Diosmin induces caspase-dependent apoptosis in human glioblastoma cells

diosmin induces caspase-dependent apoptosis in human glioblastoma Abstract: Diosmin is a flavone glycoside clinically used as the main component of Daflon for the treatment of venous diseases. Several studies demonstrated that this natural compound can induce apoptosis in different tumors. However, isolated diosmin has not been studied regarding its effects on glioblastoma so far. Since glioblastoma is a highly lethal and fast-growing brain tumor, new therapeutic strategies are urgently needed. Herein, we evaluated the role of this flavonoid against glioblastoma cells using in vitro assays. Diosmin significantly reduced the viability of GBM95, GBM02, and U87MG glioblastoma cells, but not of healthy human astrocytes, as verified by MTT assay. Vimentin immunostaining showed that diosmin induced morphological changes in GBm95 and GBm02 cells, making them smaller and more polygonal. diosmin did not inhibit GBm95 and GBm02 cell proliferation, but it caused dNA fragmentation, as verified by the TUNEL assay, and increased cleaved caspase-3 expression in these cells. In summary, diosmin is able to induce caspase-dependent apoptosis specifically in tumor cells and, therefore, could be considered a promising therapeutic compound against glioblastoma.


INTRODUCTION
Glioblastoma, a malignant brain tumor, is characterized by rapid cell proliferation, high parenchyma infiltration, and angiogenesis. Due to its fast evolution and morphological heterogeneity, glioblastoma is very aggressive (maher et al. 2001). Currently, the treatment consists in surgical removal followed by radiation and chemotherapy, mainly with temozolomide (TmZ) (Würth et al. 2014). In addition to TmZ, bevacizumab is also indicated to treat recurrent glioblastoma, but, to date, no significant impact on overall survival upon treatment with this monoclonal antibody has been achieved (mooney et al. 2019). despite therapeutic advances, glioblastoma is still lethal, as the median survival is 15 to 18 months for newly diagnosed patients, and around 10% survive 5 years (lieberman 2017).
Since current therapies are not effective in curing and/or significantly expanding life expectancy of glioblastoma-affected patients, the search for new therapeutic approaches is intensifying. In this context, natural compounds, such as flavonoids, are emerging as alternatives for treating different types of tumors with reduced side effects (Ali et al. 2012, Chen et al. 2016b, Sak 2014. Flavonoids, polyphenolic compounds found in fruits and vegetables, have been described as presenting various biological and pharmacological effects (Chahar et al. 2011, Chen et al. 2016b, Sak 2014. In vitro and in vivo studies have suggested that they can influence different phases of the cell cycle and can modulate tumor progression by inhibiting proliferation and/or inducing cell death (Chen et al. 2016a, Fantini et al. 2015, Srinivasan and Pari 2012. Several works have reported the activity of flavonoids against different cell lines of human glioblastoma. Among these flavonoids, are the flavones apigenin, luteolin, and chrysin (Chakrabarti and Ray 2015, Chen et al. 2016b, das et al. 2010, Kim et al. 2016, Santos et al. 2015, Stump et al. 2017; the flavonols quercetin and kaempferol (Kim et al. 2016, Santos et al. 2015; the flavanones naringenin and hesperetin (li et al. 2017, 2019); and the isoflavones genistein and daidzein (das et al. 2010(das et al. , Siegelin et al. 2009). A recent review pointed out the capacity of different flavonoids to reduce glioblastoma cell proliferation and to induce apoptosis (erices et al. 2018).
Diosmin (3',5,7-trihydroxy-4'-methoxyflavone 7-rutinoside) is a flavonoid glycoside constituted by a flavone named diosmetin (3',5,7-trihydroxy-4'-methoxyflavone), a unit of glucose, and a unit of rhamnose (Fig. 1). This molecule is reported to have anti-inflammatory and vascular protective effects due to its ability to inhibit prostaglandin and thromboxane synthesis (el-Shafae and eldomiaty 2001, Hitzenberger 1997, laurent et al. 1988, Srinivasan and Pari 2012. diosmin is already commercially available in the formulation of Daflon for treating chronic venous diseases, for which it is considered to be very effective (Ramelet 2001). This drug is also highly safe for both animals and humans (meyer 1994).
