval on the transcription of DHFR. Accordingly, cells were incubated in the presence or absence of Minerval for 48 h and then, the total RNA was extracted from 3 x 106 A549 or Jurkat cells using the RNeasy Mini kit in combination with the Qiagen RNase-free DNase kit (QIAGEN, Valencia, Spain) according to the manufacturer's instructions. Reverse transcription reactions were carried out using 1 g of total RNA in a final volume of 20 Î¼l, containing the following reagents (Invitrogen, Carlsbad, CA): First-Strand Buffer; oligo(dT) (2.5 Î¼M); random hexamers (2.5 Î¼M); dNTP mix (dGTP, dCTP, dATP and dTTP; each at 500 Î¼M); dithiothreitol (10 mM); "RNase OUT" (recombinant ribonuclease inhibitor, 0.4 U/Î¼l); Moloney murine leukemia virus reverse transcriptase (10 U/Î¼l) and RNase-free water. The reaction mixtures were then incubated at 65°C (5 min), 37°C (50 min) and 70°C (15 min) and the cDNA samples obtained were stored at âˆ’20°C prior to use.
For PCR amplification, the primers designed were based on the DHFR sequence in GenBank (accession N° 1719): 5'-TCACCCAGGCCATCTTAAAC-3' (DHFR forward) and 5'-GAACACCTGGGTATTCTGGC-3' (DHFR reverse). As endogenous control, the expression of 18S RNA (GenBank accession N° 100008588) was determined using the following primers: 5'-GAGGTGAAATTCTTGGACCGG-3' (18S RNA forward) and 5'- CGAACCTCCGACTTTCGTTCT-3' (18S RNA reverse). Real-time PCR amplifications were carried out in a LightCycler thermal cycler (Roche Diagnostics, Indianapolis, IN) using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics), which contained 0.5 M of each dNTP and 2.5 mM MgCl2. An initial denaturation step at 95°C for 5 min preceded thermal cycling. DNA amplification and fluorescence quantification was determined over 30 cycles with a denaturation step at 95°C for 3 s, a 7 s annealing step at 60°C for DHFR or 63°C for 18S RNA, and an extension step at 72°C for 12 s. Fluorescence quantification was carried out after each DNA extension step (72°C) and the data were analyzed using the LightCycler software. The ratio between the expression of DHFR and 18S RNA (whose expression is not modulated by Minerval) was determined by means of the equation described by Pfaffl et al (16). This value was used to calculate the relative expression in Minerval-treated cells with respect to untreated cells (control = 1). Melting curve analysis and agarose gel electrophoresis were used to further characterize the PCR products.
Statistics. The results were expressed as the mean ± S.E.M. of at least three independent experiments and the level of significance was set as P < 0.05.
Having previously demonstrated that Minerval produces potent cell cycle arrest of human lung cancer (A549) cells and apoptosis of lymphoblastic (T-lymphocyte) leukemia (Jurkat cells: 1, 2), we investigated whether a common molecular mechanism might explain these effects. Accordingly
, we first studied the structure-function effects of Minerval using its trans analogue, elaidic acid (EA), and the membrane-binding lipids, phosphatidylcholine (PC) and cholesterol (CH, Fig. 1). Minerval but not the other lipids can regulate the lipid structure of membranes, facilitating the binding and activation of important membrane signaling proteins, such as PKC (1). In this context, Minerval but not EA, PC and CH, inhibited cancer cell growth (Fig. 1), arguing against any possible non-specific effects mediated by the general disorganization of membrane lipid structure.
