Caffeine (C₈H₁₀N₄O₂) is a methylxanthine compound commonly found in coffee and tea (Figure 1A), which are the most ingested neuroactive substance in the world (34). Caffeine possesses antiinflammatory and antioxidant effect, as acetylcholinesterase inhibitors (35). Recently, it was reported that caffeine consumption might be associated with lower glioma risk (36). In fact, caffeine has been associated with rat glial cell (C6) and human GBM cell lines (U251 and U87MG) growth inhibition and apoptosis activation (16, 17, 19, 21).

PT93, a compound derived from caffeic acid (CA), has been evaluated regarding its effect on invasion and migration on GBM cell lines (T98G and U251) (22). The authors observed MMP-2 and MMP-9 inhibition in a dose-dependent way (3 and 10μM) (Figure 2B). PT93, a selective MMP inhibitor, seems to present fewer side effects when compared to the first generation of MMP inhibitors in vitro studies (61–63), as Batimastat (BB-94) and Marimastat (BB-2516) (64).

In another study, an U87MG xenotransplant mouse has treated with two other CA derivatives, FLVM and FLVZ by targeting the normally overexpressed IL17A, HIF-1α and vascular endothelial growth factor (VEGF). The reduction of these cytokines leads to inhibition of U97MG GBM cell line, mainly because the angiogenesis is also inhibited. Interestingly, the inhibition of all described factors also provides nutrient partition, leading to a lower adipocyte storage due to the glucose, triglycerides, and fat oxidation metabolism reducing (23). In addition, FLVM and FLVZ provide a reduction in the glucose and adipocyte metabolism in the CNS, inhibiting GBM development (23) due to the adipocytes and blood vessel inhibition (65, 66).

Licorice root (Glycyrhiza glabra) is widely used in traditional Chinese medicine for its pharmacological and physiological action as antiallergic, antibiotic, antiinflammatory, and antitumor effects (67, 68) (Figure 1B). Glycyrrhizic acid (GA) (C₄₂H₆₂O₁₆), a compound isolated from licorice root, has presented antiinflammatory and antitumor effects on several tumor cell lines such as human hepatoma (HLE), promyelocytic leukemia (HL-60), stomach cancer (KATO III), and prostate cancer (LNCaP e DU-145) by both DNA fragmentation and deregulating genes required for oxidative stress control (69–71). However, toxicity has been observed in in vivo models (72).

More recently, one study has exposure U251 GBM cell line to different concentrations of GA (1, 2, and 4 mM), and the authors have observed inhibition on cell proliferation and colony formation, apoptosis stimulation, and significantly decreasing in p65 protein, responsible for nuclear factor kappa B (NF-κB) pathway activating (24).

The NF-κB pathway is constantly activated in GBM, being responsible for the aggressiveness of the disease and regulation of the expression of antiapoptotic genes and cell adhesion and invasion factors (73). Thus, some studies have suggested inhibition of NF-κB pathway could decrease the resistance of tumor cells to chemotherapy and contribute to increase the survival of patients with GBM (74–77).

Following that idea, a recent study has evaluated the DPG (C₄₂H₆₀K₂O₁₆), a dipotassium salt of GA (Figure 1B), effects in GBM cell lines (25). The authors have demonstrated antitumoral effect in the GBM cell lines, U87MG and T98G, through a decrease of proliferation and an increase of apoptosis. Moreover, after both DPG and TMZ exposure, higher suppressed cell viability was observed in a dose-dependent way. Thus, even low TMZ concentration with half-maximal inhibitory concentration (IC50) of DPG was able to induce U87MG and T98G cell viability reduction (25, 50, 75, 100, 125, 150, 175, and 200 μM) for 6, 12, 18, and 24 h. Thus, a combinatorial therapy, DPG in combination with TMZ, showed a synergistic effect in U87MG and T98G cell lines (25). Interestingly, DPG was able to induce cell viability reduction even in T98G cell line, which presents both the hypermethylated MGMT promoter and mutated P53 gene, making the phenotype more aggressive and resistant to the action of TMZ than the ones with wild-type P53 (78), since p53 is fundamental in regulating the cell cycle arrest and the entry in the apoptotic process (79, 80).

