Flavonoids Synergistically Enhance the Anti-Glioblastoma Effects of Chemotherapeutic Drugs

Flavonoids are polyphenolic plant secondary metabolites with pleiotropic biological properties, including anti-cancer activities. These natural compounds have potential utility in glioblastoma (GBM), a malignant central nervous system tumor derived from astrocytes. Conventional GBM treatment modalities such as chemotherapy, radiation therapy, and surgical tumor resection are beneficial but limited by extensive tumor invasion and drug/radiation resistance. Therefore, dietary flavonoids—with demonstrated anti-GBM properties in preclinical research—are potential alternative therapies. This review explores the synergistic enhancement of the anti-GBM effects of conventional chemotherapeutic drugs by flavonoids. Primary studies published between 2011 and 2021 on flavonoid–chemotherapeutic synergy in GBM were obtained from PubMed. These studies demonstrate that flavonoids such as chrysin, epigallocatechin-3-gallate (EGCG), formononetin, hispidulin, icariin, quercetin, rutin, and silibinin synergistically enhance the effects of canonical chemotherapeutics. These beneficial effects are mediated by the modulation of intracellular signaling mechanisms related to apoptosis, proliferation, autophagy, motility, and chemoresistance. In this light, flavonoids hold promise in improving current therapeutic strategies and ultimately overcoming GBM drug resistance. However, despite positive preclinical results, further investigations are necessary before the commencement of clinical trials. Key considerations include the bioavailability, blood–brain barrier (BBB) permeability, and safety of flavonoids; optimal dosages of flavonoids and chemotherapeutics; drug delivery platforms; and the potential for adverse interactions.


The Challenges of GBM Therapy and the Potential of Flavonoids
Glioblastoma (GBM) is an astrocyte-derived solid tumor of the brain or spinal cord that occurs at an overall rate of 3.19 cases per 100,000 individuals in the United States. Its incidence varies notably between subpopulations, with males and older individuals at higher risk [1]. GBM is fatal, with median survival times under one year [2].
Currently, conventional medical and surgical interventions predominate in GBM therapy. Standard treatment regimens include (1) radiation therapy with concurrent temozolomide (TMZ) chemotherapy and (2) surgical tumor resection with radiation therapy [3,4]. Recent advances in these therapies have improved patient outcomes; the addition of TMZ, an alkylating agent, to standard radiation-only regimens after 2005 greatly increased survival rates [2]. Nevertheless, conventional interventions remain constrained by GBM's malignant properties. Surgical methods, for instance, are hindered by widespread tumor invasion and metastasis, while drug and radiation resistance-particularly associated with glioma stem cells (GSCs)-pose challenges for chemo-and radiotherapy [5,6]. Intraand intertumoral heterogeneity further complicates anti-GBM regimens [6]. Therefore, Biomolecules 2021, 11, 1841 2 of 15 a need exists for alternative and supportive therapies with the potential to overcome these challenges.
Dietary natural compounds constitute promising candidates in this regard; they have wide-ranging biological properties, including anti-cancer effects [7][8][9][10][11]. Among these compounds, flavonoids-polyphenolic plant secondary metabolites-are of interest. Flavonoids exert anti-cancer effects through chemosensitization, metabolic modulation, metastatic inhibition, and apoptotic induction [12,13]. Based on these well-evidenced oncostatic activities, flavonoids have great potential in modulating GBM cell responses to anti-cancer drugs by overcoming their therapeutic resistance. The efficacy of flavonoids in GBM is well documented in preclinical research [14]. This review aims to complement previous research by focusing on the synergistic efficacy of flavonoids and conventional chemotherapeutics in GBM therapy.

Study Methodology
Primary studies on flavonoid-chemotherapeutic synergy in GBM were obtained through a PubMed search with the keywords "flavonoid", "chemo *", "synerg *" and "glioblastoma" or "glioma." Approximately 15 articles published from 2011 to 2021 were included. Studies demonstrating the effects of flavonoids alone on GBM-without trials with chemotherapeutic drugs-were excluded.

