431
research article
Metformin enhanced in vitro radiosensitivity associates with G2/M cell cycle arrest and elevated adenosine-5'-monophosphate-activated protein kinase levels in glioblastoma
Sebastian Adeberg1-4, Denise Bernhardt1-3'4, Semi B. Harrabi134, Nils H. Nicolay1-4, Juliane Hörner-Rieber134, Laila König134, Michael Repka5, Angela Mohr134, Amir Abdollahi1 3 4 6, Klaus-Josef Weber14, Juergen Debus1-4, Stefan Rieken134
1	University Hospital of Heidelberg, Department of Radiation Oncology, Heidelberg, Germany
2	Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany
3	Heidelberg Ion-Beam Therapy Center (HIT), Heidelberg, Germany
4	Heidelberg Institute for Radiation Oncology, University Hospital of Heidelberg, Heidelberg, Germany
5	Department of Radiation Medicine, Georgetown University Hospital, Washington DC, USA
6	Tanslational Radiation Oncology, German Cancer Consortium (DKTK), National Center for Tumor Diseases German Cancer Research Center (DKFZ), Heidelberg, Germany
Radiol Oncol 2017; 51(4): 431-437.
Received 12 February 2017 Accepted 30 September 2017
Presented in part at the Annual Meeting of the German Society of Radiation Oncology (DEGRO), 15th - 18th June 2017, Berlin, Germany
Correspondence to: Sebastian Adeberg, M.D., University Hospital of Heidelberg, Department of Radiation Oncology, Im Neuenheimer Feld 400, 69120 Heidelberg,Germany. Phone: +49 6221 5635654; Fax: +49 6221 56565353; Email: sebastian.adeberg@med.uni-heidelberg.de
Disclosure: No potential conflicts of interest were disclosed.
Background. It is hypothesized that metabolism plays a strong role in cancer cell regulation. We have recently demonstrated improved progression-free survival in patients with glioblastoma who received metformin as an antidiabetic substance during chemoradiation. Although metformin is well-established in clinical use the influence of metformin in glioblastoma is far from being understood especially in combination with other treatment modalities such as radiation and temozolomide.
Materials and methods. In this study, we examined the influence of metformin in combinations with radiation and temozolomide on cell survival (clonogenic survival), cell cycle (routine flow cytometric analysis, FACScan), and phosphorylated Adenosine-5'-monophosphate-activated protein kinase (AMPK) (Phopho-AMPKalphal - ELISA) levels in glioblastoma cell lines LN18 and LN229.
Results. Metformin and temozolomide enhanced the effectiveness of photon irradiation in glioblastoma cells. Cell toxicity was more pronounced in O6-methylguanine DNA methyltransferase (MGMT) promoter non-methylated LN18 cells. Induction of a G2/M phase cell cycle block through metformin and combined treatments was observed up to 72 h. These findings were associated with elevated levels of activated AMPK levels in LN229 cells but not in LN18 cells after irradiation, metformin, and temozolomide treatment.
Conclusions. Radiosensitizing effects of metformin on glioblastoma cells treated with irradiation and temozolomide in vitro coincided with G2/M arrest and changes in pAMPK levels.
