- Department of Neurosurgery, Medical College of Georgia, Augusta, GA 30912, USA
- Department of Biochemistry, Medical College of Georgia, Augusta, GA 30912, USA
John R. Vender
Department of Neurosurgery, Medical College of Georgia, Augusta, GA 30912, USA
DOI:10.4103/2152-7806.77029© 2011 Sukumari-Ramesh S This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
How to cite this article: Sukumari-Ramesh S, Singh N, Dhandapani KM, Vender JR. mTOR inhibition reduces cellular proliferation and sensitizes pituitary adenoma cells to ionizing radiation. Surg Neurol Int 23-Feb-2011;2:22
How to cite this URL: Sukumari-Ramesh S, Singh N, Dhandapani KM, Vender JR. mTOR inhibition reduces cellular proliferation and sensitizes pituitary adenoma cells to ionizing radiation. Surg Neurol Int 23-Feb-2011;2:22. Available from: http://sni.wpengine.com/surgicalint_articles/mtor-inhibition-reduces-cellular-proliferation-and-sensitizes-pituitary-adenoma-cells-to-ionizing-radiation/
Background:Pituitary adenomas are the most frequent brain tumor in adults. Although histologically benign, pituitary tumors cause significant morbidity and mortality. Neurosurgery and medical therapeutics may lessen the morbidity and mortality associated with pituitary tumors; however, these treatments are associated with significant adverse side effects. Thus, an improved understanding of pituitary adenomas at the molecular and cellular level is needed to design novel therapeutic compounds.
Methods:To assess the effect of mammalian target of rapamycin (mTOR) inhibition on pituitary adenoma cells, rat GH3 or MMQ cells were treated with the clinically useful mTOR inhibitors, rapamycin or RAD001. Cellular proliferation and growth following exposure to mTOR inhibitors or radiation were assessed using biochemical methods.
Results:In the present study, we observed basal activation of mTOR, downstream of constitutive Akt signaling, in rat GH3 adenoma cells. Functionally, the mTOR inhibitors, rapamycin and RAD001 (500 pM–5 nM), induced G1 growth arrest within 24 hours, an effect associated with reduced cellular proliferation. Both rapamycin and RAD001 decreased the phosphorylation of mTOR at the serine 2448, a key determinant of mTOR activity. Inhibition of mTOR also radiosensitized GH3 cells such that 2.5 Gy in combination with 500 pM rapamycin or RAD001 reduced cellular viability more effectively than 2.5 or 10 Gy alone.
Conclusions:These data may support a possible therapeutic role for mTOR inhibitors in limiting the cellular proliferation and radioresistance of pituitary adenoma cells.
Keywords: Akt, everolimus, mTOR, pituitary tumor, radiation, rapamycin, RAD001
Pituitary tumors are among the most prevalent brain tumors in adults and may be associated with significant morbidity and mortality. Although histological malignancy of the pituitary is rare, adenomas may exhibit infiltrative and invasive growth patterns characteristic of malignant tumors.[
Akt (protein kinase B) is a cellular serine/threonine protein kinase that has been implicated in cellular growth and survival.[
Mammalian target of rapamycin (mTOR), a serine/threonine kinase involved in diverse cellular processes, including protein translation, mRNA turnover, and protein stability, mediates, at least in part, some of the biological actions of Akt.[
All cell culture reagents, sera, and media were purchased from Hyclone Laboratories (Logan, UT, USA). The mTOR inhibitors, rapamycin and RAD001 (everolimus), were purchased from Alexis Biochemicals (San Diego, CA, USA) and Sigma (St. Louis, MO, USA), respectively.
