Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens’ effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue’s noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.
Citation: Gentile MT, Ciniglia C, Reccia MG, Volpicelli F, Gatti M, Thellung S, et al. (2015) Ruta graveolens L. Induces Death of Glioblastoma Cells and Neural Progenitors, but Not of Neurons, via ERK 1/2 and AKT Activation. PLoS ONE 10(3): e0118864. doi:10.1371/journal.pone.0118864
Academic Editor: Gerolama Condorelli, Federico II University of Naples, Italy, ITALY
Received: July 25, 2014; Accepted: January 7, 2015; Published: March 18, 2015
Copyright: © 2015 Gentile et al. 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
Data Availability: All relevant data are within the paper.
Funding: This work was supported by Progetto Sicurezza, sostenibilità e competitività nelle produzioni Agroalimentari delle Campania “CARINA” (POR FSE 2007–2013), Network per la salvaguardia e la gestione delle risorse genetiche agro-alimentari “AGRIGENET” (PSR 2007/2013, to CC), and Programma di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN, 20109MXHMR_004, to MABM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors report no biomedical financial interests or potential conflicts of interest with regards to this manuscript.
Gliomas comprise several types of primary brain tumors accounting for approximately 50% of all neoplasms of the central nervous system (CNS) [1–3]. In particular, glioblastoma, IV grade glioma, is characterized by marked cell proliferation and heterogeneity, invasiveness and neoangiogenesis, presenting rapid progression and high frequency of recurrence [4, 5]. Therefore, the prognosis for the patients is extremely poor, with mean survival of about 14 months, even after the introduction of temozolomide [6, 7], currently the gold standard cytotoxic drug for gliomas, and few patients survive beyond five years . Other treatment options are limited, and in most cases ineffective and the survival rate for these patients remains extremely low [9–13]. The cell type that gives origin to glioblastoma is still an open issue. It has been reported that either dysregulated neural stem cells, or dedifferentiated glial and neuronal cells are involved in tumor development [14, 15]. Besides the derivation of the tumoral cells, recent evidence suggests that the malignant features of glioblastoma, including radio-chemo-resistance, relay on a subset of tumoral cells endowed with stem-like properties. Thus, this subpopulation has been named as cancer stem-like cells, tumor initiating cells, or cancer propagating cells [16–19].
A number of molecular abnormalities have been involved in the pathogenesis of glioblastoma, including growth factors (i.e. EGF, PDGF, HGF, VEGF) and growth factors receptors (EGFR and HGFR) that are often upregulated, overexpressed and/or constitutively activated. Among the intracellular signaling cascades, Ras-ERK1/2, PI3K/AKT, p53 and Rb play a key role in promoting cellular transformation. In particular, upon alterations of tyrosine kinase receptors, ERK1/2 and PI3K/AKT constitutive signaling seem to be constantly present in glioblastoma, and combined activation of RAS and AKT in neural progenitors is sufficient to induce glioblastoma in mice [20–30]. Targeting specific molecular alterations is a strategy for the development of cancer therapy. Thus, a number of selective inhibitors of molecules and/or pathways involved in the pathogenesis of glioblastoma have been developed and some of them entered clinical trials. Nevertheless, for reasons largely unclear, clinical response is poor. Therefore, there is still an urgent need for novel and effective therapies for treating these tumors.
On this issue, natural product-based molecules represent interesting therapeutic alternatives. Over the past decades, cell culture and animal studies allowed the identification of numerous dietary and botanical natural compounds with anti-cancer effects, including curcumin, epigallocatechin gallate, ellagic acid and resveratrol, extracted from the Curcuma longa, from the grape skin, from the green tea and from many fruits and vegetables, respectively [31–40]. Diet or botanical-derived compounds are also considered for cancer preventive properties, due to the fact that they are less toxic, easily available, cost-effective and better accepted by patients. Recently, several clinical trials have been started to investigate the preventive and therapeutic efficacy of natural compounds.
Ruta graveolens L. (R. graveolens) is a perennial plant, native of the Mediterranean region but cultivated throughout Europe and many Asian countries, including China, India and Japan. R. graveolens, commonly known as rue, is known as medicinal plant since ancient times and currently used, particularly in Asian countries, for treatment of various disorders such as aching pain, eye problems, rheumatism and dermatitis [41, 42]. The extract of the plant contains more than 120 compounds of different classes of natural products such as acridone alkaloids, coumarins, essential oils, flavonoids and furoquinolines . The components of R. graveolens species are of great interest in medicinal chemistry, as these compounds show a broad range of biological activities, and a number of them are already used in medicine. Alcoholic extracts of R. graveolens have been tested for anti-proliferative effect on different types of cancer cells, pointing towards a potential therapeutic effect in oncology [44–49].
The present study was aimed to assess the effects of the aqueous extract of R. graveolens on the proliferation of human glioma cells and of neural progenitors from mouse CNS, in comparison to differentiated, non-proliferating neural cells. Moreover, we evaluated the effects of two drugs, temozolomide and cisplatin, widely used in the GBM chemotherapy on proliferating and non proliferating neural cells as comparators of the R. graveolens extract. Finally, we investigated the modulation of ERK1/2 and AKT activities as molecular correlate of the biological effects of R. graveolens extract.
Materials and Methods
R. graveolens is not a protected species, leaves were collected from plants conserved at the Experimental Section of Medicinal Plants at the Botanical Garden of Naples, Italy with the permission of the “Orto Botanico” director Prof. Paolo De Luca. Whole leaves were harvested before the flowering stages, during spring 2013. 250 g of leaves were chopped, boiled in 1 L of distilled water at 110°C for 5 minutes. The extract was subsequently filtered through 0.22 μm filters (MILLEXGP, MILLIPORE, Bedford, MA), frozen under liquid nitrogen and lyophilized (VirTis-SP Scientific). When necessary for the experiments, the aqueous extract (a.e.) was diluted with MEM/F12 medium to standard concentration of 50 mg/ml.
A1 mes c-myc cells (A1) are an established cell line, generated in our laboratory as previously reported [50, 51]. A1 cells were cultured in MEM/F12 medium (Invitrogen, Milan, Italy) supplemented with 10% FBS (Invitrogen) and differentiated by serum withdrawal and stimulation with 1 mM cAMP (Sigma-Aldrich, Milan, Italy) and N2 supplement (Invitrogen). U87MG and U138 human glioblastoma cells (American Type Culture Collection, Rockville, MS, USA) [52–53] were cultured in DMEM (Invitrogen) supplemented with 10% FBS. C6 rat glioma cells  were cultured in Ham’s-F12 (Invitrogen) supplemented with 10% FBS. We investigated R. graveolens a.e. effects at the concentration of 0.01, 0.1, 1 and 10 mg/ml for 24, 48 and 72 hours. The results showed that the viability of U87MG, U138, C6 and A1 cells significantly decreased as the dose and the time increase with similar values at 1 and 10 mg/ml (data not shown). Rutin was purchased from Sigma-Aldrich and added at the indicated concentrations. For the inhibition tests, cells were pre-treated with 10 μM PD98058 (Sigma-Aldrich) or 1μM wortmannin (Sigma-Aldrich) 1h before addition of R. graveolens a.e.
