Curcumin’s Beneficial Effects on Neuroblastoma: Mechanisms, Challenges, and Potential Solutions

Curcumin, a natural polyphenolic compound derived from the South Asian turmeric plant (Curcuma longa), has well-characterized antioxidant, anti-inflammatory, anti-protein-aggregate, and anticancer properties. Neuroblastoma (NB) is a cancer of the nervous system that arises primarily in pediatric patients. In order to reduce the multiple disadvantages and side effects of conventional oncologic modalities and to potentially overcome cancer drug resistance, natural substances such as curcumin are examined as complementary and supportive therapies against NB. In NB cell lines, curcumin by itself promotes apoptosis and cell cycle arrest through the suppression of serine–threonine kinase Akt and nuclear factor kappa of activated B-cells (NF-κB) signaling, induction of mitochondrial dysfunction, and upregulation of p53 and caspase signaling. While curcumin demonstrates anti-NB efficacy in vitro, cross-validation between NB cell types is currently lacking for many of its specific mechanistic activities. Furthermore, curcumin’s low bioavailability by oral administration, poor absorption, and relative insolubility in water pose challenges to its clinical introduction. Numerous curcumin formulations, including nanoparticles, nanocarriers, and microemulsions, have been developed, with these having some success in the treatment of NB. In the future, standardization and further basic and preclinical trials will be required to ensure the safety of curcumin formulations. While the administration of curcumin is clinically safe even at high doses, clinical trials are necessary to substantiate the practical efficacy of curcumin in the prevention and treatment of NB.

stabilized, accumulated, and overexpressed [24,25]. This is a typical mechanism for the functional inactivation of p53 in NB [24][25][26]. However, the p53 signaling pathway can be activated in several NB cell lines despite its cytoplasmic localization and nuclear exclusion [26][27][28]. In addition, the expression of p53 in malignant NB cells, as opposed to non-malignant NB cells, is significantly downregulated at the physiological level [24,25,29]. This underscores the possibility that these cells can undergo apoptosis [30,31]. Interestingly, the p53 transformation process in NB cells is reversible, suggesting that it could represent a therapeutic target. Malignant NB cells are often resistant to typical radiation and chemotherapy treatments. The upregulation of transcription factors such as NF-κB and activator protein 1 (AP-1) in tumors is partly responsible for this increased resistance [32,33]. Curcumin, a strong and natural NF-κB inhibitor [34][35][36][37][38][39][40], significantly reduces cell survival and modulates the expression of apoptosis-related genes in human NB cells in combination with radiation therapy [32]. Notably, p53-dependent cell death in various tumor cells can be specifically and effectively activated by treatment with the naturally occurring polyphenol curcumin [41].
Curcumin exists as a yellow-orange solid, with a molecular weight of 368 g/mol and a melting point of 183 • C. Chemically, it is a polyphenolic compound with two aromatic rings, each with one hydroxy and one methoxy substituent ( Figure 1). A seven-carbon chain with two α-β unsaturated carbonyl groups (which are subject to tautomerization) links the rings [43]. Chemical structure of curcumin, a polyphenolic chemical constituent of turmeric with antioxidant, anti-inflammatory, and anticancer effects. Curcumin is a beta-diketone compound containing two substituted aromatic rings linked by a seven-carbon chain. Each aromatic ring has one hydroxy and one methoxy group.
The biological and physiological properties of curcumin have been extensively examined and reviewed in the context of neurodegenerative and inflammatory diseases, such as Alzheimer's disease and dementia. Curcumin exhibits notable antioxidant activities through iron chelation, inhibition of lipid peroxidation, and scavenging of reactive oxygen species (ROS) [52]. The compound also has anti-inflammatory properties in acute and chronic inflammation [53]. Beyond these functions, curcumin is widely examined as an anticancer agent. Orally administered curcumin-alone and in combination with conventional chemotherapeutics-has been clinically trialed in pancreatic, breast, and prostate cancer patients [54][55][56][57]. Curcumin also demonstrates oncologic effects by itself on murine glioblastoma xenografts and in combination with the chemotherapeutic 5-fluorouracil on colorectal cancer cells [49,[58][59][60].
Although curcumin has many positive effects on health, it is sometimes criticized for its low bioavailability-a result of its low absorption and rapid metabolism by the body. However, its bioavailability can be enhanced through combination with adjuvants. Indeed, piperine, a component of black pepper, is suitable for this purpose [61][62][63]. Notably, while curcumin's anticancer properties are frequently reported, its membrane transport mechanisms remain mostly uncharacterized. Molecular dynamics simulations and solid-state nuclear magnetic resonance experiments indicate that curcumin can insert itself into plasma membranes [64,65]. However, specific transport mechanisms for the nervous system and NB cells are unclear at this time.
Despite these shortcomings, curcumin is a major component of numerous spices and foods, and is well tolerated in relatively high concentrations as part of daily diets without side effects. As such, it has high potential as a putative chemopreventive or therapeutic agent [66].