Key words: metformin; glycolysis; metabolism; gliomas; proliferation; cell cycle
Introduction
The relationship of tumorigenesis, and tumor cell metabolism by an increased glycolysis was
first described in the early 20th century by Otto Warburg.12 Multiple modifications in cancer cell metabolism have subsequently been detected, but the influence on alterations in signaling path-
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doi: 10.1515/raon-2017-0024
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ways, cell growth, and therapy response is not yet understood. Nonetheless, tumor cell metabolism represents an intriguing potential target in the multidisciplinary treatment of cancer. The addition of metabolically active substances, particularly those with limited toxicity, with chemotherapy and radiation is an area of active research. In this context, the antidiabetic medication metformin is of particular interest as it demonstrated prolonged progression-free survival in a retrospective study in diabetic patients with glioblastoma (GBM).3 The primary glucoregulatory effects of metformin are predominantly explained through reduced hepatic glucose production and increased glucose uptake in the periphery.4 These effects lead to decreased mitochondrial-dependent ATP production and cell proliferation, increased glycolytic ATP production, induction of cell cycle arrest, autophagy, and apop-totic processes through activation of adenosine-5'-monophosphate-activated protein kinase (AMPK)5 and inhibition of the mTOR (mammalian target of rapamycin) pathway in glioblastoma cells.67
AMPK is a serine/threonine kinase that functions as a cellular energy sensor. AMPK is an obligate heterotrimer, consisting of one catalytic subunit (a) and two regulatory subunits (|3 and Y).8 Under cellular stress conditions, AMPK is activated by increased AMP-to-ATP ratios to promote catabolism and inhibit anabolism.8 High cellular activated AMPK levels, particularly by phospho-rylation (pAMPK), seems to be associated with tumor cell growth and cell survival.910 Furthermore, increased AMPK phosphorylation has been observed in cells following radiation-induced DNA damage in several studies.71112 The activation of AMPK is hypothesized to regulate irradiation-induced metabolism changes and might be a key determinant of cell survival after exposure to ionizing radiation.7 The AMPK pathway is therefore a potential objective for targeted therapies. Although the concomitant application of metformin with te-mozolomide (TMZ) and this effect is not well understood. In this study, we investigated the effects of metformin effect in combination with current standard of care therapy in glioblastoma cell lines.
Materials and methods
Cell lines and culture conditions
Two representative human GBM cell lines (ATCC; Manassas, VA, USA) were utilized in this study. LN18 is a GBM cell line with a mutant tumor sup-
pressor protein 53 (p53mut) and an un-methylated O6-methylguanine DNA methyltransferase (MGMT) promoter. LN229 is a GBM cell line with both mutant and wild type p53 (p53 mut/WT) and a methylated MGMT promoter. Both cell lines were cultured in Dulbeccos's modified Eagle medium (Biochrom, Berlin, Germany) supplemented with 10% Fetal Calf Serum (FCS) superior (Biochrom AG) and 1% penicillin/streptomycin (Gibco, Darmstadt, Germany). Cultures were maintained in exponential monolayer at incubator conditions with 37°C, 5% CO2, and 95% humidity.
Drug treatment and irradiation
We performed clonogenic assays to evaluate treatment effects on cell survival. Low passage cells were plated in T25 flasks (Becton, Dickinson, Heidelberg, Germany) with 5 ml medium as described previously.13 TMZ was obtained by Schering-Plough (Kenilworth, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Deisenhofen, Germany) at concentrations < 0.5%. Cell samples were incubated with 10 |jM or 50 |jM TMZ for 4 hours prior to irradiation. 1,1-Dimethylbiguanide hydrochloride (metform-in; 97%) was provided by Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and applied in concentrations of 1 mM or 20 mM for 24 hours prior to irradiation. Immediately prior to irradiation, all samples were rinsed twice with phosphate buffered saline and fresh medium was added. Adherent cells were irradiated using a 6MV photon linear accelerator (XRAD 320 Precision X-ray Inc., N.Bradford, USA) with single photon doses up to 6 Gy. Cells were fixed with 70% ethanol and stained with methylene blue. Colonies of > 50 were counted. Experiments were repeated in triplicate at least three times.
Cell cycle analyses
To evaluate cell cycle distributions, cells were harvested, washed and fixed in ice-cold 70% ethanol after 24, 48 or 72 hours and stained with propidium iodide (Sigma-Aldrich). 10.000 events were counted for each experimental setup with routine flow cytometric analysis (FACScan, Becton-Dickinson, Heidelberg, Germany). Histograms were created and analyzed using ModFit software (Verity Software House, Topsham, ME, USA). Each experiment was repeated at least three times on different days for validation.
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Phopho-AMPKal - ELISA
ELISA for pAMPK levels in LN229 and LN18 cell lysates was performed by commercially available DuoSet ELISA development kits (R&D Systems, Minneapolis, USA) according to the manufacturer's instruction using goat anti-human AMPKal (T183) as primary antibody and biotinylated rabbit antihuman phospho-AMPKal (T183) as secondary antibody. Recombinant human phospo-AMPKal (T183) was utilized as standard. The results were presented as relative values to the control value, which was set as 1. Data analysis was carried out using the Infinite® F50/Robotic ELISA plate reader (absorbance at 450 nm, correction wavelength at 570 nm) and Magellan for F50 software (Tecan Group Ltd, Crailsheim, Germany). Measurements were repeated at least three times on three different days.