Cell proliferation assay
For in vitro cell proliferation assay, MTT and Trypan blue exclusion tests were performed. MTT assay: cells were seeded at 3x105 cells/well in a 24 well plate in the presence or absence of 1mg/ml of R graveolens a.e., and cell proliferation assessed after 24, 48 and 72 hours. According to manufacturer’s recommendations, 50 μl of 3-(4,5 dymethylthiazol-2-il)-2,5 dyphenyl-2H-tetrazolium bromide (MTT) reagent (5mg/ml in PBS) was added to each well and, then, the cells were incubated at 37°C for three hours. One volume (500μl) of Stop mix solution (20% SDS in 50% dimethyl formamide) was added to each well and incubated at room temperature for a minimum of 1h. The plate was read at 550nm and at 630 nm as the reference wavelength. Same volume of medium without cells was used as blank. Results are expressed as OD.
Trypan blue exclusion test: cells were seeded at 3x105 cells/well in a 24 well plate in presence or not of 1mg/ml of R. graveolens a.e. Cell proliferation was assessed 24, 48 and 72 h after R. graveolens a.e. addition. Cells were harvested and resuspended in 1ml of PBS. 0.2 ml of cell suspension were added to 0.5 ml of PBS and 0.3 ml of 0.4% of Trypan blue solution (Lonza, Walkersville, MD, USA). After 5 min at room temperature, cells were counted in a Burker’s chamber.
The occurrence of necrosis and/or apoptosis was analyzed by Tali image-based cytometer (Invitrogen). Briefly, cells were seeded at 106 cells/plate in 60mm plates, treated or not with 1mg/ml of R. graveolens a.e. for 24, 48 and 72 h. Cells were harvested and resuspended in ABB (annexin binding buffer), 5 μl of Annexin V 488 Alexa Fluor was added to each sample and incubated at room temperature, in the dark, for 20 min. Cells were centrifuged and resuspended in 100 μl of ABB, 1 μl of Tali. Propidium iodide was added to each sample and incubated 5 min at room temperature, in the dark. Emitted fluorescence was analyzed by Tali image-base cytometry and expressed as number of cells/100. To confirm the data obtained by Tali image-based cytometry, a morphological apoptosis assay was performed. In particular, 48 hr after R. graveolens a.e. addition, cells were washed three times in PBS, and then fixed with 4% paraformaldehyde for 10min at room temperature (RT). Cells were rinsed with PBS, and 0.1μg/ml DAPI (4′6 diamidi-no-2-phenylindole, for nuclei staining) was added for 10 min at RT. After washing with PBS, the cells were detected with fluorescence microscopy (AxioImager M2, Zeiss, Germany), and cells with condensed and/or fragmented chromatin indicative of apoptosis were not counted as living cells. 250 fields/well were counted using a Scion Image version 4.5 software.
Caspase 3 activation was assessed by means of CPP32 Colorimetric Kit (BioVision Inc., CA, USA). Briefly, cells were treated with 1mg/ml R. graveolens a.e. for 24 and 48 hours and harvested. 1x106 cells were incubated with chilled Cell Lysis Buffer for 10 minutes. Protein concentration was determined by Bradford method. 100 μg of total protein were additioned with 5μl of 4 mM DEVD-pNA caspase 3 substrate, and incubated at 37°C for 2 hours. Samples were read at 400 nm. Fold-increase in CPP32 activity was determined by comparing samples absorbance at 400 nm to the absorbance of the control extracts.
Isolation of cytosolic protein was performed by scraping the cells in lysis buffer (25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.1 mM NaF, 0.1 mM Na3VO4). Protein concentration was determined by the Bradford method. 20 μg of protein extract were subjected to a 10% SDS-PAGE and electroblotted on nitrocellulose membranes, which were blocked at room temperature for 1 h in blocking solution (5% milk in TBS-T), incubated overnight at 4°C with anti-phsphoERK1/2 or anti-phosphoAKT antibodies (1 μg/ml; Millipore, Milan, Italy). Membrane was incubated with peroxidase-conjugated anti-rabbit antibodies (1:10,000; GE Healthcare Life Sciences, Milan, Italy). Proteins were revealed by ECL (Millipore). Normalization was performed with anti-ERK1/2 or anti AKT antibodies (1 μg/ml in TBS-T; Millipore) for 1 h at room temperature and incubated with peroxidase-conjugated anti-mouse IgG (1:10,000 in TBS-T). Proteins were revealed as above. The intensity of the bands was quantified by scanning densitometry using Scion Image version 4.5 software.
R. graveolens aqueous extract induces cell death of glioma cell lines and of proliferating mesencephalic cells.
To assess the antiproliferative effects of R. graveolens water extract, MTT reduction assay and Trypan blue exclusion test were performed on the human glioblastoma cell line U87MG. In particular, incubation for 24 h with 1 mg/ml R. graveolens a.e determined cell proliferation arrest, as compared to vehicle treated cells, and induced cell death for treatments of 48, 72 and 96 h (Fig. 1A). These results were confirmed by Trypan blue exclusion test (Fig. 1B). Similar results were also obtained in U138 and C6 human and rat glioma cells, respectively (S1 Fig). Moreover, as expected, MTT reduction assay, performed on the same cell lines in the presence of temozolomide (TMZ) or cisplatin (CIS), two alkylating agents currently used to treat human GBM, showed that the two drugs induced a significant cell death already after 24 h of treatment, (data not shown).
(A) MTT assay on proliferating U87MG human glioma cells treated with vehicle (♦) or with 1mg/ml R. graveolens a.e. (■) for 24, 48, 72, 96h, *p<0,01 vs control conditions. (B) Trypan blue exclusion test on U87MG glioma cells treated (light gray) or not (dark gray) with 1mg/ml R. graveolens a.e. for 24, 48 and 72h; *<0,01 vs control conditions.
