Siderol Inhibits Proliferation of Glioblastoma Cells and Acts Synergistically with Temozolomide

Glioblastoma (GBM) is the most aggressive primary central nervous system (CNS) tumor in adults with dismal prognosis. Currently, the therapeutic interventions include gross total resection, when possible, followed by radiotherapy and chemotherapy. However, despite treatment, tumor usually recurs within 7–9 months. The presence of glioma cells with stem-like properties and tumor’s heterogeneity have been identified as the most important factors driving recurrence. Recently, research efforts have been focused on the use of natural substances as treatment for GBM. Siderol is an ent-kaurane diterpenoid, isolated from the genus Sideritis. Sideritis extracts have already been investigated for their anti-inflammatory, antioxidant, and anticancer effects. In this study, we investigated the antitumoral effects of siderol in GBM T98 and U87 cell lines, as well as the effects of combined treatment with temozolomide (TMZ). Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue exclusion assay. Different concentrations of siderol were used in order to calculate the IC50 values at 72 h after treatment. Flow cytometry used for the DNA cell cycle analysis after treatment with siderol in concentrations of IC50 and twice the IC50 values for 72 h. Furthermore, the effect of siderol in cell’s migratory ability was tested using wound healing assay. Cell viability and proliferation, after combined treatment with siderol and TMZ, also were evaluated with the trypan blue exclusion assay and the effects of the combination treatment were analyzed with CompuSyn software. Treatment with siderol significantly reduced cell viability in T98 and U87 cell lines in a dose-dependent manner and IC50 values were calculated, 18 μM and 13 μM, respectively. Moreover, siderol induced G0/G1 cell cycle arrest in a dose-dependent manner and inhibited the migration in both cell lines. In addition, siderol and TMZ seem to have synergistic action in the majority of tested concentrations in both T98 and U87 cells. In conclusion, siderol may represent an innovative strategy for the treatment of GBM, and further studies are needed on siderol’s efficacy and mode of action.


Introduction
GBM is the most aggressive tumor of the CNS with poor clinical prognosis in adults [1]. Currently, the therapeutic interventions include surgery, radiotherapy, and chemotherapy; however, the mean survival period of patients still remains short, ranging from 14 to 16 months-only 9.8% of GBM patients can reach a survival period of up to 5 years [2,3]. Several drugs are FDA approved for GBM, with TMZ being the standard chemotherapy. Nevertheless, TMZ increases the median survival by only 2.5 months, while higher TMZ doses can cause systemic toxicity [4,5]. Additionally, the BBB, which permits the selective penetration of some low molecular weight compounds and blocks several chemotherapeutic chemotherapy. Nevertheless, TMZ increases the median survival by only 2.5 months, while higher TMZ doses can cause systemic toxicity [4,5]. Additionally, the BBB, which permits the selective penetration of some low molecular weight compounds and blocks several chemotherapeutic medications, prevents the majority of pharmaceuticals from entering the brain [6,7]. Due to the failure of classical chemotherapies and targeted drugs, research efforts have focused on the use of less toxic substances. Therefore, a variety of organic compounds are assessed for their potential to act as GBM therapy agents. These organic substances slow tumor growth and promote GBM cell death [8].
Siderol is an ent-kaurane diterpenoid, isolated from the genus Sideritis (species S. scardica), also known as "ironwort" and "mountain tea". Sideritis is a genus of flowering plants well known for their use as herbal medicine, especially in Greece, Turkey, Albania, Bulgaria, and other countries. The stems, leaves, and flowers of S. scardica are widely decocted or infused to make herbal tea (aerial parts). This herbal medicine has been used for centuries to improve immunity, support the digestive system, and lessen the symptoms of numerous ailments. According to several studies, this plant may have a beneficial impact on many common illnesses, especially when it comes to its anti-microbial, anti-inflammatory, and antioxidant properties. The main active compounds present in this genus are diterpenoids and flavonoids [9][10][11].
As a result of the high concentration of phenolic compounds, S. scardica extracts perform dose-dependent anti-inflammatory and gastroprotective activities. The control of pro-inflammatory mediators (NF-B, TNF-, IL-1, and IL-6) is key to the management of inflammation [12,13]. Studies have shown that the ethanolic extract of S. scardica has considerable anti-inflammatory properties that are comparable to those of the nonsteroidal anti-inflammatory medication indomethacine. Moreover, several studies have shown that S. scardica preparations have antibacterial action against a number of typical Gram-positive and Gram-negative bacteria, as well as the yeast Candida albicans [14]. Concerning the antioxidant properties, Sideritis extracts have the capacity to prevent reactive oxygen species from damaging DNA, to reduce the amount of damage caused, and to increase the effectiveness of DNA repair mechanisms [15,16].
Additionally, the results of certain studies seem to be promising for the use of Sideritis extracts as a treatment against different types of cancer such as liver and colon cancer [11]. The high concentration of several phenolic compounds, particularly a few flavonoids, may result in cytotoxic action against cancer cells [14]. Furthermore, the molecular weight of siderol is 346.5 Daltons that permit this agent to cross the BBB (Figure 1). In this study, we investigated the antitumoral effects of Siderol in U87 and T98 GBM cell lines, alone and in combination with TMZ.

