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. 2010 Aug;24(8):2620–2630. doi: 10.1096/fj.09-152546

Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase

Sofia Theodoropoulou *, Paraskevi E Kolovou †, Yuki Morizane *, Maki Kayama *, Fotini Nicolaou ‡, Joan W Miller *, Evangelos Gragoudas *, Bruce R Ksander †, Demetrios G Vavvas *,1
PMCID: PMC2909289  PMID: 20371623

Abstract

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP, is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. We studied the effects of AICAR on the growth of retinoblastoma cell lines (Y79, WERI, and RB143). AICAR inhibited Rb cell growth, induced apoptosis and S-phase cell cycle arrest, and led to activation of AMPK. These effects were abolished by treatment with dypiridamole, an inhibitor that blocks entrance of AICAR into cells. Treatment with the adenosine kinase inhibitor 5-iodotubericidin to inhibit the conversion of AICAR to ZMP (the direct activator of AMPK) reversed most of the growth-inhibiting effects of AICAR, indicating that some of the antiproliferative effects of AICAR are mediated through AMPK activation. In addition, AICAR treatment was associated with inhibition of the mammalian target of rapamycin pathway, decreased phosphorylation of ribosomal protein-S6 and 4E-BP1, down-regulation of cyclins A and E, and decreased expression of p21. Our results indicate that AICAR-induced activation of AMPK inhibits retinoblastoma cell growth. This is one of the first descriptions of a nonchemotherapeutic drug with low toxicity that may be effective in treating Rb patients.—Theodoropoulou, S., Kolovou, P. E., Morizane, Y., Kayama, M., Nicolaou, F., Miller, J. W., Gragoudas, E., Ksander, B. R., Vavvas, D. G. Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase.

Keywords: cancer, energy, metabolism, eye, intraocular tumors

retinoblastoma is the most common primary malignant intraocular tumor in infants, ranking third of all tumors after leukemia and neuroblastoma (1⤻, 2)⤻. Slightly more than one-half of the patients have the sporadic or noninherited form of the disease, which results from the spontaneous inactivation of the retinoblastoma gene (RB1). In the heritable form, the patient inherits usually 1 defective gene from the parents and a subsequent “hit” of the uninvolved gene results in tumor formation. The heritable form is more often bilateral than the nonheritable form of the disease. Despite progress in the treatment of retinoblastoma (3)⤻, significant problems remain unsolved. Metastatic disease is often fatal (4)⤻. Retinoblastoma can be treated by enucleation and/or combination of chemotherapy, laser, and cryotherapy. Conventional external beam radiation is used to control large tumors but has complications, including an increased appearance of secondary malignancies, such as osteosarcoma. This complication occurs more frequently in patients with hereditary retinoblastoma. The 30 yr cumulative incidence of second malignancies is >35% for patients who received external beam therapy vs. 6% for those patients without radiation (5)⤻. For this reason, external beam radiation therapy has fallen from favor and is used less often and at reduced dosages (1500 cGy compared with the old standard of 3500–4500 cGy; ref. 6⤻). Systemic chemotherapy used as a first line treatment for intraocular retinoblastoma with subsequent consolidation with either photocoagulation, cryotherapy, or radiotherapy has a recurrence rate of 24% by 5 yr (7)⤻. This increases to 50% for patients with vitreous seeds (8)⤻. Most recent analysis of chemoreduction and adjuvant consolidation therapy by the Shields and Murray groups (6⤻, 9⤻10⤻11⤻12)⤻ show success for local control approaching 100% for RE stage I–IV and ∼83% for RE stage V or 90–100% for group A–C (new international classification). However, significant morbidity with the chemotherapy has been described previously (13)⤻. In addition, 1 of the drugs used for chemotherapy (etoposide) is thought to be associated with increased incidence of acute myeloblastic leukemia, although the actual cases implicated so far have been low with ∼20 cases reported (14)⤻. For these reasons, there is still a need for alternative new treatment modalities for retinoblastoma with a better efficacy and safety profile.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase (15)⤻ that acts as a sensor of cellular energy levels and stress. Its activity is regulated by hypoxia, exercise, ischemia, heat shock, and long-term starvation (16⤻17⤻18⤻19)⤻. Its upstream protein kinase LKB1 (20⤻, 21)⤻ is known to be a tumor suppressor involved in Peutz-Jegher syndrome (22)⤻. Downstream effectors of AMPK also involve the tumor suppressor tuberous sclerosis complex (TSC2) and the mammalian target of rapamycin (mTOR). Both are important known factors in cell cycle progression and tumor formation (23⤻, 24)⤻. Pharmacologic activation of AMPK can be achieved by administration of 5-aminoimidazole-4-carboxamide riboside (AICAR), which is taken into cells and converted to the monophosphorylated form ZMP, mimicking an increase of AMP intracellular levels (25)⤻. AICAR has low or no apparent toxicity and has been shown to be a great in vivo exercise mimetic (26)⤻.