Statistical analysis
Clonogenic survival was calculated from the measured plating efficiencies. With the data of the combined treatment approaches survival curves were generated with the linear quadratic (LQ) -model as described earlier.14 Sigma Plot's (Systat Software GmbH, Erkrath, Germany) non-linear least-squares regression option was used to fit the calculated survival curves. Clonogenic survival was calculated using the sensitizer enhancement ratio (SER), comparing the radiation dose at 20% cell survival, due to the high efficiency of TMZ and metformin on cellular killing.
LN229
LN18
SER =
radiation dose without sensitizer radiation dose with sensitizer
Ctrl • 50[iM TMZ
-	Ctrl + 2 Dm M Metformin
-	Ctrl + S0;iM TMZ + 2ü iiM Metformin
-	CM + 50LLM TMZ
-	Ctrl * 20mM Metformin
-	Ctrl * SOmM TMZ + 20jnM Metformin
dose (Gy)
dose (Gyï
Lnis fj 11M TMZ Li}229 5«uM TMZ
Lnt 6 2QmM Metformin Ln229 20mM Metformin
The Student's two-sided t-test was used for comparison of cell survival curves, differences in cell cycle analysis distribution and pAMPK levels. Data are shown as mean values ± standard deviation. Statistical significance was set at p < 0.05.
Results
Metformin enhances the effectiveness of irradiation in glioblastoma cells
Metformin sensitivity of glioblastoma cell lines LN18 and LN229 was investigated by clonogenic survival assays. Increasing concentrations of metformin (1 mM and 20 mM) and TMZ (10 |jM and 50 |jM) were chosen for all baseline experiments. Representative concentrations (20 mM metform-
FIGURE 1. (A,B) Clonogenic survival assays of LN229 and LN18 glioblastoma cell lines after treatment with photon irradiation. (C,D) Clonogenic survival assays after comparing LN229 and LN18 cell lines after combined treatment with TMZ (C) or metformin (D). Error bars represent standard deviations. * shows a statistical significance (P <0.05). In LN229 enhanced cell kill was observed by TMZ and in LN18 cells lines metformin treatment resulted in increased cell toxicity compared to the control, as well as the combination of both agents itself. Combined bimodal and trimodal treatment results in superior cell toxicity.
in and 50 |xM TMZ) were chosen for subsequent combined experiments. Clonogenic survival of the MGMT promoter methylated LN229 cell line was significantly reduced after treatment with TMZ compared to the untreated control and LN18 cell lines. Combined treatment approaches with irra-
TABLE 1. Sensitizer enhancement ratio (SER 20%) for LN229 and LN18 cells after treatment with metformin, temozolomide (TMZ) and combined treatment with 2Gy irradiation
SER (20% survival) / 2 Gy
Cell line	LN18	LN229
50 pM TMZ	1.11	2.15
1mM Metformin	0.67	0.74
20 mM Metformin	3.13	0.61
50 pM TMZ + 20mM Metformin	2.57	2.69
SER values (ranging from 0.67-3.13) depending on cell line, chemofherapeufic agent and dose
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TABLE 2. Cell cycle distribution into G1, S, and G2/M phase of LN18 and LN229 cells after various treatments. Measurements were performed after 72h. * shows a statistical significance (P <0.05) of the treatment compared to the control
Ln18 24h 72h	Gl (% ) ± std.dev.	S (%) ± std.dev.	G2/M (%) ± std.dev.