Among the events triggered by Minerval, there is a marked knockdown of E2F-1, a key transcription factor that regulates the expression of a large number of cell cycle-related genes, including DHFR (17, 18). Due to the central role of this enzyme in regulating cell viability and proliferation, we investigated the effects of Minerval on DHFR expression and as a consequence, on DNA synthesis and cell cycle progression. Indeed there was a significant decrease in DHFR expression (protein and mRNA) in both A549 and Jurkat cells exposed to Minerval for 48 h (50-100 M, Fig. 2), the first evidence that Minerval produces a common effect in different cancer cells. Since DNA inhibition can induce either cell cycle arrest or apoptosis in A549 and Jurkat cells, this effect may explain the divergent anticancer effects of Minerval (see Discussion).
Induction of the cell cycle arrest and apoptosis by Minerval (100 M) was further determined by flow cytometry, studying the proportion of cells in the various phases of the cell cycle (G0/G1, S and G2/M phases or the sub-G1 peak for apoptosis). Minerval increased the proportion of A549 cells in the G0/G1 phase, consistent with cell cycle arrest (Fig. 3E). Indeed, Minerval also down-regulated the expression of cdk2 and cyclin D3 (Fig. 3B and C).
To determine whether DHFR participated in the cell cycle arrest of A549 cells, these cells were maintained in the presence of folinic acid (200 M), a molecule that is readily converted into THF without the participation of DHFR. A549 cell proliferation was restored in the presence of Minerval (100 M) and folinic acid (Fig. 3). Similarly, the levels of cdk2 and cyclin D3 as well as the number of cells in mitosis also increased significantly in the presence of folinic acid (Fig. 3).
Folinic acid also impaired the apoptosis induced by Minerval in Jurkat cells (2), as witnessed by a decrease in the sub-G1 peak and PARP degradation, as well as the concomitant recovery of Jurkat cell proliferation (Fig. 4).
These results clearly indicate the specificity of Minerval and the relevance of DHFR in inducing cell cycle arrest in A549 cells and apoptosis in Jurkat cells. However, the fact that this recovery was not complete in both cell lines suggests that other pathways are also likely to be regulated by this drug.
Pivotal function of DHFR in the action of Minerval against cancer. Minerval is a potent anticancer drug that inhibits tumor growth and induces tumor regression, markedly increasing the life span of animals with cancer (1, 2). Two apparently divergent mechanisms appear to be associated with the therapeutic effects of this drug against cancer: inhibition of cancer cell proliferation and apoptosis. To date these mechanisms have been studied in 12 different cancer cell lines, although A549 and Jurkat cells were used here as models to investigate the molecular bases underlying the induction of cell cycle arrest and apoptosis, respectively. We show that concentrations in the range of the IC50 for this drug induced marked and significant reductions of DHFR mRNA and protein expression, a pivotal enzyme necessary for DNA synthesis and hence, cell proliferation.
Analysis of the DNA content in A549 cells by flow cytometry showed that cells accumulate in the G0/G1 phase after exposure to Minerval. Induction of cell accumulation by Minerval in the G0/G1 phase was associated with the down-regulation of cdk2 and Cyclin D3, both of which are involved in G1 to S phase progression (19). These results suggest that DNA synthesis is reduced, in agreement with the reductions in S and G2/M flow cytometry peaks observed. While Minerval does not induce apoptosis of A549 cells, it does promote the death of Jurkat cells, as witnessed by the degradation of PARP and the presence of a sub-G1 flow cytometry peak. In this regard, the induction of apoptosis by a number of antifolate drugs in T cells is a well characterized phenomenon (20). These results suggest that the Minerval-induced impairment of DNA synthesis drives Jurkat cells into an apoptotic program.