Flavonoids
Bioactive flavonoids occur in fruits, vegetables, and other natural plant products and are unified by a three-ring structural backbone that includes two phenyl rings and one central heterocyclic ring. These compounds are classified based on structural differencesrelated primarily to the presence and positioning of substituents on the heterocycle ( Figure 1). A variety of flavonoids, including flavan-3-ols, flavones, isoflavones, flavonols, flavonol glycosides, and flavonolignans, demonstrate anti-GBM effects combined with chemotherapeutic drugs in vitro and/or in vivo (Table 1).
Biomolecules 2021, 11, x 2 of 17 [5,6]. Intra-and intertumoral heterogeneity further complicates anti-GBM regimens [6]. Therefore, a need exists for alternative and supportive therapies with the potential to overcome these challenges. Dietary natural compounds constitute promising candidates in this regard; they have wide-ranging biological properties, including anti-cancer effects [7][8][9][10][11]. Among these compounds, flavonoids-polyphenolic plant secondary metabolites-are of interest. Flavonoids exert anti-cancer effects through chemosensitization, metabolic modulation, metastatic inhibition, and apoptotic induction [12,13]. Based on these well-evidenced oncostatic activities, flavonoids have great potential in modulating GBM cell responses to anti-cancer drugs by overcoming their therapeutic resistance. The efficacy of flavonoids in GBM is well documented in preclinical research [14]. This review aims to complement previous research by focusing on the synergistic efficacy of flavonoids and conventional chemotherapeutics in GBM therapy.

Study Methodology
Primary studies on flavonoid-chemotherapeutic synergy in GBM were obtained through a PubMed search with the keywords "flavonoid," "chemo*," "synerg*," and "glioblastoma" or "glioma." Approximately 15 articles published from 2011 to 2021 were included. Studies demonstrating the effects of flavonoids alone on GBM-without trials with chemotherapeutic drugs-were excluded.

Flavonoids
Bioactive flavonoids occur in fruits, vegetables, and other natural plant products and are unified by a three-ring structural backbone that includes two phenyl rings and one central heterocyclic ring. These compounds are classified based on structural differences-related primarily to the presence and positioning of substituents on the heterocycle ( Figure 1). A variety of flavonoids, including flavan-3-ols, flavones, isoflavones, flavonols, flavonol glycosides, and flavonolignans, demonstrate anti-GBM effects combined with chemotherapeutic drugs in vitro and/or in vivo (Table 1).   Flavan-3-ols comprise a class of flavonoids with a hydroxyl substituent at the third position of the heterocyclic ring. One flavan-3-ol of particular interest in GBM therapy, epigallocatechin-3-gallate (EGCG), occurs predominantly in green tea and exerts proapoptotic, antiproliferative, and antioxidant effects in cancerous cells [15].
In contrast, the class of flavones and isoflavones includes flavonoid compounds with a ketone substituent at the fourth position of the heterocycle. Two flavones and one isoflavone are of interest in synergistic GBM therapy. Chrysin, a flavone found in passionflower, honey, and propolis, has anti-cancer, neuroprotective, and other beneficial properties [16]. Similarly, hispidulin, a flavone from Grindelia, Artemisia, and Salvia plants, exerts anti-cancer, antifungal, antioxidant, and anti-inflammatory effects; it is moreover a benzodiazepine (BZD) receptor ligand [17]. Finally, formononetin, an O-methylated isoflavone, and phytoestrogen in legumes and clovers, have anti-cancer properties [18].
Flavonols have both the third position hydroxyl substituent of flavan-3-ols and the fourth position ketone substituent of flavones. Flavonols and flavonol glycosides, including quercetin, rutin, and icariin, are of interest in synergistic GBM therapy. Quercetin, a flavonol found in oak, berries, apples, grapes, cilantro, and onions, exerts antioxidant, antihistamine, anti-inflammatory, and anti-cancer activities [19]. Rutin, the glycoside of quercetin, has similar biological activities and occurs in rue, apples, buckwheat, and citrus fruits [21]. Another flavonol glycoside, icariin, is commonly found in horny goat weed; in addition to its anti-cancer properties, it has aphrodisiac, neuroprotective, and anti-osteoporotic effects [20].
Finally, flavonolignans are flavonoid derivatives with both flavonoid and phenylpropanoid structural components. Silibinin, a flavonolignan of interest in synergistic GBM therapy, is found in milk thistle seeds and has broad anti-cancer and antimetastatic effects [22].

Chemotherapeutics
Conventional chemotherapeutics leverage diverse mechanistic pathways to exert their anti-cancer effects. TMZ, the canonical anti-GBM drug, is an alkylating agent that induces apoptotic cell death through the p53-dependent and O6-methylguanine-based activation of the Fas/caspase 8 pathway ( Figure 2) [23]. In addition, several noncanonical and repurposed drugs hold promise in synergistic GBM therapy ( Table 2).    Purine analogs Alkylating agent [23] One such drug, arsenic trioxide (ATO), exerts pleiotropic anti-cancer effects through ROS generation and cell cycle regulation [24]. In glioma cells, ATO induces caspaseindependent autophagic cell death [29]. Moreover, combinations of ATO and TMZ and ATO and vismodegib exert synergistic effects against GBM growth in vivo [30].