Similar experiments were performed on A1 cells. A1 cells derived from E11 mouse mesencephalon were immortalized by means of infection with a c-myc-carrying retroviral vector. These cells proliferate and remain undifferentiated when grown in the presence of serum whereas they cease to proliferate and differentiate, ensuing neurite outgrowth, neuronal electrophysiological properties, and expression of neuronal markers, when serum is withdrawn and cAMP is added  (Fig. 2A). MTT reduction assay and Trypan blue exclusion test were performed on proliferating-undifferentiated and differentiated-non proliferating A1 cells to evaluate the effects of R. graveolens a.e on cell viability. As shown in Fig. 2B, the treatment of A1 cells with 1 mg/ml R. graveolens a.e. was able to inhibit cell proliferation in proliferating A1 cells after 24h, and to induce cell death after 48 (viability decreases of about 120% as compared to vehicle treated conditions) and 72h (viability decreases of about 200% as compared to vehicle treated conditions). Trypan blue exclusion test confirmed these results showing that 1mg/ml R. graveolens a.e. significantly decreased the number of viable proliferating A1 cells, after 48 and 72 h of treatment (Fig. 2D). Conversely, both MTT assay and Trypan blue viability test performed on differentiated, non-proliferating A1 cells, revealed no statistically significant difference between control and treated cells after 48 hours from the addition of 1 mg/ml R. graveolens a.e (Fig. 2C, E). Moreover, MTT reduction assay was performed on A1 cells, in both undifferentiated and differentiated conditions, treated with TMZ and CIS. CIS and TMZ caused a significant cytotoxicity in both proliferating (Fig. 3A and B) and differentiated-non proliferating A1 cells (Fig. 3C and D).
(A) Microphotographs of the mouse mesencephalic embryonic cell line A1 mes c-myc (A1). They are proliferating/undifferentiated in the presence of serum (left panel) but acquire a neuronal phenotype upon serum withdrawal and stimulation with cAMP (right panel). (B) MTT assay on proliferating A1 cells in control conditions (♦) or treated with 1mg/ml R. graveolens a.e. (■) for 24, 48, 72 and 96 hours. *p<0.01 vs controls. (C) MTT assay on differentiated A1 cells in control conditions (♦) or treated with 1mg/ml R. graveolens a.e. (■) for 48 and 72 hours *p<0.01 vs controls. (D) Trypan blue exclusion test on proliferating A1 cells treated (light grey) or not (dark grey) with 1mg/ml R. graveolens a.e. for 24, 48 and 72h; *p<0.01 vs controls. (E) Trypan blue exclusion test on differentiated A1 cells treated (light gray) or not (dark gray) with 1mg/ml R. graveolens a.e. for 48 and 72h; *p<0.01 vs controls.
(A-B) MTT assay on proliferating A1 cells treated with increasing concentrations of temozolomide (A) and cisplatin (B) for 48 (♦) and 72 (■) hours; *p<0.01 vs controls; (C-D) MTT assay on differentiated A1 cells treated with increasing concentrations of temozolomide (C) and cisplatin (D) for 48 (♦) and 72 (■) hours; *p<0.01 vs controls.
R. graveolens aqueous extract induces activation of ERK1/2 and AKT pathways.
To investigate the molecular mechanisms by which R. graveolens a.e. promotes cell death in glioma and A1 cells, we analyzed the modulation of ERK1/2 and AKT cascades by means of Western blotting. As showed in Fig. 4, R. graveolens a.e. increased ERK1/2 phosphorylation in all the glioma cell lines analysed as well as in non-differentiated A1 cells. In particular, as compared to control conditions, ERK1/2 phosphorylation was increased by 4-folds in C6 cells, as early as 5 min after the addition of the R. graveolens extract. The activation of ERK1/2 lasted up to 30 minutes with a further 1,5 fold increase 60 min after the treatment (Fig. 4A). In U138 cells, R. graveolens induced a significant increase in ERK1/2 phosphorylation 5 and 10 minutes after treatment with a return to control levels 60 minutes after treatment (Fig. 4D). Similar results were obtained in A1 cells (Fig. 4C) and in U87MG cells (Fig. 4D) in which after a rapid activation of ERK1/2, longer treatments resulted in a partial reduction of the effect. These results suggest that the antiproliferative effects of R. graveolens could be related to activation of the ERK1/2 pathway. Being this result rather unexpected, we verified this relationship analyzing cell proliferation in the presence of the selective MEK inhibitor PD98059. MTT assay performed on U87MG, U138, C6 and A1 cells treated or not with 1mg/ml R. graveolens a.e. for 48h in the presence of PD98059 (10μM), revealed that the inhibition of ERK1/2 pathway rescued cell viability impairment induced by R. graveolens a.e. treatment (Fig. 4E-H).
Western blotting detection of p-ERK1/2 and ERK1/2 proteins in C6 glioma cells (A), in U138 glioma cells (B), in A1 cells (C) and in U87MG (D) treated with 1mg/ml R. graveolens a.e. for 5, 10, 30 and 60 minutes. Two specific bands are observed respectively at 44 and 42 kDa. Each blot is representative of three separate experiments. The graphs show the relative quantitation of p-ERK1/2 and ERK1/2 in the different cell lines. Data are expressed as ratios of p-ERK/ERK. Asterisks represent p<0.05 vs controls. (G-H) MTT assay in U87MG cells (E), A1 cells (F), C6 glioma cells (G) and U138 cells (H) treated for 48 hours with 1mg/ml R. graveolens a.e. (R48), 10μM PD98059 (PD) or in combination (PD+R48); *p<0.01 vs control conditions.°p<0.05 vs R48.
We also evaluated the role of AKT in the R. graveolens effects. AKT phosphorylation as index of kinase activation, was assessed in U87MG, C6 and proliferating A1 mes c-myc cells, by Western blot analysis. As shown in Fig. 4, the treatment with R. graveolens significantly increased also AKT activation, showing a sustained phosphorylation for all the length of the treatment (up to 60 minutes). However, while in U87MG and C6 glioma cells (Fig. 5A, B) AKT phosphorylation status remained stable at the peak levels, in A1 mes c-myc cells (Fig. 4C), after a rapid (5 minutes) 2-fold increase, AKT phosphorylation partially declined, although remaining at a significantly higher levels as compared to control conditions, up to 60 minutes.
To confirm the involvement of PI3K/AKT pathway in the antiproliferative effect of R. graveolens a.e., cell proliferation was assessed in the presence of the selective PI3K inhibitor wortmannin. MTT assay performed on U87MG, C6 and A1 cells treated or not with 1mg/ml R. graveolens a.e. for 48h in the presence of wortmannin (1μM). These experiments revealed that also the inhibition of R. graveolens-activated AKT rescued cell viability impairment as compared to cells treated with R. graveolens a.e. alone (Fig. 5 D-F). The preincubation with the combination of the two inhibitors (PD98059 and wortmannin) did not induce a higher protective effect on the cells viability (data not shown), suggesting that the two pathways do not cooperate.
(A) Western blotting detection of p-AKT and AKT proteins in U87MG (A), in C6 glioma cells (B) and in A1 cells (C) treated with 1mg/ml R. graveolens a.e. for 5, 10, 30 and 60 minutes. A specific band is observed at 60 kDa. Each blot is representative of three separate experiments. The graphs show the relative quantitation of p-AKT and AKT in the different cell lines. Data are expressed as ratios of p-AKT/AKT. Asterisks represent p<0.05 vs controls. (D-E) MTT assay in U87MG cells (D), C6 glioma cells (E) and A1 cells (F) treated for 48 hours with 1mg/ml R. graveolens a.e. (R48) or with 1μM wortmannin (W), or in combination (W+R48); *p<0.01 vs control conditions.°p<0,05 vs R48.
