Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA
© Costa et al.; licensee BioMed Central Ltd. 2014
Received: 25 February 2014
Accepted: 4 June 2014
Published: 2 July 2014
Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential.
Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype.
We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29.
These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.
Metformin (N,N-dimethylbiguanide) is an oral biguanide in clinical use since the 1950s for its hypoglycemic activity. Currently, it is the most widely used anti-type 2 diabetes drug, with nearly 120 million prescriptions worldwide filled every year . Metformin decreases hyperglycemia primarily by activating 5’-adenosine monophosphate-activated protein kinase (AMPK) to suppress glucose production in the liver, increase glucose utilization, and reduce hyperinsulinemia .
Recent population studies suggested that metformin decreases the incidence of several cancers and cancer-mortality in diabetic patients [3–6], and improves the response to chemotherapy in diabetic breast cancer patients . Additionally, this therapeutic potential has been confirmed in vitro since metformin inhibits in breast, colon, lung, prostate, and pancreas cancer cell proliferation [8–11]. These studies highlight a direct antitumoral activity of metformin, besides the possible indirect effects mediated by the improvement of the metabolic parameters and, in particular, of the hyperinsulinemia. More recently, prospective studies also demonstrated that preoperative metformin treatment of non-diabetic patients with breast (two weeks) or colorectal aberrant cryptic foci (one month) provided a reduction of the number of proliferative cells [12, 13].
Interestingly, it was shown that the antitumor effect exerted by metformin in breast cancer, glioblastoma, and hepatocellular carcinoma cells is mainly mediated by a directed and selective antiproliferative activity against the cancer stem/tumor initiating cell (TIC) fraction [14–17]. According to the cancer stem cell theory this cell subpopulation represents the main pharmacological target to obtain efficacious therapeutic responses in tumors [18–20].
In this work we address, for the first time, the possible anticancer effect of metformin in a high risk neuroblastoma (NB) cell model, including cancer cell lines displaying different levels of differentiation and stemness/tumor initiating potential.
In particular, we document a significant inhibition of NB cells proliferation and viability exerted by metformin. Interestingly, overexpression of NDM29, a NB differentiating non-coding (nc)-RNA, transcribed by RNA polymerase III, and able to reduce cell tumorigenicity [21–23], leads to an increased cell sensitivity towards metformin, while all trans-retinoic acid (ATRA)-induced differentiation reduced metformin NB cell susceptibility.
These findings provide the basis for further, deeper investigations on the possible usefulness of metformin as adjuvant/neo-adjuvant treatment for NB, and its specific role in the stemness/differentiation balance of tumor cells.
Materials and methods
Cell Cultures and metformin treatment
Cell lines: SH-SY5Y, grown in DMEM (Sigma–Aldrich), supplemented with 10% FBS (GIBCO), L-glutamine (2 mM; EuroClone), and penicillin–streptomycin (100 U/ml/ 100 μg/ml; EuroClone); SKNBE2, grown in RPMI (Sigma–Aldrich), supplemented with 10% FBS (GIBCO), L-glutamine (2 mM; EuroClone), and penicillin–streptomycin (100 U/ml/ 100 μg/ml; Euro Clone).
SKNBE2 cells were transfected using polyethylenimine (PEI; Sigma P3143) with pEGFP-N1 as control (hereafter referred to as pMock) or pEGFP-N1-NDM29 (hereafter referred to as NDM29). G418 (geneticin; Invitrogen) was used in culture medium as mean of selection up to 1000 μg/ml, until resistant clones were identified. After selection, the clones were preserved in 200 μg/ml G418 in standard culture conditions. Treatment with metformin (20 mM) was performed when cell culture reached 80% of confluence. ATRA treatment was performed in SKNBE2 and SHSY5Y neuroblastoma cells grown in RPMI or DMEM medium with 10% FBS. Cells were grown for 2 days to reach the log phase of growth. When cell cultures reached 80% of confluence the medium was replaced with RPMI or DMEM medium containing 10% FBS and ATRA (1 or 10 μM) or DMSO (0.01% or 0.1%) in control cultures. Cells were then grown for 10 days before the experiments were performed.