Chemotherapeutic Class Primary Function Reference
Chloroquine, another compound of interest, is a repurposed antimalarial drug that induces p53-dependent apoptosis and disrupts the mitochondrial membrane potential in glioma cells [25]. In conjunction with standard radiation and chemotherapeutic treatment regimen, a recent clinical trial examined its efficacy against GBM [31].
Similarly, the naturally derived topoisomerase II inhibitor etoposide was extensively clinically trialed in GBM. Etoposide induces glioma cell apoptosis through sequential ceramide formation, Bax/Bcl-2 modulation, cytochrome c release, and caspase activation [27].
Finally, sodium butyrate (NaB) is a short-chain fatty acid histone deacetylase inhibitor that reduces glioma cell proliferation, migration, and cell cycle progression [28]. While NaB has anti-GBM potential, its effects remain unsubstantiated by clinical trials at this time.

Mechanisms of GBM
GBM tumorigenesis, progression, and metastasis are driven by numerous interconnected signaling mechanisms ( Figure 3). Rapid cell proliferation, an essential process at all stages of GBM development, is mediated by the Akt/mammalian target of rapamycin (mTOR), nuclear factor κappa of activated B cells (NF-κB), and other similar pathways. Uncontrolled proliferation of this nature is enabled by the inhibition of normal cell cycle controls (such as FOXO and p53), and the downregulation of key actors in autophagic (LC3, Beclin-1, and P62) and apoptotic (caspases) cell death. Moreover, a metabolic transition to aerobic glycolysis (the Warburg effect) energetically sustains rapid GBM cell division. Angiogenic and neovascular processes-stimulated mainly by vascular endothelial growth factor (VEGF) signaling-ensure oxygen and nutrient transport to growing tumors. GBM cells may further develop chemoresistance; this often occurs through O6methylguanine methyltransferase (MGMT), which confers resistance to alkylating agents and/or P-glycoprotein (P-gp), which enhances drug efflux from the cells. Finally, Snail, Slug, and matrix metalloproteinases (MMPs) contribute to the epithelial-mesenchymal transition (EMT), which causes GBM cells to develop migratory and invasive phenotypes.
lial growth factor (VEGF) signaling-ensure oxygen and nutrient transport to growing tumors. GBM cells may further develop chemoresistance; this often occurs through O6methylguanine methyltransferase (MGMT), which confers resistance to alkylating agents and/or P-glycoprotein (P-gp), which enhances drug efflux from the cells. Finally, Snail, Slug, and matrix metalloproteinases (MMPs) contribute to the epithelial-mesenchymal transition (EMT), which causes GBM cells to develop migratory and invasive phenotypes.

TMZ and Icariin
While icariin functions primarily as an apoptotic enhancer in conjunction with TMZ, it also inhibits NF-κB-mediated proliferation and reduces migration and invasion in U87MG cells [36].

TMZ and Rutin
TMZ increases both apoptotic and autophagic cell death in GBM cells. At the same time, the flavonoid rutin shifts the balance toward apoptosis by upregulating caspases and inhibiting autophagy by downregulating light chain 3 (LC3) and c-Jun N-terminal kinase (JNK). As such, TMZ and rutin synergistically decrease tumor weight and volume in both intracranial (orthotopic) and subcutaneous (heterotopic) murine xenograft models [33].

Other Combinations of Flavonoids and Chemotherapeutics
Six additional flavonoid-chemotherapeutic combinations with promising synergistic anti-GBM effects are quercetin and chloroquine, quercetin and NaB, Gardenia jasminoides (GJ) extract and cisplatin, silibinin and etoposide, silibinin and ATO, and chrysin and ATO (Table 4).

Quercetin and Chloroquine
Co-administration of quercetin with chloroquine causes both apoptotic and autophagic cell death ( Figure 5). These compounds induce autophagy by upregulating Beclin-1, LC3, and P62 and increasing apoptosis through ER stress and mitochondrial dysfunction. ER stress, associated with the upregulation of ATF4 and CHOP and the buildup of ubiquitinated proteins, leads to calcium (Ca 2+ ) release into the cytosol. Intracellular Ca 2+ then enters mitochondria via the mitochondrial Ca 2+ uniporter (MCU); increased mitochondrial calcium concentrations ([Ca 2+ ] m ) upregulate the generation of reactive oxygen species (ROS), which in turn contribute to caspase-induced apoptosis [43]. ER stress causes the release of Ca 2+ into the intracellular space; some of this Ca 2+ enters mitochondria via the MCU, leading to mitochondrial ROS generation. In this case, both mitochondrial ROS and autophagic mechanisms contribute to apoptotic cell death.