R. graveolens aqueous extract induces apoptosis in proliferating/undifferentiated A1 mes-c-myc cells.
In order to elucidate the type of cell death induced by R. graveolens a.e., the analysis of annexin V binding and propidium iodide (PI) nuclear staining was used to discriminate between apoptotic and necrotic cell death, using Tali image-based cytometry. As shown in Fig. 6A, R. graveolens a.e. (1mg/ml), administered for 48 h, significantly increased the number of apoptotic A1 cells as compared to vehicle-treated cells. Noteworthy, no changes in the number of necrotic cells was observed in treated and control cells (Fig. 6A). These data were confirmed evaluating nuclear morphology after DAPI staining, in A1 cells before and after treatment with R. graveolens a.e. (1mg/ml) for 48 hours. As shown in Fig. 6B, the number of apoptotic nuclei (i.e. showing condensed and/or fragmented morphology) was significantly higher (+400%) in treated cells as compared to control conditions. Altogether, these data indicated that R. graveolens a.e. was able to induce cell death in proliferating A1 cells activating the apoptotic program. To corroborate this finding we measured caspase 3 activity in A1, U87MG and C6 cells. As shown in Fig. 6 (C-E), R. graveolens a.e. treatment increased caspase 3 activity of about 100% over control cells in A1 mes c-myc cells and caused a 4-fold induction in U87MG and C6 cells. Moreover, preincubation with PD98059, wortmannin, or both inhibitors completely abolished this effect in both A1 and glioma cells (Fig. 6 C-E). The treatment with either inhibitor alone did not modulate caspase 3 activity as compared to untreated cells (data not shown).
(A) Cell cycle was analyzed by means of Tali image-based cytometry on proliferating A1 cells in control conditions (dark grey) and 48h after 1mg/ml R. graveolens a.e. treatment (light grey) *p<0.01 vs controls (B) Number of apoptotic nuclei/100 cells treated (R48) or not (CTRL) with 1 mg/ml R. graveolens a.e. for 48 hours. *p<0.01 vs controls. (C-E) Caspase 3 activity expressed as absorbance at 400 nm in A1 cells (C), U87MG cells (D) and C6 cells (E) treated with vehicle (CTRL), 1mg/ml R. graveolens a.e. for 24 (R24) or 48 (R48) hours, 10μM PD98059 in combination with ml R. graveolens a.e. for 48 hours (PD+R48), 1μM wortmannin in combination with ml R. graveolens a.e. for 48 hours (W+R48) or the combination of the two inhibitors (PD+W+R48) for 48 hours. *p<0.01 vs control conditions.
Rutin, a major component of R. graveolens extract, does not affect proliferating A1 cells viability.
Rutin is a major component of R. graveolens extract . Thus, we tested the effect of rutin on A1 undifferentiated/proliferating cells. MTT (data not shown) and Trypan blue (Fig. 7) assays demonstrate that rutin treatment (3μg/ml, 30μg/ml and 300μg/ml for 24, 48 and 72h) does not affect proliferating A1 cell viability, and suggest that rutin by itself is not responsible of the cell death induced by R. graveolens a.e.
Discussion and Conclusions
A water extract from R. graveolens induces caspase 3-dependent apoptotic cell death of human and rat glioblastoma cells and of a neural progenitor cell line (A1 cells) derived from embryonic mouse CNS. Interestingly, upon differentiation and cell cycle exit, A1 cells become insensitive to the noxious effects of R. graveolens extract. GBM aggressive behaviour and poor prognosis is partly due to its peculiar ability to massively invade surrounding brain tissues preventing a complete surgical excision. The property of R. graveolens extract to discriminate between proliferating (i.e. tumor cells) and non-proliferating neural cells, as shown in this study, has a potential to be exploited as therapeutic tool in GBM. Further experiments, especially exploring at a molecular level these effects, are, however, required to definitely ascertain the absence of noxious effects on fully differentiated, non-proliferating neurons. It is worth noting that it was previously shown that R. graveolens extract or some of its components were able to exert anti tumor activity in vivo and in vitro [56, 57]. In particular, R. graveolens extract in combination with calcium phosphate was shown to induce cell death of glioma cells but proliferation of peripheral blood limphocytes  Although this study provided data showing that rue extract exerts both in vitro and in vivo therapeutic effects on brain tumors, it has been basically ignored in the literature. The reasons for that may depend upon different matters. Indeed, this study does not provide clues on the molecular mechanism(s) underlying the observed therapeutic effects. Moreover, rue extract was administered as a omeopathic solution in combination with CaPO4 and no explanation was given for that protocol. In addition, in literature there are contrasting results on the genotoxic and clastogenic effects of R. graveolens extract on healthy and tumor cells . Thus, our study may provide a new spin on the use of R. graveolens extracts as a therapeutic tool. To this regard, it is worth noting that the concentrations of rue extract used in our experiments are significantly lesser than those tested by Preethi , and, in addition, we indicate a signal transduction pathway involved in the effects. Finally, it is of note that differently from the studies of Pathak  and Preethi  who tested the pharmacological effect of the alcoholic extract of R. graveolens, our study reported the antiproliferative effect of the water extract of the plant.
Drugs used to treat GBM, including temozolomide  and cisplatin , two alkylating agents that target the DNA of both cancer and normal cells and are currently used in first- and second-line treatment, respectively, cause cognitive impairment such as loss of memory and learning, which are even more severe in the case of childhood brain. Although the underlying cellular and molecular mechanisms are largely unknown, these side-effects, often referred to as “chemo-brain”, are likely due to normal brain cells damages . Importantly, in our model these drugs, differently form R. graveolens extract, were not able to discriminate among glioma or proliferating A1 cells and differentiated A1 cells causing a significant cell death in all the cell populations analysed. Moreover, tumours treated with temozolomide or cisplatin develop chemo-resistance largely due to high methyl-guanine methyl transferase (MGMT) levels and to inactivation of mismatch repair enzymes MLH1 and MSH29 and over-expression of multidrug resistance proteins, respectively . Thus, it is important to explore new, more efficacious and less toxic therapeutic approaches. Terrestrial and marine life can generate a number of compounds, termed secondary metabolites, in response to intense ecological competition in order to survive in their environment. Most of these compounds are endowed with biological properties, including anti-cancer and/or chemo-preventive effects [36, 37]. Preclinical and clinical studies have shown that water or alcoholic natural extracts, as a whole and/or molecules isolated from such extracts, can exert a number of different effects useful to fight tumors. In particular, natural compounds can exert anti-cancer activity by means of different mechanisms: i) directly killing transformed cells; ii) acting synergistically with classical chemotherapy by improving its efficacy and, thus, allowing a lower dose of drug to be effective; iii) reverting the drug-dependent resistance; iv) providing antioxidant molecules; and iv) activating and/or enhancing endogenous anti-cancer defences, including immune system. A specific goal of anti-cancer research is to find natural products endowed with toxicity against cancer cells but harmless to healthy cells. Although many natural products are promising therapeutic tools since in preclinical studies they were shown to kill transformed cells, nevertheless few data are present about their effects on normal tissues, in particular as far as CNS is concerned .