Previous works have shown that diosmin also displays antitumor activity, such as in pulmonary melanoma (Álvarez et al. 2009 In a recent study, diosmin was identified in ethanolic extracts from Phlomis viscosa leaves, and treatment of U87mG cells with the whole plant extract significantly reduced cell viability (Yarmolinsky et al. 2019). However, there are no reports on whether isolated diosmin (or its aglycone diosmetin), like other flavones, also acts against glioblastoma. Thus, in the present study, we investigated the effects of diosmin against glioblastoma by analyzing the viability, proliferation, and morphology of different tumor cell lines. We also evaluated whether diosmin affects the viability of healthy astrocytes to ensure it has cell-specific effects. diosmin (3',5,7-trihydroxy-4'-methoxyflavone 7-rutinoside) was extracted from orange peels with methanol. The extraction was cheaper than the commercial diosmin sample. In addition, it allowed us to use a natural resource to extract diosmin, avoiding orange peel waste and allowing its use for a possible therapeutic function. The orange peel samples were dehydrated in a forced air oven at 55 ºC for 8 hours. To perform the extraction, 100 g of dried orange peel powder were weighted and then 100 ml of 80% methanol was added. The sample was placed in an ultrasound equipment for assisted extraction (60 kHz for 30 min at 40 ºC). After the extraction, methanol was evaporated in a condenser. Then, 2 m NaOH was used as basifying agent and water was used as solvent. Afterwards, acetic acid was added until the medium pH reached 7 for diosmin precipitation. The resulting mixture was centrifuged to obtain the diosmin pellet, which was lyophilized for 24 hours until the sample was completely dry. The yield was approximately 4.45 mg per 100 g of orange peel powder. The diosmin sample was dissolved in dimethyl sulfoxide/methanol and filtered using a 0.45-μm nylon membrane. Then, it was purified by high-performance liquid chromatography (HPlC The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (mTT, Sigma-Aldrich, Inc) colorimetric method was used to determine glioblastoma and astrocyte cell viability. After treatments, cells were incubated with 5 mg/ml mTT for 2 hours. The reaction product, formazan, was dissolved in dmSO. The amount of formazan was quantified at 570 nm using a Victor 3 Perkin elmer UV spectrophotometer.

edU ASSAY
The Click-iT edU Alexa Fluor 488 imaging kit (Invitrogen Corporation) was used to evaluate glioblastoma cell proliferation after diosmin treatment (at 50 and 100 μM) for 48 hours. GBM95 and GBm02 cells were cultured in 24-well tissue culture plates (TPP, Switzerland) with 13-mm cover slips (Knittel Glass, Germany) for 24 hours with dmem-F12 containing 10% FBS. After 42 hours, edU was added to the treatment medium and incubated for 6 hours. Cells were then fixed with 4% formaldehyde solution and incubated at room temperature with edU detection cocktail. Subsequently, Hoechst 33342 dye (Thermo Fisher Scientific) was used for nucleus staining.
All reagents and kit components were diluted and stored according to the manufacturer's protocol. Proliferative cells stained by edU were imaged by fluorescence microscopy (Nikon TE 300) and counted using ImageJ software (version 1.44p, Wayne Rasband National Institutes of Health, USA). Three independent experiments were performed in duplicate, and ten randomized images were analyzed. The morphology and total number of glioblastoma cells were analyzed after diosmin treatment (at 50 and 100 μM) for 24 and 48 hours. Cytoskeleton changes were assessed using ImageJ (software 1.44p, Wayne Rasband National Institute of Health, USA) by measuring the length of vimentin-stained cells. The length of treated cells was compared to the length of control cells treated with dmSO. ImageJ was also used to count glioblastoma cell nuclei. The total number of cells was assessed by the percentage of dAPI positive cells in comparison to control. Three independent experiments were performed in duplicate, and ten randomized images were analyzed.