Cell Lines and Conditions for Treatment
The human glioma cell lines U87 and T98 were provided by Dr W.K. Alfred Yung (Department of Neuro-Oncology, M.D. Anderson Cancer Center, Houston, TX, USA) and ATCC (Manassas, VA, USA), respectively. Both cell lines were grown in DMEM (Gibco BRL, Life Technologies, Grand Island, NY, USA), after being enriched with 10% FBS and 1% penicillin-streptomycin (Gibco BRL). The cell's incubation carried out in humidified conditions containing 5% CO 2 at 37 • C. Siderol isolated from the hexane extract of cultivated Sideritis scardica diluted in DMSO and storage at −80 • C. TMZ was obtained from Sigma Aldrich, diluted in DMSO and storage at −20 • C. Before every experiment, siderol and TMZ were diluted from stock solution to the final concentration with DMEM.

Viability Assay
Cell viability was evaluated by the MTT (Sigma Life Sciences, Grand Island, NY, USA) assay and trypan blue exclusion assay. Approximately 5000 cells were seeded in 96-well plates, and after 24 h were treated with siderol in concentrations of 10-300 µM. At 72 h, after siderol treatment, MTT was added. The MTT-formazan concentration was measured at 570 nm. For the trypan blue exclusion assay, 20,000 cells were seeded in 12-well plates and were incubated for 24 h. Afterward, they were treated with siderol in concentrations of 10-60 µM for 72 h. Trypan blue exclusion assay was carried out. At least three times each of both approaches were used, and the results were expressed as the mean of the three. Cell viability after treatment with TMZ, in both cell lines, was also evaluated by the trypan blue exclusion assay. T98 cells were treated with TMZ in concentrations of 100-600 µM for 72 h and U87 cells in concentrations 20-100 µM for 72 h.

Flow Cytometric Analysis of DNA Cell Cycle
For the DNA cell cycle analysis, 20,000 cells were seeded in 12-well plates, and after 24 h were treated with siderol in concentrations of IC50 and twice the IC50 values for another 72 h. Subsequently, cells were washed with PBS solution, harvested after incubation with trypsin, and held at 37 • C for 20 min with a PI working solution (50 g/mL PI, 20 mg/mL RNase A, and 0.1% Triton X-100). PI fluorescence data were collected using a flow cytometer (Omnicyt, Cytognos S.L., Grand Island, NY, USA) and were analyzed using the GraphPad Prism version 6 software and MedCalc software (Trial version).