Cell proliferation and cytotoxicity assays
A) Real time cell proliferation and cytotoxicity was assessed by xCELLigence RTCA DP System (Roche, Germany), as reported . This system monitors cellular events in real time by measuring electrical impedance across interdigitated gold micro-electrodes integrated on the bottom of tissue culture plates. The impedance measurement provides quantitative information about the biological status of the cells, including cell number, viability, and morphology. Cell-sensor impedance is expressed as an arbitrary unit called Cell Index . In order to calculate CI, cells were seeded into 100 μL of standard medium in 96X microtiter plates (E-Plate-Roche, Germany). Background impedance was determined using 50 μl of standard medium. After 24 hrs, 20 mM metformin was added to the wells and cell proliferation was monitored for 72 hrs or more. Cell adhesion, spreading and proliferation were monitored every 30 min using the xCELLigence system to produce time-dependent cell response dynamic curves. All experimental results were obtained using RTCA Software 1.2 of the xCELLigence system.
B) Cell counting studies: cell from the different lines were plated in 6-well plates, incubated in standard medium for approximately 24 hrs before being treated with 20 mM metformin. Cells were counted with a hemocytometer after 24 and 48 hrs of metformin treatment and evaluated by Trypan blue exclusion test in which live cells are able to exclude the dye from the cytosol.
C) [3H]-thymidine incorporation assay: different cell lines were plated in 24-well plates, incubated in standard medium for 24 hrs, then treated with 20 mM metformin. After 24 and 48 hrs of metformin treatment, cells were pulsed with [3H]-thymidine (0.3 μCi/500 μl/well) (GE Healthcare, New York, NY) for the last 14 hrs. Averaged proliferation rate was then calculated by the thymidine uptake assay.
D) ATPlite 1step Luminescence assay: this system measures cellular ATP levels, as a marker of cell viability (Perkin Elmer, Monza, Italy). ATPlite 1step assay system is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. The emitted light is proportional to ATP concentration. Cell suspension (100 μl) was seeded in 96-well culture plate white (Thermo Scientific). After 24 hrs, 100 μl culture medium containing metformin (20 mM) was added to the cells. Each group had 5 repeats. The plates for the ATP-Lite assay were incubated for 24 or 48 hrs at 37°C. Then 100 μl of culture medium was removed from each well using a manual multichannel pipet, followed by the addition to each well of 100 μl cell substrate solution (ATPlite kit content). The plate was shaken for 2 min followed by dark adapted for 10 min and luminescence was measured using a luminometer (TECAN Genios Pro reader).
E) SYTOX Blue dead cell stain: this is a high-affinity nucleic acid stain that easily penetrates dead cells with compromised plasma membranes but not in the healty ones. Cells were plated in 6 well-plates, incubated in standard medium for 24 hrs, and treated with 20 mM metformin for 24 or 48 hrs at 37°C. After brief incubation with SYTOX Blue stain, nucleic acids of dead cells fluoresce bright blue when excited with 405 nm violet laser light. Samples were analyzed using a Cyan ADP cytofluorimeter (Beckman-Coulter, Brea CA, USA). For each sample, 20,000 events were acquired. The data were analyzed using Summit 4.3.1 software (Beckman-Coulter, USA).
Cells were plated onto 60-mm dishes for 24 hrs before being treated. Cells were lysed in buffer containing 1% Nonidet P-40, 20 mM Tris–HCl, pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM NaF (all from Sigma), and the “Complete” protease inhibitor mixture (Roche Applied Science) for 10 min at 4°C . Nuclei were removed by centrifugation (13,200 rpm at 4°C, for 2 min), and total protein content was measured using the Bradford assay (Bio-Rad). Proteins (20 μg) were resuspended in 2X reducing sample buffer (2% SDS, 62.5 mM Tris, pH 6.8, 0.01% bromphenol blue, and 1.43 mM β-mercaptoethanol, 0.1% glycerol), electrophoresed on 10% SDS polyacrylamide gels, transferred on polyvinylidene difluoride membrane (Bio-Rad), and probed with specific antibodies directed against total and phospho-Akt (Ser-473), total and phospho-AMPk, total and phospho-mTOR, and procaspase-3 and cleaved caspase-3, all purchased from Cell Signaling Technology (Beverly, MA); α-tubulin (Sigma) was used as protein loading standard. Immunocomplex detection was performed using ECL system (Bio-Rad), as reported .