Rat GH3 and mouse MMQ pituitary adenoma cells (American Type Tissue Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum, 5% bovine growth serum, and antibiotics in a 37°C humidified incubator at 5% CO2. GH3 cells are a well-characterized model of secretory adenomas that hypersecrete both prolactin (PRL) and growth hormone (GH) and that reflect the biology of human secretory tumors.[
Cellular viability assays
Cell viability was estimated by the addition of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) followed by incubation for 4 hours at 37°C, as described by our laboratory.[
Cell cycle analysis
5 × 104 cells were plated in a 60-mm plate for 48 hours. Exponentially growing cells were synchronized in serum-free DMEM overnight, and then cells were treated for up to 72 hours in complete culture media containing rapamycin or RAD001. Cells were washed twice, and then permeabilized with 70% methanol overnight at −20°C. Cells were incubated with 0.5 mL of a 50 μg/mL propidium iodide (PI) solution containing 200 μg/mL RNaseA for 30 minutes, and the cells were immediately analyzed by flow cytometry using BD Biosciences FACS Calibur analyzer.
Following treatments, the cells were washed with phosphate-buffered saline (PBS) and whole cell lysates were collected in radioimmunoprecipitation (RIPA) buffer containing protease inhibitor cocktail, phosphatase inhibitor cocktail and phenyl methane sulfonyl fluoride (PMSF). Following sonication, cell lysates were centrifuged for 5 minutes at 14,000 rpm at 4°C, and protein concentrations were quantified by BCA protein assay kit (Pierce, Rockford, IL, USA). 30 μg of protein were resolved on 4–20% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. Blots were incubated overnight at 4°C in primary antibody (1:500 anti-mTOR antibody or 1:500 anti-phospho-mTOR) (Cell Signaling Technology, Beverly, MA, USA) followed by a 2 hour incubation with an AlexaFluor 750 secondary antibody at room temperature. Blots were visualized using the Li-Cor Odyssey near-infrared imaging system and densitometry analysis was performed using Quantity One software (Bio-Rad, Foster City, CA, USA).
3 × 104 cells/well were plated in a 24-well plate for 48 hours. Exponentially growing cells were pretreated with rapamycin or RAD001 for 3 hours and then irradiated using a Nordion Gammacell 40 Exactor Irradiator. Following irradiation, the media were replaced with complete culture media and cells incubated for 48 hours at 37°C, prior to the determination of cellular viability.
mTOR inhibition induces G1 growth arrest in pituitary adenoma cells
The mTOR inhibitors, rapamycin and RAD001, reduced the cellular viability of GH3 [
Effect of rapamycin or RAD001 on cellular viability. (a) Rapamycin or (b) RAD001 (0.1–5 nM) reduced cellular viability in GH3 pituitary adenoma cells in a time- and concentration-dependent manner. Cellular viability was assessed at 24, 48, or 72 hours following treatment, and data were normalized to the vehicle-treated group. Data were compared by one-way ANOVA followed by Dunnett’s post-hoc test (**P < 0.01, ***P < 0.001 vs. vehicle-treated cultures)
Inhibition of the mTOR pathway appeared to influence cellular proliferation; thus, the effect of rapamycin and RAD001 on cell cycle progression was investigated. Rapamycin (500 pM or 5 nM) significantly increased the percentage of cells in the G1 phase [75.4 ± 1.2%, 83.3 ± 0.8% respectively vs control 70.17± 0.7530], with a concomitant decrease in the percentage of cells within the G2/M phase (8.5 ± 0.8%, 6.7 ± 0.7%, respectively vs. control, 13.1 ± 0.8%) [
mTOR inhibition induces G1 growth arrest. GH3 cells were treated with rapamycin (0, 0.5, or 5 nM; top panels) or RAD001 (0, 0.5, or 5 nM; bottom panels) for 72 hours and then analyzed using flow cytometry. Both the compounds increased the percentage of cells in G1 phase and decreased the percentage of cells in G2/M phase of the cell cycle. Data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test (**P < 0.01 and ***P < 0.001 vs. vehicle-treated cultures)
Rapamycin and RAD001 reduce mTOR activity
Phosphorylation of serine 2448, a key determinant of mTOR activity, was basally increased in GH3 adenoma cells. Consistent with a role for Akt in the constitutive activation of mTOR, phosphorylation of serine 2448 was reduced by treatment with LY294002, an Akt pathway inhibitor [