Activation of AMPK has been shown to be both prosurvival and proapoptic depending on the stimulus and the environment. Thus, activation of AMPK has been related with protection from apoptosis in situations of ischemia/reperfusion injury (27)⤻, hyperglycemia (28)⤻, and glucose deprivation (29)⤻. AICAR-induced activation of AMPK resulted in inhibition of proliferation and induction of apoptosis in multiple myeloma cells (30)⤻, neuroblastoma cells (31)⤻, glioblastoma cells (32)⤻, childhood acute lymphoblastic leukemia cells (33)⤻, and colon cancer cells (34)⤻ by various mechanisms, including up-regulation of p53 (33)⤻, activation of MAPK-p38 pathway (22⤻, 23)⤻, increased expression of cell cycle inhibitory proteins p27 and p21 (25⤻, 33)⤻, inhibition of NF-κB activity (25⤻, 34)⤻, and inhibition of Akt/mTOR/P70S6K pathway (30⤻, 32⤻, 33)⤻. It is likely that the various effects of AMPK on survival or growth inhibition depend on cell types, cellular events following external stimuli, duration of AMPK activation, and/or downstream-regulated pathways of AMPK.

In the present study, we investigated the effects of AICAR on cell proliferation in vitro in 3 different retinoblastoma cell lines. We found that AICAR-mediated activation of AMPK was an efficient inhibitor of retinoblastoma cell proliferation through S-phase arrest, decrease of cyclins A and E, and partial inhibition of the mTOR pathway, thereby exhibiting potential as a novel nonchemotherapeutic drug for this disease.

MATERIALS AND METHODS

Chemicals

AICAR, dypiridamole, and 5-iodotubericidin (Iodo) were purchased from Sigma-Aldrich (St. Louis, MO, USA). AICAR was dissolved in PBS at a concentration of 100 mM (stock solution) and stored at −20°C until utilization. Dypiridamole and Iodo were prepared fresh from stock solutions and diluted with growth medium. Ribonuclease-A and 3-(4,5-dimethlythiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Propidium iodide was purchased from Invitrogen (Carlsbad, CA, USA). The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): phospho-ACC (Ser-79), phospho-S6 ribosomal protein (Ser-235/236), CDK2, CDK4 (DCS156), p21 Waf1/Cip1 (12D1), p27Kip1 (D37H1), phospho-Akt (Ser-473), LC3B, phospho-4E-BP1 (Ser-65), and PCNA (PC10).

Cell culture

The human retinoblastoma cells WERI, Y79, and Rb143 were grown in RPMI medium (RPMI 1640; Invitrogen), supplemented with 10% FBS (Invitrogen), penicillin (100 μg/ml)-streptomycin (100 μg/ml; Invitrogen), 2 mM l-glutamine (Invitrogen), 10 mM HEPES (Invitrogen), and 55 μM 2-mercaptoethanol (Cambrex Bioscience, Walkersville, MD, USA). Cells were incubated at 37°C in a humidified atmosphere of 95% air-5% CO2 and split when the cells reached ∼90% confluency. The WERI and Y79 cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The Rb143 cell line was established from a primary tumor explant recovered from the enucleated eye of a patient with a large tumor. The cell line was validated by sequencing the 27 exons in the Rb gene. Rb 143 has a single large deletion encompassing exons 3–17.

