AICAR was purchased from Toronto Research Chemicals (North York, ON, Canada). SB203580, a specific p38 MAPK inhibitor, was purchased from Calbiochem (San Diego, CA, USA). Activated and pan-type antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and a monoclonal antibody directed against p21 was obtained from BD Pharmingen (San Jose, CA, USA). Horseradish peroxidase-conjugated antimouse and rabbit antibodies were purchased from Vector Laboratories (Burlingame, CA, USA). All other biotechnology grade chemicals were purchased from Amresco Inc. (Solon, OH, USA).

FRO human thyroid cancer cells were cultured in RPMI 1640 containing 10% fetal bovine serum at 37℃ in 5% CO2. The cells were seeded at 1 × 10³ cells/well in 96-well culture plates or at 5 × 10⁵ cells/60-mm culture dish, and incubated for 24 hours before treatment with AICAR. In some experiments, the cells were pretreated with 20 mM SB203580, a specific p38 MAPK inhibitor, for 30 minutes before adding AICAR.

Cell viability was measured using the colorimetric Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay in accordance with the manufacturer's instructions. Cells cultured in 96-well culture plates were treated with various concentrations of AICAR, with or without SB203580, for 48 or 72 hours. The amount of formazan dye generated by cellular dehydrogenase activity, an indicator of cell viability, was determined by measuring absorbance at 450 nm using a SPECTRA MAX Microplate Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Untreated cells served as controls.

Aliquots (30 mg) of total cellular protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% to 12% (w/v) gradient NuPAGE gels (Invitrogen, Carlsbad, CA, USA), transferred to nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA), and incubated with specific antibodies. Immunoreactive proteins were detected by enhanced chemiluminescence (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and relative protein band intensity was determined by densitometric scanning using a FluorS Multi-Imager (Bio-Rad, Hercules, CA, USA). Expression levels of target proteins were normalized to that of β-actin in each sample.

The small interfering RNA (siRNA) sequence targeting the AMPK-α1 gene (GenBank accession no. NM_206907) was designed using the BLOCK-iT RNAi Designer software (Invitrogen). Three potential AMPK-α1-targeting siRNAs (Invitrogen Stealth RNAi siRNA) were used at the same concentration to screen for effective siRNAs. The sequences of selected siRNAs were 5'-UUA AGG CUU CAU CAU CAA UCA UGG U-3' and 5'-ACC AUG AUU GAU GAU GAA GCC UUA A-3'. Nonsilencing random sequence siRNAs were purchased from Invitrogen and used as negative controls. A random oligomer conjugated to green fluorescence protein (Invitrogen) was used to assess transfection efficiency.

Transfections were performed using the Lipofectamine RNAi MAX reagent (Invitrogen), according to the manufacturer's instructions. An efficiency test and negative control transfections were performed using the same reagents. After transfection, the cells were incubated for 24 hours and treated with AICAR in the presence or absence of SB203580. Changes in expression of AMPK-α1 or other molecules were evaluated using Western blot analysis.

AICAR is the best characterized AMPK activator, and promotes apoptosis in various cancer cells. AICAR acts in a cell-type-specific manner, and AMPK-independent effects of AICAR have been reported. We first investigated the effect of AICAR on cell growth and whether this correlated with AMPK activation in FRO thyroid cancer cells.

The cells were incubated with different concentrations of AICAR for 48 or 72 hours, and cell proliferation was determined using a colorimetric CCK-8 assay. AICAR significantly inhibited proliferation of FRO cells in a time- and dose-dependent manner (Fig. 1A). We used 200 µM AICAR, which corresponded roughly to the IC50 in a FRO cell viability assay, and performed a Western blot analysis to determine whether AICAR stimulated activation of AMPK and proapoptotic molecules. As shown in Fig. 1B, AICAR treatment promoted rapid AMPK activation and p21 accumulation, and also enhanced cleavage of caspase-3. These results suggest that AICAR not only triggers AMPK activation but also suppresses cell growth by inducing p21 and activating caspase-3.

To establish whether activation of AMPK by AICAR is related to MAPK activity, cells were treated with 200 µM AICAR for the indicated times. We evaluated changes in the phosphorylation status of MAPK molecules (ERK, p38, and JNK) by Western blot analysis. AICAR treatment significantly promoted activation of p38 MAPK, but not ERK or JNK in FRO cells (Fig. 2).

We examined whether inhibition of cell proliferation by AICAR was associated with activation of p38 MAPK in FRO cells. Cells were pretreated with SB203580 (20 µM), a specific p38 MAPK inhibitor, for 30 minutes, and then incubated with 200 µM AICAR for the indicated times. After incubation, cell proliferation was assessed using the colorimetric CCK-8 assay. As shown in Fig. 3, AICAR treatment suppressed relative cell proliferation in a time-dependent manner, but pretreatment with SB203580 significantly rescued cells from AICAR-induced cell death (19% increase at 48 hours and 37% increase at 72 hours, respectively). These results indicate that activating p38 MAPK is required for AICAR-induced AMPK activation and inhibition of cell proliferation in FRO thyroid cancer cells.

Given our finding that AICAR-induced AMPK activation also induced cell death by activating p38 MAPK in FRO cells, we elucidated the involvement of AMPK in AICAR-induced activation of p38 and caspase-3. We performed RNA interference experiments using siRNAs targeting AMPK in FRO cells. AMPK is a heterotrimeric complex that consists of three subunits, including catalytic serine/threonine kinase subunits (contains α1 and α2) and two regulatory subunits (β and γ) [16]. We first investigated the expression patterns of the a1 and a2 catalytic subunits of AMPK in FRO cells. As shown in Fig. 4A, AMPK-α1 was predominantly expressed, whereas AMPK-α2 was barely expressed in these cells. As a consequence, we next assessed the efficiency of siRNA-mediated silencing of AMPK-α1. Cells were transfected with AMPK-α1-targeting siRNAs or with random RNAi oligomers (negative control), and then analyzed for AMPK expression (Fig. 4B). Transfection of AMPK-α1-targeting siRNAs markedly reduced the level of AMPK protein compared with mock treatment (no oligomer) and random oligomers for up to 96 hours after transfection. We further examined the effects of AMPK-α1 on AICAR-induced p38 activation and cell death regulatory molecules in FRO cells. The cells were transfected with AMPK-α1-siRNAs for 24 hours, followed by stimulation with 200 µM AICAR for the indicated times. As expected, AMPK-α1-siRNAs inhibited AICAR-induced p38 activation, and reduced protein abundance of cell death regulators such as p21 and caspase-3 (Fig. 4C). Taken together, these findings indicate that AMPK-α1 is directly involved in AICAR-induced p38 activation and in the initiation of cell death by activating p21 and caspase-3 in FRO thyroid cancer cells.