Background
A characteristic of tumour cells is the profound re-organization of metabolic parameters in relation to their healthy counterparts, allowing them to obtain the macromolecular constituents required for their rapid de-regulated growth and also as alternative sources of energy [
1‐
3]. One of the best known modifications is the increased dependence on glucose metabolism instead of oxidative phosphorylation, even under aerobic conditions (a property known as “aerobic glycolysis” or “Warburg’’ effect). This peculiarity made possible the development of glycolysis-targeting drugs as potential anti-cancer agents. This category includes, among others, the glucose inactive analog 2-deoxy-
d-glucose (2-DG) [
4], the indazole derivative lonidamine (Lon) [
5], and the small alkylating drug 3-bromopyruvate (3-BrP) [
6]. Allowing for the disparity in chemical structure and hence in biochemical and molecular effects, these drugs target critical enzymes in the glycolytic pathway, namely hexokinase II (HKII) in the case of 2-DG and Lon [
5,
7], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and to a lower extent HKII in the case of 3-BrP [
8]. While promissory results have been obtained in clinical assays [
5,
6,
9,
10], the efficacy of these agents is different. Thus, 3-BrP is quite toxic per se, while 2-DG and Lon are well tolerated but poorly efficacious in monotherapy. Nonetheless, 2-DG and Lon may be useful as radio- and chemo-sensitizing agents, overcoming resistance and increasing cyto-reduction by conventional anti-tumour treatments [
4,
5,
9]. Using combinatory assays with the anti-leukemic agent arsenic trioxide (Trisenox), we recently demonstrated that a common effect of the anti-glycolytic drugs is the stimulation (albeit with different kinetics and intensity) of Akt/mTOR and MEK/ERK defensive pathways in several human acute myeloid leukemia (AML) cell lines, and that this stimulation restrains the apoptotic efficacy of 2-DG and Lon when used as single agents [
11,
12]. Akt and/or ERK activation by 2-DG was also observed in other tumour cell models [
13‐
15].
Polyphenols represent a large collection of molecules present in the plant kingdom. At the low doses attainable in the daily diet these compounds exert multiple protective functions (e.g., against cellular oxidation, inflammation, aging, tumour initiation…). On the other hand, at high albeit still pharmacologically attainable concentrations many polyphenols selectively induce apoptosis in tumour cells, and exhibit clinical efficacy either alone or in combination with conventional anti-cancer drugs [
16]. While the multiplicity of biochemical actions makes impossible to unequivocally ascribe their anti-cancer action to a single mechanism, a frequent effect of polyphenols is the inhibition of the PI3K/Akt defensive pathway [
17,
18]. For instance, the flavonoid quercetin (Quer) is the natural analog of 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent PI3K inhibitor commonly used in laboratory research [
19]. We previously observed that prolonged treatment (24–48 h) with sub-lethal concentrations of Quer, curcumin (Cur) and genistein (Gen) reduced the constitutive Akt phosphorylation in U937 and HL60 AML cells [
20‐
22]. On this ground, we hypothesized the pre-treatment with polyphenols might prevent Akt activation and as a consequence improve the lethality of glycolytic inhibitors. With this hypothesis in mind, in the present work we analyze the capacity of Quer, Cur and Gen to cooperate with 2-DG and Lon to induce apoptosis in HL60 and other AML cell lines. The regulatory function of Akt and other protein kinases, as well as the potential importance of other factors such as mitochondrial dysfunction and oxidative stress, are examined.