Measurement of cell growth by MTT assay

Cell viability was assessed by MTT assay. Cells were cultured in 48-well plates at a density of 50,000 cells/well in 300 μl growth medium and were incubated with the indicated drugs at 37°C for 3 and 5 d. On the day of culture, after 3 d of treatment and after 5 d of treatment, MTT (5 mg/ml in PBS) was added to each well at a 1/10 vol. After incubation for 1–2 h at 37°C, the culture medium and cells are aspirated, put in Eppendorf tubes and spun down at 3 krpm for 2 min. The supernatant was removed, and the pelleted cells were resuspended in 200 μl DMSO. The absorbance at 595 nm was measured using a microplate reader. For each treatment, cell growth was evaluated as a percentage using the following equation: (OD595 of treated sample − OD595 of sample on day of culture)/(OD595 of untreated sample − OD595 of sample on day of culture) × 100.

Flow cytometry assessment of cell cycle

Cellular DNA content was assessed by flow cytometry. Cells were cultured in 12-well plate at a density of 300,000 cells/well in 1 ml growth medium and were treated with 1 and 2 mM AICAR for 1, 3, and 5 d. After drug treatment, the cells were spun at 1200 rpm for 5 min and washed twice with 1 ml of cold PBS; 2 ml of ice-cold 75% ethanol was then added slowly while the cells were continuously vortexed, and the cells were then fixed overnight. On the day of measurement, cells were spun, resuspended in 2 ml of PBS with the addition of 100 μl of 200 μl/ml DNase-free, RNase A (Invitrogen), and incubated at 37°C for 30 min. Then, 100 μl of 1 mg/ml propidium iodide (Invitrogen) was added, and the cells were incubated at room temperature for 10 min. The samples were read on a Becton Dickinson FACScan (Becton Dickinson, Franklin Lakes, NJ, USA). The sub-G1 peak was quantified and represented the nonviable cell population.

Western blot analysis

After 24 h of incubation in the presence or absence of AICAR, cells and medium were aspirated and spun at 300 g for 30 s. The supernatant was removed, and 70 μl hot (90°C) LDS sample buffer (Invitrogen), containing 7 μl 2-mercaptoethanol (Cambrex), was added to each sample. The samples were incubated at 90°C for 5 min and sonicated. Fifty microliters of each sample per lane was loaded onto a NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and then transferred to a PVDF membrane (0.45 μm; Millipore, Billerica, MA, USA). The membranes were incubated overnight with primary antibody [1:1000 in 5% wt/vol BSA, Tween 20 (TTBS) for all antibodies, except CDK4 and PCNA, which were diluted in 5% nonfat dry milk, TTBS], at 4°C with gentle shaking. The blotted membranes were washed 3 times (5 min) with TTBS and incubated for 30 min at room temperature with horseradish peroxidase-labeled anti-rabbit or anti-mouse secondary antibody (1:10,000; Jackson ImmunoResearch, West Grove, PA, USA). The membranes were washed 3 times (5 min) in TTBS, and immunoreactive bands were visualized by ECL and exposure onto Fuji RX film (Fujifilm, Tokyo, Japan) for ∼5 min.

Separation for nuclear/cytoplasm extracts

After 24 h of incubation in the presence or absence of AICAR, cells and medium were aspirated and spun at 1200 rpm for 5 min. The supernatant was removed, and the pellet was suspended in 200 μl of lysis buffer (20 mM HEPES, pH 8.0; 12 mM KCl; 2 mM EDTA; 1 mM dithiothreitol; 1% protease inhibitor cocktail (Sigma P8340); 20mM b-glycerol 2-phosphate disodium salt hydrate; 50 mM Naf; and 1 mM Na3VO4). After incubating the suspended cells on ice for 20 min, 7.5 μl of 10% Nonidet P-40 was added, and the tubes were vortexed twice for 5 s separated by a 1 min interval, then spun in a microcentifuge at the highest speed for 30 s. The supernatant was saved in another tube as cytoplasmic extract. To the remaining pellet, 45 μl of extraction buffer [25 mM HEPES, pH 8.0; 400 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM dithiothreitol; 1% protease inhibitor cocktail (Sigma P8340); 20 mM b-glycerol 2-phosphate disodium salt hydrate; 50 mM NaF; and 1 mM Na3VO4] was added. The tubes were incubated on ice for 30 min with intermittent vigorous vortexing and then centrifuged in a microcentrifuge at highest speed for 10 min. The supernatant was collected as nuclear extract and used for Western blot analysis.