Rationale and Aims of the Study
A vast body of preclinical literature is available on the anticancer properties of curcumin. Promising results have arisen from pancreatic and colon cancer cells, and within the CNS in glioblastoma and brain tumor cells [67]. Within the previous decade, clinical trials have substantiated the potential oncologic applications of curcumin for several common cancer types (pancreatic, prostate, and breast cancers). Moreover, clinical trials have also tested the anticancer properties of curcumin in the nervous system. Its effects on glioblastoma cell lines have been widely documented and extensively reviewed. However, the literature relating curcumin and NB, a rare malignancy with poor prognosis, remains comparatively sparse.
As such, this review explores curcumin's viability as a natural supportive therapy for the prevention and treatment of NB. It first summarizes existing preclinical trials of curcumin on NB cell lines and presents overarching pathways through which curcumin may exert oncologic effects. This study then notes the synergistic and combinatorial effects of curcumin with other natural substances, chemotherapeutics, and radiotherapy on NB. Moreover, the differential effects of curcumin on various NB cell lines and types are explored. Finally, this review discusses the current challenges involved in curcumin delivery and presents some potential solutions and improvements.

Study Methodology
Core literature for this review was obtained from PubMed and Google Scholar, using the keywords "curcumin," "neuroblastoma," and "apoptosis." A PubMed search with these keywords yielded 42 literature results. Approximately 20 of these studies were utilized for this review. Studies including both curcumin and NB but concerning subjects other than oncology (e.g., Alzheimer's disease, or neurodegeneration) were excluded. Further literature (approximately 10 recent studies) on the bioavailability and absorption of curcumin was obtained using the keywords "curcumin", "bioavailability", "nervous system", or "brain delivery".

Curcumin as an Anticancer Agent in NB
Preclinical trials demonstrate that curcumin exerts a range of oncologic effects on NB cell lines. These include the induction of apoptosis and cell cycle arrest, downregulation of proliferative signaling pathways, inhibition of cell migration, and disruption of cellular glucose metabolism (Table 1).