With regard to selectivity, in a previous collaborative study, together with marine eco-physiologists and chemists, we reported that a compound extracted from microalgae exerts very different effects according to the differentiation status of the cells . In particular, 2–4, trans-trans decadienal, an aldehyde presents in the microalgae diatomee, produces its noxious effects on copepods, tiny crustaceans their natural predators, in a manner that is dependent upon cell differentiation. Thus, diatomee-derived aldehydes are harmless towards adult copepods whereas when used to feed mothers, they are teratogen to larvae, causing anatomic malformations and death of eggs. We showed that this differentiation-dependent mechanism is maintained along the phylogenies. In fact decadienal causes apoptotic cell death in the undifferentiated, proliferating mammalian neural cell line A1, generated from embryonic mouse mesencephalon, whereas, upon differentiation and cell cycle exit, the same cells become unresponsive to the noxious effects of the same compound. It seems that 2,4, trans-trans decadienal and R. graveolens share the same selective effects on proliferating/undifferentiated and non-proliferating differentiated neural cells, at least in the A1 cellular model.
Rutin, the major component of R. graveolens water extract that we administered on cells, was not able, even at high concentrations, to affect the viability of A1 cells grown under proliferating, undifferentiated cells, thus indicating that either rutin is necessary but not sufficient to elicit cell death or even that other compounds present in minor amounts in R. graveolens a.e. are responsible for most of the observed biological effects. Further experiments will assess the involvement of rutin and/or of other compounds within R. graveolens water extract in determining the biological effects observed.
Interestingly, as far as R. graveolens antiproliferative intracellular signalling, we show the R. graveolens extract stimulates the phosphorylation/activation of both ERK1/2 and AKT. A number of natural compounds are able to interfere with ERK1/2 and AKT activation. In turn, in many instances, perturbation of ERK1/2 and AKT signalling is responsible for anti-cancer effects. ERK1/2 and AKT signalling have been classically related to cell proliferation and survival. Indeed many mutated oncogenes cause the constitutive activation of these pathways and contribute to cell transformation [64–67]. In fact, selective ERK1/2 and AKT inhibitors or receptor-mediated reduction of the activity of these kinases have been shown to halt uncontrolled cell proliferation or to kill transformed cells [68–74]. Nevertheless, the biological effects of ERK1/2 and AKT are cell context- and/or stimulation mode-dependent. For instance, as far as ERK1/2 is concerned, its activation can elicit, on the one hand, either proliferation or differentiation and cell cycle exit, and on the other, either cell survival or cell death, according to the intensity and/or timing of stimulation [75, 76]. We found that R. graveolens-induced cell death decreases, in both glioma and A1 cells, upon blocking, by means of selective inhibitors, ERK1/2 or AKT, thus showing that, in our experimental conditions, ERK1/2 and AKT, at least partly, mediate the toxic effects. Similar results were obtained analyzing caspase 3 activity induced by R. graveolens. Indeed, caspase 3 was abolished upon inhibition of ERK1/2 and/or AKT activity by PD98059 or wortmannin. As a possible mechanism by which increased ERK activity may result in antiproliferative effects, it was reported, in a different cell context (i.e. CHO cells), that the antiproliferative activity of somatostatin was related to ERK1/2-dependent induction of the cell cycle inhibitor p21cip1 . Further experiments are required to identify the molecules involved also in the signalling cascade originated by R. graveolens. However, a similar paradoxical activation of both ERK1/2 and AKT was reported in glioma cell lines to mediate the antiproliferative activity of adiponectin , confirming that in glioma cells over-activation of these kinase cascades is responsible of antiproliferative effects. Moreover, in A549 lung carcinoma cells, quercetin, a metabolite in the degradation pathway of rutin, was shown to induce activation of ERK and, as a consequence, of caspase-3 and apoptosis. In turn, activation of MEK-ERK was required for quercetin-induced apoptosis .
Finally, although the precise mechanisms of the anti-proliferative action of rue extract are unknown, there are evidence suggesting that the action of flavonoids might be mediated by their interaction with aryl hydrocarbon receptor or estrogen binding sites, which have been shown to be occupied by flavonoid-like molecule with growth inhibitory properties [80–82].
In conclusion, our data show that R. graveolens a.e. is endowed with potent antitumoral activity in human glioma cells and in undifferentiated cells originated from mouse embryonic brain, whereas it results harmless towards the same differentiated, non-proliferating cell line. Thus, it represents a potential new therapeutic tool for brain cancer therapy and also for investigating the molecular events underlying the toxicity of neural cells to exogenous compounds.
S1 Fig. R. graveolens extract induces cell death of C6 and of human U138 glioma cells.
MTT assay on proliferating C6 glioma cells (A) and U138 human glioma cells (B) treated with vehicle (♦) or with 1mg/ml R. graveolens a.e. (■), *p<0,01 vs control conditions.
We are deeply grateful to Prof. Giovanni Aliotta (Naples) for sharing with us his previous work on Ruta graveolens and for illuminating discussions.
Conceived and designed the experiments: LCDA TF. Performed the experiments: MTG MGR CC MG ST. Analyzed the data: LCDA TF MTG FV. Contributed reagents/materials/analysis tools: LCDA TF FV MABM. Wrote the paper: LCDA.