Wound Healing Assay
In 6-well plates, cells (10 5 ) were plated and incubated until they achieved 70-80% confluence. Afterward, DMEM rejected, the monolayer cells were scratched using a 200 µL pipette tip at the bottom of the well and were cultivated under standard conditions in DMEM supplement with 1% FBS, after being treated with siderol at IC50 and half IC50 concentrations. Each cell line's migratory distance was evaluated at 0, 24, and 48 h after scratching using the ImageJ software. Data were analyzed using the GraphPad Prism version 6 software.

Statistical Analysis
The mean and SD were used to express the data. We employed the t-test for multiple comparisons to examine the significance of differences between the outcomes of several experimental conditions. A difference was deemed significant when the p value was less than 0.05.

Viability of GBM Cells after Treatment with Siderol and IC50 Calculation
T98 and U87 cells were cultured with escalating siderol concentrations for 72 h to test the susceptibility of GBM cells to siderol. Both cell lines responded to siderol therapy with varying degrees of sensitivity. In T98 cells and U87 cells, siderol's IC50 value for reduced viability was 18 µM and 13 µM, respectively ( Figure 2). When siderol concentrations were increased, cells underwent changes such as cell shrinkage and death, which could be seen under a microscope ( Figure 3). substances' concentrations combined proportional from lowest to highest. After 72 h, viability was calculated using the trypan blue exclusion assay.

Statistical Analysis
The mean and SD were used to express the data. We employed the t-test for multiple comparisons to examine the significance of differences between the outcomes of several experimental conditions. A difference was deemed significant when the p value was less than 0.05.

Viability of GBM Cells after Treatment with Siderol and IC50 Calculation
T98 and U87 cells were cultured with escalating siderol concentrations for 72 h to test the susceptibility of GBM cells to siderol. Both cell lines responded to siderol therapy with varying degrees of sensitivity. In T98 cells and U87 cells, siderol's IC50 value for reduced viability was 18 μM and 13 μM, respectively ( Figure 2). When siderol concentrations were increased, cells underwent changes such as cell shrinkage and death, which could be seen under a microscope ( Figure 3).  substances' concentrations combined proportional from lowest to highest. After 72 h, viability was calculated using the trypan blue exclusion assay.

Statistical Analysis
The mean and SD were used to express the data. We employed the t-test for multiple comparisons to examine the significance of differences between the outcomes of several experimental conditions. A difference was deemed significant when the p value was less than 0.05.

Viability of GBM Cells after Treatment with Siderol and IC50 Calculation
T98 and U87 cells were cultured with escalating siderol concentrations for 72 h to test the susceptibility of GBM cells to siderol. Both cell lines responded to siderol therapy with varying degrees of sensitivity. In T98 cells and U87 cells, siderol's IC50 value for reduced viability was 18 μM and 13 μM, respectively ( Figure 2). When siderol concentrations were increased, cells underwent changes such as cell shrinkage and death, which could be seen under a microscope ( Figure 3).

Viability of GBM Cells after Treatment with TMZ and IC50 Calculation
To calculate the IC50 values for TMZ, T98 and U87 cells were treated with increasing TMZ concentrations for 72 h. The IC50 values of reduced viability for TMZ were 330 µM in T98 cells and 50 µM in U87 cells (Figure 4).

Viability of GBM Cells after Treatment with TMZ and IC50 Calculation
To calculate the IC50 values for TMZ, T98 and U87 cells were treated with increasing TMZ concentrations for 72 h. The IC50 values of reduced viability for TMZ were 330 μM in T98 cells and 50 μM in U87 cells (Figure 4).

Siderol Induced G0/G1 Cell Cycle Arrest in Both Cell Lines
To investigate the effects of siderol on cell cycle progression in T98 and U87 cell lines, cells were treated with IC50 and twice IC50 values of siderol for 72 h. Siderol induced G0/G1 cell cycle arrest in both cell lines in a dose-dependent manner ( Figures 5 and 6, Table  1).

Siderol Induced G 0 /G 1 Cell Cycle Arrest in Both Cell Lines
To investigate the effects of siderol on cell cycle progression in T98 and U87 cell lines, cells were treated with IC50 and twice IC50 values of siderol for 72 h. Siderol induced G 0 /G 1 cell cycle arrest in both cell lines in a dose-dependent manner ( Figures 5 and 6, Table 1).    concentrations. An asterisk is used to indicate differences that were significant (p < 0.05).