Quantitative real-time RT-PCR

After 48 h of incubation in the presence or absence of AICAR, cells and medium were aspirated and spun at 1200 rpm for 5 min. RNA of the cells was extracted and purified with the RNeasy Micro kit (Qiagen, Valencia, CA, USA). RNA was further cleaned with an additional DNase I digestion step, according to the manufacturer’s instructions. Reverse transcription was performed for equal RNA amounts (4 μg, as measured by ultraviolet spectrophotometry) with OligodT primer (Invitrogen) and Superscript II (Invitrogen). cDNA (100 ng) was used for each of the 3 replicates for quantitative PCR. Human cyclin A1, cyclin A2, cyclin D1, cyclin D2, cyclin D3, cyclin E1, cyclin E2, and 18S (as an endogenous control) were amplified with commercially designed Taqman gene expression assays (Applied Biosystems, Foster City, CA, USA) and the Taqman universal PCR master mix (Applied Biosystems). Quantitative expression data were acquired and analyzed with a Step One Plus real-time PCR system (Applied Biosystems).

RESULTS

AICAR inhibits the growth of retinoblastoma cells

To investigate the effect of AICAR on the growth and metabolism of retinoblastoma cells, we treated 3 different Rb cell lines (WERI, Y79, and Rb143) with various concentrations of AICAR (0.25–2 mM) for 3 and 5 d. Metabolism and growth were measured by MTT assay. AICAR inhibited the growth of all 3 cell lines in a time- and dose-dependent manner with the WERI cell line being the most sensitive (P<0.05 for all cell lines; Fig. 1⤻).

Figure 1.

Figure 1.

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AICAR inhibits growth of human retinoblastoma cells. Retinoblastoma cell lines WERI (A), Y79 (B), and Rb143 (C) were treated for 3 and 5 d with various concentrations of AICAR (0.25–2 mM), and cell viability was measured by MTT assay. Results are expressed as percentage of growth (%) relative to control values, defined as 100%; data points are averages of triplicate cultures. Data represent 3 independent experiments.

AICAR enters cells through surface adenosine receptors. To confirm that inhibition of Rb cells was dependent on receptor-mediated uptake of AICAR, we pretreated cells with dypiridamole (DPY), which blocks adenosine transporters and prevents uptake of AICAR into the cells. As a negative control, DPY treatment alone did not affect cell metabolism and growth. By contrast, treatment of Rb cells with DPY plus AICAR abolished the inhibitory effect of AICAR in all 3 cell lines (P<0.05), indicating that surface adenosine receptors are expressed on Rb cells and mediate uptake of AICAR (Fig. 2⤻).

Figure 2.

Figure 2.

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DPY and Iodo effects on AICAR-mediated retinoblastoma cell growth inhibition. Retinoblastoma cell lines WERI (A), Y79 (B), and Rb143 (C) were pretreated for 30 min with 2 μM DPY or 0.1 μM Iodo. Cells were then incubated for either 3 or 5 d without or with AICAR (1 mM). An MTT assay was performed, and results are expressed as percentage of growth (%) relative to control values, defined as 100%; data points are averages of triplicate cultures. Data represent 3 independent experiments.

Antiproliferative effects of AICAR are mediated via the AMPK pathway

The following series of experiments determined whether AICAR-mediated Rb cell inhibition coincides with AMPK activation. It is important to confirm that inhibition is mediated via AMPK, since there are several reports that AICAR can inhibit cells via an AMPK-independent pathway. To determine whether AICAR treatment of Rb cells was associated with AMPK activation, we examined the phosphorylation of acetyl-CoA carboxylase (ACC), the downstream target of AMPK. Cells treated with 1 and 2 mM of AICAR clearly show a dose-dependent increase of phosphorylated ACC by Western blotting (Fig. 3A⤻). To confirm ACC phosphorylation was due to AICAR, cells were pretreated with DPY before AICAR. Blocking AICAR receptors with DPY inhibited ACC phosphorylation (Fig. 3B⤻). These data indicate that the AICAR-mediated inhibition of Rb cells coincides with activation of the AMPK pathway.

