Metformin enhanced in vitro radiosensitivity associates with G2/M cell cycle arrest and elevated adenosine-5’-monophosphate-activated protein kinase levels in glioblastoma

The relationship of tumorigenesis, and tumor cell metabolism by an increased glycolysis was first described in the early 20th century by Otto Warburg.1,2 Multiple modifications in cancer cell metabolism have subsequently been detected, but the influence on alterations in signaling pathways, 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 apoptotic 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.6,7

AMPK is a serine/threonine kinase that functions as a cellular energy sensor. AMPK is an obligate heterotrimer, consisting of one catalytic subunit (α) and two regulatory subunits (β and γ).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 phosphorylation (pAMPK), seems to be associated with tumor cell growth and cell survival.9,10 Furthermore, increased AMPK phosphorylation has been observed in cells following radiation-induced DNA damage in several studies.7,11,12 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 temozolomide (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.

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 progression-free 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 blockade15,16, inhibition of tumor growth17,18 and induction of autophagy and apoptosis19,20 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 consumption.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 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 metformin 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 antiproliferative 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, 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 mTOR6,15,24,25 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 LKB1.27,28,29 Furthermore, AMPK has been associated with p53-dependent apoptosis through p53 phosphorylation30, 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.32,33,34,35

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.6,15

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 metformin, 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.

Together with our previously published clinical findings3 and the well-established use of metformin in clinical practice, these data show that radiosensitizing effects of metformin on glioblastoma cells treated with irradiation and temozolomide in vitro coincided with G2/M arrest and changes in pAMPK levels.