Ctrl	74.5 ± 8	5.6 ± 0	19.9 ± 1
50JM TMZ	66.0 ± 8	9.5 ± 1	24.6 ± 3
1mM Metformin	71.0 ± 5	6.3 ± 1	22.7 ± 0
20mM Metformin	46.9 ± 3	9.3 ± 2	43.8 ± 2*
50|JM TMZ + 1mM Metformin	64.5 ± 0	8.6 ± 4	26.9 ± 4
50|JM TMZ + 20mM Metformin	41.2 ± 0	19.4 ± 7	39.4 ± 2
2Gy	71.7 ± 1	7.5 ± 1	20.8 ± 4
6Gy	56.2 ± 5	8.0 ± 1	35.7 ± 1
2Gy + 50jM TMZ	62.0 ± 5	6.6 ± 5	31.4 ± 4
2Gy + 1mM Metformin	68.2 ± 7	10.3 ± 1	21.5 ± 2
2 Gy + 20mM Metformin	41.3 ± 0	16.1 ± 1	42.4 ± 4*
2Gy + 50jM TMZ + 1mM Metformin	60.1 ± 7	8.6 ± 1	31.3 ± 0
2Gy + 50jM TMZ + 20mM Metformin	33.5 ± 2	20.0 ± 3	44.5 ± 4*
6Gy + 50jM TMZ	47.1 ± 7	11.6 ± 3	41.3 ± 3*
6Gy + 1mM Metformin	51.3 ± 2	10.4 ± 0	38.2 ± 3
6Gy + 20mM Metformin	36.1 ± 3	14.2 ± 0	49.7 ± 3*
6Gy + 50jM TMZ + 1mM Metformin	46.3 ± 4	10.7 ± 1	42.9 ± 2*
6Gy + 50jM TMZ + 20mM Metformin	27.9 ± 1	14.1 ± 3	58.0 ± 2*
			
Ln229 24h 72h	Gl (%)	S (%)	G2/M (%)
Ctrl	86.0 ± 2	3.6 ± 2	10.4 ± 2
50JM TMZ	37.4 ± 3	8.1 ± 2	54.4 ± 5*
1mM Metformin	87.4 ± 3	3.3 ± 1	9.3 ± 0
20mM Metformin	63.9 ± 10	14.9 ± 1	21.2 ± 9
50jM TMZ + 1mM Metformin	38.9 ± 0	6.1 ± 2	55.0 ± 4*
50jM TMZ + 20mM Metformin	25.2 ± 1	25.4 ± 2	49.4 ± 2*
2Gy	83.5 ± 5	3.3 ± 2	13.2 ± 5
6Gy	67.1 ± 2	6.2 ± 1	26.7 ± 1
2Gy + 50jM TMZ	44.4 ± 1	6.9 ± 1	48.7 ± 4*
2Gy + 1mM Metformin	82.3 ± 1	3.5 ± 1	14.2 ± 2
2 Gy + 20mM Metformin	55.6 ± 2	11.8 ± 2	32.4 ± 1 *
2Gy + 50jM TMZ + 1mM Metformin	46.0 ± 1	7.9 ± 1	44.1 ± 0*
2Gy + 50jM TMZ + 20mM Metformin	32.3 ± 3	28.9 ± 7	38.8 ± 5*
6Gy + 50jM TMZ	41.6 ± 2	8.9 ± 3	49.5 ± 3
6Gy + 1mM Metformin	55.5 ± 11	8.8 ± 4	35.7 ± 23
6Gy + 20mM Metformin	56.5 ± 18	7.8 ± 6	35.7 ± 16
6Gy + 50jM TMZ + 1mM Metformin	41.9 ± 2	5.9 ± 2	52.2 ± 2*
6Gy + 50jM TMZ + 20mM Metformin	18.4 ± 1	16.2 ± 3	45.4 ± 3*
diation, TMZ and metformin showed additive cell toxicity compared to the control (P < 0.05, Student's t-test) (Figure 1A,B and Table 1). Furthermore, clonogenic survival after metformin exposure was reduced in LN18 cells when compared to the untreated control and to LN229 cells (P < 0.05) (Figure 1C,D and Table 1). Additionally, additive cell toxicity could be reached in LN229 cells adding ionizing irradiation (2 Gy and 6 Gy) to TMZ and Metformin, whereas additional effects with ionizing irradiation could only be reached for met-formin in LN18 cells.
Metformin induces a G2/M phase block in combination with irradiation
Cell cycle assessment of glioblastoma cell lines was carried out using FACS analyses. Exposure to metformin (20 mM) resulted in accumulation at G2/M phases after 72 hours to a higher degree in LN18 cells (G2 phase cells: ctrl vs 20mM metformin: 19.9% vs 43.8%). However, the results did not reach statistical significance. Combined treatment approaches with irradiation and metformin resulted in a more pronounced G2/M block after 72 hours (P < 0.05, Student's t-test) (Table 2). Accumulations in G2/M phases after 72 hours were even more marked when using higher radiation doses (6 Gy) (Table 2) and trimodal approaches with irradiation, 50 |jM TMZ, and 20 mM metformin (P < 0.05, Student's t-test) (Table 2). Analysis of sub-G1 populations indicating apoptosis did not show any measurable results.