In addition, DPG (18 mM and 24 mM for U87MG and T98G cells, respectively) antitumoral effect was related to NF-κB pathway suppression by IRAK2 and TRAF6-mediating miR-16 and miR-146a, respectively (25) (Figure 3B), and consequently increasing the TMZ-induced apoptosis. Finally, the authors have also showed that DPG was able to inhibit the subpopulation of stem cells essential for tumor formation, survival, and recurrence (25). Further in vivo studies may elucidate the antitumor effect of DPG as an alternative treatment for GBM. The effects of GA and DPG on NF-κB pathway are presented in Figure 3B.

More recently, it was demonstrated that the cytotoxic effect of DPG was time- and dose-dependent also in U251 and U138MG and DPG (IC50: 32 mM and 20 mM for 48 h, respectively) inhibited cell viability by activating apoptosis, inhibiting cell proliferation and stem cell subpopulation formation through miR-4443 and miR-3620 upregulation. Both miRs are responsible for post-transcriptional inhibition of the NF-κB pathway by CD209 and TNC modulation on U251 and U138MG cell lines. The authors have concluded that DPG presents an antimigratory effect on GBM cell lines by inhibition of cancer stem-like cells, evidenced by inhibition of neurosphere formation (unpublished data).

Curcumin (diferuloylmethane; C₂₁H₂₀O₆) (Figure 1C) is the main compound found in turmeric, an Indian spice derived from Curcuma longa Linn (81). It has been demonstrated that the curcumin has present antioxidant and antiinflammatory effects (81), mainly in diabetes (82), Alzheimer's disease (83), and hepatitis (84).

Curcumin seems to play a role in a variety of pathways as proinflammatory cytokines (TNF-alpha, IL1, IL6, IL8) inhibition, wingless-related integration site (WNT) suppressing, mitogen-activated protein kinase pathway (MAPK) and Janus kinase/signal (JAK/STAT) activation (85–88). Furthermore, curcumin is highly lipophilic, which makes it permeable to the BBB (89, 90), making it further viable in vivo therapy.

A recent study has evaluated the curcumin effect over miRs in the GBM cell line, A172. Interestingly, the authors have observed that curcumin was able to downregulate the serine or threonine kinase pathway (AKT) and p53-BCL2 pathways by overexpressing several miRs such as miR-223-3p, miR-133a-3p, miR-181a-5p, miR-34a-5p, miR-30c-5p, and miR-1290 and also downregulating the expression of miR-27a-5p, miR-221-3p, miR-21-5p, miR-125b-5p, and miR-151-3p (31) (Figure 3B). AKT and p53-BCL2 pathways are related to proliferation and cellular growth, metabolism, apoptosis, and autophagy (31). In contrast, the authors also observed that in GBM curcumin-treated cells, the miR-27a expression level was reduced and its target gene, CHOP, was overexpressed leading the cells to paraptosis, mainly due to endoplasmatic reticulum (ER) instability (31) (Figure 3B).

Other studies have also observed that curcumin enhanced the expression of miR-326 (28), miR-378 (27), and miR-21 (29) in GBM cell lines. Thus, Yin et al. (28) have observed that U87MG and U251 cells presented a marked increase of curcumin-induced cytotoxicity and apoptosis and a decrease of proliferation and migration in GBM cells. Moreover, the authors have found that combination treatment of miR-326 mimics and curcumin caused significant inhibition of the SHH/GLI1 pathway in cells compared with either treatment alone, independent of p53 status. Furthermore, in vivo, the curcumin-induced miR-326 expression reduces tumor volume and prolonging the survival period compared with either treatment alone. The results support an important role of miR-326 in enhancing the chemosensitivity of glioma cells to curcumin. Similar results were observed in another study, in which the authors have evaluated the curcumin effect on U87MG cells stably expression miR-378. Cells were unable to form colonies compared with control cells, indicating a lower survival rate when treatment of curcumin and miR-378 was combined. In conclusion, curcumin can inhibit MAPK pathway activation by overexpressing miR-378 (27).