Apoptosis and Cell Cycle Arrest
In cancerous cells, proliferative and pro-survival effectors such as the serine-threonine kinase Akt, nuclear factor kappa light-chain enhancer of activated B-cells (NF-κB), Bcl-2, and Survivin are active, while apoptotic effectors such as cytochrome c, p53, and caspases are suppressed. Curcumin shifts the intracellular signaling mechanisms in NB to upregulate apoptosis and cell cycle arrest [41]. Mechanistically, curcumin promotes apoptosis through the downregulation of Akt and NF-κB signaling, induction of mitochondrial dysfunction, upregulation of p53, and activation of caspases ( Figure 2). Upon its in vitro application to NB cells, curcumin firstly modulates phosphatase and tension homolog (PTEN)-Akt signaling. Curcumin upregulates PTEN, which downregulates Akt and reduces Akt translocation from the cytosol to the nucleus [68]. Downregulation of Akt, in turn, leads to (1) downregulation of NF-κB; (2) upregulation of the pro-apoptotic mitochondrial protein Bad and induction of its translocation from the cytosol to the mitochondria; and (3) upregulation of the forkhead box O3a (FOXO3a) protein and induction of its nuclear translocation from the cytosol [68][69][70]. FOXO3a consequently upregulates the pro-apoptotic Bcl-2-interacting mediator of cell death (Bim), as well as the Fas ligand (Fas-L). Elevated levels of Fas-L induce the Fas pathway, in which Fas-L and Fas activate caspase 8, a proteolytic enzyme that cleaves the BH3-interacting domain death agonist (Bid) protein into its active form [68,71] (see Figure 2).
In inducing mitochondrial dysfunction, curcumin depolarizes the mitochondrial membrane potential (MMP), leading to the downregulation of Bcl-2 [72]. The upregulation of Bim and downregulation of NF-κB, as previously discussed, also contribute to the downregulation of Bcl-2 [69,73]. In turn, the downregulation of Bcl-2 causes the upregulation of the pro-apoptotic Bcl-2-associated X protein (Bax) [72]. Together, the upregulation of Bax, Fas-L, Bid, and Bad; the depolarization of the MMP; as well as the downregulation of Bcl-2 all contribute to the release of cytochrome c from the mitochondria into the cytosol [70][71][72]74]. Additionally, curcumin downregulates the mitochondrial heat shock protein Hsp60 [68].  The proliferative Akt, NF-κB, and ERK 1/2 pathways, as well as the antiapoptotic Bcl-2 and Survivin proteins, are active and upregulated. Apoptotic signals via mitochondrial dysfunction, cytochrome c release, p53, and caspases are downregulated. Cyclin-dependent kinase inhibitors (p21 and p27) are inactive, allowing for cell cycle progression. (B) Curcumin modulates intracellular signaling in NB cells in favor of apoptosis and cell cycle arrest. Curcumin upregulates PTEN, which in turn downregulates Akt and NF-κB and upregulates FOXO3a and Fas pathway signaling. Moreover, curcumin-induced ROS generation elevates intracellular ROS levels and supports both FOXO3a and p53 n signaling. Together, p53 n , Fas pathway, and Akt signaling, along with mitochondrial membrane potential (MMP) depolarization, promote the release of cytochrome c from the mitochondria into the cytosol, where it supports caspase activation. Mitochondrial dysfunction also involves the downregulation of Hsp60, which ultimately leads to the downregulation of Survivin-a protein that ordinarily inhibits caspase activity. Caspase 3 cleaves PARP-1, causing apoptosis, while FOXO3a and p53 activate cyclin-dependent kinase inhibitors, which induce cell cycle arrest.
Interestingly, curcumin activates the degradation of amyloidogenic proteins, including the amyloid β precursor protein and α-synuclein, via translocation of the transcription factor EB-autophagy signaling pathway. This activity is regulated by the inhibition of GSK-3β signaling, which thereby increases antioxidant gene expression in human NB cells [81]. Bavisotto and co-workers demonstrated that curcumin decreases heat shock protein (Hsp60) levels and upregulates Hsp60 mRNA expression, and thereby causes apoptosis. Curcumin downregulates the ubiquitination and nitration of Hsp60 and increases chaperonin levels, indicating that it may disrupt NB progression through a protective pathway involving chaperonin [82].

Effect
Cell

Curcumin Applied Simultaneously with Other Natural Substances
While curcumin is independently effective in suppressing NB, it also exhibits synergistic effects in conjunction with several conventional and natural therapies ( Table 2). Compared to other natural substances, curcumin and berberine (BBR) together demonstrate more significant cytotoxic effects on NB cells than either substance alone [90]. BBR, a plant-derived alkaloid, is a common dietary supplement. However, some clinical trials reported adverse reactions or toxicity at high doses, with 14.3% incidence of adverse effects at dosages of 0.9 to 1.5 g/day and 10.6% incidence at 1.5 g/day [91]. Therefore, while the application of 100 µM BBR is possible in vitro, clinical attainment of such a high concentration may induce potentially dangerous side effects.

Curcumin Applied Simultaneously with Conventional Chemotherapeutics
Another potential application of curcumin in NB therapy is in combination with conventional chemotherapeutic drugs. These drugs, particularly cisplatin (a platinum compound) and doxorubicin (an anthracycline), share several anti-NB pathways with curcumin ( Figure 3). Similarly to curcumin, cisplatin downregulates Bcl-2 (in SH-SY5Y cells) and upregulates Bax (in B50 cells), thereby inducing mitochondrial dysfunction [93,94]. Moreover, both curcumin and doxorubicin upregulate components of the Fas signaling pathway. Curcumin upregulates Fas-L and induces the Fas signaling pathway in LAN5 cells [68]. Doxorubicin, in contrast, upregulates Fas but not Fas-L in SH-EP-1 and SK-N-AS cells [95]. Therefore, an interesting contrast emerges, as while curcumin enhances mitochondrial dysfunction via the Fas pathway, doxorubicin does not because said pathway does not proceed without the ligand (Fas-L). Mitochondrial dysfunction leads to the release of cytochrome c, as observed in SK-N-SH cells treated with doxorubicin and B50 cells treated with cisplatin [94,96]. Furthermore, in another pathway shared with curcumin, cisplatin upregulates p53 in SH-SY5Y cells [93]. Finally, curcumin, cisplatin, and doxorubicin all induce the activation of caspases 9 and 3, leading to apoptosis. Synergistically, curcumin potentiates the cytotoxic and pro-apoptotic effects of doxorubicin in SH-SY5Y and SK-N-AS cells [69,80]. This effect may be of clinical relevance, since it occurred at relatively low doses (0.05 µg/mL; 0.5-1.5 µM) of doxorubicin, which may feasibly be obtained in physiological environments. Moreover, Fonseka et al. (2020) evaluated a combination treatment of curcumin with doxorubicin in in vitro and in vivo NB models. The in vitro and consequent in vivo results demonstrate the oncostatic efficacy of this drug combination. Notably, curcumin and doxorubicin increased the survival and reduced the tumor volume of mice implanted with SK-N-Be(2) cells [97]. Curcumin also enhances the cytotoxic effects of 15, 30, and 45 µM cisplatin [69]. Similar drug concentrations are physiologically achievable via cisplatin infusion; as such, curcumin-cisplatin therapy has potential clinical applicability [98].