Methods
Reagents and antibodies
All components for cell culture were obtained from Invitrogen, Inc. (Carlsbad, CA). Dichlorodihydrofluorescein diacetate (H2DCFDA) and monochlorobimane were obtained from Molecular Probes, Inc. (Eugene, OR). The kinase inhibitors 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 2′-Amino-3′-methoxyflavone (PD98059), 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), 5-Dihydro-5-methyl-1-β-d-ribofuranosyl-1,4,5,6,8-pentaazaacenaphtylen-3-amine hydrate (triciribine hydrate, Akt inhibitor V), 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), and the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F (z-VAD-fmk), were obtained from Calbiochem (Darmstad, Germany), and 1-5-tert-Butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalene-1-yl] urea (BIRB 796) from Shelleck (Houston, TX). Rabbit anti-human p44/42 MAP kinase, phospho-p44/p42 MAP kinase (Thr202/Tyr204), Akt, phospho-Akt (Ser473) (D9E) XP™, p38 MAP kinase, phospho-p38 MAP kinase (Thr180/Tyr182), phospho-GSK-3α/β (Ser21/9), and phospho-S6 ribosomal protein (Ser235/236) polyclonal antibodies, were obtained from Cell Signaling Technology Inc. (Danvers, MA). Mouse GSK-3α/β monoclonal antibody (0011-A) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Peroxidase-conjugated immunoglobulin G antibodies were from DAKO Diagnostics, S.A. (Barcelona, Spain). All other non-mentioned reagents and antibodies were from Sigma (Madrid, Spain).
Cells and treatments
HL60 myeloblastic cells [
23] and THP-1 promononocytic cells [
24] were obtained from our institutional repository (CIB), and NB4 promyelocytic cells [
25] were kindly supplied by Profs. M.D. Delgado and J. León (Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, Santander, Spain). These cell lines represent distinct subtypes of human AML cells (HL60, M2; NB4, M3; THP-1, M5, according to the classification of the French-American-British (FAB) cooperative group), with substantial differences in molecular and biochemical parameters, and hence in the capacity of response to anti-cancer agents. Absence of mycoplasma contamination, and authentication by STR analysis, specific antigen expression, and PML-RARα fusion protein expression (NB4 cells) were corroborated by us or our technical staff. Cell handling (and all experimental procedures in general) was carried out strictly following the regulations of the Bioethics and Biosafety Commission of our Institution (Centro de Investigaciones Biológicas, CSIC). Conditions of cell growth and treatment were described in detail in preceding publications [
11,
12]. For glucose starvation (Glu−), cells were extensively washed with phosphate-buffered saline (PBS) and then seeded at the appropriate concentration in glucose-lacking RPMI medium supplemented with 10 % (v/v) serum. For good comparison, the corresponding controls were subject to the same manipulation, but finally seeded in complete medium (Glu+).
Calcein-AM was commercially obtained as a 4 mM solution in dimethyl sulfoxide. Rhodamine 123 (R123, 1 mg/ml) was prepared in ethanol. Stock solutions of Lon (100 mM), Quer (100 mM), Gen (50 mM), Cur (20 mM), H2DCFDA (5 mM), monochlorobimane (200 mM), U0126 (2.63 mM), PD98059, LY294002 and triciribine (20 mM each), SB203580 (13,2 mM), SB216763 (50 mM), BIRB 796 (0.1 mM) and z-VAD-fmk (25 mM) were prepared in dimethyl sulfoxide. 3(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was dissolved at 5 mg/ml in PBS. All these solutions were stored at −20 °C. A stock solution of propidium iodide (PI, 1 mg/ml) was prepared in phosphate buffered saline (PBS), and stored at 4 °C. 2-DG and DL-buthionine-(S,R)-sulfoximine (BSO) were freshly prepared at 250 and 50 mM, respectively, in PBS. 3-Bromopyruvate was freshly prepared at 30 mM in PBS, and the pH of the solution was adjusted at 7.2 with NaOH.
Flow cytometry
The analysis of samples was carried out on an EPICS XL flow cytometer (Coulter, Hialeah, FL) equipped with an air-cooled argon laser tuned to 488 nm. The specific fluorescence signal corresponding to fluorescein isothiocyanate, H2DCFDA, calcein-AM and R123 was collected with a 525-nm band pass filter, and the signal corresponding to PI with a 620-nm band pass filter. A total of 104 cells were scored in each determination.