DHFR knockdown in response to Minerval was concentration-dependent in both A549 and Jurkat cells. Moreover, the pharmacological action of Minerval was reversed in both cell lines in the presence of folinic acid (5-formyl-THF), a molecule that is readily converted to THF without the intervention of DHFR. These results indicate that DHFR knockdown is a key event in the induction of the cell cycle arrest (A549 cells) or apoptosis (Jurkat cells). In addition, they suggest that DNA synthesis is in part responsible for either the inhibition of cell growth in A549 cells or the induction of apoptosis in Jurkat cells. However, since two different anticancer mechanisms were triggered by Minerval and given that its anticancer action was not fully reversed, simultaneous or sequential mechanisms would appear to be involved in the pharmacological effect of this molecule (see Fig. 5). This second signal or pathway would define the final and complete anticancer effect of the drug (either inhibition of cell growth or apoptosis), and it might originate from the plasma membrane, from a downstream molecular event leading to DHFR reduction or from both. An example of such simultaneous membrane signals might be the induction of Fas receptor clustering in Jurkat cells (2). By contrast, the divergent effect of impairing cyclin and cdk activity that leads to cell cycle arrest in A549 or apoptosis in Jurkat cells, may also reflect the downstream cross-talk mechanisms (2, 3, 21-23).
A number of antineoplasic drugs alter the organization of the plasma membrane (4, 5, 8) and strongly influence the localization and activity of key signaling proteins. Such modifications can be used for therapeutic purposes, an approach termed "membrane-lipid therapy" (7, 24). Indeed, lipid molecules such as edelfosine and mitelfosine are potent antineoplastic drugs that target the membrane (25, 26) and they induce apoptosis in cancer cells through mechanisms that alter membrane lipid raft organization (26). While we showed that oleic acid was a very active compound in regulating membrane lipid structure (8), this compound is readily metabolized by cells and therefore, it lacks relevant pharmacological activity. By contrast, alpha-hydroxy derivatives of fatty acids (e.g., Minerval) are less prone to be used by cells and they are therefore likely to display better pharmacological properties (27).
Role of lipid structure in the effects of Minerval. The structural alterations to the cell membrane induced by Minerval favor the translocation of PKC to the cell membrane and a series of events that provoke the down-regulation of E2F1 (1, 3). E2F1 is an important transcription factor that regulates the expression of dozens of genes required for cell cycle progression (28). Among others, E2F1 controls the expression of DHFR (17, 18) and hence, it seems likely that this signaling pathway initiated at the plasma membrane accounts for the knockdown of DHFR. We investigated the effect of different lipids on cancer cell growth and we found that, EA, the trans deoxylated isomer of Minerval, did not significantly affect the growth rate of A549 and Jurkat cells. This result argues in favor of the specificity of the effects of Minerval, supporting the structural bases of its influence on membranes. In this context, the lamellar-prone lipid phosphatidylcholine and the fluidity regulator cholesterol did not change the growth rate of cancer cells either. In turn, oleic acid has similar effects on the modulation of membrane lipid structure, E2F1 expression and cell growth inhibition (3), although its metabolism reduces its pharmacological potency and limits its use in the treatment of cancer in humans.
On the whole, the data presented support and extend previous studies indicating that membrane lipid structure is crucial in the mode of action of Minerval. However, new studies will be required to fully understand the different effects of this drug on cancer and normal cells. Accordingly, we are currently investigating the changes in membrane lipid composition after treatments with this compound, and how these changes regulate the physicochemical and functional properties of lipid bilayers.
Specificity of Minerval. An important issue regarding Minerval's therapeutic effects is related to the specificity of this compound. In contrast to most drugs designed on a rational structural bases, Minerval does not target proteins but rather membrane lipids. Our current knowledge about protein structure, structure-function relationships and the role of proteins in different signaling pathways makes it possible to understand the rationale for protein-targeted drugs. However, fatty acids are mainly considered as cellular fuel and their effects on membranes are mostly associated with changes in membrane fluidity.