Figure 3.

Figure 3.

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AICAR treatment of retinoblastoma cells is associated with activation of AMPK. A) Western blot analysis of phosphorylated ACC (Ser-79 expression in WERI, Y79, and Rb143 cells that were treated with AICAR at a concentration of either 1 or 2 mM for 24 h. B) Western blot analysis of phosphorylated ACC expression in WERI, Y79, and Rb143 cells pretreated with DPY for 30 min before addition of AICAR at a concentration of either 1 or 2 mM for 24 h. C) Western blot analysis of phosphorylated ACC expression in WERI, Y79, and Rb143 cells pretreated with Iodo for 30 min before addition of AICAR at a concentration of either 1 or 2 mM for 24 h. Density values of phosphorylated ACC bands are graphically expressed relative to control. Multiple bands represent separate biological samples.

Other laboratories have reported that once AICAR enters a cell, it can be converted to either inosine or ZMP. Inosine inhibits cells by raising the adenosine concentration, which is independent of AMPK. By contrast, ZMP inhibits cells by activating the AMPK pathway. AICAR is converted to ZMP by adenosine kinase, but this conversion is blocked by Iodo. To determine whether AICAR inhibition of Rb cells coincides with the conversion of AICAR to ZMP, Rb cells were treated with AICAR plus Iodo. Activation of AMPK was assessed by phosphorylation of ACC. Although activation of AMPK was shown to be effectively blocked by Iodo treatment as judged by phosphorylated ACC immunoblots (phosphorylated ACC±Iodo; inhibition at P<0.05; Fig. 3C⤻), a significant but not complete reversal of AICAR-inhibited Rb cell growth was observed (Fig. 2⤻), indicating that AMPK kinase activation by ZMP is only partially responsible for the inhibitory effects of intracellular AICAR.

AICAR causes cell cycle arrest in S phase and an increase in sub-G0/G1 population of retinoblastoma cell lines

Previous reports have shown variable effects of AICAR on cell cycle and apoptosis depending on the cell type studied. To examine the effect of AICAR on retinoblastoma cell cycle, cells were treated with AICAR (1 and 2 mM) for 1, 3, and 5 d, and cell cycle phase was analyzed for nuclear DNA content by propidium iodide staining and flow cytometry. Compared with control cells, AICAR treatment had 2 major effects on cell cycle: it resulted in accumulation of cells in sub-G0/G1 phase (suggestive of apoptosis) as well as in S phase (Fig. 4⤻). This effect of AICAR was time and dose dependent and similar for all cell lines. The proportion of sub-G1-phase cells/apoptotic cells increased from 1.63, 4.63, and 11.8% in untreated cells to 8.3, 15.07, and 42.13% in 3 d AICAR-treated WERI, Y79 and Rb143 cells, respectively (Fig. 4⤻ and Supplemental Fig. 1), while S phase increased from 11.77, 13.8, and 7.9% to 39.33, 31.27, and 12.83%, respectively (P<0.05; Fig. 4⤻).

Figure 4.

Figure 4.

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AICAR induces apoptosis and blocks cell cycle progression at S phase in human retinoblastoma cells. WERI (A), Y79 (B), and Rb143 (C) retinoblastoma cells were treated with AICAR 1 and 2 mM for 1, 3, and 5 d. After overnight fixation, cells were suspended in PBS with RNase A and propidium iodide and acquired for DNA content by flow cytometry. All the data are graphically represented as percentage of cells in apoptosis, S phase, and G2/M phase. Data represent 3 independent experiments.