Tan et al. (29) have transfected deoxycholic acid-conjugated polyethylenimine (DP) micelles containing curcumin and miR-21 antisense oligonucleotide (ASO) into C6 GBM cell line, aiming knockdown the miR and enhance the expression of proapoptotic target genes. The authors have significantly observed cell viability decreases and apoptosis stimulation when compared to control cells (DP-curcumin). The anticancer effect of DP-curcumin-mir-21ASO complex was also evaluated in in vivo essays and it was noticed a significantly tumor growth decrease compared with miR-21ASO and curcumin alone in animals. Curcumin seems increasing miR-21 and consequently inhibiting PDCD4 and PTEN target genes, resulting in cell death.

Glioma stem cells (GSCs) were transfected with lentivirus-GFP-miR-145, upregulating miR-145 and treated with demethoxycurcumin (DC), a curcumin compound. It was observed a greater inhibition in tumor growth and stimulation of apoptosis in both in vivo and in vitro when compared to control and to monotherapy (30). Combined lentivirus-GFP-miR-145 plus DC was able to inhibit miR-145 target gene SOX2, leading to downstream beta-catenin downregulation, responsible for transcriptional activation of CCND1 and C-MYC (30).

Interestingly, curcumin appears to suppress AP-1 and NF-kB pathways leading to chemosensitization (91). Therefore, Wu et al. (26) have treated U87MG cells with curcumin and TMZ alone, and a higher expression of miR-146a was observed in curcumin-treated cells, in a dose-dependent way. The enhancement of miR-146a leads to inhibition of p65 and phosphorylation IκBα and consequently, suppressing NF-kB, increasing the TMZ-induced apoptosis (26).

The effects of curcumin on several pathways at GBM cell lines are summarized in Figures 3A,B. In addition, this study indicated that only DPG and curcumin have an antitumoral effect on GBM cell lines through miR modulation (Table 2, Figure 3).

Furthermore, curcumin in combination with radiation has presented a synergistic effect in U87MG and T98G GBM cell lines, using different concentrations (ranging from 5 to 25 μM and 3.25–26 μM in U87 and T98 cells, respectively) and dosages (2 Gy or 4 Gy of irradiation). In U87 cells, curcumin and radiation exerted synergism in most of the tested combinations, and the highest synergy was monitored when curcumin was given at its IC50 (10 μM). In T98 cells, the highest levels of synergy were observed at higher curcumin concentrations, particularly at 26 μM, possibly due to the resistance of those cells to both chemotherapy and radiotherapy. The combinatorial treatment arrested both cell lines at the G2/M phase to a higher extent than radiation or curcumin treatment alone. In addition, it was also observed a synergistic effect of curcumin when combined with TMZ resulting in increased tumor cell death (92).

In accordance, Yin et al. (93) have treated U87MG cell line with curcumin in combination with TMZ. The authors have observed that apoptosis was enhanced, in vitro and in vivo, in the GBM cells by both generation of reactive oxygen species production and phosphorylated AKT and mTOR suppression. These data indicated that blockage of AKT/mTOR signaling appeared to contribute to the elevated apoptosis caused by the combination treatment of curcumin and TMZ. Further, in the U87MG xenograft mouse model, the combination treatment with curcumin and TMZ showed a significantly enhanced inhibition of tumor growth compared with single treatments. A similar trend was observed by the authors in the measurement of tumor weight.