Real time quantitative RT-PCR analysis
Total RNAs from samples were extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, DNAseI-digested and subjected to reverse transcription by Transcriptor High Fidelity cDNA Synthesis Kit (Roche 05081955001) following manufacturer’s instructions. The total RNA from samples was measured by real-time quantitative RT-PCR using PE ABI PRISM@ 7700 Sequence Detection System (Perkin Elmer Corp./Applied Biosystems, Foster City, CA) and SybrGreen method. The sequences of forward and reverse primers were: NDM29, 5’-GGCAGGCGGGTTCGTT-3’ and 5’-CCA CGCCTGGCTAAGTTTTG-3’; neurofilament-68 (NF-68), 5’-CAAGGACGAGGTGTCCG AG-3’ and 5’-CCCGGCATGCTTCGA; Integrin beta-1 (CD29), 5’-AACGAGGTCATGGTT CATGTTG-3’ and 5’-ACCACACCAGCTACAATTGGAA-3’. For endogenous control, the expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene was examined. The sequences for human GAPDH rRNA forward and reverse primers were 5’-GAAGGTGAAG GTCGGAGTC-3’ and 5’-GAAGATGGTGATGGGATTTC-3’. Relative transcript levels were determined from the relative standard curve constructed from stock cDNA dilutions, and divided by the target quantity of the calibrator following manufacturer's instructions.
Experiments were performed at least in triplicate. Data are reported as mean values ± standard error (SE). Statistical significance of observed differences among different experimental groups was examined using the unpaired Student's t-test, as reported . Statistical analysis were performed before normalization (metformin vs control).
Metformin inhibits neuroblastoma cell growth
In order to evaluate whether the reduction in Cell Index was directly associated to a decrease of cell proliferation, control and metformin-treated cells were counted using an hemocytometer. Results confirmed a significant reduction of the cell number 48 hrs after metformin administration (Figure 1B). In addition, a statistically significant reduction of the mitotic rate of metformin-treated SKNBE2 cells was confirmed by a statistically significant decrease in DNA synthesis 24 hrs after the treatment, measured by [3H]-thymidine incorporation (Figure 1C). Next, metformin effects on cell viability were tested using the ATPlite assay, which, measuring intracellular ATP content in metabolically active cells, is an indirect index of cell viability. In agreement with the previously described experiments, we found that, after 48 hrs of treatment, metformin leads to a statistically significant decrease in ATP content (Figure 1D).To strengthen these results, and verify whether metformin effects were merely cytostatic or cytotoxicity was also induced, we quantified, by FACS analysis, the percentage of dead cells by SYTOX blue incorporation assay, in which the intracellular accumulation of the dye is an index of dead cells. Again, results showed that metformin increased the number of cell death after 48 hrs of treatment (Figure 1E).In order to assess the mechanism of cell death induced by metformin we tested by Western blot the possible activation of the apoptotic machinery measuring the expression of the cleaved/activated caspase-3. However, 8, 24 and 48 hours of treatment with metformin 20 mM did not increase the cleaved caspase-3 intracellular content and unaltered caspase-3 pro-enzyme expression was detected (Figure 1F). In addition, in treated cells, we observed neither the up-regulation of the pro-apoptotic protein Bax, nor the presence of nuclear piknosis, both common features of apoptotic cells (data not shown). Altogether these results exclude apoptosis from the major causes of cell death induced by metformin.