AICAR decreases the levels of cyclins A and E in retinoblastoma cells

Cell cycle progression is controlled by specific cyclins. Given the effect of AICAR on the cell cycle, we wanted to see whether that was mediated by changes in the levels of the appropriate cyclins. After treatment with AICAR (1 and 2 mM) for 24 h, quantitative RT-PCR analysis showed decrease of cyclins A1, E1, and E2 in a dose-dependent manner (Fig. 5⤻). Our results suggest that AICAR-induced S-phase arrest in retinoblastoma cells might be associated with decreasing levels of cyclin A and E protein.

Figure 5.

Figure 5.

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AICAR decreases the levels of cyclins A and E in retinoblastoma cells. WERI (A), Y79 (B), and Rb143 (C) retinoblastoma cells were treated with AICAR at a concentration of either 1 or 2 mM for 24 h. Quantitative RT-PCR analysis showed decrease of cyclin A1, E1, and E2 in a dose-dependent manner in treated cells in comparison with control cells. RQ, respiratory quotient.

AICAR does not affect the levels of the cyclin-dependent kinases cdk-2 and cdk-4, cdk inhibitor p27, Akt pathway, autophagy marker LC3, and NF-κB p65 activity, while it down-regulates p21

We wanted to examine whether other regulators of cell cycle progression are affected by AICAR as has been shown in other cell types. We thus examined its effects on cdk-2, cdk-4, cdk inhibitor p21, cdk inhibitor p27, and the expression of PCNA. As shown in Fig. 6⤻, AICAR had little or no effect on the expression of cdk-2 and cdk-4, as well as on the expression of cell cycle protein inhibitor p27 and of PCNA (Fig. 6⤻). We did not see down-regulation of the important tumor progression Akt pathway or changes in the autophagy marker LC3 that have been observed in other tumors. In addition, Western blot analysis of nuclear extract of retinoblastoma cells after 1–2 d AICAR treatment showed that AICAR does not change nuclear translocation of NF-κB p65. Unexpectedly, AICAR down-regulated the expression of the cip/kip protein cdk inhibitor p21 in all 3 cell lines (65–85% at 2 mM P<0.05; Fig. 7A⤻). This is in contrast with previous reports that AICAR inhibited tumor cell growth by increasing p21, which is known to be a tumor suppressor. These data suggest that p21 may have a unique role in regulating Rb growth and could possibly function as an oncogene.

Figure 6.

Figure 6.

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AICAR does not affect levels of cell cycle progression in retinoblastoma cells. Western blot analysis of cdk-2, cdk-4, cdk inhibitor p27, P-Akt, LC3, PCNA, and nuclear translocation of NF-κB p65 in WERI, Y79, and Rb143 cells treated with AICAR at a concentration of either 1 or 2 mM for 24 h. Multiple bands represent separate biological samples.

Figure 7.

Figure 7.

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Antiproliferative effect of AICAR on retinoblastoma cells is mediated via inhibition of mTOR pathway and down-regulation of p21. A) Western blot analysis of the cdk inhibitor p21 in WERI, Y79, and Rb143 cells treated with AICAR at a concentration of either 1 or 2 mM for 24 h. B) Western blot analysis of phosphorylated ribosomal protein S6 (P-S6, Ser-235/236) and of phospho-4EBP1 (Ser-65 expression in WERI, Y79, and Rb143 cells treated with AICAR at a concentration of either 1 or 2 mM for 24 h. Density values of the bands are graphically expressed relative to control. Multiple bands represent separate biological samples.

AICAR inhibits the mTOR pathway in retinoblastoma cell lines

It has been well established that AMPK activation leads to inhibition of the mTOR pathway, resulting in dephosphorylation of ribosomal protein S6 that causes decreased initiation of translation and protein synthesis. We treated Rb cells with AICAR and examined the activity of the mTOR pathway by Western blot analysis of ribosomal protein S6, as a measure of mTOR activity. Treatment with 2 mM AICAR for 24 h caused a strong decrease in the phosphorylation of ribosomal S6 protein (ranging from 53.5 to 85% in the different cell lines, P<0.05), while the treatment with 1 mM had no significant effect on P-S6 phosphorylation (Fig. 7B⤻), indicating that only part of the inhibitory effects of AICAR in these cell lines is mediated through inhibition of the mTOR pathway. Examination of the downstream effector of S6K 4E-BP1 showed down-regulation of its phosphorylation, most consistently observed among all cell lines at the 2 mM concentration of AICAR treatment (Fig. 7B⤻).