Siderol Inhibited Cell Migration in Both Cell Lines
In order to determine if siderol could influence the migration of the T98 and U87 cell lines into a scratch-induced wound, a cell monolayer demonstrated that siderol strongly hindered both cell lines' ability to heal their wounds at concentrations of IC50 and half IC50 values (Figures 7 and 8). concentrations. An asterisk is used to indicate differences that were significant (p < 0.05). Table 1. Cell-cycle distribution assessed by flow cytometry in T98 (a) and U87 (b) glioblastoma cells after treatment with siderol. The experiment was carried out three times and the values are means of three repetitions. Significant differences (p < 0.05) are marked with an asterisk.

Siderol Inhibited Cell Migration in Both Cell Lines
In order to determine if siderol could influence the migration of the T98 and U87 cell lines into a scratch-induced wound, a cell monolayer demonstrated that siderol strongly hindered both cell lines' ability to heal their wounds at concentrations of IC50 and half IC50 values (Figures 7 and 8).

Siderol and TMZ Have Synergistic Effect in Both Cell Lines
The effect of siderol in combination with TMZ, in both cell lines, is summarized in Tables 2 and 3, as the concentrations of each substance combined. In both cell lines, siderol and TMZ have synergistic effect in the majority of tested combinations. In T98 cells, the highest synergism was monitored when siderol and TMZ were given between half IC50 and IC50 values (13.5 μM Siderol and 247 μM TMZ), while antagonistic effect was monitored only at the lowest concentrations. Contrariwise, in U87 cells, antagonistic behavior showed up only at the highest concentrations (Figures 9 and 10). However, further studies are required.

Siderol and TMZ Have Synergistic Effect in Both Cell Lines
The effect of siderol in combination with TMZ, in both cell lines, is summarized in Tables 2 and 3, as the concentrations of each substance combined. In both cell lines, siderol and TMZ have synergistic effect in the majority of tested combinations. In T98 cells, the highest synergism was monitored when siderol and TMZ were given between half IC50 and IC50 values (13.5 µM Siderol and 247 µM TMZ), while antagonistic effect was monitored only at the lowest concentrations. Contrariwise, in U87 cells, antagonistic behavior showed up only at the highest concentrations (Figures 9 and 10). However, further studies are required.