STATISTICAl ANAlYSIS
Statistical analyses were executed in GraphPad Prism (version 7.0, California, USA). All values were expressed as mean ± Sem. data were analyzed by one-way ANOVA followed by post hoc comparison (Tukey's test). Statistical significance was set at *p<0.05; **p<0.01; ***p<0.001.

In Silico PHARmACOKINeTIC eVAlUATION
The chemical structure of diosmetin, the aglycone form of diosmin, was drawn using Chemdraw (version Ultra 12.0). Several in silico analyses of physicochemical and pharmacokinetic properties were performed on AdmeT Predictor Tm software (version 9.0, Simulations Plus, CA). The parameters analyzed were molar weight (mW, g/mol), octanol/water partition coefficient (logP), octanol/ water distribution coefficient at pH 7.4 (logd), topological polar surface area (TPSA), number of hydrogen bond donors (HBd), the ionization constant (pKa) for the most basic center, brain penetration (BP), brain/blood partition coefficient (logBB), and the probability of being Pgp substrate (PgpS). Regarding the accuracy of such analyses, . To evaluate its effects on the viability of glioblastoma cells and healthy astrocytes, the mTT colorimetric assay was performed. GBm95, GBm02, U87mG, and astrocyte cells were incubated with diosmin at 10, 50, 75, 100, and 150 μM for 24 and 48 hours. These treatment times and concentrations were selected based on previous studies with flavonoids, such as luteolin and apigenin, which belong to the same flavonoid class as that of diosmin (Chakrabarti and Ray 2015, Stump et al. 2017). In relation to control, diosmin decreased GBm95 and GBm02 cell viability in almost all conditions. even at the lowest concentration (10 μM), diosmin was able to reduce glioblastoma cell viability. However, the intermediate concentrations (50 and 100 μM) provided more satisfactory results. GBm95 cells treated with diosmin at 50 and 100 μM exhibited, respectively, a viability reduction of 50.6% and 54% after a 24-hour treatment and 49.9% and 60.2% after a 48-hour treatment (Fig. 2a). Under the same conditions, diosmin reduced GBm02 cell viability in 30% after a 24-hour treatment and in 33% after a 48-hour treatment (Fig. 2b). At the concentrations of 50 and 100 μM, diosmin reduced U87mG cell viability in 40%, but this cytotoxicity was only seen after a 48-hour treatment (Fig. 2c).
In summary, diosmin significantly decreased the viability of all three glioblastoma cell lines, acting as a cytotoxic compound.
Healthy astrocytes were also subjected to treatment with diosmin to assess whether the toxicity of this compound is cell-specific. We only observed a small decrease (less than 15%) in astrocyte viability after diosmin treatment at the concentrations of 10 and 100 μM for 48 hours (Fig.  2d). None of the other treatment conditions caused a significant decrease in astrocyte viability. This result indicates that diosmin was significantly more toxic against glioblastoma cells than for healthy astrocytes, highlighting diosmin cell-specific effects. The commercial and well-established U87mG cell line was used herein for comparison, and the following in vitro evaluations were conducted using only GBm95 and GBm02 cells, which have known origin and number of passages, since these cell lines have been fully characterized by our group (Faria et al. 2006 Our first analysis revealed that diosmin reduced glioblastoma cell viability. Therefore, we decided to evaluate whether a combination of diosmin with TmZ, the reference chemotherapeutic agent for glioblastoma treatment (Würth et al. 2014), would be better than the individual treatments. GBm95 and GBm02 cells were treated with diosmin at 50 and 100 μM in combination with 200 μM TMZ. Cells were also treated with TmZ at 50, 100, and 200 μM. After a 48-hour treatment, TMZ alone at 200 μM was able to reduce GBM95 and GBM02 cell viability by only 20% and 14%, respectively (Fig. 3a,b). When we performed a 24-hour treatment with 50 μM diosmin in combination with 200 μM TmZ, there was an approximate viability reduction of 23% and 16% for GBm95 and GBm02 cells, respectively. At the same concentrations, the 48hour treatment reduced the viability of both cell lines in 29% (Fig. 3a,b). A 48-hour treatment of diosmin at 100 μM combined with TMZ at 200 μM decreased the viability of GBM95 and GBm02 cells in 25% and 19%, respectively (Fig.  3a,b). These results showed that the association of diosmin and TMZ is more effective than only TMZ. Controversially, diosmin alone was more effective than TmZ and the combination of diosmin with TMZ, indicating that there is no synergistic effect between these drugs.