Cell proliferation, cell cycle, apoptosis and necrosis
Total cell proliferation was measured by cell counting of trypan-blue excluding cells, or by means of the MTT colorimetric assay. This later procedure gives an indirect estimation of the relative number of viable cells in the culture, based on changes in mitochondrial metabolic activity. Cell cycle phase distribution was routinely determined by cell permeabilization followed by PI staining and flow cytometry analysis. When convenient, the resulting histograms were analyzed with the FlowLogic program (Inivai, Victoria, Canada). This technique also provided an estimation of the frequency of apoptotic cells, characterized by low (sub-G
1) DNA content. The criterion used for necrosis was the loss of plasma membrane integrity, as determined by free PI uptake into non-permeabilized cells and flow cytometry analysis. In addition, apoptosis and necrosis were determined simultaneously by double labeling with annexin V-FITC and PI followed by flow cytometry measurement using an Annexin V-FITC Apoptosis Detection Kit (Immunostep, Salamanca, Spain). This procedure allows the distinction between viable cells (annexin V-negative/PI-negative), early apoptotic cells (annexin V-positive/PI-negative), late apoptotic or necrotic cells (annexin V-positive/PI-positive), and genuine necrotic cells (annexin-V negative/PI-positive). Since the loss of plasma membrane integrity leading to free PI penetration is compatible with both genuine necrosis and late apoptosis (also termed “secondary” necrosis), the pan-caspase inhibitor z-VAD-fmk was occasionally used to discriminate between these two possibilities. A detailed description of all these techniques can be found in our preceding works [
21,
26, and references therein].
Mitochondrial membrane permeabilization and membrane potential dissipation
Inner mitochondrial membrane permeabilization (mIMP) was determined using the calcein-AM/CoCl
2 method, originally reported by Petronilli et al. [
27]. Our adaptation for flow cytometry using HL60 cells was already described in a preceding article [
11]. Mitochondrial membrane potential (ΔΨm) was determined using the cationic agent R123 and flow cytometry analysis, as previously described [
26].
Reactive oxygen species and reduced glutathione levels
The intracellular accumulation of reactive oxygen species (ROS) was measured by flow cytometry using the ROS-sensitive probe H
2DCFDA. The intracellular level of reduced glutathione (GSH) was measured in a Varioskan Flash microplate reader (Thermo Fisher Scientific Inc, Waltham, MA) at excitation wavelength of 390 nm and emission wavelength of 520 nm, using the fluorescent probe monochlorobimane. The detailed procedures were described in a previous publication [
26].
Immunoblotting
Cells were collected by centrifugation, washed with PBS and total protein extracts were obtained by lysing them for 20 min at 4 °C in a buffer consisting of 20 mM Tris–HCl (pH 7.5) containing 137 mM NaCl, 2 mM EDTA, 10 % (v/v) glycerol, and 1 % Nonidet P-40, and supplemented with a protease inhibitor cocktail, 1 mM sodium orthovanadate, and 10 mM NaF. After brief sonication and centrifugation for 15 min at 10,000×
g at 4 °C, the supernatants were collected, and samples containing equal amounts of proteins were resolved by SDS–polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes and immunodetected, as previously described [
28]. When convenient, the relative band intensities were quantified using the Quantity One 1-D Analysis Software, version 4.6 (Bio-Rad Laboratories, Inc., Hercules, CA).
Data analysis and presentation
Except when indicated, all experiments were repeated at least three times, and as a rule the results are expressed as the mean value ± SD. Statistical analyses were carried out using one way ANOVA with Dunnett or Bonferroni post-test, using SAS version 9.4 (SAS Institute, Cary NC). The Dunnett’s method was followed when comparing different treatments with controls, and Bonferroni’s when pairwise comparisons were performed. The symbols used were: &, to compare treatment vs. control; *, to compare pairs of single treatments; and #, to indicate that the value in a combined treatment is higher than the sum of values in the corresponding single treatments. Sum of values were obtained by considering single treatment as independent random variables. In all cases, single symbol means p < 0.05, double symbol p < 0.01, and triple symbol p < 0.001. n.s., non-significant.
