EF1α, caspase-3, GAPDH, AMPKα2, and GAPDH antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA) and New England BioLabs, Inc. (Ipswich, MA, USA). Phospho-specific AMPK (Thr¹⁷²) and anti-AMPK antibodies were purchased from Upstate Biotechnology (Temecula, CA, USA). A FOXO3a/FKHRL1 (Ser²⁵³) antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). A phospho-specific FOXO3a/FKHRL1 (Ser²⁵³) antibody was purchased from Millipore Corp. (Billerica, MA, USA). An anti-p21^Waf1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HRP-conjugated secondary antibodies were obtained from the Kirkegaard and Perry Laboratory (Gaithersburg, MD, USA). Paclitaxel was purchased from Sigma-Aldrich. 5-Amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) and 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine (compound C) were obtained from Calbiochem (San Diego, CA, USA).

The MCF7 cell line was grown in a 1:1 mixture of RPMI-1640 (Gibco, Auckland, New Zealand) containing 0.584 g/l of L-glutamate, 4.5 g/l of glucose, 100 g/ml of gentamicin, 2.5 g/l of sodium carbonate and 10% heat-inactivated fetal bovine serum (FBS).

Cells were grown in 6-well plates and serum-starved for 24 h prior to the treatment with indicated agents. The medium was aspirated and the cells were washed twice in ice-cold phosphate-buffered saline (PBS) and lysed in 100 μl of lysis buffer [50 mM Tris-Cl (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA and 150 mM NaCl]. The lysed samples were briefly sonicated, heated for 5 min at 95°C, and centrifuged for 5 min. The supernatants were electrophoresed on 8% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The blots were incubated overnight at 4°C with a primary antibody. They were then washed 6 times in Tris-buffered saline/0.1% Tween-20 and probed for 1 h with an HRP-conjugated secondary antibody at room temperature. The blots were visualized using an ECL detection system (Amersham Biosciences, Buckinghamshire, UK).

The MTT assay was performed as a crude measure of cell viability. MCF7 cells were seeded at a density of 5×10⁴/ml in 96-well plates and allowed to grow for 24 h. The growth medium was replaced with serum-free medium 24 h prior to treatment. Subsequently, MTT reagents (10 μl/well; 7.5 mg/ml in PBS) were added and the cultures were incubated for 30 min at 37°C. The reaction was stopped by addition of acidified Triton buffer [0.1 M HCl in 10% (v/v) Triton X-100; 50 μl/well], and tetrazolium crystals were dissolved by mixing on a plate shaker at room temperature for 20 min. The samples were then measured on a plate reader (Bio-Rad 450; Bio-Rad Laboratories, Richmond, CA, USA) at a test wavelength of 595 nm and a reference wavelength of 650 nm. The results are representative of experiments repeated at least in triplicate.

MCF7 cells were seeded in 6-well plates and allowed to grow to 70% confluence for 24 h. Transient transfections were performed with the transfection reagent (Lipofectamine 2000; Invitrogen, Paisley, UK) as per the manufacturer's instructions. In brief, EF1α (NM_001013367; Dharmacon, Lafayette, CO, USA), AMPKα1, and non-targeted control siRNAs were designed. siRNA (5 μl) and transfection reagent (5 μl; Lipofectamine 2000) were each diluted in 95 μl of the reduced serum medium (Opti-MEM; Invitrogen) and then mixed. The mixtures incubated for 30 min at room temperature and then added in drops to each culture well containing 800 μl of the reduced serum medium (Opti-MEM; final siRNA concentration, 100 nM). The medium was replaced with a fresh medium at 4 h after transfection. The cells were cultivated for 24 h, lysed, and the expression of AMPKα1 protein was assayed by western blotting.

First-strand cDNA synthesis was performed using 1 μg of total RNA isolated from frozen tissues at 55°C for 20 min using a ThermoScript II One-Step RT-PCR kit (Invitrogen). Amplification of cDNA was performed in the same tube using the ABI GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Warrington, UK). The reverse transcriptase was inactivated by heating to 94°C. The following PCR conditions were used: 27 cycles for 30 sec at 94°C, 30 sec at 56°C and 30 sec at 72°C, followed by 7 min at 72°C. The number of PCR cycles used was optimized to ensure amplification in the exponential phase. Samples (10 μl) of each RT-PCR product were analyzed by agarose gel electrophoresis. The bands were stained with ethidium bromide and visualized under ultraviolet light. Band intensity quantification was determined by a gel documentation system (Gene Genius; Syngene, UK). The following primers were used: EF1α sense, 5′-GATATGGTTCCTGGCAAGCCC-3′ and antisense, 5′-CATTTAGCCTTGTGAGCTTTC-3′; GAPDH sense, 5′-ATTTGGTCGTATTGGGCGCCTGGTCACC-3′ and antisense, 5′-GAAGATGGTGATGGGATTTC-3′.

The data are expressed as mean ± SEM Statistical analyses were conducted using SigmaStat (SPSS Inc., Chicago, IL, USA). Differences were considered significant at P-value <0.05.