Curcumin in Conjunction with Radiation Therapy
Finally, curcumin also enhances the cytotoxic, apoptotic, and anticolonigenic effects of radiation therapy on NB in vitro. Potential side effects of radiation therapy include the induction of antiapoptotic genes such as Bcl-2 and the suppression of pro-apoptotic effectors such as caspases 3 and 7. However, curcumin can effectively attenuate these negative effects. Importantly, inflammation plays a central role not only in tumor resistance, but also in the incidence of side effects following radiotherapy [99]. Curcumin is not only a radiosensitizer for various malignancies, but also a radioprotector for normal tissues. As such, it exerts protective effects against the following radiotherapy toxicities: dermatitis, pneumonitis, cataractogenesis, neurocognition, myelosuppression, secondary malignancies, and mucositis or enteritis [100]. Moreover, curcumin modulates and downregulates the radiation-activated genes of the TNF superfamily, as well as the expression of NF-κB-and NF-κB-mediated pro-survival signaling in NB cells [32]. Overall, the synergistic potential of curcumin for treating NB is promising based on the results of primary studies, but is not yet supported by clinical trials.
Once sufficient basic and preclinical studies have been conducted, clinical trials will be necessary to substantiate both the isolated and synergistic effects of curcumin on NB. The combination of curcumin with radiation therapy is a promising avenue of investigation, as a low dose of 100 nM curcumin promotes apoptosis and reverses the detrimental side effects of radiation [32]. Significantly, curcumin-mediated radiosensitization and downregulation of radiation-induced antiapoptotic effectors have already been demonstrated in prostate and colorectal cancers [37,101]. Curcumin notably exerts protective effects against radiation-induced inflammation and consequent lung damage [102]. In fact, curcumin was clinically investigated for its anti-inflammatory, anticarcinogenic, and free radical scavenging properties [44]. Table 2. Synergistic anticancer effects of curcumin and other natural substances, conventional chemotherapeutic drugs, or radiation therapy on NB cell lines. While solid lipid curcumin particles (SLCP) were trialed with BBR and CPZChN with piperine, pure (or "standard") curcumin was trialed with doxorubicin, cisplatin, and radiation therapy. Downregulations, decreases, and inhibitions are in bold. "Curc." refers to "Curcumin", "Conc." refers to "Concentration", and "Subs." refers to "Substance".