Discussion
The present results indicate that that pre-treatment with low lethal concentrations of the flavonoid Quer strongly potentiates the anti-proliferative and apoptotic action of the glycolytic inhibitor 2-DG in HL60 AML cells. Apoptosis was assessed using different markers, namely DNA loss, phosphatidyl serine translocation, and ΔΨm decrease, and the protective action of z-VAD-fmk proves that it is in fact a caspase-mediated response. Our precedent studies demonstrated that Quer and 2-DG, as well as the other agents used here (Lon, Cur and Gen) activated apoptosis throughout the mitochondrial (“intrinsic”) executioner pathway [
11,
12,
20,
21,
29]. In this study we show that 2-DG and Quer, both of them characterized as mitochondria-targeting drugs [
7,
33], cause the rapid induction of mIMP (4 h), which may therefore represent a trigger or at least a necessary condition for apoptosis. Unfortunately the cause-effect relationship between mIMP and apoptosis could not be corroborated, due to the elevated toxicity of commonly used permeability transition pore modulators (cyclosporine A, bongkrekic acid) in leukemia cell models ([
45], and our data not shown).
Additional experiments corroborated the cooperative effect using other polyphenols (Cur, Gen), anti-glycolytic agents (Lon), and leukemia cell models (NB4 promyelocytic, THP-1 promonocytic), although with different efficacy. For instance, Gen was less efficacious than Quer and Cur, and a moderately lethal concentration of the isoflavone was required to obtain good cooperation with 2-DG. As possible explanations, Gen causes cell arrest at G
2/M (see Additional file
3: Fig. S3) and also stimulates myeloid cell differentiation [
22], which might temporarily restrain the trigger of the apoptotic response. In the same manner, the efficacy of cooperation between Quer and 2-DG was lower in NB4 and THP1 than in HL60 cells. This might be explained by intrinsic differences in molecular and biochemical properties and in the maturation stage of these cell lines. For instance, in addition to the above indicated different susceptibility to 2-DG, THP-1 promonocytic cells are more resistant to Quer [
46] (an in our experience also to other cytotoxic agents) than the less mature HL60 and NB4 cells. On the other hand, the finding that Quer plus 2-DG and Quer plus Lon induced apoptosis with similar efficacy allows to exclude that apoptosis potentiation may be a trivial consequence of polyphenol-provoked inhibition of GLUT 1 activity and 2-DG uptake, and hence of subsequent biochemical responses (e.g., 2-DG-provoked activation of defensive kinases, as discussed later). By contrast, the apoptotic efficacy of Quer was only marginally increased when 2-DG treatment was substituted by glucose starvation. This indicates that the partial ATP depletion provoked by both treatments may affect cell proliferation, but it is not a determinant of drug lethality. Finally, a cautionary note must be expressed. We centered the attention on the apoptotic response, and for convenience (better observation of drug cooperation) selected sub-lethal drug concentrations. Nonetheless these concentrations caused appreciable anti-proliferative effects, as measured by the MTT assay (Fig.
1a) or by cell counting (the proliferation inhibition rates at 24 h, in relation to the control, were: 43 % for 5 mM 2-DG; 5 % by 100 μM Lon; 44 % by 20 μM Quer; 21 % by 8 μM Cur; and 65 % by 50 μM Gen). This may be explained in some cases by cell cycle disruption (e.g., almost total G
2/M arrest by Gen, and with lower intensity by Quer: see Additional file
3: Fig. S3). The molecular mechanisms responsible for Gen-provoked cycle arrest were investigated in a preceding article [
22]. In addition, a possible activation of autophagy, which opposes apoptosis, may not be discarded.
The generation of apoptosis and/or necrosis by anti-tumour drugs is frequently associated to oxidative stress, two manifestations of which are the increase in intracellular ROS accumulation and the decrease in anti-oxidant molecules such as GSH. For instance, we earlier demonstrated that the potentiation of arsenic trioxide (Trisenox)-provoked apoptosis by Lon and Gen was mediated by the stimulation of ROS production [
11,
29], probably due to the interference of Lon and Gen with the mitochondrial respiratory chain [
47,
48]. On the other hand, 2-DG was reported to decrease ROS [
49,
50], while Quer may exert either inhibitory [
33] stimulatory [
37,
38] effects. Our present results confirm the drug-dependent variability, and above all prove that the increase in lethality in the combined treatments (Quer plus 2-DG and Quer plus Lon) may not be adequately explained by ROS over-production. Thus, Quer not only reduced the basal ROS content, but exacerbated the decrease caused by 2-DG, and reversed the increase caused by Lon to levels lower than in untreated cells. In another study we observed that prolonged (14–24 h) treatment with Quer decreased the intracellular GSH content, and as a consequence potentiated the lethality of the GSH-sensitive drug arsenic trioxide [
20]. However a regulatory role of GSH may be excluded in the present conditions. In fact, Quer did not cause early changes in GSH content, and the impact on apoptosis of a potential long-term alteration may be discarded, in view of the lack of effects of GSH specific inhibitor BSO.