dIOSmIN RedUCeS CellUlARITY ANd PROmOTeS mORPHOlOGICAl CHANGeS IN GlIOBlASTOmA CellS
Cell size reduction is closely associated with cytotoxic effects, such as apoptosis (Elmore 2007). This type of morphological change in glioblastoma cells has been associated with treatment with flavonoids (Santos et al. 2015). To evaluate whether the cytotoxic effect of diosmin caused changes in glioblastoma cytoskeletal architecture, we analyzed vimentin, an intermediate filament protein, by immunocytochemistry. After diosmin Figure 2 -diosmin reduced the viability of GBm95, GBm02, and U87mG cells, but did not reduce the viability of human astrocytes. Cell viability was assessed by mTT reduction assay and expressed as percentage of viable cells, compared to control (DMSO residue). Glioblastoma cells and healthy human astrocytes were treated with diosmin for 24 and 48 hours at different concentrations (10 to 150 μM). Representative graphs of the viability of GBM95 (a), GBm02 (b), U87mG (c), and healthy human astrocytes (d). Significant viability reduction was observed in glioblastoma cells under almost all conditions. Results represent the mean ± Sem of three independent experiments, each performed in triplicate (*p <0.05; **p <0.01; ***p <0.001). (2019) 91(4) e20191031 8 | 16 treatment (at 50 and 100 μM) for 24 and 48 hours, cells with a different phenotype could be observed, as they became smaller and more polygonal (Fig.  4a,b). However, no cytoplasmic retraction was seen in the controls (Fig. 4). Cell length measurements indicated that diosmin decreased the size of GBm95 and GBm02 cells. diosmin reduced GBm95 length by more than 50% after a 24-hour treatment and by almost 60% after a 48-hour treatment (Fig.  4c). diosmin also changed GBm02 morphology by reducing cell length in 50% after a 24-hour treatment and in 27% after a 48-hour treatment (Fig. 4e).

An Acad Bras Cienc
We also noticed a reduction in GBm95 and GBm02 cellularity. GBm95 culture exposed to diosmin (at 50 and 100 μM) for 24 hours underwent a significant reduction in cell number (40%) (Fig.  4d). The 48-hour treatment significantly reduced the number of GBM95 cells (37.7%) only at 50 μM. GBm02 cells treated with diosmin (at 50 and 100 μM) for 24 and 48 hours underwent a cellularity decrease of more than 30% (Fig. 4f). Our data indicate that diosmin decrease the population of GBm95 and GBm02 cells and induces their size reduction, suggesting a potential cytotoxic effect.

dIOSmIN-INdUCed deCReASe IN GlIOBlASTOmA CellUlARITY IS NOT ASSOCIATed WITH RedUCed Cell PROlIFeRATION
According to previous reports, reduced cellularity may be associated with a decrease in cell proliferation (Ouhtit et al. 2013). Therefore, the Click-it edU imaging kit was used to assess GBm95 and GBm02 cell proliferation after diosmin treatment (at 50 and 100 μM) for 48 hours; it was done by analyzing these cells at the dNA synthesis phase. At none of the concentrations tested, diosmin significantly decreased the number of proliferative cells in relation to control (Fig.  5), suggesting that diosmin-mediated reduction in cellularity was not linked to changes in the proliferation rate of glioblastoma cells.