GJ and Cisplatin
Flavonoid-rich GJ extract synergistically enhances cisplatin-induced apoptotic cell death through the upregulation of active caspases. However, GJ-cisplatin synergy differs from quercetin-chloroquine synergy. GJ inhibits cisplatin-induced autophagy in favor of apoptosis in a manner consistent with that of rutin-TMZ synergy (Figure 3) [44].

Quercetin and NaB
Similarly to GJ extract, quercetin synergistically enhances apoptosis by upregulating caspases and downregulating Survivin and Bcl-2, and concurrently inhibits NaB-induced autophagy by downregulating LC3 and Beclin-1 [45]. Cellular senescence is another option for GBM therapy; NaB and quercetin together induce senescence-like growth arrest in U87 and C6 cells [48]. ER stress causes the release of Ca 2+ into the intracellular space; some of this Ca 2+ enters mitochondria via the MCU, leading to mitochondrial ROS generation. In this case, both mitochondrial ROS and autophagic mechanisms contribute to apoptotic cell death.

GJ and Cisplatin
Flavonoid-rich GJ extract synergistically enhances cisplatin-induced apoptotic cell death through the upregulation of active caspases. However, GJ-cisplatin synergy differs from quercetin-chloroquine synergy. GJ inhibits cisplatin-induced autophagy in favor of apoptosis in a manner consistent with that of rutin-TMZ synergy (Figure 3) [44].

Quercetin and NaB
Similarly to GJ extract, quercetin synergistically enhances apoptosis by upregulating caspases and downregulating Survivin and Bcl-2, and concurrently inhibits NaB-induced autophagy by downregulating LC3 and Beclin-1 [45]. Cellular senescence is another option for GBM therapy; NaB and quercetin together induce senescence-like growth arrest in U87 and C6 cells [48].