Finally, the present results corroborate the capacity of 2-DG and Lon to stimulate Akt activation in AML cells, and demonstrate that the stimulation is abrogated or attenuated by co-treatment with Quer, Gen and Cur, which at the same time potentiate apoptosis. The cause-effect relationship between Akt inhibition and apoptosis potentiation was supported by experiments using standard pharmacologic protein kinase inhibitors, and was also strengthened by the results obtained using glucose-free medium, where the lack of Akt activation correlates with poor Quer lethality. There are nevertheless some quantitative differences: thus, Akt activation by Lon was delayed in relation to 2-DG; a prolonged pre-treatment with Cur (14 h) was required to abrogate 2-DG-provoked Akt activation; and 50 μM Gen sufficed to block 2-DG-provoked Akt activation, although the pro-apoptotic action of this Gen concentration was poor. The discrepancies might be explained by the particular action mechanisms of the used drugs, and also indicate that Akt inhibition is an important factor but not the only one accounting for apoptotic potentiation in the combined treatments. Noteworthy, prolonged pre-treatment with Cur also abrogated 2-DG-provoked ERK activation, indicating that at least in some circumstances this kinase also regulates apoptosis potentiation in the combined treatments. Nonetheless the function of ERKs in the present experiments is less clear, mainly because the disparity of effects caused by polyphenols. Thus, ERK phosphorylation was strongly stimulated by Quer, slightly stimulated by Gen, and unaffected by Cur. Of note, co-treatment with MEK/ERK inhibitors increased the apoptotic efficacy of Quer alone. It seems therefore that ERK activation serves to restrain excessive polyphenol toxicity, in the same manner as 2-DG toxicity. In addition Quer, alone or with 2-DG, caused p38-MAP activation, but this response seems irrelevant for apoptosis as judged by the null effect of kinase inhibitors. This conclusion is consistent with results obtained by other authors using a Quer analogue [
43]. Finally, earlier reports indicated that 2-DG elicits Akt-dependent GSK-3β phosphorylation [
13] while quercetin increases [
51] or does not affect [
52] kinase phosphorylation. Using pharmacologic inhibitors, other articles proved that GSK-3 regulates cell growth and/or apoptosis in leukemia cells [
53‐
55]. Our present results indicate that Quer and 2-DG cause hyper-phosphorylation (Ser21/9)/inactivation of GSK-3α/β in HL60 cells, although with certain drug-specificity. Thus, Quer (which only activated ERKs) preferentially stimulated the α isoform, while 2-DG (which activated Akt and ERKs) stimulated both α and β isoforms. Most studies in the literature only centered the attention on GSK-3β, but some reports call attention on the functional importance of the α isoform. As an example, GSK-3α knock-down reduced proliferation and caused spontaneous apoptosis [
56], and potentiated bortezomib-induced toxicity [
57], in leukemia cells. Our results show that the GSK-3 inhibitor SB216763 (10 μM) does no cause per se significant lethality, but potentiates apoptosis induction by Quer and 2-DG, alone and in combination, in HL60 cells. Since as commented above Quer preferentially stimulates GSK-3α, we might postulate that this isoform is the most important for regulation of Quer lethality. Unfortunately the lack of specificity of the up to date available pharmacologic inhibitors, and the difficulty to perform satisfactory knock-down procedures in the used leukemia cell model, impeded us until now to obtain more clear conclusions.