Membranes have complex structural features that are defined by the lipids they contain, and the various lipid structures found within them (membrane rafts, caveolae, synaptosomes, coated pits, etc.) are affected distinctly by each molecular entity capable of interacting with lipid bilayers. Furthermore, subtle structural changes in lipids have a strong impact on the structural properties of membranes and on their pharmacological effects. For instance, cis (oleic acid, Minerval) and trans (EA) isomers of octadecenoic acid (·-9) have similar or identical chemical compositions but since their structure differs, so does their effect on membrane structure and on the localization and activity of peripheral membrane proteins (e.g. G proteins, PKC: 1, 8). Likewise, we show here that their potency against cancer is also distinct. In fact, the biophysical, molecular and cellular effects of EA are closer to those of stearic acid, which lacks double bonds but has a closer structure to that of EA, in accordance with the proposed structural basis of the effects of lipids on membranes. In addition, the presence of an oxygen atom on carbon 2 makes Minerval far more effective against cancer growth than its precursor, oleic acid (2). These data demonstrate the structural basis underlying the cellular, physiological and pharmacological effects of fatty acids.
Although the above data clearly demonstrate the specificity of Minerval, there are other arguments in support of this phenomenon, such as Minerval's lack of general or cytological toxicity in animals. In addition to the absence of side effects in animals at a dose of up to 3 g/kg (3), this fatty acid appears to be about 100-fold less potent in killing normal cells when compared to cancer cells (2). From a therapeutic point of view, this is the strongest evidence of specificity and it establishes an important difference between Minerval and most anticancer drugs.
The small number of genes modulated by this drug is further evidence of Minerval's specificity (about 100 genes are up- or down-regulated: 2). In addition, many of the proteins regulated by this lipid have been characterized, and those whose activity is regulated by protein-lipid interactions have been shown to display marked differences in their interactions with membranes that contain Minerval (2).
After oral administration, Minerval reaches all organs and it is incorporated into membranes. In this sense, it does not differ from other drugs that are widely distributed in the organism following their clinical administration. The basic difference is that the types of signals triggered in normal cells by Minerval are compatible with cell survival, whereas the signals that it triggers in cancer cells are not. Initially, signals that inhibit DNA synthesis (e.g., knockdown or DHFR) or cell cycle progress (e.g., down-regulation of cyclins and cdks) should not markedly affect mature cells. Conversely, these types of signals will promote the death of cancer cells. The lack of toxicity and high efficacy of this drug indicates that Minerval is more closely associated with the modulation of cell signaling than with cytotoxic phenomena, justifying its specific effects on cancer but not on normal cells.
DHFR activity modulation in cancer therapy.
Methotrexate was one of the first antimetabolites, if not the first, used to treat cancer. Developed in the 1940s, it was synthesized as a DHFR inhibitor to treat human leukemia, although it is also used to treat a wide variety of solid tumors (29). Like many other enzyme inhibitors, methotrexate induces an increase in DHFR expression as an adaptive mechanism to its binding and lowering of enzyme's activity (30), and its overexpression frequently leads to drug resistance (31). Methotrexate analogues, such as pemetrexed (32), have been developed as alternative drugs, but these antimetabolites do not fully overcome the phenomenon of the drug resistance developed by cancer cells exposed to methotrexate. By contrast to these antifolates, Minerval reduces the activity of this pivotal enzyme by diminishing its expression. The way that Minerval reduces DHFR activity may have a number of therapeutic advantages over these molecules, including the evasion of drug resistance phenomena triggered by conventional antifolates.
This regulatory action on DHFR is probably involved in the high efficacy exhibited by this molecule against cancer. In addition, its oral administration and lack of toxicity suggest that Minerval could be used in first-line treatments and combinatory therapies.
This work was supported by Grants BFU2007-61071 (Ministerio de Ciencia e Innovación, Spain), PROGECIC-3C and PCTIB2005-GC4-07 (Govern de les Illes Balears, Spain). R.A. and M.A.N. were supported by fellowships from the Ministerio de Ciencia e Innovación and Conselleria d'Economia, Hisenda i Innovació, respectively. S.T. holds a Torres-Quevedo contract.
(A) Structures of Minerval, , phosphatydilcholine (PC) elaidic acid (EA) and cholesterol (CH). Effects of these lipids on A549 (B) and Jurkat (C) cell proliferation after 24 h at the concentrations indicated.