dIOSmIN INdUCeS GlIOBlASTOmA Cell deATH BY APOPTOSIS As the decrease in the viability and number of GBm95 and GBm02 cells was not associated with modifications in their proliferation rate, we hypothesized that it could be related to cell death. morphological changes, such as cell shrinkage, were previously associated with apoptosis (elmore 2007). Therefore, we analyzed cell death by dNA fragmentation using the TUNEL assay to confirm whether diosmin caused morphological changes and cytotoxic effects on glioblastoma cells. This evaluation showed an increase in GBm95 and GBm02 cell death after treatment with diosmin (at  vimentin immunostaining (red) were used to evaluate cell morphology and cytoskeleton. dAPI (blue) was used for nuclei staining to count the total number of cells. GBM95 and GBM02 were treated with diosmin (at 50 and 100 μM) for 24 and 48 hours. DMSO was used as control. Phase contrast and vimentin representative images of GBm95 (a) and GBm02 (b), representative graphs of the length (micrometers) of GBm95 (c) and GBm02 (e), and representative graphs of total number of GBm95 (d) and GBm02 (f) cells are shown. Results represent mean ± Sem of three independent experiments, each performed in duplicate (*p <0.05; **p <0.01; ***p <0.001). Scale bar: 50 μm. 50 and 100 μM) for 24 and 48 hours (Fig. 6a,b). Diosmin at 50 μM increased GBM95 cell death by 47.5% after a 24-hour treatment and by 31.1% after a 48-hour treatment (Fig. 6c). A similar effect was observed for GBm02 cells, as diosmin increased cell death by 27.5% after 24 and 48-hour treatments (Fig. 6e). Subsequently, cleaved caspase-3 immunostaining was performed to understand whether cell death was being triggered by caspasedependent apoptosis. A 48-hour treatment with 100 μM diosmin increased the expression of cleaved caspase-3 in GBm95 and GBm02 cells (Fig. 6a,   b). We noticed that 5% of GBm95 cells (Fig.   6d) and 11% of GBm02 cells (Fig. 6f)  Intestinal bacteria hydrolyze diosmin to its flavone aglycone, named diosmetin, which is then absorbed (lyseng-Williamson and Perry 2003). diosmetin, which is expected to be the active component of diosmin, must in turn reach the central nervous system (CNS). Therefore, we evaluated the ability of diosmetin to cross the blood-brain barrier (BBB) and reach the CNS by using four different in silico parameters: (i) the CNS multiparameter Optimization (CNS mPO) algorithm, (ii) a binary parameter of brain penetration (BP), (iii) a brain/ blood partition coefficient (logBB), (iv) and the probability of being a P-glycoprotein substrate (PgpS). The CNS mPO algorithm (i) is based on six physicochemical properties: (a) lipophilicity, measured by the partition coefficient (logP); (b) distribution coefficient at pH 7.4 (logD); (c) molecular weight (mW); (d) topological polar surface area (TPSA); (e) number of hydrogen bond donors (HBd); and (f) ionization constant (pKa) for the most basic center (Wager et al. 2016). All the 6 properties used in the CNS mPO algorithm were calculated on AdmeT Predictor Tm software. The total CNS mPO score ranges from 0 to 6, and the higher the score, the greater the probability of a certain molecule to cross the BBB (Wager et al. 2016). Most CNS-acting drugs have a score ≥ 4.0 (Wager et al. 2016). diosmetin had a score of 4.71, which suggests it can cross the BBB.
The BP parameter (ii) simply predicts whether a molecule is able to cross the BBB. The logBB parameter (iii) predicts whether a molecule will be retained in the CNS once it gets in. The PgpS parameter (iv) predicts whether a molecule will be transported back from the CNS to the bloodstream by P-glycoprotein (Pgp), which would impair the distribution to the CNS. The results for these three parameters were provided directly by AdmeT Predictor Tm software. diosmetin was predicted to have low permeability through the BBB. However, its logBB value was -0.81, which is higher than the minimum value of -1.0 for CNS-acting drugs (information provided in the software manual). Glioblastoma cells were exposed to diosmin (at 50 and 100 μM) for 48 hours. DMSO was used as control. Representative microscopy images of GBm95 (a) and GBm02 (c) stained with EdU (green) and the quantification of GBM95 (b) and GBm02 (d) proliferative cells labeled by edU are shown. Results represent mean ± Sem of three independent experiments, each performed in duplicate. No statistical significance (p <0.05) was observed. Scale bar: 50 μm.
diosmetin was also predicted not to undergo PgPmediated efflux. In summary, these results suggest that, after oral administration of diosmin, its flavone aglycone would likely reach the CNS.