In traditional medicine, the base extracts of species of the genus Euphorbia (Euphorbiaceae) are often used as a form of treatment for ulcers and warts (32, 94–96). The euphol (C₃₀H₅₀O), a tetracyclic triterpene alcohol, is the main constituent of the Euphorbia tirucalli known as aveloz (Figure 1D) and it has been observed to have antiinflammatory effects as antiviral, analgesic, and nociceptive properties. Recently, a potential antitumor activity has been also noticed by euphol (97–99). Thus, euphol was able to decrease cell viability in CS12 gastric cancer cell line (100, 101).

Silva et al. (32) have evaluated the euphol antitumor effect in 12 human gliomas and GBM cell lines comprising seven adults (U87MG, U373, U251, GAMG, SW1783, SNB19), five pediatric glioma cell lines (RES186, RES259, KNS42, UW479, and SF188), two primary cultures (HCB2 and HCB149), and one normal astrocyte cell line (NHA) for cytotoxic assays. The pediatric cell lines have showed more euphol sensibility than adult and primary cultures. Moreover, euphol had a higher selective cytotoxicity index (0.64-3.36) than TMZ (0.11-1.13). When combined, euphol and TMZ treatments seem to have a synergistic effect [combination index (CI <1) in 67% (8/12) of the glioma cell lines investigated (mean CI values: range: 0.48–0.96)]. However, no effect was found on cell cycle distribution, invasion, and colony cell formation (90). In addition, the authors have compared both drug-sensitive (GAMG) and drug-resistant (U373) cell lines using euphol dose at 15 μM, which was able to inhibit GAMG and U373 proliferation by 35.44 and 28.71%, respectively. Moreover, at 15 μM euphol suppressed cell viability of GAMG cells by 88.86% and U373 cells by 13.9%. These data suggest that euphol seems to have predominantly cytotoxic effects on the anchorage-dependent growth of both malignant glioma cell lines (32). Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic TMZ interactions in most above GBM cell lines (32). In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM.

In addition to euphol, the genus Euphorbia also has diterpenes as important bioactive constituents some already approved for precancerous conditions (33, 102–104). One diterpene that was approved for human use for the treatment of actinic keratosis, ingenol-3-angelate (I3A) (Picato®), from Euphorbia peplus demonstrated great antineoplastic potential evaluated in clinical trials for the effective treatment of basal cell carcinoma and squamous cell carcinoma through the modulation of PKC signaling (105–109). Some studies have also revealed diterpenes as promising modulators of multidrug resistance (MDR) in tumor cells and also showing in vivo antiinflammatory activity (110).

Recently, the cytotoxic potential of a new esters of semisynthetic ingenol from E. tirucalli, the derivative ingenol-3-dodecanoate (Ingenol C-IngC) was reported. IngC showed higher efficacy when compared to I3A and ingenol 3,20-dibenzoate (IDB) from E. esula L on esophageal cancer cell lines, two important ingenol diterpenes that can promote PKC activation and anticancer activity (33, 108, 111). In a panel of 11 glioma cell lines, IngC acted as a potent inhibitor of protein kinase C (PKC) activity by PDK1 inhibiting and consequently tumor invasion and migration impairment through Wnt/β-catenin (β-C) pathways by lower concentration the effector protein β-C (93) (Figure 2B). The different models of glioma cell lines exhibited a heterogeneous profile of response to IngC. At a fixed dose of 10 μM, 9.1% (1/11) of cell lines were resistant, 36.4% (4/11) were moderately sensitive, whereas 54.5% (6/11) were classified as highly sensitive. These findings identify IngC as a promising lead compound for the development of new cancer therapy and they may guide the search for additional PKC inhibitors.

Despite euphol's antitumor action, some reports have demonstrated that exposure to crude Euphorbia tirucalli may be a risk factor for Burkitt's lymphoma, as a result its performance as a genotoxic agent (97, 112) indicating that further studies are needed to define the potential therapeutic use of euphol. The effects of IngC and euphol on different GBM cell pathways are summarized in Figures 2B, 3A, respectively.