Effect of Minerval on DHFR in A549 and Jurkat cells. (A) Effect of Minerval on DHFR protein levels as determined by quantitative immunoblotting (a representative immunoblot is also shown). A549 and Jurkat cells were incubated in the presence or absence (C) of Minerval (50 M, M50; 75 M, M75; 100 M, M100). (C) Effects of Minerval on DHFR mRNA expression in A549 and Jurkat cells. Bars correspond to mean ± SEM values of five independent experiments. *, P<0.05; **, P<0.01; ***,P<0.001.
Effects of Minerval and folinic acid on A549 cells. (A) A549 cell proliferation in the presence or absence (C, control) of Minerval (M), or Minerval plus folinic acid (M+F). (B) Effects of Minerval on Cdk2 and (C) on cyclin D3 were determined by quantitative immunoblotting (a representative immunoblot is also shown).The bars correspond to mean ± SEM values of five independent experiments of A549 cells incubated for 48 h (100 M Minerval, 200 M folinic acid; ***, P<0.001 with respect to untreated cells; ###, P<0.001 with respect to Minerval-treated cells). (D) Representative phase-contrast micrographs (100 Ã- magnification) of A549 cells in the presence or absence (C) of Minerval (M), or Minerval plus folinic acid (M+F). (E) DNA content in A549 cells incubated in the presence or absence (C, control) of Minerval (M, 100 M) or Minerval plus folinic acid (M+F, 100 M Minerval, 200 M folinic acid). From left to right, the bars and indicated values correspond to the percentage of cells in the subG1 (apoptosis), G1, S and G2+M phases of the cell cycle (for further details see materials and methods).
Effects of Minerval and folinic acid on Jurkat cells. (A) Jurkat cell proliferation in the presence or absence (C, control) of Minerval (M) or Minerval plus folinic acid (M+F). (B) Effects on PARP. (C) Representative immunoblot showing the effect of M and M+F on PARP. The bars correspond to the mean ± SEM of five independent experiments of Jurkat cells incubated for 48 h (75 M Minerval, 200 M folinic acid; ***, P<0.001 with respect to untreated cells; ###, P<0.001 with respect to Minerval-treated cells). (D) Representative phase-contrast micrographs (100 Ã- magnification) of Jurkat cells in the presence or absence (C) of Minerval (M), or Minerval plus folinic acid (M+F). (E) DNA content in Jurkat cells incubated in the presence or absence (C, control) of Minerval (M, 75 M), or Minerval plus folinic acid (M+F, 75 M Minerval and 200 M folinic acid). From left to right, the bars correspond the percentage of cells in the subG1 (apoptosis), G1, S and G2+M phases of the cell cycle (for further details see materials and methods).
Proposed mechanism for the anticancer effects of Minerval. Minerval modifies the membrane lipid structure (Signal 1), inducing PKC to translocation to the membrane and its subsequent activation. This event is followed by activation of p21CIP1, cyclin-cdk inhibition, pRb hypophosphorylation and E2F1 knockdown (1, 3). E2F1 is a critical transcription factor that regulates several genes involved in cell proliferation, such as DHFR. Finally, DHFR downregulation impairs DNA synthesis, which results in either the cell cycle arrest (A549 cells) or apoptosis (Jurkat cells). The particular molecular features of each cell line are responsible for the selection of one or another anticancer mechanism. Indeed, other molecular events may participate in the generation of a second signal (right side of the figure), Signal 2, which could originate directly at the membrane or as a consequence of cross-talk with the Signal 1 cascade. An example of the former is the clustering of the Fas receptor after altering membrane lipid structure (2), which might be involved in induction of apoptosis in Jurkat cells. By contrast, the latter may reflect the differential effect of cyclin-cdk inhibition, which induces the cell cycle arrest in A549 cells and apoptosis in Jurkat cells (2, 3, 22-24).