DISCUSSION
Since there is no effective treatment for glioblastoma so far, the discovery of new strategies and targeted agents are emerging (li et al. 2017). In this context, flavonoids can act against different events associated with cancer (Batra and Sharma 2013). In vitro experiments have shown that flavonoids can inhibit glioblastoma proliferation and/or induce apoptosis (Kim et al. 2016, li et al. 2017, Santos et al. 2015. Apigenin and luteolin, like diosmin, belong to the flavone class and are known for their anti-cancer properties (Chen et al. 2016b, Singh et al. 2014. In vitro studies have shown that apigenin induces apoptosis in U373, T98G, and U87mG cells (das et al. 2010, Kim et Figure 6 -diosmin induced glioblastoma cells death by cleaved caspase-3 dependent apoptosis. It was evaluated whether diosmin treatment (at 50 and 100 μM) for 24 and 48 hours could induce glioblastoma cell death by TUNEL assay. Immunostaining for cleaved caspase-3 was performed to evaluate whether cell death occurred by caspase-dependent apoptosis. dmSO was used as control. Representative microscope images of GBm95 (a) and GBm02 (d) stained with TUNel (red) and cleaved caspase-3 (green) and representative graphs of GBm95 (c, d) and GBm02 (e, f) cell death assessed by TUNel and cleaved caspase-3 staining, respectively, are shown. Results represent mean ± Sem of three independent experiments, each performed in duplicate (*p <0.05; **p <0.01). Scale bar: 50 μm.
al. 2016) and that luteolin decreases the viability of U87mG and T98G cells (Chakrabarti and Ray 2016). Previous work demonstrated that the flavonoids apigenin, luteolin, and silibinin were able to induce apoptosis in glioblastoma cells and to reduced their proliferation (Chakrabarti and Ray 2015, Stump et al. 2017).
In this study, we evaluated the potential use of diosmin in the therapy for human glioblastoma using cell-based assays and a complementary in silico analysis. diosmin caused a significant decrease in the viability of GBm95, GBm02, and U87mG cells, especially after 48-hour incubation. Notably, diosmin did not significantly change the viability of healthy human astrocytes, indicating that it acts selectively on tumor cells. A similar selectivity was observed for rat carcinoma cells, wherein diosmin reduced the viability of HA22T cells without impairing the viability of healthy liver cells (dung et al. 2012).
The viability of glioblastoma cells was evaluated after the combination of diosmin with TmZ. Previous studies demonstrated that TmZ acts against glioblastoma cells at a minimum concentration of 200 μM (Matias et al. 2017, Zhang et al. 2017. Recently, our group calculated TmZ potency against GBm02 and GBm95 cells, evidencing IC 50 values above 600 µm (da Silva et al. 2019). The cytotoxicity of TmZ is associated with the formation of O 6 -methylguanine in dNA that mispairs with thymine during dNA replication (Fan et al. 2013). Consequently, glioma cells respond by undergoing G2/m arrest and die by autophagy or apoptosis (Fan et al. 2013, Kanzawa et al. 2004. moreover, recent studies suggested that the combination with natural compounds could increase TMZ efficiency. Quercetin, for example, was able to sensitize glioblastoma cells to TmZ, reducing treatment resistance (Hu et al. 2016, Jakubowicz-Gil et al. 2013, Sang et al. 2014. Quercetin and TMZ together induced a potent apoptotic effect in glioblastoma cells by endoplasmic reticulum stress, followed by activation of the mitochondrial death pathway (Jakubowicz-Gil et al. 2013).