Discussion
GBM is the most aggressive primary brain tumor. Surgery, radiotherapy, and chemotherapy are the most common treatments against GBM, but the mean survival period still remains short [3,17,18]. In addition, both radio and chemo resistance constitute inhibitory factors for the successful treatment of GBM [19]. Natural compounds constitute an important research field since most of these substances are of low toxicity [5]. Novel products from natural compounds protect glial cells by decreasing oxidative stress and neuroinflammation. They also restrain proliferation, activating apoptosis pathways and inhibiting pro-oncogene events [20,21]. These products could contribute to the development of new therapeutic options that improve patient quality of life and extend survival [22]. In addition, many natural substances have been investigated for their synergistic effect with TMZ or/and radiation and the results seem to be encouraging [23,24].
Siderol is an ent-kaurane diterpenoid obtained from the hexane extract of cultivated Sideritis scardica (Lamiaceae) [25]. Sideritis involves around 150 species, of which many are popular herbal medicines in Mediterranean folk medicine [26,27]. "Ironwort" is the common name for the aerial parts of various species of Sideritis plants. According to EMA, ironwort preparations can be used for the relief of cough associated with a cold and for the relief of mild stomach and gut discomfort. Furthermore, there is evidence that they have been used safely for at least 30 years, although there is insufficient evidence from clinical trials.
In previous studies, Sideritis extracts have been investigated for their interesting biological activities, such as antioxidant, anti-inflammatory, anti-neuropathic, and antimicrobial properties [28][29][30][31]. Their ability to safeguard the DNA from reactive oxygen species, to curtail the inflicted damage and improve the efficiency of the DNA repair mechanisms, has been evaluated [15,16]. Moreover, plants of this genus, that are rich in polyphenolic compounds, such as flavonoids, can produce a therapeutic effect by reducing the symptoms of inflammatory processes. The management of inflammation and neuropathic pain mainly succeed by the management of pro-inflammatory mediators (NF-κB, TNF-α, IL-1β, and IL-6) [12,13]. Other studies have demonstrated the anti-aging effect of Sideritis extracts, by anti-hyaluronidase activity [32,33] and the antimicrobial activities [34,35].
Furthermore, several studies have focused on the anti-cancer activity of Sideritis extracts. DLD1 (human colon adenocarcinoma), HL60 (human promyelocytic leukemia), and ARH77 (human myeloma) cell lines, have been investigated, after treatment with methanolic extracts of Sideritis species. The data revealed that the used extracts had cytotoxic activity against all applied cancer cell types in a dose and time-dependent manner. The cell death occurs by apoptosis. The effect of Sideritis extracts on the expression level of the pro-apoptotic gene CASPASE 3, evaluated and found to have positive effect [36][37][38]. Other genes, which are involved at the apoptotic mechanism in cells, such as BAX, BCL2, p53, and TNF have also been investigated in DLD1 cell lines and the results confirmed that Sideritis extracts lead cells to apoptosis [11]. Additionally, in MCF-7 (human breast cancer) cell line, cell proliferation and viability reduction was demonstrated after treatment with Sideritis methanol extracts [39].
In this study, we investigated the cytotoxic effect of siderol, an ent-kaurane diterpenoid isolated from the hexane extract of cultivated Sideritis scardica, in GBM cell lines. The results determine that siderol caused a significant cell viability and proliferation inhibition in a dose-dependent manner, and the IC50 values in both applied cell lines are relatively low. Furthermore, siderol induced G 0 /G 1 phase cell cycle arrest in both cell lines. These results confirm the cell proliferation inhibition by blocking the cell cycle development [40,41].
Another important result of our research is that siderol inhibited the cell migration. The migratory ability of cancer cells constitutes an important failure reason in GBM treatments. Even after a successful surgery, cancer cells, which have already migrated, can recreate a new tumor [42]. Siderol inhibited the migratory ability of T98 and U87 cell lines in a dose and time-dependent manner and there are significant differences compared to controls.
Concerning the combined treatment of siderol and TMZ, the experiments proved a synergistic effect in both cell lines. T98 cells are resistant to TMZ and high concentration for treatment is required. Nevertheless, higher doses of TMZ may cause higher risk of adverse effects. The synergistic effect with siderol may be a possible solution using lower doses of TMZ and/or overcoming the resistance of cells. Summarizing, the results of combined treatment may be promising at GBM treatment as the TMZ's dose could be decreased. Therefore, further experiments are needed to fully determine the synergy between siderol and TMZ in GBM cells.
The present study has several limitations. Siderol's exact mechanism of action should be investigated, as well as the synergistic mechanism with TMZ. In addition, siderol should be evaluated with in vivo models to determine if there is toxicity [43,44]. Proliferation data can be further verified using other methods such as clonogenic assay, cell-cycle analysis, or mRNA/protein expression analysis of proliferative markers. The effect of siderol and temozolomide, alone and in combination, on cell apoptosis should be also investigated. Concerning the synergistic action, siderol could be examined in combination with radiation, as, today, radiotherapy and chemotherapy are the standard therapeutic interventions in GBM treatment. Furthermore, the investigation of siderol's action on other GBM cell lines would be a contribution to determine the further possible effects of siderol.
The development of new natural products as a treatment for GBM constitute important goal for researchers, as these products reduce the probability of toxicity, which other chemotherapy drugs bring out. Siderol may represent an innovative strategy for the treatment of GBM.

Conflicts of Interest:
The authors declare no conflict of interest.