DISCUSSION

In the present study, we demonstrated that AICAR, a pharmacological activator of AMPK, induces growth inhibition, apoptosis, and S-phase cell cycle arrest in human retinoblastoma cell lines. These effects were abolished if AICAR was not allowed to enter the cell by the adenosine transporter inhibitor DPY. Once AICAR enters the cell, it may be converted to inosine, increase the levels of adenosine, and/or be converted to the AMP analog ZMP. Iodotubericidin, an inhibitor of the enzyme responsible for the conversion of AICAR to ZMP, abrogated AMPK activation as judged by ACC phosphorylation and significantly blocked the growth inhibition by AICAR, indicating that the growth inhibition is mediated by intrinsic mechanisms and at least partially by AMPK activation.

Research by other laboratories indicates the effects of AICAR on cell cycle depend on the specific type of cell that is tested. Treatment of various cancer cell lines with AICAR has demonstrated arrest either in the G1 phase (33⤻, 35)⤻ or S phase (25⤻, 30)⤻ and/or an increase in the sub-G0/G1 population (34)⤻. When retinoblastoma cell lines were treated with AICAR, we observed an increase in the S-phase population as well as increases in the sub-G0/G1 population, suggestive of apoptosis. Moreover, treatment with AICAR resulted in the down-regulation of cyclins A and E, consistent with their S-phase arrest, as cyclins A and E control progression through S phase.

In other cancer cell lines, AICAR inhibited cell growth by a variety of mechanisms. Induction of AMPK activity was found to be dispensable in studies of neuroblastoma cell lines, myelogenous leukemia cell lines, and Jurkat cells (31⤻, 36⤻, 37)⤻, whereas it was shown that AMPK activity was up-regulated and/or necessary in several other studies of prostate, colon, breast, hepatic, and multiple myeloma cell lines (25⤻, 30⤻, 34⤻, 38)⤻. Our results showed that AMPK activity was at least partially needed for the inhibitory effects of AICAR in retinoblastoma cells. Further investigation of the mechanism of growth inhibition by AICAR revealed again more differences depending on the cell lines studied. AICAR treatment inhibited glioblastoma cells by inhibiting lipogenesis (32)⤻, triggered apoptosis by inhibiting NF-κB pathway in colon cancer cells (34)⤻, and inhibited proliferation by up-regulating the cell cycle inhibitor proteins p21 in C6 glioma cells and acute lymphoblastic leukemia cells (25⤻, 33)⤻. It increased p27 and decreased PCNA in C6 glioma cells (25)⤻. In contrast to these studies, we observed that AICAR-treated retinoblastoma cells were not inhibited by any of these mechanisms.

We paradoxically observed down-regulation of p21, which is most often thought of as a tumor suppressor. This paradoxical down-regulation of p21 has not been reported in any previous study of AICAR effects on cancer cells. Two possible explanations are that either p21 was down-regulated as a compensatory mechanism or p21 acts as an oncogene in retinoblastoma cells. Recent evidence suggesting that p21 can function as an oncogene was reported in some tumors (lymphoma, esophageal squamous cell carcinoma with p53 gene mutations). In these studies, p21 promotes tumor growth by inhibiting apoptosis and/or promoting cell proliferation (reviewed in ref. 39⤻). The studies of Gartel and Radhakrishnan (40)⤻ suggest that p21 may act as a positive regulator of the cell cycle. In fact, mitogenic stimuli result in transient p21 induction during G1-S progression. Thus, when p21 is repressed in such a context, it will lead to impairment of cell cycle progression due to decreased complex formation of cyclin D-cdk4/cdk6. This may be one of the mechanisms of AICAR inhibition of Rb cells and their arrest in S phase. Together, these data suggest that depending on the cell environment, p21 may function as either a tumor suppressor or an oncogene.