To ascertain whether the activity of AMPK affects EF1α expression, MCF7 cells were treated with AICAR, a pharmacologic activator of AMPK, and then analyzed by RT-PCR. We observed that EF1α mRNA levels were decreased in a time-dependent manner following AICAR treatment (Fig. 1A). In addition, examination of EF1α protein levels in AICAR-treated MCF7 cells by western blotting revealed that EF1α expression was significantly decreased following incubation with AICAR compared with the expression under control conditions (Fig. 1B). Phosphorylation of AMPK was increased by AICAR treatment, confirming the appropriate conditions of our assay. Taken together, these results indicate that AMPK can suppress the expression of EF1α in MCF7 cells.

EF1α is known to be critical for breast tumor cell maintenance. Thus, we hypothesized that AMPK-mediated downregulation of EF1α has an anticancer effect. To test this hypothesis, we determined whether EF1α is involved in paclitaxel-stimulated breast cancer MCF7 cell apoptosis. The phosphorylation status of AMPK in pacli-taxel-treated MCF7 cells was examined to determine whether the effects of paclitaxel are mediated by AMPK activation. We performed western blotting using a phosphorylation-specific (Thr¹⁷²) AMPK antibody and found that the level of AMPK phosphorylation in paclitaxel-treated MCF7 cells increased in a time- and dose-dependent manner compared the level in the controls (Fig. 2A and B). This observation provided further indirect evidence of increased AMPK phosphorylation following the treatment of MCF7 cells with paclitaxel.

EF1α not only functions as a translation factor but also binds to and severs microtubules (17,18). To better understand the role of EF1α in MCF7 cells, we determined EF1α levels in paclitaxel-treated MCF7 cells by western blotting. We found that EF1α expression was decreased in a dose-dependent manner by paclitaxel (Fig. 3A). Paclitaxel also downregulated the expression of EF1α in a time-dependent manner (Fig. 3B). Then, the effect of paclitaxel on cell viability was assessed. Treatment with paclitaxel resulted in a reduction of cell viability in a dose-dependent manner (Fig. 3C). Paclitaxel also activated caspase-3, a key mediator of apoptosis (Fig. 3D). These results suggest that paclitaxel suppresses MCF7 cell viability through the downregulation of EF1α expression.

FOXO3a belongs to the forkhead family of transcription factors, which are characterized by a distinct forkhead DNA-binding domain. This protein functions as a trigger for apoptosis and is known to be a tumor suppressor. To evaluate the activation of FOXO3a by paclitaxel, we analyzed the changes in FOXO3a levels in paclitaxel-treated MCF7 cells. The expression of FOXO3a and p21^Waf1, its downstream effector molecule, was induced by paclitaxel treatment (Fig. 4A). A similar pattern of FOXO3a expression was also observed after AICAR treatment (Fig. 4B). Furthermore, FOXO3a phosphorylation was transiently increased by paclitaxel treatment (Fig. 4C). To determine whether AMPK activity was involved in the effect of paclitaxel, we investigated FOXO3a phosphorylation following treatment with 10 μM compound C, an AMPK inhibitor. Paclitaxel-induced phosphorylation of FOXO3a was inhibited in cells pretreated with compound C (Fig. 4D). These results indicate that FOXO3a is involved in the paclitaxel-stimulated signaling pathway in an AMPK-dependent manner.

To explore the role of AMPK in the paclitaxel-activated biochemical pathway, we investigated the expression of EF1α and FOXO3 in AMPK-silenced cells. The expression of AMPKα1 was downregulated by AMPKα1 siRNA (Fig. 5A). The expression of EF1α and FOXO3a were suppressed in AMPK knockdown cells (Fig. 5B), suggesting the involvement of these molecules in AMPK-mediated signaling. To confirm the role of AMPK in the regulation of FOXO3a and EF1α, we examined the effect of AMPK silencing on FOXO3a and EF1α levels in paclitaxel-treated cells. We found that paclitaxel failed to upregulate FOXO3a in AMPKα knockdown cells (Fig. 5B). Similarly, paclitaxel did not decrease EF1α levels in AMPKα knockdown cells. These results indicated that AMPK plays an important role in the regulation of EF1α and FOXO3a levels via paclitaxel-activated signaling.

To confirm the role of EF1α in the regulation of cell viability and to exclude unrelated effects due to EF1α, we transfected MCF7 cells with double-stranded EF1α siRNA oligonucleotides. The cells were harvested 24 h after transfection, and EF1α expression was analyzed by western blotting. EF1α-specific siRNA completely suppressed EF1α expression, whereas scrambled siRNA (negative control) had no effect on EF1α levels (Fig. 6A). In addition, EF1α silencing increased the expression of caspase-3 and FOXO3a (Fig. 6A), key molecules for apoptosis, suggesting that EF1α may regulate apoptotic cell death. Moreover, knockdown of EF1α potently enhanced the induction of FOXO3a by paclitaxel (Fig. 6B). These results indicate that EF1α is essential for activation of the FOXO3a pathway by paclitaxel.

To ascertain the role of AMPK in maintaining the viability of cancer cells, MTT assays were performed. The viability of MCF7 cells was inhibited by paclitaxel treatment (Fig. 7A). Moreover, co-treatment with AICAR further inhibited the viability of MCF7 cells (Fig. 7A). To gain insight into the mechanism by which paclitaxel suppresses MCF7 viability, FOXO3a expression levels were examined. We observed that FOXO3a expression was further increased by co-application of AICAR and paclitaxel compared with the expression following treatment with paclitaxel alone (Fig. 7B). In conclusion, these results indicate that AMPK activation increased the susceptibility of MCF7 cancer cells to the adverse effects of paclitaxel on cell viability.