Irradiation, TMZ and metformin enhance activated AMPK levels in glioblastoma cells
Phosyphorylated serine/threonine kinase AMPK levels induced by irradiation, TMZ, and metformin exposure were investigated as a potential mechanism for the described metformin sensitivity of glioblastoma cell lines. Increased pAMPK levels were demonstrated in LN229 cells after treatment with the following regimens: 2 Gy + 50 |jM TMZ, 2 Gy + 20 mM metformin, 2 Gy + 50 |jM TMZ + 20 mM metformin, 6 Gy, 6 Gy + 50 |jM TMZ, 6 Gy + 20 mM metformin, and 6 Gy + 50 |jM TMZ + 20 mM metformin (P<0.05, Student's t-test) (Figure 2A). All other treatment approaches showed a trend towards higher pAMPK levels in LN229 cells (P<0.1, Student's t-test). Higher radiation doses were associated with increased pAMPK levels (P<0.05, Student's t-test) (Figure 2A). Interestingly, LN229
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demonstrated a twofold higher level of pAMPK after treatment with 6 Gy compared to the untreated control (P<0.05, Student's t-test). pAMPK measurements obtained from LN18 cell lines did not show a significant increase after each treatment combination (Figure 2B).
Discussion
Standard of care multidisciplinary management of GBM entails surgical resection followed by radiotherapy with concomitant and adjuvant TMZ, resulting in overall survival rates of approximately one year. Given this dismal prognosis, the need to improve the efficacy of chemoradiation for these common primary brain tumors is urgent. Recently, we demonstrated improved progressionfree survival rates in diabetic patients receiving metformin3, a biguanide that is commonly used and well tolerated in patients with type II diabetes. Accordingly, combined approaches targeting cell metabolism became attractive. Metformin is known to exhibit anticancer effects via LKB1/ AMPK/mTOR/S6K1 pathway blockade1516, inhibition of tumor growth1718 and induction of au-tophagy and apoptosis1920 in various cancer cell lines. Accordingly, integration of approaches targeting cell metabolism into standard therapy is an attractive area of investigation. In the present study, we examined the interaction of metformin in combination with photon irradiation and the alkylating agent TMZ. Furthermore, we demonstrated that metformin has antitumoral effects and increases sensitivity to ionizing radiation, which was particularly pronounced in a non-MGMT methylated cell line (LN18).
Of note, both the LN18 and LN229 cell lines express wildtype for PTEN, which is associated with increased sensitivity to metformin.6 This susceptibility may be explained by opposing PI3K signaling, thus leading to a down-regulated AKT survival pathway and decreased glucose consump-tion.6 Therefore, the higher metformin sensitivity for LN18 cannot solely be explained by the more effective deactivation of AKT in these cells. The authors believe the MGMT promoter methylation in metformin sensitive LN18 cells is rather a coincident than the cause of this finding. In fact, glioma cells are known to have a high intrinsic radiation sensitivity caused by several intrinsic factors such as high efficient radiation damage repair, a high ratio of hypoxic cell fraction and rapid repopulation following irradiation.21 The intrinsic sensitivity of
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LN229
LN18
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FIGURE 2. (A) In LN229 pAMPK measurements were significantly increased after treatment with Metformin, 2 Gy and temozolomide and 6 Gy combined with Metformin compared to the control if not otherwise specified (* p<0.05). (B) No significant increase of pAMPK levels were observed in cell line LN18. Error bars represent standard deviation.