We are extremely interested in determining whether the effects of AICAR on Rb cells are specific to only Rb cells and/or tumor cells with Rb null mutations. As far as we know, none of the previous studies on AICAR-treated tumor cells were performed with cells that were carefully analyzed for Rb gene mutations. Therefore, the published data on the AICAR response in other tumors may, or may not, include tumors that are Rb null. This makes it difficult to compare our data with the published literature and determine which of our data are potentially related to Rb null gene mutations. However, Rb tumors express other mutations in addition to the loss of Rb, including p130, p170, and p53 (41⤻42⤻43)⤻. Therefore, our results could be related to other mutations present in Rb cells. Future experiments will study which AICARs are specific to either Rb tumor cells and/or Rb null mutations.

AICAR has been shown to be an exercise mimetic (26)⤻ to have anti-inflammatory properties (44)⤻ and anticancer properties as well as prosurvival effects of normal cells under stress (25⤻, 30⤻, 31⤻, 33⤻, 34)⤻. The mechanisms responsible for these effects are not fully understood, but they likely involve activation of AMPK. It is possible that the various effects of AICAR and AMPK depend on the specific cell type, cellular events following external stimuli, duration of AMPK activation, and/or downstream-regulated pathways of AMPK. Research on the antitumor effect of AICAR-induced AMPK activation is becoming an important area of investigation because of its link with tumor suppressors. The tumor suppressor LKB1 is an upstream activator of AMPK and the gene mutated in Peutz-Jeghers syndrome, an autosomal dominant disease characterized by hamartomatous polyp growth and predisposition to cancers of the gastrointestinal tract. Downstream of activated AMPK is known to be TSC2. TSC2 forms a complex with TSC1 and inhibits mTOR, leading to negative regulation of cell growth (23)⤻. Mutations of TSC are associated with tuberous sclerosis, which in humans is associated with hamartomas and an increased risk of cancers. Rattan et al. (25)⤻ reported that AICAR arrests C6 glioma cells in the S phase and is associated with the inhibition of the mTOR pathway. Similar to that study, we also observed decreased phosphorylation of ribosomal protein S6, a downstream effector of mTOR and a regulator of protein synthesis. AICAR also resulted in the decreased phosphorylation of 4E-BP1, a downstream effector of S6K. Given that the inhibition of the mTOR pathway was observed most consistently at the higher dose of AICAR, the possibility remains open that other mechanisms are responsible for the inhibition of Rb cells at the lower dose (1 mM). Given that the effects of AICAR on cyclins and the mTOR pathway were much more robust at the higher concentrations, we presume that the down-regulation of cyclins A and E and the mTOR pathway is only partially responsible for the cell cycle inhibition observed by AICAR.

Although advances in therapy for retinoblastoma have led to significant increases in the life expectancy of patients, significant problems still remain, such as the increased incidence of secondary malignancies after radiation and significant morbidity with current chemotherapeutic agents. This underscores the need for the development of new targets and less toxic therapies. In summary, our results indicate that after AICAR enters retinoblastoma cells, it induces AMPK activation through conversion to ZMP by adenosine kinase and inhibits the growth of retinoblastoma cells through inhibition of the mTOR pathway, down-regulation of cyclins A and E, and inhibition of p21, which in Rb cells may act as an oncogene (Fig. 8⤻). Moreover, other studies indicate that when AICAR is administered in nonchronic situations, it displays low toxicity, anti-inflammatory properties, and exercise mimetic features (26)⤻. Together, these data indicate that AICAR has tremendous potential as a novel targeted therapy with low toxicity.

Figure 8.

Figure 8.

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Proposed mechanism of action for AICAR in human retinoblastoma cells. AICAR, on entering the cell, is converted by adenosine kinase to ZMP, which activates AMPK. On activation, AMPK inhibits mTOR pathway. In addition, induction of AMPK by AICAR decreases the levels of cyclins A and E, which control the progression through S phase, as well as the levels of p21, which was recently found to inhibit apoptosis and promote cell proliferation. Overall signaling leads to loss of viability due to apoptosis and proliferation block.

Acknowledgments

D.G.V. was supported by an Onassis Foundation Scholars grant, the Massachusetts Lions Eye Research Fund, and Research to Prevent Blindness. B.R.K. was supported by U.S. National Institutes of Health grant NIH EY009294.

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