Challenges in the Use of Curcumin in the Prevention and Treatment of NB
The application of curcumin as a preventive and therapeutic agent in NB is contingent upon its clinical delivery in sufficient doses. While a 2008 study demonstrated statistically significant decreases in the viability of SK-N-SH, Kelly, and LAN-5 NB cells treated with 0.001 µM curcumin, contemporary studies continue to utilize much higher concentrations [86]. As such, many recent studies report anticancer effects in NB cells after exposure to around 10-100 µM pure curcumin (Table 1).
While such concentrations are attainable in vitro, numerous physiological challenges may hamper their clinical feasibility.
A major disadvantage of pure curcumin is its low water solubility. Organic solvents may overcome this problem; however, these solvents are rarely appropriate (or safe) for clinical use. Therefore, numerous synthetic curcumin formulations, including curcumin-alginate conjugated micelles and whey protein nanofibril carriers, have been developed to enhance the compound's solubility [103,104].
Beyond its low water solubility, the poor oral bioavailability and rapid metabolism and elimination of curcumin have been extensively characterized and reviewed [105]. The data in Table 3 indicate that curcumin has low oral bioavailability in humans, which is improved in murine and rat models. In Wistar rats (WR), peak curcumin concentrations after oral administration are reached slowly (6 h after ingestion) in the liver and kidney, even after relatively high doses. In contrast, intravenous (IV) injection substantially decreases the time to peak concentration in the same organs. Finally, maximal curcumin concentrations in the nervous system (brain and spinal cord) are attainable relatively quickly (sometimes in a matter of minutes) after oral, IV, or intranasal administration in WR, Institute for Cancer Research (ICR) mice (ICRM), and Sprague-Dawley rats (SDR). The rapid delivery of curcumin to nervous system organs underscores the compound's potential value as an anticancer agent in NB. In considering clinical administration, it is essential to note that curcumin lacks adverse side effects at dosages of up to 8000 mg/day [106]. Table 3. Bioavailability of pure curcumin and various curcumin formulations after oral, intravenous, and intranasal delivery in humans, mice, and rats. Within the sample column, "H" refers to "humans", "WR" to "Wistar rats", "ICRM" to "ICR mice", and "SDR" to "Sprague-Dawley rats".

Novel Curcumin Encapsulation and Delivery Modalities
As categorized in Tables 3-5, numerous experimental curcumin formulations have been developed to improve its delivery, oral bioavailability, and in vivo absorption. These formulations are diverse and include curcumin-protein complexes, nanoparticles, nanosuspensions, and microemulsions.

Curcumin-Protein Complexes
Curcumin-protein complexes leverage the properties of certain biomacromolecules to stabilize curcumin and optimize their delivery. Mirzaee et al. developed complexes of curcumin with bovine serum albumin (BSA), casein, and beta-lactoglobulin (β-lg). In particular, native BSA and a modified form of β-lg significantly attenuated water-mediated degradation of curcumin [89]. These curcumin-protein systems could improve the half-life of curcumin under physiological conditions, and thereby enhance its bioavailability and absorption.

Curcumin Microemulsions
In contrast with curcumin-protein complexes, microemulsions combine curcumin with oil and water. Within these microemulsions, curcumin can be combined with Capmul mono-diglyceride of medium-chain fatty acids (MCM) only (CUR Capmul ME), or both docosahexaenoic acid (DHA)-rich oil and Capmul MCM (CUR DHA ME). The injection or intranasal delivery of either microemulsion in SDR improves brain and plasma concentrations relative to standard curcumin at 1 mg/kg [111]. DHA is an essential native component of the CNS, and as such can substantially enhance the brain uptake of curcumin.

Curcumin Nanosuspensions
Nanosuspensions, which stabilize nanoscale droplets of curcumin with biopolymers or surfactants, constitute a further delivery modality. Examples of these formulations include D-α-tocopheryl polyethylene glycol 1000 succinate-coated curcumin nanosuspension (TPGS-NS) and Tween-80-coated curcumin nanosuspension (Tween-NS). TPGS-NS and Tween-NS exhibit rapid and complete dissolution in phosphate-buffered saline, compared to the slower and incomplete dissolution of pure curcumin. Moreover, the IV administration of these nanosuspensions increases curcumin concentrations in the plasma, brain, heart, and digestive system in comparison to standard curcumin at 10 mg/kg [112].

Curcumin Nanoparticles
Finally, nanoparticles comprise a widely investigated category of curcumin formulations. These encapsulate curcumin or other hydrophobic substances in solid nanostructures and may be conjugated with proteins to facilitate cell uptake. Examples include cerium oxide nanoparticles (CNP-Cur), which are pH-sensitive, and therefore selectively induce apoptosis in cancerous cells with minimal disruption to healthy cells, even at high concentrations. Application of a dextran coating to CNPs (Dex-CNP-Cur) enhances their biocompatibility and controls the release of curcumin over time [72]. Similarly to CNPs, silk fibroin nanoparticles (Curc-SFN) target cancer cells without harming healthy cells. Moreover, Montalban et al. synthesized SFNs using physical adsorption and coprecipitation-methods that could feasibly be scaled-up [113].
A further curcumin formulation utilizes polylactic-co-glycolic acid (PLGA) nanoparticles. In ICRM, these nanoparticles substantially increase curcumin concentrations in the brain, spinal cord, and plasma after oral ingestion as compared to pure curcumin at the same dosage of 20 mg/kg [109]. Additional modalities of curcumin delivery utilize lipid nanostructures, including solid lipid nanoparticles (C-SLN), stearic acid nanoparticles (C-SALN), and nanostructured lipid carriers (C-NLC) [88,110]. C-SLN and C-NLC improve brain concentrations compared to standard curcumin at 4 mg/kg in SDR, with a better brain absorption of C-NLC than C-SLN [110].