GBM cells is probably independent of the MGMT promotor methylation status. The mechanistic effects of these findings are beyond the scope of this manuscript and will be evaluated in future experiments. An inverse effect was shown for TMZ, where the MGMT promoter-methylated LN229 cells were more sensitive to TMZ compared to MGMT promoter non-methylated LN18 cells. This finding can be explained by the lack of the MGMT DNA-repair protein in LN229 cells which normally removes O6-MG-DNA and counteracts the antineoplastic effect of TMZ.22
We performed cell cycle analyses in order to further investigate the antiproliferative effects of met-formin on glioblastoma cell lines. In PTEN wildtype cells, cytotoxic effects have been described starting at 48 hours.6 However, in LN18 and LN229 cell lines, significant increase of G2/M block rates started delayed, chiefly 72 hours after irradiation. This effect was pronounced in combined treatment approaches with higher radiation doses and TMZ administration. These results indicate that the anti-proliferative effects of metformin on glioblastoma cell lines might be mediated trough cell cycle arrest starting at 72 hours post-exposure. These results are in line with results from in vitro data of Yu et al. who observed G2/M cell cycle arrests after TMZ and metformin. A combined treatment showed synergistic effects.23 In a former study G1 arrests were described after metformin exposure,
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whereas mainly G2/M phase arrests were observed in the current study. This effect might be due to the two-fold metformin dose (10 nM vs 20 nM) with varying impact. Furthermore metformin effects were mainly observed after 72 hours but the observation period of Sesen et al. ended after 48 hours.6 Nevertheless, molecular mechanisms of metformin are far from understood and further research to examine its role in tumor cell metabolism is necessary.
Previous studies have demonstrated that activation of AMPK leads to an inhibition of mTOR615'24'25 and is essential for glioma proliferation by promoting cell cycle progression in vitro and in vivo.26 These findings are supported by reports indicating a major AMPK phosphorylation and activation through the tumor suppressor LKB1.27-29 Furthermore, AMPK has been associated with p53-dependent apoptosis through p53 phosphoryla-tion30, underlining the potential function of AMPK activation as an "energy checkpoint".31 This proposed mechanism permits proliferation and cell growth in cells with intact AMPK signaling only in favorable metabolic cell conditions. Conversely, cancer cells with deficient AMPK signaling might be capable of receiving a metabolism-independent growth stimulus. In these cases, induction of AMPK activation could present a valuable therapeutic approach.26 Although the role of AMPK as a metabolic sensor in homeostasis is well described, its function in cancer remains opaque. In vitro studies have shown highly efficient inhibition of tumor cell growth across multiple glioblastoma cell lines with several AMPK agonists, including 5-amino-imidazole-4-carboxamide ribonucleotide (AICAR) and activated AMPK adenovirus.32-35
In this study, we demonstrated a dose-dependent increase of AMPK in LN229 glioblastoma cells following radiation in combination with metformin and TMZ. Although AMPK level changes did not show statistically significant changes in the LN18 cell line, the radiosensitizing effect of metformin was more pronounced in these cells. Another reason for the absent of significant findings could be attributed to a high standard deviation masking subtle changes (Figure 4B). The authors carefully performed pAMPK level measurements with at least n=3. Even though, future in-depth analyses may help to unmask subtle changes. Interestingly, the failure of LN18 to phosphorylate AMPK compared to LN 229 cells could be a reason for the enhanced radiosensitivity in the latter, since AMPK activation might be a keyregulator for glioma cell proliferation.26 On the other hand, it is likely that
metformin exhibits both AMPK dependent and AMPK-independent effects which are contingent on molecular tumor characteristics.615
Taken together, the present finding that activated AMPK levels are elevated after treatment with radiation, TMZ, and metformin contributes to the understanding of GBM metabolism following therapeutic intervention. However, more detailed knowledge of the antitumoral effects of met-formin, the role of AMPK, and tumor cell biology is necessary to establish a novel multidisciplinary approach to glioblastoma therapy. We planned to perform mechanistic in vitro metformin experiments in the future based on the current baseline results. Additional challenges, including the ability of AMPK activating agents such as AICAR to cross the blood brain barrier more effective, are ongoing. Nonetheless, our results suggest that the development of an AMPK activating agent with high central nervous system bioavailabity may be a promising new therapeutic avenue in the treatment of this aggressive malignancy.
Conclusions
Together with our previously published clinical findings3 and the well-established use of metform-in in clinical practice, these data show that radio-sensitizing effects of metformin on glioblastoma cells treated with irradiation and temozolomide in vitro coincided with G2/M arrest and changes in pAMPK levels.
Acknowledgement
The authors thank Mrs Lena Orschiedt and Mrs Sigrid Daffinger for excellent technical assistance.