- 1. Preusser M, de Ribaupierre S, Wöhrer A, Erridge SC, Hegi M, Weller M, et al. Current concepts and management of glioblastoma. Ann Neurol. 2011;70: 9–21. doi: 10.1002/ana.22425. pmid:21786296
- 2. Johnson BE, Mazor T, Hong C, Barnes M, Aihara K, McLean CY, et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science. 2014;343: 189–193. doi: 10.1126/science.1239947. pmid:24336570
- 3. Mielcke TR, Mascarello A, Filippi-Chiela E, Zanin RF, Lenz G, Leal PC, et al. Activity of novel quinoxaline-derived chalcones on in vitro glioma cell proliferation. Eur J Med Chem. 2012;48: 255–264. doi: 10.1016/j.ejmech.2011.12.023. pmid:22209415
- 4. Youland RS, Schomas DA, Brown PD, Nwachukwu C, Buckner JC, Giannini C, et al. Changes in presentation, treatment, and outcomes of adult low-grade gliomas over the past fifty years. Neuro Oncol. 2013;15: 1102–1110. doi: 10.1093/neuonc/not080. pmid:23814262
- 5. Shibahara I, Sonoda Y, Shoji T, Kanamori M, Saito R, Inoue T, et al. Malignant clinical features of anaplastic gliomas without IDH mutation. Neuro Oncol. 2014;17: 136–144. doi: 10.1093/neuonc/nou112. pmid:24958096
- 6. Cheng YP, Lin C, Lin PY, Cheng CY, Ma HI, Chen CM, et al. Midkine expression in high grade gliomas: Correlation of this novel marker with proliferation and survival in human gliomas. Surg Neurol Int. 2014;5: 78. doi: 10.4103/2152-7806.133205. pmid:24949221
- 7. Kamson DO, Mittal S, Robinette NL, Muzik O, Kupsky WJ, Barger GR, et al. Increased tryptophan uptake on PET has strong independent prognostic value in patients with a previously treated high-grade glioma. Neuro Oncol. 2014:16: 1373–1383. doi: 10.1093/neuonc/nou042. pmid:24670609
- 8. Vredenburgh JJ, Desjardins A, Reardon DA, Friedman HS. Experience with irinotecan for the treatment of malignant glioma. Neuro Oncol. 2009;11: 80–91. doi: 10.1215/15228517-2008-075. pmid:18784279
- 9. Van den Bent MJ, Hegi ME, Stupp R. Recent developments in the use of chemotherapy in brain tumours. Eur J Cancer. 2006;42: 582–588. pmid:16427778
- 10. Hilario A, Sepulveda JM, Perez-Nuñez A, Salvador E, Millan JM, Lagares A, et al. A prognostic model based on preoperative MRI predicts overall survival in patients with diffuse gliomas. AJNR Am J Neuroradiol. 2014;35: 1096–1102. doi: 10.3174/ajnr.A3837. pmid:24457819
- 11. Yi L, Hou X, Zhou J, Xu L, Ouyang Q, Liang H, et al. HIF-1α Genetic Variants and Protein Expression Confer the Susceptibility and Prognosis of Gliomas. Neuromolecular Med. 2014;16: 578–586. doi: 10.1007/s12017-014-8310-1. pmid:24929654
- 12. Rème T, Hugnot JP, Bièche I, Rigau V, Burel-Vandenbos F, Prévot V, et al. A Molecular Predictor Reassesses Classification of Human Grade II/III Gliomas. PLoS One. 2013;8: e66574. pmid:23805239
- 13. Oszvald Á, Quick J, Franz K, Güresir E, Szelényi A, Vatter H, et al. Resection of gliomas in the cingulate gyrus: functional outcome and survival. J Neurooncol. 2012;109: 341–348. doi: 10.1007/s11060-012-0898-0. pmid:22660921
- 14. Friedmann-Morvinski D, Bushong EA, Ke E, Soda Y, Marumoto T, Singer O, et al. Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. Science. 2012;338: 1080–1084. doi: 10.1126/science.1226929. pmid:23087000
- 15. Friedmann-Morvinski D, Verma IM. Dedifferentiation and reprogramming: origins of cancer stem cells. EMBO Rep. 2014;15: 244–253. doi: 10.1002/embr.201338254. pmid:24531722
- 16. Ilkanizadeh S, Lau J, Huang M, Foster DJ, Wong R, Frantz A, et al. Glial progenitors as targets for transformation in glioma. Adv Cancer Res. 2014;121: 1–65. doi: 10.1016/B978-0-12-800249-0.00001-9. pmid:24889528
- 17. Tamura K, Aoyagi M. Stem cells and the origin of different glioma subtypes. Response. J Neurosurg. 2014;120: 1010–1011. pmid:24809083
- 18. Gronych J, Pfister SM, Jones DT. Connect four with glioblastoma stem cell factors. Cell. 2014;157: 525–527. doi: 10.1016/j.cell.2014.04.001. pmid:24766799
- 19. Florio T, Barbieri F. The status of the art of human malignant glioma management: the promising role of targeting tumor-initiating cells. Drug Discov Today. 2012;17: 1103–1110. doi: 10.1016/j.drudis.2012.06.001. pmid:22704957
- 20. Li L, Puliyappadamba VT, Chakraborty S, Rehman A, Vemireddy V, Saha D, et al. EGFR wild type antagonizes EGFRvIII-mediated activation of Met in glioblastoma. Oncogene. 2015;34: 129–34. doi: 10.1038/onc.2013.534. pmid:24362532
- 21. Xie J, Ma YH, Wan M, Zhan RY, Zhou YQ. Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes. Oncol Lett. 2014;7: 131–136. pmid:24348834
- 22. Auf G, Jabouille A, Delugin M, Guérit S, Pineau R, North S, et al. High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor. BMC Cancer. 2013;13: 597. doi: 10.1186/1471-2407-13-597. pmid:24330607
- 23. Burrell K, Singh S, Jalali S, Hill RP, Zadeh G. VEGF regulates region-specific localization of perivascular bone marrow-derived cells in Glioblastoma. Cancer Res. 2014;74: 3727–3739. doi: 10.1158/0008-5472.CAN-13-3119. pmid:24820020
- 24. Clara CA, Marie SK, de Almeida JR, Wakamatsu A, Oba-Shinjo SM, Uno M, et al. Angiogenesis and expression of PDGF-C, VEGF, CD105 and HIF-1α in human glioblastoma. Neuropathology. 2014;34: 343–352. doi: 10.1111/neup.12111. pmid:24612214
- 25. McNamara MG, Sahebjam S, Mason WP. Emerging biomarkers in glioblastoma. Cancers (Basel). 2013;5: 1103–1119. doi: 10.3390/cancers5031103. pmid:24202336
- 26. Höland K, Boller D, Hagel C, Dolski S, Treszl A, Pardo OE, et al. Targeting class IA PI3K isoforms selectively impairs cell growth, survival, and migration in glioblastoma. PLoS One. 2014;9: e94132. doi: 10.1371/journal.pone.0094132. pmid:24718026
- 27. Lee JS, Xiao J, Patel P, Schade J, Wang J, Deneen B, et al. A novel tumor-promoting role for nuclear factor IA in glioblastomas is mediated through negative regulation of p53, p21, and PAI1. Neuro Oncol. 2014;16: 191–203. doi: 10.1093/neuonc/not167. pmid:24305710
- 28. Zheng H, Ying H, Yan H, Kimmelman AC, Hiller DJ, Chen AJ, et al. p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation. Nature. 2008;455: 1129–1133. doi: 10.1038/nature07443. pmid:18948956
- 29. Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet. 2000;25: 55–57. pmid:10802656
- 30. Sun Y, Zhang W, Chen D, Lv Y, Zheng J, Lilljebjörn H, et al. A glioma classification scheme based on coexpression modules of EGFR and PDGFRA. Proc Natl Acad Sci U S A. 2014;111: 3538–3543. doi: 10.1073/pnas.1313814111. pmid:24550449
- 31. Sallam AA, Mohyeldin MM, Foudah AI, Akl MR, Nazzal S, Meyer SA, et al. Marine natural products-inspired phenylmethylene hydantoins with potent in vitro and in vivo antitumor activities via suppression of Brk and FAK signaling. Org Biomol Chem. 2014;12: 5295–5303. doi: 10.1039/c4ob00553h. pmid:24927150
- 32. Mace TA, King SA, Ameen Z, Elnaggar O, Young G, Riedl KM, et al. Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling. Cancer Immunol Immunother. 2014;63: 889–900. doi: 10.1007/s00262-014-1564-5. pmid:24893859
- 33. Li L, Leung PS. Use of herbal medicines and natural products: An alternative approach to overcoming the apoptotic resistance of pancreatic cancer. Int J Biochem Cell Biol. 2014;53C: 224–236.