Cell Uptake and Anti-NB Efficacy of Curcumin Formulations
Protein-conjugated curcumin nanoparticles may leverage specific cellular uptake mechanisms for enhanced delivery; cellular uptake data for some formulations are detailed in Table 4. Unfortunately, only limited data specific to NB are available. Transferrin (Tf)-conjugated solid lipid nanoparticles (Tf-C-SLN) leverage Tf-receptor-mediated endocytosis to enter SH-SY5Y cells; these nanoparticles are of particular relevance, as SH-SY5Y cells overexpress the Tf receptor. Tf-C-SLN achieves a higher cellular uptake than non-Tf-conjugated curcumin SLN (C-SLN) and curcumin-solubilized surfactant solution (CSSS) [114]. Similarly, apolipoprotein-E3 curcumin-loaded poly(butyl)cyanoacrylate (ApoE3-C-PBCA) nanoparticles enter SH-SY5Y cells via endocytosis, mediated by the overexpressed low-density lipoprotein receptor (LDL-R). As such, ApoE3-C-PBCA nanoparticles achieve higher cellular uptake than non-ApoE3-conjugated curcumin PBCA nanoparticles (C-PBCA) and CSSS [115]. Finally, in the human NB cell line SH-SY5Y695 APP, a novel curcumin formulation exhibits significantly better quality and cytotoxic efficacy due to its improved cellular uptake. This formulation is well tolerated and without side effects when administered orally [116]. Table 4. Cellular uptake of several curcumin formulations, including suspension, nanoparticles, and protein-conjugated nanoparticles. The protein-conjugated nanoparticles (ApoE3-C-PBCA and Tf-C-SLN) utilize receptor-mediated endocytosis to enter NB cells and exhibit greater uptake over time than curcumin-solubilized surfactant solution (CSSS) and the corresponding non-protein-conjugated nanoparticles. The anti-NB efficacy of several curcumin formulations is demonstrable in primary experiments ( Table 5). Some of these formulations, including C-SLN, Tf-C-SLN, C-PBCA, and ApoE3-C-PBCA, exhibit statistically significant oncologic effects at low concentrations (0.5, 1, 2, 4 µM), indicating their potential clinical viability. Interestingly, other formulations, such as cerium oxide nanoparticles (CNP-Cur) and dextran-CNP-Cur (Dex-CNP-Cur), have only been tested or demonstrate significant effects at higher (and possibly clinically inviable) concentrations of 100 and 250 µM. Finally, the ability of some formulations, including CNP-Cur and Curc-SFN, to target NB cells with minimal collateral damage will be valuable. In the meantime, clinically relevant studies continue. Table 5. Anticancer effects of various curcumin formulations, including protein-conjugated nanoparticles and curcumin-protein complexes, on NB cell lines. While a variety of novel curcumin formulations successfully induce NB cell death, their specific mechanistic actions remain unclear. Downregulations, decreases, and inhibitions are in bold.

Effect
Cell

Patterns in NB Cell Line Usage in Curcumin Studies
Recent preclinical studies examined the effects of curcumin on diverse human and murine NB cell lines. As enumerated in Table 1, studies utilizing pure curcumin were conducted with a wide range of cell lines, some of which exhibit MYCN amplification or 11q aberration. A similar breadth can be observed for the reviewed synergistic experiments (Table 2). However, all cell uptake data arise from a single cell line, SH-SY5Y (Table 4). Finally, the data in Table 5 indicate that studies with curcumin formulations were performed with both MYCN-amplified and non-MYCN-amplified cell lines.