Rapamycin and RAD001 reduce mTOR activity. Treatment with rapamycin (1–20 nM) or RAD001 (1–20 nM) for 24 hours reduced mTOR phosphorylation on serine 2448, an important residue involved in mTOR activity (top panel). Neither rapamycin nor RAD001 affected total mTOR protein. β -actin was used as a loading control. Densitometric analysis of phospho-mTOR/mTOR is depicted in the bottom panel. Data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test (*P < 0.05 and **P < 0.01 vs. vehicle-treated cultures)
mTOR inhibition reduces radioresistance in GH3 adenoma cells
G1 growth arrest may increase cellular sensitivity to radiation; thus, the effect of mTOR inhibition on radiation-induced cell death was next assessed. 2.5 and 10 Gy alone reduced cellular viability by 15 and 19%, respectively. In contrast, cellular viability was reduced by 45 and 27% when 2.5 Gy was combined with a 3h pretreatment with either rapamycin (0.5 nM) or RAD001 (0.5 nM), respectively [Figure
mTOR inhibition radiosensitizes GH3 adenoma cells. Cells were pretreated with (a) rapamycin (0.5 nM) or (b) RAD001 (0.5 nM) for 3 hours and then irradiated (2.5 or 10 Gy). Cell viability was assessed 48 hours following irradiation. Data were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated cultures)
Aberrant activation of mTOR, an important serine/threonine protein kinase involved in protein translation, cellular proliferation, cellular motility, and cellular survival,[
Pituitary adenoma accounts for approximately 15% of primary intracranial tumors.[
Increased expression and activity of the serine/threonine kinase, Akt, was observed in human pituitary adenomas, as compared to the normal pituitary gland; however, the functional significance of Akt activation in the progression of pituitary adenoma remains unknown. Similarly, the downstream effectors of Akt signaling remain poorly defined within pituitary adenoma. Recent work done in our laboratory found that Akt was constitutively activated in GH3 adenoma cells, as was observed in human pituitary tumors, and that attenuation of this signaling pathway significantly reduced cellular viability. The effect of Akt was partially mediated by the NFkB transcription factor, although other signaling pathways also contributed to the effect on cellular growth.[
Basal mTOR functionally contributed toward the cellular growth of pituitary adenoma cells as pharmacological inhibition of mTOR limited cellular proliferation. Unfortunately, rapamycin is highly lipophilic and exhibits poor stability in vivo. These properties potentially limit the clinical usefulness of rapamycin in the management of pituitary tumors; however, more stable and soluble rapamycin analogues, such as RAD001, may retain biological activity against mTOR, and thus, could be more amendable to clinical use. In support of this possibility, oral administration of RAD001 was approved by the Food and Drug Administration (FDA) in early 2009 for the treatment of advanced renal carcinoma. In the present study, both rapamycin and RAD001 attenuated mTOR activation and increased acute G1 growth arrest in pituitary adenoma cell lines. In GH3 cells, 410 pM rapamycin was the IC50 concentration, whereas 986 pM RAD001 was required to exert a similar effect. Similarly, 4.7 μM rapamycin and 8.6 μM RAD001 were the IC50 concentrations following a 48 hour treatment in MMQ cells (data not shown). These findings are entirely consistent with a previous study which reported a 2–3 fold greater immunosuppressive effect of rapamycin, as compared to RAD001.[
Rapamycin at a dose of 15 ng/mL (>15 nM serum concentration) is clinically well tolerated as an immunosuppressive agent to present transplant rejection;[
Radiation remains a frontline and generally effective treatment option for pituitary adenoma patients; however, radiotherapy may be associated with hypopituitarism in ~33% of patients at 2 years and close to 100% of patients at 10 years. Similarly, radiosurgery is limited by the high sensitivity of surrounding structures, such as the optic apparatus, to radiation injury.[
As a whole, these data suggest a possible use for mTOR inhibitors, either as a single agent to limit tumor growth and/or as an adjunct to radiation therapy. These findings therefore warrant further investigation in a pre-clinical animal model of pituitary tumors.
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