To investigate at molecular level the intracellular pathways altered by metformin to affect SKNBE2 cells proliferation and survival, we measured its effects on Akt phosphorylation/activation. Akt inhibition, known to correlate with impaired cell survival, was recently shown to represent a molecular correlate of metformin direct antitumoral effects . In Western blot experiments, we report that metformin (20 mM) induced a long-lasting (5–60 min) decline in Akt phosphorylation/activation (Figure 1G), suggesting that metformin effects in SKNBE2 cells were mediated by the inhibition of Akt activity. Conversely, AMPK/mTOR pathways was not affected (see below).
Altogether these results suggest that metformin is an effective antiproliferative agent towards NB cell lines with different malignant potential.
Effects of metformin on differentiated neuroblastoma cell growth
NB is composed by extremely heterogeneous cell populations, including cells at different maturation stages. Thus, we investigated the efficacy of metformin as antiproliferative agent in NB cells at different level of differentiation and malignancy.
Since in differentiated hepatic cells metformin hypoglycemic effects are mainly mediated by the modulation of the AMPK/mTOR pathway, we examined whether this may occur also in differentiated NB cells. By western blot analysis we compared the effects of metformin on AMPK and mTOR phosphorylation before and after the induction of ATRA-mediated differentiation in SKNBE2 cells. As reported in Figure 3F, no differences on the activity (phosphorylation) of AMPk or mTOR has been found in both differentiated and undifferentiated cells. Altogether, these results show that the susceptibility to the effects of metformin is directly correlated to NB differentiation level, as reported in glioblastoma cells  and confirmed that AMPK seems not involved in the antiproliferative effects of the drug.Next we investigated the occurrence of this effect in NB cells highly responsive to ATRA-induced differentiation. We found that, although less sensitive to metformin than SKNBE2 cells, SHSY5Y cells were highly influenced by ATRA treatment (1 μM, for 10 days) that completely abolished the ability of metformin to reduce cell proliferation (Figure 3G,H). Higher ATRA concentrations (10 μM, for 10 days) did not lead to additional changes in metformin susceptibility (data not shown).
To delve deeper into the mechanisms by which differentiation may affect metformin antiproliferative effects in NB cells, we took advantage of a novel in vitro model of NB differentiation, we recently developed, that was based on the expression level of a nc-RNA, named NDM29, in SKNBE2 cells [21, 22, 30]. In this system the level of differentiation of NB cells, as well as the reduction of the stemness/tumor initiating potential, is directly related to the level of synthesis of NDM29 [21, 22, 30]. Here, we compared the effects of metformin in mock transfected cells, that express low levels of endogenous NDM29 RNA, with established cell lines in which different levels of NDM29 are overexpressed. The overexpression of ncRNA NDM29 represent a novel model of NB cell differentiation, resulting in the acquisition of neuronal electrophysiological properties as previously reported .
These results highlight an unexpected different modulation of the response to metformin after NB cell differentiation, which is not correlated to differentiation per se but directly depend on the expression level of NDM29 ncRNA.
We previously reported that NDM29 sensitizes SKNBE2 cells to several cytotoxic drugs (cisplatin, doxorubicin) by powerfully down-regulating MDR1 expression . Thus, we verified whether the increased sensitivity of metformin observed in S1 cells might be related to NDM29-dependent alterations of the expression of molecules involved in the metformin interaction with the cells. In particular, we focused on the expression of the organic cation transporter 1 (OCT-1) that represents one of the main regulators of metformin cell internalization. However, in Western blot analysis we did not find significant differences in OCT-1 expression in pMock, S1 and S2 cells (data not shown), suggesting that different mechanisms should be responsible of the higher sensitivity to metformin of NDM29 expressing cells. Conversely, we observed that in SH-SY5Y OCT-1 expression was slightly reduced after metformin treatment (data not shown), possibly representing the molecular correlate for the lower sensitivity of these cells as compared to SKBNE2.
Besides being a first-line antidiabetic drug, metformin is currently under consideration for additional anticancer properties [33–35]. Recent reports evidenced TICs from different cancer types as the preferential targets of this drug [14–16]. These studies are in line with reports showing that also other antidiabetic drugs, such as PPAR-γ agonists exert cytostatic effects [36, 37].