Apoptotic Mechanisms
Curcumin exhibits a wide variety of apoptotic effects when applied to NB cell lines; however, many of the compound's individual mechanistic effects are only documented in a few cell lines (Table 1). For example, the modulation of several mitochondrial proteins (Hsp60, Bad, and Bim), the downregulation of HKII, and the upregulation of PTEN, FOXO3a, Fas-L, and p27 are reported only in the MYCN-amplified LAN5 cell line. Induction of the Bex genes and downregulation of proliferative ERK 1/2 signaling are demonstrated only in murine Neuro2a cells. The activation of caspases 9 and 3 is an essential downstream apoptotic mechanism; however, caspase 3 activation is reported in only three NB cell lines (Neuro2a, SH-SY5Y, and GL-LI-N), and caspase 9 activation in only Neuro2a cells. Finally, reductions in cell viability and upregulation of apoptosis are broadly documented.

Synergistic Effects
The synergistic effects of curcumin with natural substances, anticancer drugs, and radiation therapy are documented in only five NB cell lines (Table 2). At present, the synergistic efficacy of curcumin-cisplatin is confirmed only in SK-N-AS cells, while the synergy of curcumin-BBR and curcumin-piperine is limited to SH-SY5Y cells. Moreover, curcumin was trialed with radiation therapy only in SK-N-MC cells. Curcumin-doxorubicin treatment has a greater breadth of efficacy, with demonstrable effects in the MYCN-amplified and 11q-abberated SK-N-Be(2) and IMR-32 cell lines, as well as in the non-MYCN-amplified SK-N-AS and SH-SY5Y lines.

Curcumin Formulations
Broadly, novel curcumin formulations demonstrate efficacy against a wide variety of NB types and cell lines. The breadth of cell uptake studies with these formulations is highly limited-many formulations have not yet been evaluated, while the two reviewed studies utilized SH-SY5Y cells exclusively (Table 4). Moreover, preclinical oncologic studies with these formulations yielded promising general outcomes, but few specific details and limited mechanistic insights.  [72]. These results are of particular interest, as MYCN amplification typically enhances NB tumorigenesis but does not independently lead to drug resistance [117].

Repetition of Mechanistic Trials and Synergistic Studies Across NB Cell Lines
Specific insights on curcumin's isolated and synergistic anti-NB activities remain relatively sparse. In particular, important apoptotic mechanisms such as PTEN, FOXO3a, and Fas-L upregulation; ERK 1/2 downregulation; Bex gene induction; several components of mitochondrial dysfunction; and caspase 9 activation have each only been reported in a single NB cell line. Among these, confirmation of Bex gene induction and ERK 1/2 downregulation is limited to murine Neuro2a cells. Similarly, synergy between curcumin and BBR, piperine, cisplatin, and radiotherapy are also each limited to one cell line. As such, cross-validation of curcumin's effects between different types of NB cell lines (e.g., murine, human, (non-)MYCN-amplified, (non-)11q aberrated, etc.) is significantly lacking. Further experiments are therefore necessary to evaluate the generalizability of curcumin's apoptotic pathways across NB cell lines.

Further Testing and Standardization of Curcumin Formulations
While curcumin formulations are promising, their safety and efficacy, as well as the generalizability of their effects across NB cell lines, merit special consideration. As discussed in Section 9.1 above, curcumin formulations should be tested on a variety of NB cell lines and types. Moreover, each research group or organization currently synthesizes formulations according to its own internal procedures. As such, these formulations may vary substantially between laboratories and even between different batches from the same laboratory. While curcumin should be further investigated as an anticancer agent in NB, it is important to achieve an agreement on one or several standardized curcumin formulations before the commencement of clinical trials.

Investigation of the Effects of Curcumin on NB Cell Metabolism
Given recent developments in curcumin delivery systems and their efficacy against NB cell lines in primary studies, curcumin is a promising candidate for the clinical treatment of NB. However, many more experiments are needed before the introduction of curcumin for clinical trials. In particular, the effects of curcumin on NB cell glucose metabolism merit further investigation. Cancerous cells notably exhibit altered metabolic processes, which favor aerobic glycolysis over oxidative phosphorylation; this is known as the Warburg effect and has been demonstrated in NB [84]. Curcumin suppresses the Warburg effect in lung, breast, cervical, and prostate cancer cell lines, but this has not been demonstrated in NB [85]. The literature on curcumin and glucose metabolism in NB is sparse. D'Aguanno and colleagues reported that curcumin downregulates biosynthesis, glucose uptake, glycolysis, and metabolic enzymes specific to glycolysis, such as LDH, ALDOA, GAPDH, TPI1, and ENO1 [83]. Notably, epigenetic modifications that occur during tumor progression are potentially reversible, with consequent diminishment of the Warburg effect [118,119]. These results suggest that the Warburg effect, in which decreased vascularization, nutrient deprivation, and hypoxia lead to a tumor microenvironment with low pH, is a reversible process that may be targeted by curcumin. These observations could potentially also explain the anabolic and catabolic actions of phytochemicals in healthy and tumor cells, respectively. Overall, additional studies are necessary to fully elucidate the interactions between curcumin and the Warburg effect in NB.