- 34. Haghighitalab A, Matin MM, Bahrami AR, Iranshahi M, Saeinasab M, Haghighi F. In vitro investigation of anticancer, cell-cycle-inhibitory, and apoptosis-inducing effects of diversin, a natural prenylated coumarin, on bladder carcinoma cells. Z Naturforsch C. 2014;69: 99–109. pmid:24873030
- 35. Wang CY, Bai XY, Wang CH. Traditional chinese medicine: a treasured natural resource of anticancer drug research and development. Am J Chin Med. 2014; 42: 543–559. doi: 10.1142/S0192415X14500359. pmid:24871650
- 36. Skropeta D, Wei L. Recent advances in deep-sea natural products. Nat Prod Rep. 2014;31: 999–1025. doi: 10.1039/c3np70118b. pmid:24871201
- 37. Luo F, Gu J, Chen L, Xu X. Systems pharmacology strategies for anticancer drug discovery based on natural products. Mol Biosyst. 2014;10: 1912–1917. doi: 10.1039/c4mb00105b. pmid:24802653
- 38. Velázquez KT, Enos RT, Narsale AA, Puppa MJ, Davis JM, Murphy EA, et al. Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice. J Nutr. 2014;144: 868–875. doi: 10.3945/jn.113.188367. pmid:24759931
- 39. Seo BR, Min KJ, Cho IJ, Kim SC, Kwon TK. Curcumin significantly enhances dual PI3K/Akt and mTOR inhibitor NVP-BEZ235-induced apoptosis in human renal carcinoma Caki cells through down-regulation of p53-dependent Bcl-2 expression and inhibition of Mcl-1 protein stability. PLoS One. 2014;9: e95588. doi: 10.1371/journal.pone.0095588. pmid:24743574
- 40. Zhuang W, Long L, Zheng B, Ji W, Yang N, Zhang Q, et al. Curcumin promotes differentiation of glioma-initiating cells by inducing autophagy. Cancer Sci. 2012;103: 684–690. doi: 10.1111/j.1349-7006.2011.02198.x. pmid:22192169
- 41. Conway GA, Slocumb JC. Plants used as abortifacients and emmenagogues by Spanish New Mexicans. J Ethnopharmacol. 1979;1: 241–261. pmid:232204
- 42. Kuzovkina I, Al'terman I, Schneider B. Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens. Phytochemistry. 2004;65: 1095–100. pmid:15110689
- 43. Gutiérrez-Pajares JL, Zúñiga L, Pino J. Ruta graveolens aqueous extract retards mouse preimplantation embryo development. Reprod Toxicol. 2003;17: 667–72. pmid:14613818
- 44. Ratheesh M, Shyni GL, Sindhu G, Helen A. Inhibitory effect of Ruta graveolens L. on oxidative damage, inflammation and aortic pathology in hypercholesteromic rats. Exp Toxicol Pathol. 2011;63: 285–90. doi: 10.1016/j.etp.2010.01.007. pmid:20163942
- 45. Ratheesh M, Sindhu G, Helen A. Anti-inflammatory effect of quinoline alkaloid skimmianine isolated from Ruta graveolens L. Inflamm Res. 2013;62: 367–376. doi: 10.1007/s00011-013-0588-1. pmid:23344232
- 46. Fadlalla K, Watson A, Yehualaeshet T, Turner T, Samuel T. Ruta graveolens extract induces DNA damage pathways and blocks Akt activation to inhibit cancer cell proliferation and survival. Anticancer Res. 2011;31: 233–241. pmid:21273604
- 47. Brower V. Back to nature: extinction of medicinal plants threatens drug discovery. J Natl Cancer Inst. 2008;100: 838–839. doi: 10.1093/jnci/djn199. pmid:18544733
- 48. Li JW and Vederas JC. Drug discovery and natural products: end of an era or an endless frontier? Science. 2009;325: 161–165. doi: 10.1126/science.1168243. pmid:19589993
- 49. Pollio A, De Nauale A, Appetiti E, Aliotta G, Touwaide A. Continuity and change in the Mediterranean medical tradition: Ruta spp. (rutaceae) in Hippocratic medicine and present practices. J Ethnopharmacol. 2008;116: 469–482. doi: 10.1016/j.jep.2007.12.013. pmid:18276094
- 50. Colucci-D'Amato GL, Tino A, Pernas-Alonso R, ffrench-Mullen JM, di Porzio U. Neuronal and glial properties coexist in a novel mouse CNS immortalized cell line. Exp Cell Res. 1999;252: 383–91. pmid:10527628
- 51. Gentile MT, Nawa Y, Lunardi G, Florio T, Matsui H, Colucci-D'Amato L. Tryptophan hydroxylase 2 (TPH2) in a neuronal cell line: modulation by cell differentiation and NRSF/rest activity. J Neurochem. 2012;123: 963–970. doi: 10.1111/jnc.12004. pmid:22958208
- 52. Bastida E, Ordinas A, Escolar G, Jamieson GA. Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. Blood. 1984;64: 177–184. pmid:6733271
- 53. Rehemtulla A, Murphy P, Dobson M, Hart DA. Purification and partial characterization of a plasminogen activator inhibitor from the human glioblastoma, U138. Biochem Cell Biol. 1988;66: 1270–1277. pmid:3149898
- 54. Benda P, Lightbody J, Sato G, Levine L, Sweet W. Differentiated rat glial cell strain in tissue culture. Science. 1968;161: 370–371. pmid:4873531
- 55. Chen CC, Huang YL, Huang FI, Wang CW, Ou JC. Water-soluble glycosides from Ruta graveolens. J Nat Prod. 2001;64: 990–992. pmid:11473445
- 56. Pathak S, Multani AS, Banerji P, Banerji P. Ruta 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer. Int J Oncol. 2003;23: 975–982. pmid:12963976
- 57. Réthy B, Zupkó I, Minorics R, Hohmann J, Ocsovszki I, Falkay G. Investigation of cytotoxic activity on human cancer cell lines of arborinine and furanoacridones isolated from Ruta graveolens. Planta Med. 2007;73: 41–48. pmid:17109253
- 58. Preethi KC, Nair CK, Kuttan R. Clastogenic potential of Ruta graveolens extract and a homeopathic preparation in mouse bone marrow cells. Asian Pac J Cancer Prev. 2008;9: 763–769. pmid:19256773
- 59. Ruggiero A, Rizzo D, Attinà G, Lazzareschi I, Maurizi P, Ridola V. Phase I study of temozolomide combined with oral etoposide in children with malignant glial tumors. J Neurooncol. 2013;113: 513–518. doi: 10.1007/s11060-013-1145-z. pmid:23666235