Key Considerations and Challenges
While recent preclinical findings on flavonoid-chemotherapeutic synergy in GBM therapy are promising, many mechanistic unknowns, intricacies, and challenges remain. One major limitation of current knowledge is inherent in the literature: all of the reviewed studies are in vitro or in vivo preclinical studies utilizing statistical significance as a threshold for treatment efficacy. However, statistical significance does not necessarily correspond to clinical significance, and laboratory studies are often insufficient to predict outcomes under genuine (and highly variable) physiological conditions.
Another pertinent consideration related to the preclinical literature is the justification of synergistic effects. The data in Tables 3 and 4 represent synergism as defined in the reviewed primary studies. However, it is worth noting that synergism is poorly defined at present, with limited consensus across the scientific and biomedical communities; this ambiguity leads to the mischaracterization of additive and other combined effects as synergistic effects in some cases. As such, standardized measures of synergism have been proposed. One auspicious measure developed by Chou and Talalay evaluates synergism as a mass action-rather than statistical-phenomenon, using a combination index (CI) rather than p values [49]. Notably, a significant proportion of the reviewed studies utilized CI to measure synergism (or lack thereof). Zhang et al. presented CI < 1 for combinations of 40-320 µM formononetin and 250-2000 µM TMZ, indicating synergy between the two compounds [39]. Similarly, Wang et al. demonstrated synergy between hispidulin and TMZ, with CI = 0.584 [37]. Synergistic effects of EGCG-TMZ, quercetin-chloroquine, quercetin-NaB, chrysin-ATO, and silibinin-ATO combinations were likewise justified with CI < 1 [32,43,45,46].
Concerning the flavonoids themselves, their consideration as medicinal agents necessitates evaluating their toxicity, blood-brain barrier (BBB) permeability, bioavailability, and potential adverse effects under said physiological conditions. Most of the flavonoids included in this review are nontoxic: chrysin at up to 400-500 mg per day, EGCG at 338 mg, quercetin at 5000 mg, rutin at 1000 mg, and silibinin at 20 mg/kg [16,[50][51][52][53]. Icariin is well tolerated at lower doses; however, gastrointestinal side effects may occur at 1,680 mg [54]. Importantly, formononetin administration poses a risk of allergic immune responses through pro-inflammatory cytokines such as interleukin 4 (IL-4) [55]. Finally, the toxicity profile of hispidulin requires further assessment [56].
Beyond toxicity, the potential physiological side effects of flavonoids-both beneficial and detrimental-merit consideration. Hispidulin, for instance, is a BZD receptor antagonist with anti-convulsive effects in vivo [57]. Another flavonoid, formononetin, is a phytoestrogen. While this flavonoid exerts neuroprotective effects through estrogen receptor βeta (ERβ)-dependent inhibition of NF-κB activity and microglia-induced neuroinflammation, it may also promote angiogenesis and endothelial cell proliferation (both potentially detrimental) via estrogen receptor αlpha (Erα) [58,59].
Nontoxicity and a favorable side effect profile constitute the baseline for human consumption; however, effective anti-GBM agents must have high bioavailability (to be present in sufficient doses following oral administration) and BBB permeability (to enter the brain from the bloodstream). Flavonoids and other natural compounds are significantly limited by their low bioavailability and poor aqueous solubility; the bioavailabilities of chrysin, EGCG, formononetin, hispidulin, icariin, rutin, and silibinin are accordingly poor [16,18,22,52,56,[60][61][62]. Extensive metabolism in the intestine, colon, and liver (with the participation of gut microbiota) further limits the bioavailability of these flavonoids [13]. In this regard, a cooperative gut microbiome is essential for their bioavailability and absorption [63]. Quercetin's bioavailability is comparatively better but remains constrained by intestinal efflux and biliary excretion [64]. More promisingly, EGCG, hispidulin, icariin, quercetin, and rutin can cross the BBB; silibinin cannot, while the permeability of chrysin and formononetin remains unclear [17,[65][66][67][68]. In this light, developing novel formulations to enhance the bioavailability and brain delivery of flavonoids is of key interest in advanc-ing synergistic anti-GBM therapy. Current research particularly highlights the potential of nanotechnology approaches to this end [12].
Although flavonoids are associated with some challenges, especially in the clinical sphere, they can confront GBM drug resistance, which hinders current conventional therapies. TMZ's introduction, for instance, improved therapeutic outcomes; however, TMZ resistance in GBM-mediated by the overexpression of MGMT and alkylpurine-DNA-N glycosylase (APNG), which repair TMZ-induced DNA lesions and thereby prevent apoptosis-is now well documented [69]. Cisplatin resistance via hypoxia-inducible factors 1 and 2 (HIF-1/2) and cluster of differentiation 133 (CD133) is also reported in GBM cell lines [70]. Moreover, an etoposide-resistant glioma cell line has been established [71]. Flavonoids hold promise in overcoming these types of resistance, as they downregulate key factors such as MGMT and P-gp and can therefore serve as chemosensitizers.
Taken together, the criteria of efficacy, nontoxicity, BBB permeability, and bioavailability suggest that (1) rutin and TMZ and (2) EGCG and TMZ are auspicious combinations. Rutin and EGCG are nontoxic, have favorable side effect profiles, and can cross the BBB. However, further preclinical experiments and eventually clinical trials are necessary to substantiate the efficacy and safety of these and other flavonoid-chemotherapeutic combinations.

Conclusions and Outlook
Despite recent medical advances, GBM's prognosis remains poor. Extensive tumor invasiveness and therapeutic resistance hinder conventional drug, radiation, and surgical therapies. In this regard, flavonoids hold potential as supportive agents that can mitigate the numerous challenges posed by GBM. The flavonoids chrysin, EGCG, formononetin, hispidulin, icariin, quercetin, rutin, and silibinin demonstrate synergistic anti-GBM effects in conjunction with TMZ, cisplatin, chloroquine, etoposide, NaB, and ATO. These beneficial effects are mediated by the enhancement of apoptosis and the reduction of proliferation, migration, and chemoresistance. As such, flavonoids could enhance individual outcomes of GBM therapy, especially by overcoming therapeutic resistance.
While these findings are promising, supportive evidence for flavonoid-chemotherapeutic synergy is currently limited to the preclinical literature. It is additionally worth noting that although many flavonoids exert anti-GBM effects, only some have been evaluated as potential synergistic agents. As such, forward-looking studies should clarify the synergistic effects of promising yet underinvestigated flavonoids. Furthermore, rigorous evaluation of the physiological properties of flavonoids-including toxicity, side effects, bioavailability, and BB permeability-is necessary on the path toward clinical implementation. If and when appropriate, clinical trials should investigate and confirm the safety and therapeutic efficacy of flavonoid-chemotherapeutic combinations.