Investigation of the Effects of Curcumin on NB Drug Resistance
Cancer drug resistance is a major detrimental side effect of conventional chemotherapeutic regimens. A wide variety of cellular factors, including microRNAs, regulate NB resistance to cisplatin and doxorubicin [120,121]. Correspondingly, NB drug resistance tends to increase with the strength of chemotherapeutic regimens [117]. In this light, supplementation of such regimens with curcumin, a complementary and supportive agent, could theoretically reduce the necessary dosage(s) of anticancer drug(s), and thereby decrease cancer drug resistance. However, further combinatory trials of curcumin with anticancer drugs are necessary to characterize curcumin's potential impact on drug resistance.

Clinical Trials of Curcumin in NB
Curcumin has been clinically trialed in combination with chemotherapy against several cancer types, such as breast cancer, pancreatic cancer, and chronic myeloid leukemia [122]. Although the concept is promising, no clinical trials for curcumin-chemotherapy in NB are available at this time. Moreover, in situ factors, including a tumor's specific location within the body, the tumor microenvironment, and tumor homogeneity or heterogeneity, are not easily investigated in vitro. However, they could significantly influence curcumin's clinical efficacy.
As an independent agent or in conjunction with radiation or chemotherapy, curcumin can theoretically be administered in very high doses. Nevertheless, patient convenience and preference could be a limiting factor in oral administration, as the safe dosage of 8000 mg per day would necessitate the swallowing of sixteen 500 mg capsules per day or an equivalent dose. Despite curcumin's safety and lack of side effects, patients may not be comfortable ingesting such large amounts.

Conclusions and Outlook
Curcumin, a naturally occurring chemical constituent of turmeric, is a promising chemopreventive and chemotherapeutic agent for a wide variety of cancers, including NB. In physiological systems, curcumin achieves its anticancer effects through the downregulation of proliferative Akt and NF-κB signaling, induction of mitochondrial dysfunction and cytochrome c release, upregulation of p53, and caspase activation, all of which lead to apoptosis. Curcumin also demonstrates synergistic effects with other natural substances, conventional chemotherapeutic drugs, and radiation therapy against NB cells.
Clearly, curcumin, as a multitargeting agent, exhibits strong activity against established tumors, with negligible toxicity to normal cells. It, therefore, has a high potential for clinical applications. Notable shortcomings, such as single-targeting and adverse toxicity, exist for current chemo-and radiotherapy treatments for NB. As such, NB treatment should be combined with more effective or safer compounds. Current evidence indicates that multitargeted curcumin may be a safe and highly effective supplement in clinical tumor control. In combination with curcumin, lower doses of chemotherapeutic drugs and radiation could achieve high antitumor efficacy, as well as low toxicity and drug resistance. In addition, curcumin has potential as an agent for safe and effective cancer prevention, treatment, and prophylaxis after chemotherapy and radiation. As such, the compound may be relevant in preventing tumor recurrence and metastasis.
Despite curcumin's anti-NB efficacy, many of its mechanistic and synergistic activities are only demonstrated in one or several cell lines at present. As such, further testing and cross-validation with various types of NB cell lines is necessary to elucidate the impact of risk factors such as MYCN amplification on the efficacy of curcumin treatment.
Moreover, while curcumin has the potential to attenuate NB progression and counter cancer drug resistance, its clinical viability is limited by its poor oral bioavailability, low water solubility, and rapid metabolism. Numerous novel curcumin formulations-including nanoparticles, lipid carriers, nanosuspensions, and microemulsions-have been proposed to overcome these challenges. While pure curcumin is clinically safe and has minimal side effects, further basic and preclinical trials are necessary to ensure the safety of novel curcumin formulations. Ultimately, clinical trials could support or refute the viability of curcumin as an anticancer agent in NB treatment.  Brain-Expressed X-linked (gene) 1 Bex2 Brain-Expressed X-linked (gene) 2 Bex3 Brain-Expressed X-linked (gene) 3 Bex4 Brain-Expressed X-linked (gene) 4 Bex6 Brain-Expressed X-linked (gene)