Although other studies demonstrated some benefits of the association of TMZ with flavonoids, our results showed that the mixed treatment is less efficient than that with diosmin alone, indicating that no synergistic effect takes place. However, the combination was more effective than the treatment with TmZ alone, perhaps because TmZ is not so effective in cell culture (da Silva et al. 2019 diosmin also caused GBm95 and GBm02 cells to have their sizes reduced and to be less elongated and more polygonal. Other flavonoids can induce similar morphological changes (Isoda et al. 2014, Touil et al. 2009). morphological alterations of glioblastoma cells induced by natural compounds were previously associated with cytotoxic effects, reduction in cell migration, and inhibition of tumor progression (matias et al. 2017(matias et al. , Santos et al. 2015. Chrysin, kaempferol, apigenin, and quercetin have been shown to induce cytoplasmic retraction in glioblastoma Gl-15, which has been mentioned as one of the features of cells which were dying by apoptosis (Santos et al. 2015).
Our morphological analysis suggested that diosmin decreased GBm95 and GBm02 cellularity. Reduction in cell number is closely linked to inhibition of proliferation and death induction (Ouhtit et al. 2013). However, diosmin did not show any anti-proliferative effect. This could be explained by glioblastoma high proliferation rate (Faria et al. 2006, Santos et al. 2015, which makes the therapy even more difficult. Since diosmin did not reduce GBm95 and GBm02 proliferation, we evaluated whether the decrease in viability and An Acad Bras Cienc (2019) 91(4) e20191031 13 | 16 cytoskeleton alterations were associated with a specific death process, and we found that diosmin caused a significant increase in DNA fragmentation in GBm95 and GBm02 cells, leading to increased cell death. We also demonstrated that cell death was associated with a caspase-dependent apoptosis. diosmin increased the expression of cleaved caspase-3, a cysteine protease considered a marker of apoptosis, as its activity is required for biochemical and morphological events associated with cell death (Crowley and Waterhouse 2016).
Other studies demonstrated that diosmin could induce apoptosis in breast, prostate, carcinoma, and melanoma cancer cells under similar treatment conditions. The apoptosis induced by diosmin has also been linked to an increase in reactive oxidative species (ROS) and to chromosomal dNA damage and methylation (lewinska et al. 2015). In the present study, the inhibition of tumor progression via apoptosis by diosmin is remarkable, especially considering the capacity of glioblastoma to evade this process (Stump et al. 2017). It is very interesting to have such a significant effect against glioblastoma using a flavonoid, as these natural compounds are less expensive and have much lower toxicity and side effects compared to classical chemotherapy (Alves et al. 2017). Because diosmin is absorbed as diosmetin after oral administration (lyseng-Williamson and Perry 2003), we performed an in silico evaluation of the aglycone accessibility to the CNS and found that it could likely cross the BBB. Nevertheless, it has been previously described that diosmin may also be directly absorbed from the digestive tract , while numerous works have reported that free diosmetin and diosmetin glycosidesincluding diosmin-are able to reach the CNS, as they have been shown to possess sedative effects (Fernández et al. 2006), antiautistic effects (Tan et al. 2013), central anti-hyperalgesic effects (Carballo-Villalobos et al. 2018), and memoryenhancing and anxiolytic-like effects (Oliveira et al. 2018). Furthermore, the brain penetration of drugs is facilitated in glioblastoma, since the high rate of parenchyma infiltration causes vascular fragility and weakens the BBB (dubois et al. 2014). The permeability could also be enhanced by nanostructure delivery systems (Renukuntla et al. 2013). Additionally, it is also possible to administer diosmin directly into the patient's brain after tumor removal (Perry 2007). This strategy has already been used in glioblastoma patients who received local administration of carmustine to guarantee direct drug arrival and avoid solubility problems (Akiyama et al. 2018).
In summary, we evaluated the antitumor effects of diosmin against different glioblastoma cell lines. The results showed that this flavonoid is a promising natural compound against glioblastoma. However, further studies are needed to better understand diosmin mechanism of action to design new protocols for its application in the treatment of glioblastoma. Since diosmin is already in clinical use and is considered safe, acute toxicity studies would not be required prior to investigating diosmin efficacy in vivo.