- 60. Zustovich F, Lombardi G, Della Puppa A, Rotilio A, Scienza R, Pastorelli D. A phase II study of cisplatin and temozolomide in heavily pre-treated patients with temozolomide-refractory high-grade malignant glioma. Anticancer Res. 2009;29: 4275–4279. pmid:19846986
- 61. Gong X, Schwartz PH, Linskey ME, Bota DA. Neural stem/progenitors and glioma stem-like cells have differential sensitivity to chemotherapy. Neurology. 2011;76: 1126–1134. doi: 10.1212/WNL.0b013e318212a89f. pmid:21346220
- 62. Spencer D. Fighting brain tumors while protecting the brain: the stem cell story. Neurology. 2011;76: e69–70. doi: 10.1212/WNL.0b013e318215b914. pmid:21444897
- 63. Ianora A, Miralto A, Poulet SA, Carotenuto Y, Buttino I, Romano G. et al. Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom. Nature. 2004;429: 403–407. pmid:15164060
- 64. Califano D, D'Alessio A, Colucci-D'Amato GL, De Vita G, Monaco C, Santelli G, et al. A potential pathogenetic mechanism for multiple endocrine neoplasia type 2 syndromes involves ret-induced impairment of terminal differentiation of neuroepithelial cells. Proc Natl Acad Sci U S A. 1996;93: 7933–7937. pmid:8755580
- 65. Colucci-D'Amato GL, D'Alessio A, Califano D, Cali G, Rizzo C, Nitsch L. et al. Abrogation of nerve growth factor-induced terminal differentiation by ret oncogene involves perturbation of nuclear translocation of ERK. J Biol Chem. 2000;275: 19306–19314. pmid:10858459
- 66. Califano D, Rizzo C, D'Alessio A, Colucci-D'Amato GL, Cali G, Bartoli PC et al. Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells. J Biol Chem. 2000;275: 19297–19305. pmid:10748077
- 67. Rizzo C, Califano D, Colucci-D'Amato GL, De Vita G, D'Alessio A, Dathan NA, et al. Ligand stimulation of a Ret chimeric receptor carrying the activating mutation responsible for the multiple endocrine neoplasia type 2B. J Biol Chem. 1996;271: 29497–29501. pmid:8910618
- 68. Ye Q, Cai W, Zheng Y, Evers BM, She QB. ERK and AKT signaling cooperate to translationally regulate survivin expression for metastatic progression of colorectal cancer. Oncogene. 2014;33: 1828–1839. doi: 10.1038/onc.2013.122. pmid:23624914
- 69. Jing Y, Liu LZ, Jiang Y, Zhu Y, Guo NL, Barnett J, et al. Cadmium increases HIF-1 and VEGF expression through ROS, ERK, and AKT signaling pathways and induces malignant transformation of human bronchial epithelial cells. Toxicol Sci. 2012;125: 10–19. doi: 10.1093/toxsci/kfr256. pmid:21984483
- 70. Chappell WH, Steelman LS, Long JM, Kempf RC, Abrams SL, Franklin RA, et al. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR inhibitors: rationale and importance to inhibiting these pathways in human health. Oncotarget. 2011;2: 135–164. pmid:21411864
- 71. Brzezianska E, Pastuszak-Lewandoska D. A minireview: the role of MAPK/ERK and PI3K/Akt pathways in thyroid follicular cell-derived neoplasm. Front Biosci (Landmark Ed). 2011;16: 422–439. pmid:21196179
- 72. Shi Z, Hodges VM, Dunlop EA, Percy MJ, Maxwell AP, El-Tanani M, et al. Erythropoietin-induced activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK pathways promotes malignant cell behavior in a modified breast cancer cell line. Mol Cancer Res. 2010;8: 615–626. doi: 10.1158/1541-7786.MCR-09-0264. pmid:20353997
- 73. Barbieri F, Pattarozzi A, Gatti M, Porcile C, Bajetto A, Ferrari A, et al. Somatostatin receptors 1, 2, and 5 cooperate in the somatostatin inhibition of C6 glioma cell proliferation in vitro via a phosphotyrosine phosphatase-eta-dependent inhibition of extracellularly regulated kinase-1/2. Endocrinology. 2008;149: 4736–4746. doi: 10.1210/en.2007-1762. pmid:18566118
- 74. Massa A, Barbieri F, Aiello C, Arena S, Pattarozzi A, Pirani P, et al. The expression of the phosphotyrosine phosphatase DEP-1/PTP dictates the responsivity of glioma cells to somatostatin inhibition of cell proliferation. J Biol Chem. 2004;279: 29004–29012. pmid:15123617
- 75. Deschênes-Simard X, Kottakis F, Meloche S, Ferbeyre G. ERKs in Cancer: Friends or Foes? Cancer Res. 2014;74: 412–419. doi: 10.1158/0008-5472.CAN-13-2381. pmid:24408923
- 76. Colucci-D'Amato L, Perrone-Capano C, di Porzio U. Chronic activation of ERK and neurodegenerative diseases. Bioessays. 2003;25: 1085–1095. pmid:14579249
- 77. Florio T, Yao H, Carey KD, Dillon TJ, Stork PJS. Somatostatin activation of mitogen-activated protein kinase via somatostatin receptor 1 (SSTR1). Mol Endocrinol. 1999;13: 24–37. pmid:9892010
- 78. Porcile C, Di Zazzo E, Monaco ML, D'Angelo G, Passarella D, Russo C, et al. Adiponectin as novel regulator of cell proliferation in human glioblastoma. J Cell Physiol. 2014;229: 1444–1454. doi: 10.1002/jcp.24582. pmid:24648185
- 79. Nguyen TT, Tran E, Nguyen TH, Do PT, Huynh TH, Huynh H. The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis. 2004;25: 647–659. pmid:14688022
- 80. Ranelletti FO, Ricci R, Larocca LM, Maggiano N, Capelli A, Scambia G, et al. Growth-inhibitory effect of quercetin and presence of type-II estrogen-binding sites in human colon-cancer cell lines and primary colorectal tumors. Int J Cancer. 1992;50: 486–492. pmid:1735617
- 81. Ashida H, Fukuda I, Yamashita T, Kanazawa K. Flavones and flavonols at dietary levels inhibit a transformation of aryl hydrocarbon receptor induced by dioxin. FEBS Lett. 2000;476: 213–217. pmid:10913616
- 82. Markaverich BM, Roberts RR, Alejandro MA, Clark JH. An endogenous inhibitor of (3H) estradiol binding to nuclear type II estrogen binding sites in normal and malignant tissues. Cancer Res. 1984;44: 1515–1519. pmid:6704964