References
1.	Warburg O, Wind F, Negelein E. The metabolism of tumors in the Body. J Gen Physiol 1927; 8: 519-30.
2.	Warburg O. On the origin of cancer cells. Science 1956; 123: 309-14.
3.	Adeberg S, Bernhardt D, Harrabi SB, Bostel T, Mohr A, Koelsche C, et al. Metformin influences progression in diabetic glioblastoma patients. Strahlentherapie Onkol 2015; 191: 928-35. doi: 10.1007/s00066-015-0884-5
4.	Goodarzi MO, Bryer-Ash M. Metformin revisited: re-evaluation of its properties and role in the pharmacopoeia of modern antidiabetic agents. Diabetes Obes Metab 2005; 7: 654-65. doi: 10.1111/j.1463-1326.2004.00448
5.	Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108: 1167-74. doi: 10.1172/JCI13505
Radiol Oncol 2017; 51(4): 431-437.
437	Adeberg S et al. / Metformin enhances G2/M arrest and cell death in glioblastoma cells

6.	Sesen J, Dahan P, Scotland SJ, Saland E, Dang VT, Lemarie A, et al. Metformin inhibits growth of human glioblastoma cells and enhances therapeutic response. PLoS One 2015; 10: e0123721. doi: 10.1371/journal.pone.0123721
7.	Zannella VE, Cojocari D, Hilgendorf S, Vellanki RN, Chung S, Wouters BG, et al. AMPK regulates metabolism and survival in response to ionizing radiation. Radiother Oncol 2011; 99: 293-9. doi: 10.1016/j.radonc.2011.05.049
8.	Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Review 2009; 89: 1025-78. doi: 10.1152/physrev.00011.2008
9.	Jang T, Calaoagan JM, Kwon E, Samuelsson S, Recht L, Laderoute KR. 5'-AMP-activated protein kinase activity is elevated early during primary brain tumor development in the rat. Int J Cancer 2011; 128: 2230-9. doi: 10.1002/ijc.25558
10.	Park HU, Suy S, Danner M, Dailey V, Zhang Y, Li H, et al. AMP-activated protein kinase promotes human prostate cancer cell growth and survival. Mol Cancer Ther 2009; 8: 733-41. doi: 10.1158/1535-7163
11.	Sanli T, Rashid A, Liu C, Harding S, Bristow RG, Cutz JC, et al. Ionizing radiation activates AMP-activated kinase (AMPK): a target for radiosensitization of human cancer cells. Int J Radiat Oncol Bool Phys 2010; 78: 221-9. doi: 10.1016/j.ijrobp
12.	Zhang WB, Wang Z, Shu F, Jin YH, Liu HY, Wang QJ, et al. Activation of AMP-activated protein kinase by temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition. J Biol Chem 2010; 285: 40461-71. doi: 10.1074/jbc.M110.164046
13.	Bischof M, Abdollahi A, Gong P, Stoffregen C, Lipson KE, Debus JU, et al. Triple combination of irradiation, chemotherapy (pemetrexed), and VEGFR inhibition (SU5416) in human endothelial and tumor cells. Int J Radiat Oncol Biol Phys 2004; 60: 1220-32. doi: 10.1016/j.ijrobp
14.	Combs SE, Bohl J, Elsasser T, Weber KJ, Schulz-Ertner D, Debus J, et al. Radiobiological evaluation and correlation with the local effect model (LEM) of carbon ion radiation therapy and temozolomide in glioblastoma cell lines. Int J Radiat Biol 2009; 85: 126-37. doi: 10.1080/09553000802641151
15.	Liu X, Chhipa RR, Pooya S, Wortman M, Yachyshin S, Chow LM, et al. Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK. Proc Natl Acad Sci U S A 2014; 111: E435-44. doi: 10.1073/ pnas.1311121111
16.	Wurth R, Pattarozzi A, Gatti M, Bajetto A, Corsaro A, Parodi A, et al. Metformin selectively affects human glioblastoma tumor-initiating cell viability: A role for metformin-induced inhibition of Akt. Cell Cycle 2013; 12: 145-56. doi: 10.4161/cc.23050
17.	Janjetovic K, Harhaji-Trajkovic L, Misirkic-Marjanovic M, Vucicevic L, Stevanovic D, Zogovic N, et al. In vitro and in vivo anti-melanoma action of metformin. Eur J Pharmacol 2011; 668: 373-82. doi: 10.1016/j.ejphar
18.	Scotland S, Saland E, Skuli N, de Toni F, Boutzen H, Micklow E, et al. Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells. Leukemia 2013; 27: 212938. doi: 10.1038/leu
19.	Feng Y, Ke C, Tang Q, Dong H, Zheng X, Lin W, et al. Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by down-regulating Stat3 signaling. Cell Death Dis 2014; 5: e1088. doi: 10.1038/cddis
20.	Tomic T, Botton T, Cerezo M, Robert G, Luciano F, Puissant A, et al. Metformin inhibits melanoma development through autophagy and apop-tosis mechanisms. Cell Death Dis 2011; 2: e199. doi: 10.1038/cddis
21.	Taghian A, Suit H, Pardo F, Gioioso D, Tomkinson K, DuBois W, et al. In vitro intrinsic radiation sensitivity of glioblastoma multiforme. Int J Radiat Oncol Biol Phys 1992; 23: 55-62.