In this study we report, for the first time, an antiproliferative effect of metformin in a high-risk NB cell model. We show that metformin induces a significant reduction in the proliferation rate in two different NB cell lines (SKNBE2 and SH-SY5Y) characterized by different N-myc expression, although a higher sensitivity was observed in the first one. While we cannot provide a definitive answer for this difference, the observation that in SH-SY5Y cells, OCT-1 expression, which control the cellular internalization of this metformin, is down-regulated upon metformin treatment, could account for this difference.
In line with previous studies , this effect was related to a powerful inhibition of Akt activation, suggesting that the treatment with metformin could directly act on cell viability. In fact, beside an effect on cell cycle progression, as suggested by the reduction of DNA synthesis in [3H]-thymidine incorporation experiments, also increased cell death was observed in SYTOX blue staining experiments. Interestingly, metformin effects on cell survival did not were the results of activation of apoptosis since we did not detect nuclear shrinkage, caspase 3 activation or Bax up-regulation. Thus, from our data metformin elicits mainly a cytostatic effect that could indirectly cause cell death via apoptosis-independent pathways.
Interestingly, no changes in AMPK activity was observed in NB cells, although this kinase was reported to represent a key intracellular mechanism in metformin effects . However, several studies suggested that mechanisms, others than AMPK activation, could mediate antiproliferative activity of metformin [39–41]. In particular, although several different mechanisms were identified in different tumoral cells, down-regulation of IGF-1 or other growth factor mediated autocrine signalling was suggested as main antiproliferative mechanisms regulated by metformin [42, 43]. In turn, this effect could be responsible for the inhibition of pro-survival intracellular pathways, such as Akt, as we show in this study in NB cells and previously reported in GBM TICs . However, further studies are still required to definitely address this issue.
Importantly, as shown in several other tumor cell types [14, 15], metformin antitumoral activity was significantly higher in less differentiated NB cells, with a lower effect in differentiated cells (here we used ATRA-dependent differentiation).
Conversely, we found that metformin sensitivity is highly increased in NB cells in which differentiation is induced by NDM29 overexpression. This effect was strictly dependent on the expression levels of this ncRNA, which also correlate with the induction of NB cell differentiation. These data, and in particular the direct relationship between NDM29 expression and the reduction of NB cell viability induced by metformin, strongly suggest the possibility of a causal effect between the two events. At present we do not know the exact molecular mechanisms by which NDM29-dependent differentiation increases NB cell susceptibility to metformin, at odd with ATRA effects. In particular, since we previously reported that NDM29 overexpression inhibits MDR1 expression, representing a molecular mechanism that increases NB cell vulnerability to antimitotic cytotoxic drugs , we hypothesized that NDM29 expression might affect susceptibility to metformin inducing OCT-1 expression, thus increasing the amount of drug internalized into the cells and able to interfere with survival signals, such as Akt activity. However, here we report that SKBNE2 cells express similar OCT-1 content independently from the levels of NDM29 expression, and thus a different molecular mechanism needs to be identified.
Our study for the first time demonstrates that metformin exerts antitumor activity against high risk NB cells, reducing cell proliferation and viability, via inhibition of Akt phosphorylation, showing higher sensitivity for less differentiated, highly proliferative cells. These data represent the starting point for further studies aimed to test the possible application of metformin in NB therapy. Moreover, as already shown for other cytotoxic drugs, the overexpression of NDM29, although inducing neuronal differentiation, powerfully sensitizes NB cells to metformin antiproliferative effects, suggesting that the pharmacological modulation of the expression of this ncRNA may represent a potential goal in the NB therapy. The molecular determinants by which the differentiation induced by NDM29, but not by retinoic acid, increases the antiproliferative activity of metformin will also represent a future goal in the translational research of novel NB therapies.
Delfina Costa and Arianna Gigoni share first authorship.
A.P. was supported by the Associazione Italiana Ricerca sul Cancro (2009 AIRC Program n° IG9378), by the IRCCS-AOU San Martino-IST, Genova-Italy (Progetto 5 × 1000) and by the Associazione Italiana per la Lotta al Neuroblastoma/Fondazione Neuroblastoma (Genoa, Italy).
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