22.	Harrabi S, Combs SE, Brons S, Haberer T, Debus J, Weber KJ. Temozolomide in combination with carbon ion or photon irradiation in glioblastoma multiforme cell lines - does scheduling matter? Int J Radiat Biol 2013; 89: 692-7. doi: 10.3109/09553002
23.	Yu Z, Zhao G, Li P, Li Y, Zhou G, Chen Y, et al. Temozolomide in combination with metformin act synergistically to inhibit proliferation and expansion of glioma stem-like cells. Oncol Lett 2016; 11: 2792-800. doi: 10.3892/ ol.2016.4315
24.	Shi WY, Xiao D, Wang L, Dong LH, Yan ZX, Shen ZX, et al. Therapeutic met-formin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy. Cell Death Dis 2012; 3: e275. doi: 10.1038/cddis
25.	Zheng B, Jeong JH, Asara JM, Yuan YY, Granter SR, Chin L, et al. Oncogenic B-RAF negatively regulates the tumor suppressor LKB1 to promote melanoma cell proliferation. Mol Cell 2009; 33: 237-47. doi: 10.1016/j.mol-cel.2008.12.026
26.	Rios M, Foretz M, Viollet B, Prieto A, Fraga M, Costoya JA, et al. AMPK activation by oncogenesis is required to maintain cancer cell proliferation in astrocytic tumors. Cancer Res 2013; 73: 2628-38. doi: 10.1158/0008-5472
27.	Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, et al. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2003; 2: 28. doi: 10.1186/1475-4924-2-28
28.	Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 2004; 101: 3329-35. doi: 10.1073/pnas.0308061100
29.	Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, et al. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 2003; 13: 2004-8.
30.	Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, et al. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 2005; 18: 283-93. doi: 10.1016/j.molcel
31.	Fogarty S, Hardie DG. Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer. Biochim Biophys Acta 2010; 1804: 581-91. doi: 10.1016/j.bbapap
32.	Guo D, Cloughesy TF, Radu CG, Mischel PS. AMPK: A metabolic checkpoint that regulates the growth of EGFR activated glioblastomas. Cell Cycle 2010; 9: 211-2. doi: 10.4161/cc
33.	Isakovic A, Harhaji L, Stevanovic D, Markovic Z, Sumarac-Dumanovic M, Starcevic V, et al. Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis. Cell Mol Life Sci 2007; 64: 1290-302. doi: 10.1007/s00018-007-7080-4
34.	Kisfalvi K, Eibl G, Sinnett-Smith J, Rozengurt E. Metformin disrupts crosstalk between G protein-coupled receptor and insulin receptor signaling systems and inhibits pancreatic cancer growth. Cancer Res 2009; 69: 6539-45. doi: 10.1158/0008-5472.CAN-09-0418
35.	Sato A, Sunayama J, Okada M, Watanabe E, Seino S, Shibuya K, et al. Glioma-initiating cell elimination by metformin activation of FOXO3 via AMPK. Stem Cell Trans Med 2012; 1: 811-24. doi: 10.5966/sctm.2012-0058
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